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Home , Chromerida, Cori (lunar crater), Fotis Kafatos, Hammondia, Keith Gull, Laverania

MPM XXX 2019

15-19 S E P T

molecular parasitology

meeting

marine biological laboratory woods hole, ma

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2019 - 30th Annual Molecular Parasitology Meeting

Sunday, September 15 2:00-5:00p Registration Swope Center 5:00-6:30p Dinner Swope Dining Hall 6:45-7:00p Welcome to MPM Lillie Auditorium 7:00-9:00p Session I Lillie Auditorium 9:00-11:00p Mixer Swope Meigs Room

Monday, September 16 7:00-8:30a Breakfast Swope Dining Hall 9:00-10:10a Session II Lillie Auditorium 10:10-10:30a Coffee Break 10:30-11:30a Session III Lillie Auditorium 11:30a-1:00p Lunch Swope Dining Hall 1:00-2:15p Session IV Lillie Auditorium 2:15-2:30p Coffee Break 2:30-3:30p Session V Lillie Auditorium 3:30-5:00p Free Time/ Parasite Games with Selina & Omar 5:00p-7:00p BBQ Dinner Swope Dining Hall 7:00-9:00p Poster Session: A Swope Center 8:00-10:00p Mixer Swope Meigs Room

Tuesday, September 17 7:00-8:30a Breakfast Swope Dining Hall 9:00-10:15a Session VI Lillie Auditorium 10:15-10:30a Coffee Break 10:30-11:30a Session VII Lillie Auditorium 11:30-1:00p Lunch Swope Dining Hall 1:00-2:15p Session VIII Lillie Auditorium 2:15-2:30p Coffee Break 2:30-3:30P Session IX Lillie Auditorium 3:30-5:00p Free Time Power hour: 3pm-4pm with Kirk Deitsch and Nina Papavasiliou

An informal session for all attendees to discuss challenges women face in science: Data on disparity will be presented, and then assigned to breakout groups to discuss and solicit solutions for group discussion.

5:00p-7:00p Dinner Swope Dining Hall 7:00-9:00p Poster Session: B Swope Center 8:00-10:00p Mixer Swope Meigs Room

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Wednesday, September 18 7:00-8:30a Breakfast Swope Dining Hall 9:00-10:15a Session X Lillie Auditorium 10:15-10:30a Coffee Break 10:30-11:30a Session XI Lillie Auditorium 11:30-1:00p Lunch Swope Dining Hall 1:00-3:00p Free Time 3:00-5:00p Poster Session: C Swope Center 4:00-5:30p Mixer Swope Meigs Room 5:30p-7:00p Keynote Lecture Lillie Auditorium 7:30-9:00p Lobster Banquet Swope Dining Hall 9:00-12:00a Dance Party/Mixer Captain Kidd

Thursday, September 19 7:00-8:30a Breakfast Swope Terrace 10:00a Housing Checkout by 10:00a 9:00-10:10a Session XII Lillie Auditorium 10:10-10:30a Coffee Break 10:30-11:30a Session XIII Lillie Auditorium 11:30-1:00p Lunch & Departure Swope Dinning Hall

*Note: Please remove your Poster by Lunch time the day following your presentation.

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Science at the 2019 Molecular Parasitology Meeting Code of Conduct PRINCIPLES: Scientific meetings are important mechanisms for rapid advancement and dissemination of new ways of thinking, particularly for pressing public health challenges such as parasitology. Clarity and openness from meeting participants is necessary for useful community feedback, appreciation, and debate. THE CODE: To meet the goals of this meeting, it is each presenter’s responsibility and duty to make claims that are exact and transparent. These claims should be supported with as much specific documentation as possible, within the time frame of the presentations. SPECIFIC EXAMPLES: Conclusive functional genetic studies should be accompanied by gene names and database references. Discovery of new inhibitors or drug candidates should be accompanied by structures and quantitative descriptions of potency and selectivity. New diagnostic approaches should be accompanied by actual names, structures, or signatures of key analytes, when known. It should be possible to evaluate the value of new experimental methods for manipulating cells or analyzing their content, based on the experimental details provided. WHY A CODE? The organizers merely offer some reminders for scientific conduct based on some time-proven principles. The 2007 MPM involved passionate pleas from many for more openness in some basic research presentations. Others argued for the need to protect potentially important intellectual property (IP). This simple code was assembled in close consultation with key leaders in the molecular parasitology community. INTELLECTUAL PROPERTY AND CHOICES: In an age of translational research, the community fully recognizes the need for IP protection. Important data, or an experimental approach, that is not ready for disclosure can be protected until it is time. Yet, one cannot have it both ways: Vague descriptions of critical data or method should not be the central part of one’s premature published abstract or public presentation. REWARDS AND PENALTIES: The MPM is organized and supported by practicing scientists who choose to engage. No individual has judicial powers. Abstracts and talks that lack specifics, and merely posture, may meet community-wide disappointment. In contrast, detailed presentations which offer new insights of general use have always been promoted and celebrated. We hope an understanding and regular observance of this code of conduct will add to another exciting scientific meeting.

Boris Striepen, Manoj Duraisingh, Nina Papavasiliou

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ACKNOWLEDGEMENTS:

The organizers appreciate and thank the following:

• Susan Waddington-Pilder (Penn) for organization • Omar Harb (Penn) and Selina Bopp (Harvard) for polling and activities • Caroline Keroack (Harvard) for T-Shirt design Site support at the MBL, Woods Hole, MA: • Nipam Patel, Director, Marine Biological Laboratory; • Kerri Mills and Paul Anderson for Conference support; • Cathy Ham, Suzanne Livingstone, Beth Sexany, Barbara Stackhouse and Nancy Hadway for Housing and the Front Office; • Erin Hummetolgu and Sodexo Dining Services staff; • Chad Baptiste, Jessica Berrios, Crystal Santiago, Cathy Hannigan, and all of the staff in Housekeeping; • Richard Boudreau of the IT/AV Department for Web booths and other support, and all the staff in the Information Technology Department.

Award sponsors: • Travel Awards: Special thanks to all the participants who donated during registration. • The American Society for Tropical Medicine and Hygiene and Burroughs Wellcome Fund facilitated travel and speaking slots at ASTMH meetings. • The Bill and Melinda Gates Foundation supported travel in particular for attendees from disease endemic countries • Travel and Cash awards: PLos Pathogens and PLos Biology

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In Memoriam: Paul T. Englund, Ph.D. 1938 - 2019

“Paul was the consummate scientist, mentor, colleague and human being.”

Dr. Paul T. Englund, professor emeritus and Johns Hopkins scientist, passed away January 12, 2019. While most of Paul’s fifty- year career focused on research involving the African trypanosome, the many contributions he made had implications beyond the field of molecular and biochemical parasitology and have altered the way we think about important cellular processes. These include advances in understanding the biosynthetic pathway of GPI anchors and fatty acids and deep insight into DNA replication, particularly of mitochondrial DNAs in kinetoplastid parasites. Paul was a brilliant and creative scientist whose excitement and enthusiasm for research never diminished. The impact of this work has been recognized by his many awards including Paul being elected to the National Academy of Sciences in 2012.

Paul's educational efforts included leadership, for seven years, of the NIH- funded , Cellular and (BCMB) training program at John Hopkins, and co-directorship, of the Marine Biological Laboratory summer course in the Biology of (1993-1994). In 2016, the remarkable contributions of his trainees and colleagues established an endowed Paul and Christine Englund Professorship in the Department of Biological Chemistry and an endowed lectureship in the Biology of Parasitism course at MBL.

Paul’s influence and impact on molecular parasitology was immeasurable, not only because of his expertise and passion for science, but most importantly because of his thoughtful, nonjudgmental, supportive mentoring style. For many, the deep and booming voice of a question from the audience in Lilly auditorium was the first encounter they had with Paul. While this could initially be intimidating, the legion of trainees that passed through Paul’s lab as fellows or students will attest to the depth of Paul’s warmth and generosity. He encouraged curiosity, and celebrated successes, and he will be missed greatly.

Michele Klingbell, James Morris and Jayne Raper 6

Molecular Parasitology Keynote Lectures

2019: When you come to a fork in the road, take it! New paradigms emerging from glycobiology and virology Stephen Beverley, Washington University, St Louis

2018: The Hidden Life of African Trypanosomes Stephen L Hajduk, Department of Biochemistry & Molecular Biology University of Georgia

2017: African trypanosomes and the art of being social Isabel Roditi, Institute of Cell Biology, University of Bern

2016: sexual development: A challenging journey from the shadows into the spotlight Robert Sinden Emeritus Proefssor, Imperial College, UK

2015: A Physician-scientist’s investigtion of amebiasis Bill Petri University of Virginia, Charlottesville, VA.

2014: Molecular Malariology and Global Health: Discoveries, Predictions, Tests of Time Thomas Wellems NIAID, NIH,

2013: Navigating the trypanosome RNA jungle. Elisabetta Ullu Yale School of Medicine,

2012: Pit bull or poodle? How are different breeds and developmental forms of Toxoplasma perfectly suited (or not.) to the challenge de jour. John Boothroyd Stanford School of Medicine

2011: : it's all about persistence. George Cross The Rockefeller University,

2010: The Major Challenges to Global Health in the Tropics & Beyond— from Vectors of Malaria & of Other Parasitic or Viral Diseases , London.

2009: Understanding Parasites: A Foundation for their Elimination. Ken Stuart Seattle Biomedical Research Institute,

2008: J, etcetera Piet Borst The Netherlands Cancer Institute, Netherlands

2007: Rationale for the development of a malaria vaccine Victor Nussensweig New York University,

2006: From genes to genomes: Insights into parasite biology Dyann Wirth ,

2005: How trypanosomes make fatty acids. Paul Englund The Johns Hopkins University, Baltimore, MD.

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MPM Organizers 1990 – 2019

Molecular Parasitology Meeting XXX, September 2019 Meeting organizers: Boris Striepen, Manoj Duraisingh, Nina Papavasiliou

Molecular Parasitology Meeting XXIX, September 2018. Meeting organizers: Markus Meissner, Jayne Raper, Kirk Deitsch

Molecular Parasitology Meeting XXVIII, September 2017. Meeting organizers: Jayne Raper, Kirk Deitsch, Markus Meissner

Molecular Parasitology Meeting XXVII, September 2016. Meeting organizers: Kirk Deitsch, Jayne Raper, Markus Meissner

Molecular Parasitology Meeting XXVI, September 2015. Meeting organizers: Isabel Roditi, Upi Singh, Akhil Vaidya

Molecular Parasitology Meeting XXV, September 2014. Meeting organizers: Upi Singh, Isabel Roditi, Akhil Vaidya

Molecular Parasitology Meeting XXIV, September 2013. Meeting organizers: Akhil Vaidya, Upi Singh, Isabel Roditi

Molecular Parasitology Meeting XXIII, September 2012. Meeting organizers: Artur Scherf, Kami Kim, Noreen Williams

Molecular Parasitology Meeting XXII, September 2011. Meeting organizers: Noreen Williams, Kami Kim, Artur Scherf

Molecular Parasitology Meeting XXI, September 2010. Meeting organizers: Kami Kim, Noreen Williams, Artur Scherf

Molecular Parasitology Meeting XX, September 2009. Meeting organizers: Pradip Rathod, Dominique Soldati, Jay Bangs

Molecular Parasitology Meeting XIV, September 2008. Meeting organizers: Dominique Soldati, Pradip Rathod, Jay Bangs

Molecular Parasitology Meeting XVIII, September 2007. Meeting organizers: Jay Bangs, Dominique Soldati, Pradip Rathod

Molecular Parasitology Meeting XVII, September 2006. Meeting organizers: David Sibley, Meg Phillips, Andy Waters

Molecular Parasitology Meeting XVI, September 2005. Meeting organizers: David Sibley, Meg Phillips, Andy Waters

Molecular Parasitology Meeting XV, September 2004. Meeting organizers: David Sibley, Meg Phillips, Andy Waters

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Molecular Parasitology Meeting XIV, September 2003. Meeting organizers: Steve Hajduk, David Roos, Debbie Smith

Molecular Parasitology Meeting XIII, September 2002. Meeting organizers: Steve Hajduk, David Roos, Debbie Smith

Molecular Parasitology Meeting XII, September 2001 Meeting organizers: Steve Hajduk, David Roos, Debbie Smith

Molecular Parasitology Meeting XI, September 2000. Meeting organizers: Christine Clayton, Kasturi Haldar, and Buddy Ullman

Molecular Parasitology Meeting X, September 1999. Meeting organizers: Christine Clayton, Kasturi Haldar, and Buddy Ullman

Molecular Parasitology Meeting IX, September 1998. Meeting organizers: Christine Clayton, Kasturi Haldar, and Buddy Ullman

Molecular Parasitology Meeting VIII, September 1997. Meeting organizers: Daniel Goldberg, Marilyn Parsons, and Elisabetta Ullu

Molecular Parasitology Meeting VII, September 1996. Meeting organizers: Daniel Goldberg, Marilyn Parsons, and Elisabetta Ullu

Molecular Parasitology Meeting VI, September 1995. Meeting organizers: Daniel Goldberg, Marilyn Parsons, and Elisabetta Ullu

Molecular Parasitology Meeting V, September 1994. Meeting organizers: Stephen Beverley, Paul Englund, and Barbara Sollner- Webb

Molecular Parasitology Meeting IV, September 1993. Meeting organizers: Stephen Beverley, Paul Englund, and Barbara Sollner- Webb

Molecular Parasitology Meeting III, September 1992. Meeting organizers: Jeffrey Ravetch, Lex Van der Ploeg, and Dyann Wirth

Molecular Parasitology Meeting II, September 1991. Meeting organizers: Jeffrey Ravetch, Lex Van der Ploeg, and Dyann Wirth

Molecular Parasitology Meeting I, September 1990. Meeting organizers: Jeffrey Ravetch, Lex Van der Ploeg, and Dyann Wirth

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Travel Awardees

Redox metabolomics and gametocytogenesis in the malaria parasite Beri, Divya (Indian Institute of Science); Tatu, Utpal (Indian Institute of Science, Bangalore, India)

A Lysophospholipase involved in malaria parasite development: Potential targets for drug development Mohmmed, Asif (International Centre for Genetic Engineering and Biotechnology.); Kumar, Pradeep (International Centre For Genetic Engineering and Biotechnology New Delhi); Botté, Yoshiki Yamaryo (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes, la tronche.); Thakur, Vandana (International Centre for Genetic Engineering and Biotechnology.); Botté, Cyrille (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes, la tronche.)

Functional characterizacion of Trypanosoma cruzi surface mucins in the of the invertebrate host Camara, Maria de los Milagros; Balouz, Virginia (IIB UNSAM, Buenos Aires, Argentina); Centeno Camean, Camila (IIB UNSAM, buenos aires, Argentina); Cori, Carmen (CHIDECAR UBA, buenos aires, Argentina); Lobo, Maite Mabel (IIB UNSAM, buenos aires, Argentina); Lederkremer, Rosa (CHIDECAR, buenos aires, Argentina); Gallo-Rodriguez, Carola (CHIDECAR UBA, buenos aires, Argentina); Buscaglia, Carlos Andres (IIB UNSAM, buenos aires, Argentina)

Trypanosoma cruzi mitochondrial pyruvate carrier (MPC) subunits do not form homodimers and are essential for pyruvate- driven respiration Negreiros, Raquel S. (State University of Campinas); Vercesi, Anibal E. (State University of Campinas, Campinas, Brazil); Docampo, Roberto (University of Georgia, Athens, GA.)

BioPalMar Best Talks – Registration Awards

Plasmodium falciparum immature gametocytes development in human primary erythroblasts Neveu, Gaëlle (INSERM, Cochin Institute); Richard, Cyrielle; Volpe, Fiona; Dupuy, Florian (INSERM, Cochin Institute); Annamalai, Pradeep (UPMC Faculté de Medecine); Naissant, Bernina (INSERM, Cochin Institute); Martins, Raphael M (CNRS5290, IRD224, University Montpellier 1 & 2, MIVEGEC); Vallin, Patrice; Andrieu, Muriel (INSERM, Cochin Institute); Lopez-Rubio, José-Juan (CNRS5290, IRD224, University Montpellier 1 & 2, MIVEGEC); Mazier, Dominique (UPMC Faculté de Medecine); Verdier, Frédérique; Lavazec, Catherine (INSERM, Cochin Institute)

IPS cell technology: A great tool to study hypnozoites and pave the road towards malaria elimination Pellisson, Melanie Anne-Marie Zeeman4, Thierry Doll, Annemarie Voorberg-van der Wel, Lucy Kirchhofer-Allan, Sven Schuierer, Guglielmo Roma, Erika Flannery, Sebastian Mikolajczak, Clemens H. M. Kocken, Pascal Mäser, Matthias Rottmann, Matthias Müller 10

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Session I: I’m in Love with the Shape of You: From Structure to Function Chair: Alexis Kaushansky and Chris de Graffenried Sunday, September 15th, 2019 7:00pm – 9:00pm

1 - Nanoscale Elucidation of the Invasion Apparatus of Apicomplexan Parasites Segev Zarko, Li-av (Stanford University); Sun, Stella; Dahlberg, Peter (Stanford University); Pelt, Danile (University of California Berkeley); Chen, Jian-Hua (Lawrence Berkeley National Laboratory, Berkeley.); Schmid, Michael; Galaz Montoya, Jesus; Moerner, W.E. (Stanford University); Larabell, Carolyn (University of California San- Francisco); Sethian, James (University of California Berkeley); Chiu, Wah; Boothroyd, John (Stanford University)

2 - An ORFeome-based Gain-of-Function library for suggests mitochondria as a key player melarsoprol killing and resistance Carter, McKenzie R. (The George Washington University); Kim, Hee-Sok (Rutgers NJ Medical School); Gomez, Stephanie; Gritz, Sam (The George Washington University); Schulz, Danae (Harvey Mudd College); Hovel-Miner, Galadriel (The George Washington University)

3 - Bottom-up structural proteomics: cryoEM of protein complexes enriched from malaria parasites Ho, Chi-Min (UCLA Dept. of Microbiology, & Molecular Genetics); Li, Xiaorun (California NanoSystems Institute); Lai, Mason (UCLA Dept. of Microbiology, Immunology & Molecular Genetics); Terwilliger, Thomas C. (Los Alamos National Laboratory and the New Mexico Consortium); Beck, Josh R. (Iowa State University Dept. of Biomedical Sciences); Wohlschlegel, James A. (UCLA Dept. of Biological Chemistry); Goldberg, Daniel E. (Washington University School of Medicine in St. Louis Depts. of Medicine and Microbiology); Fitzpatrick, Anthony WP (Zuckerman Institute, Columbia University Medical School); Zhou, Z. Hong (UCLA Dept. of Microbiology, Immunology & Molecular Genetics)

4. - Going “forward” with Leishmania classical genetics Alves-Ferreira, Eliza (NIH); Inbar, Ehud (NIH); Ferreira, Tiago (NIH); Khan, Asis (NIH); Lack, Justin; Sacks, David; E. Grigg, Michael (NIH)

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5t - New toolkit for high throughput imaging and image analysis of the chronic stage of Winiger, Rahel R. (Laboratory of Molecular Parasitology); Fisch, Daniel (Host- Toxoplasma Interaction Laboratory, London.); Yakimovich, Artur (MRC-Laboratory for Molecular Cell Biology); Hammoudi, Pierre-Mehdi (Department of Microbiology and Molecular Medicine, Geneva); Ramakrishnan, Chandra (Laboratory of Molecular Parasitology, Zürich); Soldati-Favre, Dominique (Department of Microbiology and Molecular Medicine, Geneva); Hehl, Adrian B. (Laboratory of Molecular Parasitology)

6t - Three-dimensional ultrastructure of falciparum throughout cytokinesis Rudlaff, Rachel M. (Harvard Medical School); Kraemer, Stephan (Harvard University); Marshman, Jeffrey (Carl Zeiss Microscopy); Dvorin, Jeffrey D. (Harvard Medical School)

7t - VSG mosaics: when the color of your shoes determines the shape of your hat. silva (Deutsches Krebsforschungzentrum); Erben, Esteban; Zeelen, Johan P.; Stebbins, Erec C.; Papavasiliou, Nina F. (Deutsches Krebsforschungzentrum, Heidelberg.)

8t - Analysis of the Toxoplasma mitochondrial ATP synthase structure reveals how its shapes the unique cristae and highlights the role of specific subunits Sheiner, Lilach (University of Glasgow)

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Session II: Will the Circle be Unbroken: Cell Cycle - I Chair: Paul Sigala and Galadriel Hovel-Miner Monday, September 16th, 2019 9:00am – 10:30am

9 - A divergent cyclin/cyclin-dependent kinase complex controls progression through S- and M- phases during gametogony Balestra, Aurélia (Université de Genève); Rea, Edward; Zeeshan, Mohammad (University of Nottingham); Klages, Natacha (Université de Genève); Brusini, Lorenzo (University of Nottingham); Ferguson, David (Nuffield Department of Clinical Laboratory Science, .); Mourier, Tobias (Computational Bioscience Research Centre); Brady, Declan (University of Nottingham); Pain, Arnab (Computational Bioscience Research Centre); Tewari, Rita (University of Nottingham); Brochet, Mathieu (Université de Genève)

10 - Deciphering the spatiotemporal organization of DNA replication during schizogony Ganter, Markus (Center for Infectious Disease, Parasitology, Heidelberg University); Klaus, Severina; Machado, Marta; Klaschka, Darius; Guizetti, Julien (Center for Infectious Disease, Parasitology, Heidelberg University)

11 - Calcium Influx through a Transient Receptor Potential Channel and its impact in the Toxoplasma lytic cycle Márquez-Nogueras, Karla (University of Georgia); Kuo, Ivana (Loyola University Chicago); Moreno, Silvia (University of Georgia)

12 - A Trypanosoma brucei orphan kinesin uses an ATP-dependent sorting mechanism to re-organize into a parallel bundle to form the new posterior end during cytokinesis Sladewski, Thomas , Paul; Sinclair, Amy; de Graffenried, Christopher (Brown University)

13t - Mapping of origins of replication in Plasmodium falciparum Gomes, Ana Rita (DIMNP, Université de Montpellier) CHU de Montpellier); Méchali, Marcel (Institute de Génétique Humaine, Montpellier.); Lopez-Rubio, Jose-Juan (DIMNP, Université de Montpellier)

14t - Single-cell transcriptomes of parasites reveal extensive but continuous changes in gene expression during their intraerythrocytic cycle Serre, David (University of Maryland School of Medicine); , Matthew (University of Maryland School of Medicine); Caleon, Ramon; Wellems, Thomas; Sa, Juliana (Laboratory of Malaria and Vector Research) 16

Session III: Under Pressure: Host-Parasite Metabolicsm Chairs: Galadriel Hovel-Miner and Paul Sigala Monday, September 16th, 2019 10:30am – 11:30am

15 - LIPIN: a pivotal nexus in Toxoplasma lipid metabolism, channelling host-derived FAs to storage, preventing Lipotoxicity Dass, Sheena (Apicolipid Team, IAB, CNRS UMR5309) Berry, Laurence (UNIVERSITÉ DE MONTPELLIER UMR 5235 (DIMNP)); Katris, Nicholas; Yamaryo- Botté, Yoshiki; Botté, Cyrille Y. (Apicolipid Team, IAB, CNRS UMR5309)

16 - Impact of Leishmania donovani infection on host macrophage mitochondrial metabolism, integrity and function Acevedo Ospina, Hamlet (INRS-Armand-Frappier Santé Biotechnologie); Descoteaux, Albert (INRS-Armand-Frappier Santé Biotechnologie, Laval, Canada)

17 - Mitochondrial acetyl-coA biosynthesis is essential during the red blood stages of human malaria parasite Plasmodium falciparum. Nair, Sethu C. (Johns Hopkins University); Munro, Justin; Llinas, Manuel (Penn State University, University Park.); Prigge, Sean T. (Johns Hopkins University, Baltimore.)

18 - Divergent Acyl Carrier Protein Coordinates Mitochondrial Fe-S Cluster Metabolism in Malaria Parasites Falekun, Seyi P. (Department of Biochemistry, University of Utah); Jami, Yasi (Department of Biological Chemistry, University of California Los Angeles); Park, Hahnbeom (Institute for Protein Design, University of Washington); Wohlschlegel, James A. (Department of Biological Chemistry, University of California Los Angeles); Sigala, Paul A. (Department of Biochemistry, University of Utah)

19t - Trypanosoma congolense cytoadheres to the brain vasculature Silva Pereira, Sara (Instituto de Medicina Molecular - João Lobo Antunes); De Niz, Mariana; Figueiredo, Luisa M. (Instituto de Medicina Molecular - João Lobo Antunes)

20t - Functional study of two Trypanosoma cruzi receptors (TcIP3R and TczAC) involved in signal transduction and important for parasite survival Lander, Noelia (University of Georgia); Chiurillo, Miguel A.; Docampo, Roberto (University of Georgia)

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Session IV: Another One Bites the Dust: Drug Targets Chairs: Vicki Jeffers and Adam Sateriale Monday, September 16th, 2019 1:00pm – 2:15pm

21 - Finding novel therapies against amebic parasites by screening the ReFRAME library Marcolino, Monica Kangussu (Stanford University); Ehrenkaufer, Gretchen (Stanford University); Chen, Emily (Calibr Institute); Debnath, Anjan (University of California, San Diego); Singh, Upinder (Stanford University)

22 - Pharmacological Disruption of an ApiAP2 Transcription Factor in the Human Malaria Parasite Plasmodium falciparum Russell, Timothy J. (Department of Biochemistry and Molecular Biology and Center for Malaria Research (CMaR), Pennsylvania State University); De Silva, Erandi K. (Myonexus Therapeutics, Inc.); Crowley, Valerie M. (University Health Network); Shaw-Saliba, Kathryn; McClean, Kyle J. (Molecular Biology and Immunology, Johns Hopkins Bloomberg School of Public Health); Josling, Gabrielle (Department of Biochemistry and Molecular Biology and Center for Malaria Research (CMaR), Pennsylvania State University); Panagiotou, Gianni (Leibniz Institute for Natural Products Research and Infection Biology, Hans Knöll Institute); Jacobs-Lorena, Marcelo (Molecular Biology and Immunology, Johns Hopkins Bloomberg School of Public Health); Llinás, Manuel (Department of Biochemistry and Molecular Biology and Center for Malaria Research (CMaR), Department of Chemistry, Pennsylvania State University)

23 - From Phenotypic Screens to Mode of Action: A Metabolomics Approach to Guide the Development of Novel Anti-Trypanosomal Drugs Srivastava, Anubhav (Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne) Creek, Darren J. (Monash Institute of Pharmaceutical Sciences, Monash University)

24 - Comparative chemical genomics in species identifies a novel apicomplexan drug target Keroack, Caroline D. (Harvard T. H. Chan School of Public Health); Elsworth, Brendan; Tennessen, Jacob A.; moreira, Cristina K.; Paul, Aditya S.; Duraisingh, Manoj T. (Harvard T. H. Chan School of Public Health)

25t - Differential Expression and Localization of Cytochrome b5 in Giardia intestinalis during Nitrosative Stress Response Mowat, Kaitlyn (Trent Univeristy);

26t - A Lysophospholipase involved in malaria parasite development: Potential targets for drug development Mohmmed, Asif (International Centre for Genetic Engineering and Biotechnology.); Kumar, Pradeep (International Centre For Genetic Engineering and Biotechnology New Delhi); Botté, Yoshiki Yamaryo (Institute for Advanced Biosciences, University Grenoble Alpes); Thakur, Vandana (International Centre for Genetic Engineering and Biotechnology.); Botté, Cyrille (Institute for Advanced Biosciences, University Grenoble Alpes.) 18

Session V: Highway to Hell: Functional Analysis Chairs: Adam Sateriale and Vicki Jeffers Monday, September 16th, 2019 2:30pm – 3:30pm

27 - Divide and conquer: using FACS and RNAi screening to identify novel factors for mtDNA replication/segregation in trypanosomes Miskinyte, Migla (University of Edinburgh); Ivens, Alasdair; Waterfall, Martin; Schnaufer, Achim (University of Edinburgh)

28 - A genome-scale reverse genetic screen to identify functional determinants controlling Toxoplasma gondii sexual cycle Sardinha-Silva, Aline (Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH); Yanes, Olivia (Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH); Vavrušková, Zuzka; Lukeš, Julius (Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Ceské Budejovice (Budweis), Ceské Budejovice.); Grigg, Michael E. (Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH)

29 - Identification of the Plasmodium falciparum Acetyl-CoA Synthetase as an emerging antiplasmodial drug target Summers, Robert L. (Harvard T.H. Chan School of Public Health); Vanaerschot, Manu; Murithi, James M. (Columbia University); Pasaje, Charisse FJ (Massachusetts Institute of Technology); Luth, Madeline R. (University of California, San Diego); Magistrado-Coxen, Pamela (Harvard T.H. Chan School of Public Health); , Emma F. (Wellcome Sanger Institute, Hinxton.); Rubiano, Kelly; Striepen, Josefine; Bath, Jade (Columbia University); Munro, Justin T. (Pennsylvania State University); Pisco, Joao P.; Punekar, Avinash S.; Baragaña, Beatriz; Gilbert, Ian H. (Drug Discovery Unit, University of Dundee); Llinás, Manuel (Pennsylvania State University); Niles, Jacquin C. (Massachusetts Institute of Technology); Ottilie, Sabine; Winzeler, Elizabeth A. (University of California, San Diego, La Jolla, CA.); Lee, Marcus CS (Wellcome Sanger Institute, Hinxton.); Fidock, David A. (Columbia University); Lukens, Amanda K. (The Broad Institute); Wirth, Dyann F. (Harvard T. H. Chan School of Public Health)

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30t - Developing a scalable pipeline for functional genetic analysis in the malaria parasite, Plasmodium falciparum Esherick, Lisl Y. (Massachusetts Institute of Technology) McGuffie, Bryan (Boston Children's Hospital); Cardenas, Pablo; Dey, Sumanta; Nasamu, Sebastian; Pasaje, Charisse F. (MIT); Dvorin, Jeffrey D. (Boston Children's Hospital); Niles, Jacquin C. (MIT)

31t - Development of resistance in vitro reveals novel mechanisms of artemisinin tolerance in Toxoplasma gondii. Rosenberg, Alex (Washington University in St. Louis); Luth, Madeline R.; Winzeler, Elizabeth A. (University of California, San Diego); Behnke, Michael S. (Louisiana State University); Sibley, L David (Washington University in St. Louis)

32t - Genetic screens reveal a central role for heme biosynthesis in artemisinin sensitivity Sidik, Saima (Whitehead Institute); Harding, Clare (Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, University of Glasgow); Petrova, Boryana; Markus, Benedikt; Lourido, Sebastian (Whitehead Institute)

33t - DNA uptake agonists improve transfection efficiency and provide insights into plasmid uptake mechanism in malaria parasites Crater, Anna K. (NIAID, National Institutes of Health); Garriga, Meera; Desai, Sanjay A. (National Institutes of Health)

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Session VI: We’ve Gotta in to Get Out: Host-Parasite Interactions Chairs: Catherine Merrick and Dawn Wetzel Tuesday, September 17th, 2019 9:00am – 10:15am

34 - Toxoplasma Ferlin1 facilitates trafficking and exocytosis of micronemes Tagoe, Daniel A. (Boston College) Drozda, Allison; Coleman, Bradley (Boston College); Coppens, Isabelle (Johns Hopkins Bloomberg School of Public Health); Gubbels, Marc-Jan (Boston College)

35 - The first rhoptry bulb protein identified in is secreted during invasion and localized to the host cortical actin Guerin, Amandine (UPENN); Roy, Nathan (Children's Hospital of Philadelphia); Berry, Laurence (University of Montpellier); Burkhardt, Janis K. (Children's Hospital of Philadelphia); Striepen, Boris (UPENN)

36 - Unraveling the mechanism of microsporidia polar tube firing Bhabha, Gira (Skirball Institute, NYU School of Medicine); Jaroenlak, Pattana (Skirball Institute, NYU School of Medicine); Cammer, Michael (NYU School of Medicine); Ekiert, Damian (Skirball Institute, NYU School of Medicine)

37 - Human CD55 mediates parasite entry during the process of erythrocyte invasion by Plasmodium falciparum Egan, Elizabeth - Shakya, Bikash (Stanford University) Patel, Saurabh D. (Zuckerman Mind Brain Behavior Institute, Columbia University); Tani, Yoshihiko (Japanese Red Cross Kinki Block Blood Center); Egan, Elizabeth S. (Stanford University)

38t - An ER CREC family protein regulates the egress proteolytic cascade in malaria parasites Fierro, Manuel A. (The University of Georgia); Asady, Beejan; Brooks, Carrie F.; Cobb, David W.; Villegas, Alejandra; Moreno, Silvia N. J.; Muralidharan, Vasant (The University of Georgia)

39t - An unconventional myosin, TgMyoF is an organizer of the endosome-like compartments in Toxoplasma gondii Heaslip, Aoife (University of Connecticut); Carmeille, Romain; Schiano, Irio (University of Connecticut)

21

Session VII: Dancing with the Devil: Host-Parasite Interactions Chair: Dawn Wetzel and Catherine Merrick Tuesday, September 17th, 2019 10:30am – 11:30am

40 - Cell death mediated by the human trypanolytic serum protein Apolipoprotein L-1 is preceded by an accumulation of cytoplasmic calcium Verdi, Joey (Hunter College, City University of New York); Giovinazzo, Joseph; Khalizova, Nailya; Weiselberg, Jessica; Thomson, Russell (Hunter College, City University of New York); Schreiner, Ryan (Weill Cornell Medical College); Raper, Jayne (Hunter College)

41 - Modulation of host cell responses and virulence by T.gondii's ROP55 Ruivo, Margarida; Penarete-Vargas, Diana (UMR5235 Universite de Montpellier); Hamie, Maguy; El Hajj, Hiba (American University of Beirut); Lebrun, Maryse (UMR5235 Universite de Montpellier)

42 - Identification of a novel protein complex essential for erythrocyte invasion by human-infective malaria parasites Knuepfer, Ellen (The Francis Crick Institute); Wright, Katherine E. (Imperial College London); Green, Judith L. (The Francis Crick Institute); Kumar Prajapati, Surendra (Uniformed Services University of Health Sciences); Howell, Steven A. (The Francis Crick Institute); , Robert W. (London School of Hygiene and Tropical Medicine); , Simon J. (The Jenner Institute); Rosanas- Urgell, Anna (Institute of Tropical Medicine); Higgins, Matthew K. (Department of Biochemistry, Oxford University); Baum, Jake (Imperial College); Holder, Anthony A. (The Francis Crick Institute)

43t - Transcriptional modification of host cells harbouring Toxoplasma gondii bradyzoites prevents IFNgamma-mediated cell death Seizova, Simona

44t - EXP1 is required for organization of the Plasmodium falciparum parasitophorous vacuole membrane Nessel, Timothy (Iowa State University); Beck, John M. (Iowa State University); Goldberg, Daniel E. (Washington University School of Medicine); Beck, Josh R. (Iowa State University)

45t - Systematic identification of diversity-encompassing variants of a new malaria vaccine target Niare, Karamoko, Timo Chege, James Tuju, Bourema Kouriba, Gordon Awandare1, Julian Rayner, Faith Osier

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Session VIII: Born to Be Wild: Infection Dynamics and Reservoirs Chairs: Sabrina Absalon and Aoife Heaslip Tuesday, September 17th, 2019 1:00pm – 2:10pm

46 - Plasmodium falciparum dry season reservoir Portugal, Silvia (Heidelberg University)

47 - Malaria synchrony is not driven by the host Rijo-Ferreira, Filipa (HHMI, UT Southwestern Medical Center); Acosta-Rodriguez, Victoria (UT Southwestern Medical Center); Abel, John (Massachussets Institute of Technology); Kornblum, Izabela (HHMI, UT Southwestern Medical Center); Bento, Ines (Instituto Medicina Molecular); Matthews, Krista; Kilaru, Gokhul (UT Southwestern Medical Center); Mota, Maria M. (Instituto Medicina Molecular); Takahashi, Joseph S. (HHMI, UT Southwestern Medical Center)

48 - Using Toxoplasma gondii to redefine the neuronal innate immune response Sambamurthy, Chandrasekaran (University of Arizona); Kochanowsky, Joshua (University of Arizona); Burciu, Camelia (Barrow Neurological Institute); Sattler, Rita (Barrow Neurological Institute); Koshy, Anita (University of Arizona)

49 - Into the MIST: Adaptation to facilitate persistent infection of splenic macrophages enables malaria infection through stress erythropoiesis transmigration (MIST) and represents a novel exo-erythrocytic stage of the Plasmodium life cycle. Fraser, James W. (Penn State University); Sim, Derek G. (Penn State University); Dey, Adwitia (Harvard Medical School); Jones, Matthew; Hart, Kevin J.; Llinas, Manuel; Lindner, Scott E.; Read, Andrew F.; Paulson, Robert F. (Penn State University)

50t - Whole genome sequence comparative analyses of North African Leishmania infantum may reveal genetic and molecular determinants of the parasite tissue tropism. Chakroun, Ahmed S. (Institut Pasteur de Tunis, Université Tunis El Manar); Guerbouj, Souheila (Institut Pasteur de Tunis, Université Tunis El Manar); Leprohon, Philippe (Centre de Recherche du CHU de Québec, Université Laval); Harigua, Emna (Institut Pasteur de Tunis, Université Tunis El Manar); Fathallah Mili, Akila (Université de Sousse); Ouellette, Marc (Centre de Recherche du CHU de Québec, Université Laval); Guizani, Ikram (Institut Pasteur de Tunis, Université Tunis El Manar)

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Session IX: Changes, Turn and Face the Strange: Developmental Stages Chairs: Aoife Heaslip and Sabrina Absalon Tuesday, September 17th, 2019 2:30pm – 3:30pm

51 - IPS cell technology: A great tool to study hypnozoites and pave the road towards malaria elimination Pellisson, Melanie Anne-Marie Zeeman4, Thierry Doll, Annemarie Voorberg-van der Wel, Lucy Kirchhofer-Allan, Sven Schuierer, Guglielmo Roma, Erika Flannery, Sebastian Mikolajczak, Clemens H. M. Kocken, Pascal Mäser, Matthias Rottmann, Matthias Müller

52 - Multiple aneuploidies are not the norm in natural, uncultured canine Leishmania infantum, but can be induced by in vitro culture-associated stresses. Dobson, Deborah (Washington University); Shaik, Jahangheer (Washington University); Petersen, Christine (University of Iowa); Beverley, Stephen (Washington University)

53 - Plasmodium falciparum immature gametocytes development in human primary erythroblasts Neveu, Gaëlle (INSERM, Cochin Institute); Richard, Cyrielle; Volpe, Fiona; Dupuy, Florian (INSERM, Cochin Institute); Annamalai, Pradeep (UPMC Faculté de Medecine); Naissant, Bernina (INSERM, Cochin Institute); Martins, Raphael M (CNRS5290, IRD224, University Montpellier 1 & 2, MIVEGEC); Vallin, Patrice; Andrieu, Muriel (INSERM, Cochin Institute); Lopez-Rubio, José-Juan (CNRS5290, IRD224, University Montpellier 1 & 2, MIVEGEC); Mazier, Dominique (UPMC Faculté de Medecine); Verdier, Frédérique; Lavazec, Catherine (INSERM, Cochin Institute)

54t - A novel Babesia bovis secreted protein responsible for binding of infected erythrocyte to endothelial cells Hakimi, Hassan (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University) Sakaguchi, Miako (Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University); Yamagishi, Junya (Research Center for Zoonosis Control, Hokkaido University); Yahata, Kazuhide; Kaneko, Osamu; Asada, Masahito (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University)

55t - Targeting effector proteins at the schizont membrane responsible for host cell transformation Brühlmann, Francis (University of Bern); Woods, Kerry; Olias, Philipp (University of Bern)

56t - Epigenetic regulation of gametocytogenesis in P. berghei; the roles of HAT1, HDA1, and differential histone PTMs. Power, B. Joanne J. (Pennsylvania State University); Burchmore, Richard (Glasgow Polyomics); Waters, Andrew P. (Wellcome Centre for Integrative Parasitology) 24

Session X: And the Seasons They Go Round and Round: Life- Cycle Differentiation Chairs: Laura Kirkman and Diego Huet Wednesday, September 18th, 2019 9:00am – 10:15am

57 - Functional characterizacion of Trypanosoma cruzi surface mucins in the infection of the invertebrate host camara, maria de los Milagros; Balouz, Virginia (IIB UNSAM); Centeno Camean, Camila (IIB UNSAM); Cori, Carmen (CHIDECAR UBA); Lobo, Maite Mabel (IIB UNSAM,); Lederkremer, Rosa (CHIDECAR); Gallo-Rodriguez, Carola (CHIDECAR UBA); Buscaglia, Carlos Andres (IIB UNSAM)

58 - Transcription in Trypanosoma brucei: daring to be different Florini, Francesca (IZB, University of Bern); Naguleswaran, Arunasalam (IZB, University of Bern); Heller, Manfred (DBMR, University of Bern); Roditi, Isabel (IZB, University of Bern)

59 - Identification of metabolic pathways that regulate sexual differentiation in P. falciparum. Sollelis, lauriane (WCIP); Achcar, Fiona; Barrett, Michael; Marti, Matthias (WCIP)

60t - Characterization of a Novel Sensing Mechanism Governing Antigenic Variation and Immune Evasion in P. falciparum Schneider, Victoria (The Rockefeller University); Ben Mamoun, Choukri (); Rhee, Kyu; Harris, Chantal; Kafsack, Bjorn; Deitsch, Kirk (Weill Cornell Medical College)

61t - Examining trans-regulators of the Leishmania lifecycle Walrad, Pegine B. (York Biomedical Research Institute, Univ. of York); R. Ferreira, Tiago; Pablos, Luis; Dowle, Adam; Forrester, Sarah; Parry, Ewan; V. C. Alves-Ferreira, Eliza; Newling, Katherine; Kolokousi, Foteini; Larson, Tony; Plevin, Michael (University of York); K.Cruz, Angela (University São Paulo)

62t - Redox metabolomics and gametocytogenesis in the malaria parasite Beri, Divya (Indian Institute of ScienceTatu, Utpal (Indian Institute of Science) 25

Session XI: Epress Yourself: Molecular Biology Chair: Diego Huet and Laura Kirkman Wednesday, September 18th, 2019 10:30am – 11:30am

63 - A Novel PNUTS-PP1 Phosphatase Complex Orchestrates RNA Pol II Transcription Termination in T. brucei Kieft, Rudo (University of Georgia); Zhang, Yang; Marand, Alexandre P.; Moran, Jose Dagoberto; Bridger, Robert; Wells, Lance; Schmitz, Robert J.; Sabatini, Robert

64 - Identification of a master regulator of differentiation in Toxoplasma Waldman, Benjamin S. (Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology); Schwarz, Dominic (Whitehead Institute for Biomedical Research and Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg); Wadsworth II, Marc H. (Institute for Medical Engineering & Science (IMES), Department of Chemistry, and Koch Institute for Integrative Cancer Research, MIT); Saeij, Jeroen P. (Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis); Shalek, Alex K. (Institute for Medical Engineering & Science (IMES), Department of Chemistry, and Koch Institute for Integrative Cancer Research, MIT); Lourido, Sebastian S. (Whitehead Institute for Biomedical Research and Department of Biology)

65 - Regulation of membrane trafficking by Giardia lamblia's sole RhoGTPase, GlRac Thomas, Elizabeth B. (University of Washington)

66t - Functional and biochemical characterization of SPY- catalysed nucleocytosolic O-fucosylation in Toxoplasma gondii Bandini, Giulia (Boston University); Agop-Nersesian, Carolina (Boston University); van der Wel, Henke; West, Christopher M. (University of Georgia); Samuelson, John (Boston University)

67t - The novel EhHSTF7 transcription factor binds to HSE and regulates the multidrug resistant Ehpgp5 gene expression in Entamoeba histolytica. Bello, Fabiola (Centro de Investigación y Estudios Avanzados, IPN); Orozco, Esther (Centro de Investigación y Estudios Avanzados, IPN); Pérez, Guillermo; Gómez, Consuelo (Escuela Nacional de Medicina y Homeopatía, IPN)

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Session XII: Mamma Mia, Here We Go Again: Cell-Cycle II Chairs: Ellen Yeh and Rob Moon Thursday, September 19th, 2019 9:00am – 10:00am

68 - The Plasmodium falciparum RNA-binding protein plasmei2 plays an essential role in the completion of liver stage development Goswami, Debashree (Seattle Children's Research Institute); Betz, William; Locham, Navinder K. (Seattle Childrens Research Institute); Murphy, Sean C. (Department of Microbiology); Vaughan, Ashley M.; Kappe, Stefan H.I. (Seattle Childrens Research Institute)

69 - Toxoplasma F-box Protein 1 Is Critically Required for Daughter Cell Scaffold Function During Parasite Replication Baptista, Carlos G. (University at Buffalo); Lis, Agnieszka (University at Buffalo); Gas- Pascual, Elisabet (University of Georgia); Dittmar, Ashley; Sigurdson, Wade (University at Buffalo); West, Christopher M. (University of Georgia); Blader, Ira J. (Universty at Buffalo)

70 - ESCRTing material to the invader: Mechanisms underlying Toxoplasma gondii endocytosis of host cytosolic protein Rivera-Cuevas, Yolanda (University of Michigan in Ann Arbor); McGovern, Olivia L.; Lawrence, Anna-Lisa; Carruthers, Vern B. (University of Michigan)

71 - A genetic trap: Resistance towards pyrazoleamide PA21A092 imparts hypersensitivity to the spiroindolone KAE609 Bhatnagar, Suyash (Drexel University College of Medicine); Morrisey, Joanne M.; Bergman, Lawrence W. (Drexel University College of Medicine); Painter, Heather J.; Llinas, Manuel (Penn State University); Fidock, David A. (Columbia University); Vaidya, Akhil B. (Drexel University College of Medicine)

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Session XIII: Save the Best for Last: Host-Parasite Metabolism - II Chairs: Rob Moon and Ellen Yeh Thursday, September 19th, 2019 10:30am – 11:30am

72 - Trypanosoma cruzi intracellular amastigote glutamine metabolism and its impact on susceptibility to ergosterol biosynthesis inhibitors Dumoulin, Peter C. (Harvard T.H. Chan School of Public Health) Vollrath, Joshua; Pagura, Lucas; Burleigh, Barbara (Harvard T.H. Chan School of Public Health)

73 - Defining the metabolic network of the malaria parasite reveals one-carbon metabolism is necessary for normal mitochondrial function Cobbold, Simon A. (University of Melbourne); Marapana, Danushka S. (Walter and Eliza Hall Institute); Ralph, Stuart A. (University of Melbourne); Cowman, Alan F. (Walter and Eliza Hall Institute); Tilley, Leann; McConville, Malcolm J. (University of Melbourne)

74 - Trypanosoma cruzi mitochondrial pyruvate carrier (MPC) subunits do not form homodimers and are essential for pyruvate-driven respiration Negreiros, Raquel S. (State University of Campinas); Vercesi, Anibal E. (State University of Campinas); Docampo, Roberto (University of Georgia)

75 - Membrane contact sites mediate lipid exchange at the Plasmodium – red blood cell interface Garten, Matthias (NIH/NICHD); Beck, Josh R. (Iowa State University, Ames, IA.); Roth, Robyn (Washington University); Bleck, Christopher KE (NIH / NHLBI); Heuser, John; Tenkova-Heuser, Tatyana (NIH / NICHD); Goldberg, Daniel E. (Washington University); Zimmerberg, Joshua (NIH / NICHD)

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Poster Session A Monday, September 16, 2019 7:00 pm – 9:00 pm

76 - New toolkit for high throughput imaging and image analysis of the chronic stage of Toxoplasma gondii Winiger, Rahel R. (Laboratory of Molecular Parasitology); Fisch, Daniel (Host- Toxoplasma Interaction Laboratory, London.); Yakimovich, Artur (MRC-Laboratory for Molecular Cell Biology); Hammoudi, Pierre-Mehdi (Department of Microbiology and Molecular Medicine, Geneva); Ramakrishnan, Chandra (Laboratory of Molecular Parasitology, Zürich); Soldati-Favre, Dominique (Department of Microbiology and Molecular Medicine, Geneva); Hehl, Adrian B. (Laboratory of Molecular Parasitology) For Abstract See Session I, 5t

77 - Three-dimensional ultrastructure of Plasmodium falciparum throughout cytokinesis Rudlaff, Rachel M. (Harvard Medical School); Kraemer, Stephan (Harvard University); Marshman, Jeffrey (Carl Zeiss Microscopy, Thornwood, NY.); Dvorin, Jeffrey D. (Harvard Medical School) For Abstract See Session I, 6t

78 - VSG mosaics: when the color of your shoes determines the shape of your hat. Gkeka, Anastasia (Deutsches Krebsforschungzentrum); Erben, Esteban; Zeelen, Johan P.; Stebbins, Erec C.; Papavasiliou, Nina F. (Deutsches Krebsforschungzentrum) For Abstract See Session I, 7t

79 - Analysis of the Toxoplasma mitochondrial ATP synthase structure reveals how its shapes the unique cristae and highlights the role of phylum specific subunits Sheiner, Lilach (University of Glasgow) For Abstract See Session I, 8t

80 - Mapping of origins of replication in Plasmodium falciparum Gomes, Ana Rita (DIMNP, Université de Montpellier) CHU de Montpellier); Méchali, Marcel (Institute de Génétique Humaine); Lopez-Rubio, Jose-Juan (DIMNP, Université de Montpellier) For Abstract See Session II, 13t

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81 - Single-cell transcriptomes of Plasmodium vivax parasites reveal extensive but continuous changes in gene expression during their intraerythrocytic cycle Serre, David (University of Maryland School of Medicine); Cannon, Matthew (University of Maryland School of Medicine); Caleon, Ramon; Wellems, Thomas; Sa, Juliana (Laboratory of Malaria and Vector Research) For Abstract See Session II, 14t

82 - Divergent Acyl Carrier Protein Coordinates Mitochondrial Fe-S Cluster Metabolism in Malaria Parasites Falekun, Seyi P. (Department of Biochemistry, University of Utah); Jami, Yasi (Department of Biological Chemistry, University of California Los Angeles); Park, Hahnbeom (Institute for Protein Design, University of Washington); Wohlschlegel, James A. (Department of Biological Chemistry, University of California Los Angeles); Sigala, Paul A. (Department of Biochemistry, University of Utah) For Abstract See Session III, 18t

83 - Trypanosoma congolense cytoadheres to the brain vasculature Silva Pereira, Sara (Instituto de Medicina Molecular - João Lobo Antunes); De Niz, Mariana; Figueiredo, Luisa M. (Instituto de Medicina Molecular - João Lobo Antunes) For Abstract See Session III, 19t

84 - Functional study of two Trypanosoma cruzi receptors (TcIP3R and TczAC) involved in signal transduction and important for parasite survival Lander, Noelia (University of Georgia); Chiurillo, Miguel A.; Docampo, Roberto (University of Georgia) For Abstract See Session III, 20t

85 - Differential Expression and Localization of Cytochrome b5 in Giardia intestinalis during Nitrosative Stress Response Mowat, Kaitlyn (Trent Univeristy); For Abstract See Session IV, 25t

86 - A Lysophospholipase involved in malaria parasite development: Potential targets for drug development Mohmmed, Asif (International Centre for Genetic Engineering and Biotechnology.); Kumar, Pradeep (International Centre For Genetic Engineering and Biotechnology New Delhi); Botté, Yoshiki Yamaryo (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes); Thakur, Vandana (International Centre for Genetic Engineering and Biotechnology.); Botté, Cyrille (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes) For Abstract See Session IV, 26t

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87 - Developing a scalable pipeline for functional genetic analysis in the malaria parasite, Plasmodium falciparum Esherick, Lisl Y. (Massachusetts Institute of Technology) McGuffie, Bryan (Boston Children's Hospital); Cardenas, Pablo; Dey, Sumanta; Nasamu, Sebastian; Pasaje, Charisse F. (MIT); Dvorin, Jeffrey D. (Boston Children's Hospital); Niles, Jacquin C. (MIT) For Abstract See Session V, 30t

88 - Development of resistance in vitro reveals novel mechanisms of artemisinin tolerance in Toxoplasma gondii. Rosenberg, Alex (Washington University in St. Louis); Luth, Madeline R.; Winzeler, Elizabeth A. (University of California, San Diego); Behnke, Michael S. (Louisiana State University); Sibley, L David (Washington University in St. Louis) For Abstract See Session V, 31t

89 - Genetic screens reveal a central role for heme biosynthesis in artemisinin sensitivity Sidik, Saima (Whitehead Institute); Harding, Clare (Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, University of Glasgow); Petrova, Boryana; Markus, Benedikt; Lourido, Sebastian (Whitehead Institute) For Abstract See Session V, 32t

90 - DNA uptake agonists improve transfection efficiency and provide insights into plasmid uptake mechanism in malaria parasites Crater, Anna K. (NIAID, National Institutes of Health); Garriga, Meera; Desai, Sanjay A. (National Institutes of Health) For Abstract See Session V, 33t

91 - Selection and biochemical characterization of novel leishmanicidal molecules by virtual and biochemical screenings targeting Leishmania eukaryotic translation initiation factor 4A ABDELKRIM ÉP. GUEDICHE, Yosser zina (Institut Pasteur de Tunis); Harigua, Emna; Bassoumi, Imen (Institut Pasteur de Tunis); Zakraoui, Ons (Children's Hospital Oakland Research Institute); Banroques, Josette (Institut de Biologie Physico-chimique de Paris); Essafi-Benkhadir, Khadija; Barhoumi, Mourad (Institut Pasteur de Tunis); Munier-Lehmann, He lène; Nilges, Michael; Blondel, Arnaud (Institut Pasteur de Paris); Tanner, N. Kyle (Institut de Biologie Physico-chimique de Paris); Guizani, Ikram (Institut Pasteur de Tunis)

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92 - When a protein changes jobs: novel noncanonical functions of Atg8 in malaria parasites Walczak, Marta M. (Stanford University); Ganesan, Suresh M.; Niles, Jacquin C. (MIT); Yeh, Ellen (Stanford University)

93 - Depletion of cholesterol from the trophozoite stage of P. falciparum leads to bizarre consequences: Exploration with novel antimalarials. Ahiya, Avantika I.(Drexel university, College of Medicine); Bhatnagar, Suyash; Morrisey, Joanne M. (Drexel University, College of Medicine); Beck, Josh R. (Department of Biomedical Sciences); Vaidya, Akhil B. (Drexel University, College of Medicine)

94 - Consequence of Babesia microti infection on susceptible mice in the absence or presence of Lyme spirochetes Parveen, Nikhat (Rutgers-New Jersey Medical School)

95 - Comparative mapping of Plasmodium proteomes provides new insights into erythrocyte remodeling Preiser, Peter (Nanyang Technological University); Anthony Siau1, Regina Hoo1*, Omar Sheriff1*, Donald Tay1*, Xue Yan Yam1, Han Ping Loh1, Marek Mutwil1, Meng Wei1, Siu Kwan Sze 1, Peter Preiser11Nanyang Technological University, School of Biological Sciences Singapore,2

96 - Charting the Basis for Toxoplasma gondii Virulence Traits Through Lab Adaptation Primo, Vincent (Boston College); Rezvani, Yasaman (Department of Mathematics, University of Mass.); Farrell, Andrew; Marth, Gabor T. (Department of Human Genetics and USTAR Center for Genetic Discovery, Eccles Institute of Human Genetics, University of Utah School of Medicine); Zarringhalam, Kourosh (Department of Mathematics, University of Mass); Gubbels, Marc-Jan (Boston College)

97 - Functional investigation of a nuclear binding partner of the bromodomain protein PfBDP1 Quinn, Jennifer (Institute of Microbiolgy - University Clinic Erlangen); Petter, Michaela (Institute of Microbiology - University Clinic Erlangen)

98 - Reversible glucan phosphorylation reveals a function for amylopectin in Toxoplasma gondii central carbon metabolism Murphy, Robert D. (University of Kentucky); Dhara, Animesh; Watts, Elizabeth; Brizzee, Corey; Sun, Ramon; Sinai, Anthony; Gentry, Matthew (University of Kentucky)

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99 - Investigating paralogous ApiAP2 proteins with similar DNA binding specificities in Plasmodium falciparum Bonnell, Victoria A. (The Pennsylvania State University) Josling, Gabrielle A. (The Pennsylvania State University); Gordân, Raluca (Duke University); Painter, Heather J. (Food and Drug Administration); Llinás, Manuel (The Pennsylvania State University)

100 - Division and adaptation to host nutritional environment of apicomplexan parasites depend on apicoplast lipid metabolic plasticity and host organelles remodeling Botté, Cyrille Y. (Apicolipid Team, Institue for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes) Katris, Nicholas J.; Amiar, Souad (Apicolipid Team, Institute for Advanced Biosciences); Berry, Laurence (Dynamique des interactions Membranaires normales et pathologiques, UMR5235, Université MontpellierII); Dass, Sheena (Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes); Shears, Melanie J. (McFadden Lab, School of Biosciences, University of Melbourne); Touquet, Bastien (Team Cell and membrane dynamics of parasite-host interaction, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR5309, Université Grenoble Alpes); Hakimi, Mohamed-Ali (Team Host-pathogen interactions and immunity to , Institute for Advanced Biosciences, INSERM 1209, CNRS UMR5309, Université Grenoble Alpes); McFadden, Geoffrey I. (McFadden Laboratory, School of Biosciences, University of Melbourne); Yamaryo-Botté, Yoshiki (Apicolipid Team, Institute for adavanced Bioscience, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes)

101 - Toxoplasma gondii RON13 is a rhoptry neck kinase critical for rhoptry discharge and invasion Ben Chaabene, Rouaa (UNIGE); Lentini, Gaelle; Mukherjee, Budhaditya; SOLDATI- FAVRE, Dominique (UNIGE)

102 - SLOPE: A two-part enrichment method effective on ring stage Plasmodium parasites Brown, Audrey C. (University of Virginia) Guler, Jennifer L. (University of Virginia)

103 - Unravelling the role of Pf113; novel PTEX accessory protein? Bullen, Hayley (Burnet Institute); Palmer, Catherine (Bio21); Sanders, Paul; Jonsdottir, Thorey (Burnet Institute); Riglar, David (Harvard Medical School); Dans, Madeleine (Burnet Institute); Charnaud, Sarah (The Walter and Eliza Hall Institute); Baum, Jacob (Imperial College London); Cowman, Alan (The Walter and Eliza Hall Institute); de Koning-Ward, Tania (Deakin University); Gilson, Paul; Crabb, Brendan (Burnet Institute)

104 - A Putative calcium proton antiporter of Toxoplasma gondii Calixto, Abigail; Dykes, Eric J.; Moreno, Silvia NJ (University of Georgia)

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105 - Targeting the Human Malaria Parasite P. falciparum with Kalihinol Analogues Chahine, Zeinab M. (UC Riverside); Prudhomme, Jacques M. (UC Riverside); Daub, Mary E. (UC Irvine); Ben Mamoun, Choukri (Yale School of Medicine); Vanderwal, Chris M. (UC Irvine); Le Roch, Karine M. (UC Riverside)

106 - Crystal structure of a metacyclic Variant Surface Glycoprotein (mVSG) from Trypanosoma brucei Chandra, Monica (German Cancer Research Center (DKFZ)); Branco, Francisco A.; Papavasiliou, Nina; Stebbins, Erec (German Cancer Research Center (DKFZ))

107 - First Insights into the Proteome and Endocytic Mechanism of the Cytostome/Cytopharynx Complex of Trypanosoma cruzi Chasen, Nathan (Center for Tropical and Emerging Global Diseases, University of Georgia) Tarleton, Rick; Etheridge, Drew (Center for Tropical and Emerging Global Diseases, University of Georgia)

108 - PfBTP3 reveals a novel role for the basal complex in sexual-stage malaria parasites Clements, Rebecca L. (Harvard University); Streva, Vincent; Dvorin, Jeffrey D. (Boston Childrens Hospital)

109 - Plasmodium falciparum MCMBP depletion unveils the lack of spindle assembly checkpoint and the critical role of the DNA replicative machinery for nuclear division during schizogony. Absalon, Sabrina (Indiana University School of Medicine) Dvorin, Jeffrey, Daniel (Boston Children's Hospital)

110 - Combined genetic and chemical approaches to uncover the essential ER redox network of the human malaria parasite Plasmodium falciparum Cobb, David W. (University of Georgia) Kudyba, Heather M. (NIH); Hoopmann, Michael (Institute for Systems Biology); Bruton, Baylee W.; Krakowiak, Michelle W. (University of Georgia); Moritz, Robert (Institute for Systems Biology); Muralidharan, Vasant W. (University of Georgia)

111 - Novel Antiplasmodial Compounds From Fungi , Jennifer (University of Central Florida) Cai, Shengxin; King, Jarrod; Wendt, Karen; Lee, Jinwoo; Cichewicz, Robert (University of Oklahoma); Chakrabarti, Debopam (University of Central Florida)

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112 - Epigenetic control of Plasmodium falciparum gametocyte development through unique repressive markers of gene expression Connacher, Jessica I. (University of Pretoria) Josling, Gabrielle A.; Llinas, Manuel (Pennsylvania State University); Birkholtz, Lyn-Marie (University of Pretoria)

113 - FLIM and Imaging Analysis of Plasmodium falciparum Exposed to Artemisinin Connelly, Sean (Laboratory of Malaria and Vector Research, NIAID); Levine, Zoë (Laboratory of Malaria and Vector Research, NIAID); Manzella-Lapeira, Javier; Brzostowski, Joe; Krymskaya, Ludmila (Laboratory of Immunogenetics, NIAID); Rahman, Rifat; Sá, Juliana; Wellems, Thomas (Laboratory of Malaria and Vector Research, NIAID,)

114 - Defining the functional secretory proteome during host invasion by the obligate intracellular parasite Toxoplasma gondii Costa, Elizabeth A. (Whitehead Institute); Shortt, Emily; Lourido, Sebastian (Whitehead Institute)

115 - The Acute Challenge Model: Sensitive moderate- throughput assessment of malaria T cell vaccine in mice Cruz Talavera, Irene (University of Washington); Stone, Brad; Murphy, Sean C. (University of Washington)

116 - Genetic inferences from sequencing of Pvdbp gene in Indian isolates of Plasmodium vivax Kar, Sonalika (ICMR-National Institute of Malaria Research) Sinha, Abhinav (ICMR- National Institute of Malaria Research)

117 - Functional role of the HSP40 Protein in gametocytogenesis of Malaria parasite Plasmodium berghei. Kashif, Mohammad (National Institute of Immunology); Singh, Agam P. (National Institute of Immunology)

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118 - Conoid function in is linked to a hybrid P- Type ATPase/Guanylate cyclase that controls lipid signalling required for activation of motility Katris, Nicholas (Apicolipid Team, IAB, CNRS UMR5309); Yamaryo-Botte, Yoshiki (ApicoLipid Team, IAB, Universite Grenoble Alpes, La Tronche.); Janouskovec, Jan (Department of Genetics, Evolution and Environment, University College London); Arnold, Christophe S. (ApicoLipid Team, IAB, Universite Grenoble Alpes, La Tronche.); Yang, Annie SP; Sauerwein, Robert (Radboud University Medical Centre); McFadden, Geoff I. (School of Biosciences, University of Melbourne); Tonkin, Christopher J. (Walter and Eliza Hall Institute); Waller, Ross F. (Dept. of Biochemistry, University of Cambridge); Botte, Cyrille Y. (Apicolipid Team, IAB, CNRS UMR5309)

119 - Gain of function of human mechanoreceptor PIEZO1 inhibits replication of Plasmodium falciparum in dehydrated erythrocytes Kegawa, Yuto (NIH); Waters, Hang; Bezrukov, Ludmila (NIH); Beck, Josh (Iowa State University); Glushakova, Svetlana; Zimmerberg, Joshua (NIH)

120 - Identification of Secretory Pathway Proteins within Toxoplasma gondii using RNA-Seq and CRISPR/Cas9 Kellermeier, Jacob A. (University of Connecticut) Carmeille, Romain; Heaslip, Aoife T. (University of Connecticut)

121 - Deciphering the role of CD44 as a host factor for Plasmodium falciparum invasion Kim, Chi Yong (Stanford University); Baro, Bàrbara (Stanford University); Salinas, Nichole (National Institutes of Health); Doerig, Christian D. (Monash University); Tolia, Niraj H. (National Institutes of Health); Egan, Elizabeth S. (Stanford University)

122 - The highly-unusual yet evolutionarily conserved fragmented mitochondrial genome of the coccidian, Toxoplasma gondii Kissinger, Jessica C. (University of Georgia); Namasiviyam, Ranjani; Xiao, Wenyuan; Baptista, Rodrigo P.; Hall, Erica M. (University of Georgia); Troell, Karin (National Veterinary Institute); Doggett, Joseph S. (Oregon Health Sciences University)

123 - Functional annotation of Plasmodium falciparum serine hydrolases through chemoproteomics Klemba, Michael (Virginia Tech) Elahi, Rubayet; Ray, Keith; Dapper, Christie; Liu, Jiapeng; Helm, Rich (Virginia Tech)

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124 - CD8 T cell IFN? responses are modulated by multiple T. gondii effectors. Kongsomboonvech, Angel K. (UC Merced)

125 - Functional analysis of putative circumsporozoite protein of Plasmodium vivax Kumari, Sanju (National Institute of Malaria Research) Dash, Manoswini; Sinha, Abhinav (National Institute of Malaria Research)

126 - A mutagenesis screen to identify genes involved in T. gondii autophagy during chronic infection Wang, Fengrong; Carruthers, Vern B. (University of Michigan)

127 - APPL (Apple) and EEF (Eve) : Early endosome protein in liver stage malaria Lahree, Aparajita (Instituto de Medicina Molecular- Joao Lobo Antunes);

128 - Exploring the content of noncoding RNAs, putative regulatory elements, in the protozoan parasite Leishmania Cruz, Angela K. (University of Sao Paulo); Monteiro-Teles, Natalia M. (University of York); Dias, Leandro; Freitas-Castro, Felipe; Diniz, Juliana A. (University of Sao Paulo); Lorenzon, Lucas (University of Sao Paulo); Magalhaes, Rubens (University of Sao Paulo); Vasconcelos, Elton (University of Leeds); Myler, Peter (bCenter for Infectious Disease Research); Ruy, Patricia (University of Sao Paulo)

129 - Challenges and Opportunities in Isolating Mitochondrial Ribosomes from Plasmodium falciparum Dass, Swati (Drexel University College of Medicine); Mulaka, Maruthi; Ling, Liqin (Drexel University); Gutierrez-Vargas, Christina (2Department of Biochemistry and Molecular Biophysics and Department of Biological Sciences, Columbia University); Mather, Michael (Drexel University); Dvorin, Jeffery (Division of Infectious Diseases, Boston Children’s Hospital, Boston); Vaidya, Akhil (Drexel University); Frank, Joachim (Columbia University, New York); Ke, Hangjun (Drexel University)

130 - The -associated protein PAVE1 regulates the dynamics of the subpellicular microtubule array in Trypanosoma brucei de Graffenried, Christopher L. (Brown University); Sinclair, Amy N.; Sladewski, Thomas (Brown University)

131 - Interspecies interaction in Plasmodium : opportunities and threats Deora, Nimita (ICMR-National Institute of Malaria Research); Sinha, Abhinav (ICMR-National Institute of Malaria Research, Dwarka sector-8)

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132 - Cryptosporidium parvum exports proteins into the cytoplasm of the epithelial host cell Dumaine, Jennifer E (University of Pennslyvania); Sateriale, Adam; Gibson, Alexis (University of Pennsylvania); Reddy, Amita (University of Georgia); Striepen, Boris (University of Pennsylvania)

133 - “Comparing luciferase expression in transfected stages of Plasmodium falciparum and Plasmodium knowlesi" Ellis, Angela C. (NIAID); Sa, Juliana M.; Mu, Jianbing (NIAID); Barros, Roberto M. (Universidade Federal de São Paulo); Wellems, Thomas E. (NIAID)

134 - Giardia lamblia: Increased expression of long-chain fatty acid elongase alters global ceramide and phosphatidylcholine levels in cysts Ellis, (University of Texas El Paso) Enriquez, Vanessa; Pence, Breanna; Almeida, Igor C.; , Siddhartha (University of Texas at El Paso)

135 - Babesia species parasites can acquire resistance to heparin through the use of alternative invasion pathways Elsworth, Brendan (Harvard School of Public Health); Keroack, Caroline D.; Tennessen, Jacob A.; Duraisingh, Manoj T. (Harvard T. H. Chan School of Public Health)

136 - Arginine enables the adaptive proline response to halofuginone in P. falciparum Fagbami, Lola (Harvard University)

137 - Generation of in vitro Leishmania hybrids Ferreira, Tiago R. (National Institute of Allergy and Infectious Diseases, National Institutes of Health); Louradour, Isabelle; Sacks, David (National Institute of Allergy and Infectious Diseases, National Institutes of Health)

138 - Determining how Toxoplasma gondii bradyzoites co-opt their host cell Ferrel, Abel; Branon, Tess C. (Dept. of Molecular and Cell Biology, University of California, Berkeley); Ting, Alice Y (Departments of Genetics, Biology, & by courtesy, Chemistry, Stanford University); Boothroyd, John C. (Department of Microbiology and Immunology, Stanford University)

139 - m6A methylation of the polyA-tail regulates VSG transcript stability in trypanosomes Figueiredo, Luisa M. (Instituto de Medicina Molecular, University of Lisbon); Viegas, Idalio; Rodrigues, Joao; Macedo, Juan P. (Instituto de Medicina Molecular, University of Lisbon, Lisboa, Portugal); Aresta-Branco, Francisco (German Cancer Research Center (DKFZ); Jaffrey, Samie (Weill Medical College of )

140 - Functional characterisation of the chromatin remodeling enzyme Snf2L in Plasmodium falciparum Watzlowik, Maria (University of Regensburg) 41

141 - Revising the karyotype of Toxoplasma gondii and synteny with caninum using single molecule sequencing Xia, Jing (University of Pittsburgh); Bainbridge, Rachel (University of Pittsburgh); Venkat, Aarthi; Ay, Ferhat (La Jolla Institute for Allergy & Immunology); LeRoch, Karine (University of California Riverside); Boyle, Jon (University of Pittsburgh)

142 - Characterizing insulinase-like proteases in Cryptosporidium parvum Xu, Rui (Washington University in St. Louis; East China University of Science and Technology); Xiao, Lihua (South China Agricultural University); Feng, Yaoyu (South China Agricultural University; East China University of Science and Technology); Sibley, L. Daivd (Washington University in St. Louis)

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Poster Session B Tuesday, September 17, 2019 7:00 pm – 9:00 pm

143 - An ER CREC family protein regulates the egress proteolytic cascade in malaria parasites Fierro, Manuel A. (The University of Georgia); Asady, Beejan; Brooks, Carrie F.; Cobb, David W.; Villegas, Alejandra; Moreno, Silvia N. J.; Muralidharan, Vasant (The University of Georgia) For Abstract See Session VI, 38t

144 - An unconventional myosin, TgMyoF is an organizer of the endosome-like compartments in Toxoplasma gondii Heaslip, Aoife (University of Connecticut); Carmeille, Romain; Schiano, Irio (University of Connecticut) For Abstract See Session VI, 39t

145 - Transcriptional modification of host cells harbouring Toxoplasma gondii bradyzoites prevents IFNgamma-mediated cell death Seizova, Simona For Abstract See Session VII, 43t

146 - EXP1 is required for organization of the Plasmodium falciparum parasitophorous vacuole membrane Nessel, Timothy (Iowa State University); Beck, John M. (Iowa State University); Goldberg, Daniel E. (Washington University School of Medicine); Beck, Josh R. (Iowa State University) For Abstract See Session VII, 44t

147 - Systematic identification of diversity-encompassing variants of a new malaria vaccine target Niare, Karamoko1,2,3, Timo Chege2, James Tuju2, Bourema Kouriba3, Gordon Awandare1, Julian Rayner4, Faith Osier2. 1 West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, 2Kenya Medical Research Institute – Wellcome Trust Research Programme, Kilifi, Kenya, 3Malaria Research and Training Center. 4Wellcome Sanger Institute. For Abstract See Session VII, 45t

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148 - Whole genome sequence comparative analyses of North African Leishmania infantum may reveal genetic and molecular determinants of the parasite tissue tropism. Chakroun, Ahmed S. (Institut Pasteur de Tunis, Université Tunis El Manar); Guerbouj, Souheila (Institut Pasteur de Tunis, Université Tunis El Manar); Leprohon, Philippe (Centre de Recherche du CHU de Québec, Université Laval); Harigua, Emna (Institut Pasteur de Tunis, Université Tunis El Manar); Fathallah Mili, Akila (Université de Sousse); Ouellette, Marc (Centre de Recherche du CHU de Québec, Université Laval); Guizani, Ikram (Institut Pasteur de Tunis, Université Tunis El Manar) For Abstract See Session VIII, 50t

149 - A novel Babesia bovis secreted protein responsible for binding of infected erythrocyte to endothelial cells Hakimi, Hassan (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University) Sakaguchi, Miako (Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University); Yamagishi, Junya (Research Center for Zoonosis Control, Hokkaido University); Yahata, Kazuhide; Kaneko, Osamu; Asada, Masahito (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University) For Abstract See Session IX, 54t

150 - Targeting Theileria effector proteins at the schizont membrane responsible for host cell transformation Brühlmann, Francis (University of Bern); Woods, Kerry; Olias, Philipp (University of Bern) For Abstract See Session IX, 55t

151 - Epigenetic regulation of gametocytogenesis in P. berghei; the roles of HAT1, HDA1, and differential histone PTMs. Power, B. Joanne J. (Pennsylvania State University); Burchmore, Richard (Glasgow Polyomics); Waters, Andrew P. (Wellcome Centre for Integrative Parasitology) For Abstract See Session IX, 56t

152 - Copper metabolism in Naegleria spp. Ženíšková, Katerina (Department of Parasitology, Faculty of Science, Charles University in Prague); Grechnikova, Maria; Mach, Jan; Šuták, Róbert (Department of Parasitology, Faculty of Science, Charles University in Prague)

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153 - High-throughput functionalization of the Toxoplasma gondii proteome Smith, Tyler (Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology); Lopez-Perez, Gabriella (University of Puerto Rico at Mayaguez); Lourido, Sebastian (Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Biology)

154 - Identification of the F-actin capping protein in the regulation of morphological transition of Trichomonas vaginalis Hsu, Hong Ming (The Division of Tropical Medicine and Parasitology, College of Medicine, National Taiwan University); Wang, Kai-Hsuan (The Division of Tropical Medicine and Parasitology, College of Medicine, National Taiwan University)

155 - From comes order: how an intrinsically disordered Toxoplasma effector subverts interferon signaling HUANG, ZHOU (Washington University in St. Louis); Liu, Hejun; Amarasinghe, Gaya K.; Sibley, L. David (Washington University in St. Louis)

156 - Characterizing the phylum-specific adaptations of the apicomplexan ATP synthase Huet, Diego (University of Georgia and Whitehead Institute for Biomedical Research) Lourido, Sebastian (Whitehead Institute for Biomedical Research, MIT Department of Biology)

157 - Towards characterization of protein export machinery and effectors throughout Plasmodium development Hussain, Tahir (Iowa State University); Beck, John M. (Iowa state university); Beck, Josh R. (Iowa State University)

158 - Coactosin phosphorylation controls Entamoeba histolytica cell membrane protrusions and cell motility Huston, Christopher D. (University of Vermont); Teixeira, Jose E.; Hasan, Muhammad M.; Lam, Ying Wai (University of Vermont)

159- Fast-killing inhibitors of the malaria parasite, Plasmodium falciparum, that disrupt cytosolic and digestive vacuole pH Imlay, Leah S. (UT Southwestern Medical Center); White, John (University of Washington); Hollibaugh, Ryan (UT Southwestern Medical Center); Palmer, Michael J. (Medicines for Malaria Venture); Rathod, Pradipsinh K. (University of Washington); De Brabander, Jef K.; Phillips, Margaret A. (UT Southwestern Medical Center)

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160 - A role for the conserved var gene var2csa in coordinating antigenic variation by malaria parasites Zhang, Xu (Weill Cornell Medical College); Deitsch, Kirk (Weill Cornell Medical College)

161 - EuPathDB resources: tools for analysis, integration and discovery Harb, Omar S.; Brunk, Brian (University of Pennsylvania); Warrenfeltz, Susanne; Kissinger, Jessica (University of Georgia); Roos, David (University of Pennsylvania)

162 - A combination of reverse genetics and live microscopy shows distinctive roles for the P. knowlesi Duffy binding protein and normocyte binding protein families during erythrocyte invasion. Hart, Melissa (LSHTM); Mohring, Franziska; Charleston, James (LSHTM, London.); Hall, Joanna; Almond, Neil (NIBSC, Potters Bar.); Saibil, Helen (Birkbeck, University of London); Moon, Robert (LSHTM)

163 - Probing Cryptosporidium sexual differentiation with small molecules Hasan, Md Mahmudul (University of Vermont) Jumani, Rajiv S. (University of Vermont); Klopfer, Connor; Donnelly, Liam; Franco, Sebastian (University of Vermont); Love, Melissa S.; Mcnamara, Case W. (Calibr); Huston, Christopher D. (University of Vermont)

164 - Thioredoxin peroxidase-I and its nuclear role in Plasmodium falciparum Heinberg, Adina (Hebrew University); Amit-Avraham, Inbar; Thompson, Emilie; Dzikowski, Ron (Hebrew University)

165 - The role of RNA-binding proteins abundant in Apicomplexans (RAPs) in mitochondrial biogenesis of the human malaria parasite, P. falciparum Hollin, Thomas (University of California, Riverside); Prudhomme, Jacques (University of California, Riverside); Pasaje, Charisse F.; Falla, Alejandra (Massachusetts Institute of Technology); Saraf, Anita; Florens, Laurence (Stowers Institute for Medical Research); Niles, Jacquin C. (Massachusetts Institute of Technology); Le Roch, Karine G. (University of California, Riverside)

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166 - I-BET is an anti-Toxoplasma agent that targets TgBDP3, a bromodomain protein in the TFIID complex Jeffers, Vicki (University of New Hampshire); Hanquier, Jocelyne; Sullivan Jr., William J. (Indiana University School of Medicine)

167 - PCR-HRM illustrates diverse malaria parasites circulating in urban-restricted non-human primates in Kenya Jeneby, Maamun (International Centre of Insect Physiology and Ecology- icipe); Villinger, Jandouwe (International Centre of Insect Physiology and Ecology- icipe)

168 - GPI Anchored Micronemal is required across multiple stages of the Plasmodium life cycle Jennison, Charlie Armstrong, Janna M.; Patel, Hardik (SCRI); Minkah, Nana (SCRI, Seattle.); Wilder, Brandon K. (SCRI); Sheikh, Amina; Vaughan, Ashley M.; Kappe, Stefan HI (SCRI)

169 - Contractile vacuole and flagellar homeostasis are regulated by mechanosensitive channels. Jimenez, Veronica (Center for Applied Biotechnology Studies and Department of Biological Science, College of Natural Sciences and Mathematics, California State Universi); Fonbuena, Joshua; Feldman, Marc (1Center for Applied Biotechnology Studies and Department of Biological Science, College of Natural Sciences and Mathematics, California State Universi); Augusto, Ingrid; Miranda, Kildare (2Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro)

170 - Phenotypic Characterization of an Essential Mitochondrial Protein with Unknown Function in Plasmodium falciparum Lamb, ian M. (Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine) Morrisey, Joanne; , Thomas (Drexel University College of Medicine); Jenkins, Bethany M.; Mather, Michael M. (Drexel University College of Medicine); Bergman, Lawrence (Drexel University College of Medicine); Vaidya, Akhil M. (Drexel University College of Medicine)

171 - High level resistance to P. falciparum cytochrome B inhibitors Lane, Kristin D. (NIAID/NIH); Mu, Jianbing (NIAID/NIH); Lu, Jinghua; Liu, Anna; Remcho, Parks (NIAID/NIH); Windle, Sean (University of Washington); Wellems, Thomas (NIAID/NIH)

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172 - Activation of erythrocyte permeability enhances drug uptake by malaria sexual parasites Lavazec, Catherine (Inserm U1016/CNRS UMR8104); Barbieri, Daniela (Inserm,); Bouyer, Guillaume (CNRS); Dupuy, Florian; Marteau, Anthony (Inserm); Sissoko, Abdoulaye (Université de Paris); Neveu, Gaelle; Bedault, Laurianne (Inserm); Roman, Diana (Université de Paris); Siciliano, Giulia; Alano, Pietro (Istituto Superiore di Sanita); Martins, Rafael; Lopez Rubio, Jose Juan (CNRS); Clain, Jérome; Duval, Romain (Université de Paris); Egée, Stéphane (CNRS)

173 - In Search of Novel Antimalarials: Chemical Starting Points from a Natural Product Library Phenotypic Screen. Lawong, Aloysus (UT Southwestern Medical Center); Margaret, Phillips (UT Southwestern Medical Center); Shingare, Rahul (UC Santa Cruz); MacMillan, John (UC Santa Cruz)

174 - Dynamic properties of the Leishmania cap-binding protein IF4E-1 Leger-Abraham, Melissa (Harvard Medical School);

175 - Distinct roles of small subunit proteins of the mitochondrial ribosome in Plasmodium falciparum Ling, Liqin (1, Department of Microbiology and Immunology, Drexel University College of Medicine; 2, West China Hospital, Sichuan University) Mulaka, Maruthi; Dass, Swati; Mather, Michael (Center for Molecular Parasitology, Department of Microbiology and Immunology Drexel University College of Medicine); Riscoe, Michael (Portland VA Medical Center); Zhou, Jing (Department of Laboratory Medicine, West China Hospital, Sichuan University); Ke, Hangjun (Center for Molecular Parasitology, Department of Microbiology and Immunology Drexel University College of Medicine)

176 - Evidence that transcription blockage is associated with signalling to the death in Trypanosoma cruzi BERTOLDO, WILLIAN R. (UFMG); MACEDO, ANDREA M. (UFMG); FRANCO, GLORIA R. (UFMG); MACHADO, CARLOS R. (UFMG)

177 - Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome Bhanot, Purnima (Rutgers - New Jersey Medical School) K. Govindasamy, R. Khan, M. Snyder, H. J. Lou, P. Du, Kudyba, Heather M. (NIH), V. Muralidharan, B. E. Turk, P. Bhanot

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178 - Conserved heterodimeric structure & glycan specificity of C-type lectins Anopheles gambiae CTLMA2 and CTL4 BISHNOI, RITIKA

179 - A novel amino acid substitution in the AP2 of Pfap2-g is associated with a gametocyte-deficient phenotype Prajapati, Surendra K. (Uniformed Services University of the Health Sciences); Morahan, Belinda J. (Monash University); Barbeau, Michelle C.; Hupalo, Daniel (Uniformed Services University of the Health Sciences); Wilkerson, Matthew; Dalgard, Clifton L. (Uniformed Services University of the Health Sciences); Williamson, Kim C. (Uniformed Services University of the Health Sciences)

180 - Plasticity of iron metabolism of brain eating Mach, Jan (Charles University) Ženíšková, Katerina; Arbon, Dominik; Malych, Ronald; Grechnikova, Maria; Sutak, Robert (Charles University)

181 - Utilizing collateral sensitivity to dihydroorotate dehydrogenase inhibitors as a strategy to suppress pathways to resistance Mandt, Rebecca E.K. (Harvard T.H. Chan School of Public Health); Lafuente-Monasterio, Maria Jose (Tres Cantos Medicines Development Campus, Tres Cantos, Madrid.); Luth, Madeline R. (University of California San Diego, School of Medicine); Reynolds, Matthew (Harvard College); Ottilie, Sabine; Winzeler, Elizabeth A. (University of California San Diego, School of Medicine); Gamo, F. Javier (Tres Cantos Medicines Development Campus); Wirth, Dyann F.; Lukens, Amanda K. (Harvard T.H. Chan School of Public Health)

182 - Guanabenz Reverses a Key Behavioral Change Caused by Latent Toxoplasmosis in Mice by Reducing Neuroinflammation Martynowicz, Jennifer. (Indiana University School of Medicine); Augusto, Leonardo; Wek, Ronald C. (Indiana University School of Medicine); Boehm II, Stephen L. (Indiana University Purdue University Indianapolis); Sullivan Jr, William J. (Indiana University School of Medicine.)

183 - Unveiling the role of UIS4 during malaria liver stage of infection M'Bana, Viriato (Instituto de Medicina Molecular, João Loo Antunes, Lisboa); Lahree, Aparajita; Slavic, Ksenija; Mota, Maria (Instituto de Medicina Molecular)

184 - Evolution of a new pathway of reserve carbohydrate biosynthesis in Leishmania parasites McConville, Malcolm j. (University of Melbourne); Serne, Fleur (University of Melbourne); Ralton, Julie (University of Melbourne); Nero, Tracy (University of Melbourne); Kloehn, Joachim; Cobbold, Simon; Hanssen, Eric; Parker, Michael; Williams, Spencer (University of Melbourne); Davies, Gideon (University of York) 52

185 - Identification of antigens and important for protection conferred by radiation-attenuated Plasmodium falciparum sporozoite vaccination McDermott, Suzanne M. (Seattle Children's Research Institute); Osman, Rahwa; Stuart, Ken (Seattle Children's Research Institute)

186 - A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites Meister, Thomas R. (Stanford University); Tang, Yong (Stanford University); Walczak, Marta (Stanford Universtiy); Pulkoski-Gross, Michael J. (Stanford University); Hari, Sanjay B.; Sauer, Robert T. (Massachusetts Institute of Technology); Amberg-Johnson, Katherine (Stanford University); Yeh, Ellen (Stanford Universtiy)

187 - A novel putative telomere-binding protein in Plasmodium falciparum Merrick, Catherine J. (Cambridge University); Edwards-Smallbone, James (Keele University); Jensen, Anders L.; Totanes, Francis I.G.; Roberts, Lydia E. (Cambridge University); Hart, Sarah R. (Keele University)

188 - Plasmodium histone deacetylase 1 (HDA1) plays important, dual, sex specific roles in gametocyte maturation and viability Millar, Scott (University of Glasgow); Power, B. Joanne; Martin, Julie; Hughes, Katie; Waters, Andy (University of Glasgow)

189 - Investigation of transcriptional regulation in Plasmodium ookinetes Modrzynska, Katarzyna K. (University of Glasgow) Morton, Rachel; Kirchner, Sebastian (University of Glasgow); Nyarko, Prince (West African Centre for Cell Biology of Infectious Pathogens (WACCBIP); Waters, Andrew P. (University of Glasgow)

190 - Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research Mohring, Franziska (London School of Hygiene and Tropical Medicine) Hart, Melissa N. (London School of Hygiene and Tropical Medicine); Rawlinson, Thomas A. (The Jenner Institute, University of Oxford); Henrici, Ryan; Charleston, James A.; Diez Benavente, Ernest; Patel, Avnish (London School of Hygiene and Tropical Medicine); Hall, Joanna (National Institute of Biological Standards and Control, Potters Bar.); Almond, Neil (National Institute for Biological Standards and Control); Campino, Susana; , Taane G.; Sutherland, Colin J.; Baker, David A. (London School of Hygiene and Tropical Medicine); Draper, Simon J. (The Jenner Institute, University of Oxford); Moon, Robert W. (London School of hygiene and Tropical Medicine)

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191 - Targeting parasite-produced MIF as an anti-virulence strategy to reduce tissue damage Moonah, Shannon. (University of Virginia); Ghosh, Swagata; Padalia, Jay (University of Virginia)

192 - Interactions between ribosomal proteins and fragmented rRNAs in the mitochondrial ribosome of Plasmodium falciparum Mulaka, Maruthi (Drexel University College of Medicine); Das, Swati (Drexel University College of Medicine); Ling, Liqin (Drexel Univeristy College of Medicine); Ke, Hangjun (Drexel University College of Medicine)

193 - Using metabolomics to understand the role of acetyl-CoA in asexual Plasmodium falciparum parasites. Munro, Justin (Pennsylvania State University); Allman, Erik (Pennsylvania State University); Nair, Sethu; Prigge, Sean (Bloomberg School of Public Health); Llinás, Manuel (Pennsylvania State University)

194 - Targeting Aurora-like Kinases in Plasmodium falciparum Murillo_Solano, Claribel; Vienneau-Hathaway, Jannelle (UCF); Murithi, James (Columbia University Medical Center); Collins, Jennifer; Shaw, Justin (UCF); Buskes, Melissa; Ferrins, Lori (Northeastern University); Chakrabarti, Ratna (UCF); Pollastri, Michael (Northeastern University); Fidock, David A. (Columbia University Medical Center); Chakrabarti, Debopam (UCF)

195 - Compound stage-specificity and metabolic profiling to advance malaria drug discovery Murithi, James (CUIMC); Owen, Edward (Penn State University); Otillie, Sabine (UCSD); Chibale, Kelly (UCT); Winzeler, Elizabeth (UCSD); Llinás, Manuel (Penn State); Vanaerschot, Manu; Fidock, David (CUIMC)

196 - Discovery and Characterization of Antiplasmodial Compounds Targeting Protein Kinases Bohmer, Monica (University of Central Florida) Barq, Alya (University of Central Florida); Sausman, William (University of Texas at San Antonio); Chakrabarti, Ratna (University of Central Florida); Hanson, Kirsten (University of Texas at San Antonio); Chakrabarti, Debopam (University of Central Florida)

197 - Host adaptation in Toxoplasma gondii - What makes this parasite so “successful”? Naor, Adit (Stanford); Boothroyd, John C. (Stanford) 54

198 - Tadalafil clears gametocytes from blood circulation in humanized mice N'DRI, Marie-Esther (Inserm U1016, Institut Cochin, CNRS, UMR8104, Université Paris ); BARBIERI, Daniela; GOMEZ, Lina; DUPUY, Florian (Inserm U1016, Institut Cochin, CNRS, UMR8104, Université Paris Descartes); FRANETICH, Jean François; MAZIER, Dominique (Sorbonne Université, INSERM, CNRS, Centre d'Immunologie et des Maladies Infectieuses, CIMI); MORENO-SABATER, Alicia (Sorbonne Université, Inserm U1135, CNRS ERL 8255, Centre d'Immunologie et des Maladies Infectieuses (Cimi-Paris); LAVAZEC, Catherine (Inserm U1016, Institut Cochin, CNRS, UMR8104, Université Paris Descartes)

199 - Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites Okada, Megan (University of Utah); Okada, Amanda L.; Maschek, John A. (University of Utah); Swift, Russell (Johns Hopkins); Prigge, Sean T. (John Hopkins); Sigala, Paul A. (University of Utah)

200 - Fatty acid elongases are critical for maintaining membrane lipid homeostasis in Trypanosoma cruzi Pagura, Lucas (Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health); Dumoulin, Peter C. (Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health); Mendes, María Tays; Ellis, Cameron C.; Almeida, Igor C. (Biological Sciences Department, University of Texas at El Paso); Burleigh, Barbara A. (Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health)

201 - The Plasmodium Clp protease system controls plastid biogenesis via degradation of specific substrates Florentin, Anat (University of Georgia); Stephens, Dylon; Anaguano, David; Brooks, Carrie; Muralidharan, Vasant (University of Georgia)

202 - Roles of calcium signaling and cyclic nucleotide signaling in control of invasion and egress of bradyzoites Fu, Yong (Washington University School of Medicine); Brown, Kevin; Jones, Nathaniel; Sibley, L. David (Washington University School of Medicine)

203 - Exploring COP9 signalosome as a novel therapeutic target for protozoan parasites Ghosh, Swagata (University of Virginia) Moonah, Shannon (University of Virginia)

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204 - Genome Wide CRISPR/Cas9 Knockout Screen Identifies Host Genes Important for Cryptosporidium parvum Infection Gibson, Alexis R. (University of Pennsylvania); Sateriale, Adam R.; Beiting, Daniel R.; Striepen, Boris R. (University of Pennsylvania)

205 - Elucidating spatially-resolved changes in host signaling during Plasmodium liver-stage infection Glennon, Elizabeth (Seattle Children's Research Institute); Pan, Liuliu; Liang, Yan (NanoString Technologies); Kaushansky, Alexis (Seattle Children's Research Institute)

206 - Trypanosoma brucei and Trypanosoma congolense cathepsin L proteolyse human TLF and free recombinant APOL1 Gonzalez, Bernardo (Hunter College); Raper, Jayne (Hunter College); Verdi, Joe (Hunter College); Thomson, Russell; Sanchez, Alan (Hunter College)

207 - The Canonical Histone H2A, of the Malaria Parasite Plasmodium falciparum is Phosphorylated and Recruited to Foci of Damaged DNA Goyal, Manish (Hebrew University) Singh, Brajesh Kumar; Dzikowski, Ron (Hebrew University)

208 - Full development of tsetse-transmitted trypanosomes in advanced human skin tissue models Engstler, Markus (University of Wuerzburg)

209 - Complexome profiling reveals Plasmodium falciparum mitochondrial protein dynamics (during sexual differentiation) Evers, Felix (Radboud Institute for Molecular Life Sciences); Cabrera-Orefice, Alfredo; Brandt, Ulrich (Radboud University Medical Center); Kooij, Taco W.A. (Radboud Institute for Molecular Life Sciences)

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Poster Session C Wednesday, September 18, 2019 3:00 pm – 5:00 pm

210 - Characterization of a Novel Sensing Mechanism Governing Antigenic Variation and Immune Evasion in P. falciparum Schneider, Victoria (The Rockefeller University); Ben Mamoun, Choukri (Yale University); Rhee, Kyu; Harris, Chantal; Kafsack, Bjorn; Deitsch, Kirk (Weill Cornell Medical College) For Abstract See Session X, 61t

211 - Examining trans-regulators of the Leishmania lifecycle Walrad, Pegine B. (York Biomedical Research Institute, Univ. of York); R. Ferreira, Tiago; Pablos, Luis; Dowle, Adam; Forrester, Sarah; Parry, Ewan; V. C. Alves-Ferreira, Eliza; Newling, Katherine; Kolokousi, Foteini; Larson, Tony; Plevin, Michael (University of York, University of York.); K.Cruz, Angela (University São Paulo) For Abstract See Session X, 62t

212 - Redox metabolomics and gametocytogenesis in the malaria parasite Beri, Divya (Indian Institute of Science) Tatu, Utpal (Indian Institute of Science) For Abstract See Session X, 63t

213 - Functional and biochemical characterization of SPY- catalysed nucleocytosolic O-fucosylation in Toxoplasma gondii Bandini, Giulia (Boston University); Agop-Nersesian, Carolina (Boston University); van der Wel, Henke; West, Christopher M. (University of Georgia); Samuelson, John (Boston University) For Abstract See Session XI, 66t

214- The novel EhHSTF7 transcription factor binds to HSE and regulates the multidrug resistant Ehpgp5 gene expression in Entamoeba histolytica. Bello, Fabiola (Centro de Investigación y Estudios Avanzados, IPN); Orozco, Esther (Centro de Investigación y Estudios Avanzados, IPN); Pérez, Guillermo; Gómez, Consuelo (Escuela Nacional de Medicina y Homeopatía) For Abstract See Session XI, 67t

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215 - Decoding combinatorial patterns of histone post- translational modifications of Plasmodium falciparum. von Grüning, Hilde (University of Pretoria); Coradin, Mariel; Mendoza, Mariel G. (University of Pennsylvania); Sidoli, Simone (Albert College of Medicine); Garcia, Benjamin A. (University of Pennsylvania); Birkholtz, Lyn-Marie (University of Pretoria)

216 - The regulation of translational control during human-to- transmission of Plasmodium falciparum Bennink, Sandra (Division of Cellular and Applied Infection Biology, RWTH Aachen University); von Bohl, Andreas; Ngwa, Che Julius; Pilch, Nicole (Division of Cellular and Applied Infection Biology, RWTH Aachen University); Minns, Allen; Orchard, Lindsey; Lindner, Scott; Llinás, Manuel (Department of Biochemistry and Molecular Biology, The Pennsylvania State University); Pradel, Gabriele (Division of Cellular and Applied Infection Biology, RWTH Aachen University)

217 - Genetic mapping of strain specific differences in autophagy effector recruitment to the Toxoplasma gondii parasitophorous vacuole Radke, Joshua (Washington University School of Medicine); Sibley, David (Washington University School of Medicine)

218 - Cells in tandem: A novel essential “basal body” protein in Trypanosoma brucei Ramanantsalama, Miharisoa Rijatiana MR (UMR5234 CNRS University of Bordeaux, MFP); Sahin, Annelise; Bonhivers, Mélanie; Robinson, Derrick DR; Dacheux, Denis (UMR5234 CNRS University of Bordeaux, MFP)

219 - Oligomerization of Anti-malarial drug target PfATP4 is essential for its function Ramanathan, Aarti A. (Drexel university); Morrisey, Joanne; Mather, Michael W.; Bergman, Lawrence W.; Vaidya, Akhil B (Drexel university)

220 - Plasma membrane is repaired by acid sphingomyelinase in E. histolytica Ramirez-Montiel, Fatima (Universidad de Guanajuato) Mendoza-Macias, Claudia; Andrade-Guillen, Sairy; Rangel-Serrano, Angeles; Paramo-Perez, Itzel; Rivera-Cuellar, Paris (Universidad de Guanajuato); España-Sanchez, Liliana (Centro de Investigación y Desarrollo en Electroquímica); Anaya-Velazquez, Fernando; Franco, Bernardo; Padilla- Vaca, Felipe (Universidad de Guanajuato)

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221 - PfGCN5, a global regulator of stress responsive genes, modulates artemisinin resistance in Plasmodium falciparum Rawat, Mukul (Indian Institute of Science Education and Research, Pune) Kanyal, Abhishek; Sahasrabudhe, Aishwarya (Indian Institute of Science Education and Research); Vembar, Shruthi S. (Institute for and Applied Biotechnology); Lopez-Rubio, Jose-Juan (Dynamique des Interactions Membranaires Normales et Pathologiques); Karmodiya, Krishanpal (Indian Institute of Science Education and Research)

222 - Evaluation of WR99210 from different manufacturers for selection of transfected Plasmodium falciparum Remcho, T. Parks. Liu, Anna; Wellems, Thomas E.; Lane, Kristin D. (NIAID/NIH)

223 - RNA secondary structure determination of translationally repressed transcripts in P. falciparum gametocytes Rios, Kelly T. (Pennsylvania State University) Lindner, Scott E. (Pennsylvania State University)

224 - Quantifiable imaging-based physiological parameters to define chronic Toxoplasma infections in vivo Watts, Elizabeth (University of Kenucky College of Medicine); Murphy, Robert; Dhara, Animesh (University of Kentucky College of Medicine); Patwardhan, Abhijit (University of Kentucky College of Enginnering)

225 - Characterization of a Novel Protein Phosphatase 1 Complex Involved in RNA Polymerase II Transcription Termination in Kinetoplastids Zhang, Yang

226 - Show me your ID and come in - Targeting of the lysosomal degradome in Trichomonas vaginalis Zimmann, Nadine (BIOCEV); Rada, Petr; Hrdý, Ivan; Tachezy, Jan (BIOCEV)

227 - Dissecting the role of plasmepsin II and III in piperaquine resistant P. falciparum lines Walsh, Breanna (Harvard T.H. Chan School of Public Health); Summers, Robert; Volkman, Sarah; Wirth, Dyann (Harvard T.H. Chan School of Public Health); Bopp, Selina (Harvard T.H. Chan School of Public Health)

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228 - An intravenous preparation of the antimalarial OZ439 for nonhuman primate laboratory studies Sa, Juliana M. (NIAID) Salzman, Rebecca E. (NIAID); Melendez-Muniz, Viviana A. (NIAID); Figan, Christine E.; Moraes-Barros, Roberto R. (NIAID); Burrows, Jeremy (Medicines for Malaria Venture); Walker, Larry (Campbell University); Wellems, Thomas E. (NIAID)

229 - A novel Plasmodium yoelii serine threonine kinase related to exflagellation center formation Ishizaki, Takahiro (The Institute of Tropical Medicine (NEKKEN), Nagasaki University); Chaiyawong, Nattawat; Asada, Masahito; Yahata, Kazuhide; Culleton, Richard; Kaneko, Osamu (The Institute of Tropical Medicine (NEKKEN), Nagasaki University)

230 - The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development Devendra Kumar Gupta1, Laurent Dembele1, Annemarie Voorberg Vander-wel2, Stefan H. I. Kappe3, Erika L. Flannery1, Sebastian A. Mikolajczak1, Pablo Bifani1, Clemens H. M. Kocken2, Thierry T. Diagana1

231 - G-quadruplex interactome in Plasmodium falciparum Gurung, Pratima (LPHI, University of Montpellier); Gazanion, Elodie (Université de Montpellier); Gomes, Ana-Rita; Guitard, Vincent (LPHI, University of Montpellier); Paeschke, Katrin (University Clinic Bonn Biomedical Center (BMZ); Alberti, Patrizia (Nucleic Acid Structures, Telomeres and Evolution,MNHN,CNRS,UMR 7196); Urbach, Serge (BioCampus Montpellier, CNRS UMR 5203); Lopez-Rubio, Jose-Juan (LPHI, University of Montpellier)

232 - Translocation of dense granule effectors across the parasitophorous vacuole membrane in Toxoplasma-infected cells requires the activity of ROP17, a rhoptry protein kinase. Panas, Michael W. (Stanford University); Ferrel, Abel; Naor, Adit (Stanford); Tenborg, Elizabeth (UC Davis); Lorenzi, Hernan (JCVI); Boothroyd, John (Stanford)

233 - Comparative transcriptomics of three lizard Plasmodium species Pangburn, Sarah (CUNY Graduate Center); Borner, Janus; Perkins, Susan (American Museum of Natural History)

234 - Cellular signaling in the regulation of Giardia cyst formation Paredez, Alexander (University of Washington); Shih, Han-Wei; Alas, Germain (University of Washington)

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235 - Characterization of a Plasmodium falciparum acetyl-CoA synthetase and targeted discovery of inhibitors using functional genomics Pasaje, Charisse Flerida A. (Massachusetts Institute of Technology); Cárdenas Ramirez, Pablo (Massachusetts Institute of Technology); Hollin, Thomas; Abel, Steven (University of California, Riverside); Summers, Robert L. (Harvard T.H. Chan School of Public Health); Lukens, Amanda K. (Harvard T.H. Chan School of Public Health); Vanaerschot, Manu; Murithi, James (Columbia University Irving Medical Center); Ruth, Madeline R.; Ottilie, Sabine (University of California, San Diego); Le Roch, Karine (University of California, Riverside); Fidock, David A. (Columbia University Irving Medical Center); Winzeler, Elizabeth A. (University of California, San Diego); Wirth, Dyann F. (Harvard T.H. Chan School of Public Health); Niles, Jacquin C. (Massachusetts Institute of Technology)

236 - Quantitative method for PK/PD studies of DHODH inhibitors in vivo Pontikos, Michael; Leija, Christopher; Williams, Noelle S.; Phillips, Margaret A. (University of Texas Southwestern Medical Center)

237 - The impact of organelle dynamics on mitochondrial structure and function in kinetoplastids Povelones, Megan L. (Penn State Brandywine); DiMaio, John (Penn State Brandywine); Ruthel, Gordon (University of Pennsylvania); Malfara, Madeline F.; Iatsenko, Ekaterina; Pitts, Joseph; Jackson, Jasmine (Penn State Brandywine)

238 - Identification of a mechanism of resistance to itraconazole in Toxoplasma gondii Alday, Phil H. (VA Portland Healthcare System); Zhicheng, Dou (Clemson Universoty); Carruthers, Vern (University of Michigan Medical School); Doggett, Joseph S. (VA Portland Medical Center)

239 - Understanding how var2csa uORF signals for cellular localization in Plasmodium falciparum Assaraf, Shany (Hebrew university) Fastman, Yair; Dzikowski, Ron (Hebrew university)

240 - Elucidating the nutrient acquisition strategies of Cryptosporidium. SOMEPALLI, MASTANBABU (University of Pennsylvania); Berry, Laurence (Université de Montpellier); Striepen, Boris (University of Pennsylvania)

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241 - Ablation of the TSSA (Trypomastigote Small Surface Antigen) gene causes infection impairment in Trypanosoma cruzi trypomastigotes Balouz, Virginia (Institute for Research in Biotechnology); Cámara, María de M; Rodriguez, Matías E. (Institute for Research in Biotechnology); Cruz-Bustos, Teresa (Center for Tropical and Emerging Global Diseases (CTEGD); Masip, Yamil E. (Institute for Research in Biotechnology); Berná, Luisa (Institut Pasteur de Montevideo); Burasi, Florencia; Lobo, Maite; Centeno Camean, Camila (Institute for Research in Biotechnology); Robello, Carlos (Institut Pasteur de Montevideo); Docampo, Roberto (Center for Tropical and Emerging Global Diseases (CTEGD); Tekiel, Valeria; Buscaglia, Carlos A. (Institute for Research in Biotechnology)

242 - Functional mapping of the ap2-g upstream sequence in Plasmodium falciparum Basson, Travis (Swiss TPH, University of Basel) Voss, Till S. (Swiss TPH, University of Basel)

243 - Analysis of interacting partners of the nuclear Giardia intestinalis cytochrome b5 Batoff, William

244 - Plasmodium falciparum Merozoite surface proteome and Post-Translational Modifications (PTMs) Saini, Ekta (ICGEB,New Delhi) kaur, Inderjeet (ICGEB); chauhan, charu; Sampathkumar, Gopalan (National Institute of Immunology); Malhotra, Pawan (ICGEB.)

245 - Characterizing the Myosin F interactome in Toxoplasma gondii using BioID Schiano, Irio Heaslip, Aoife T. (University of Connecticut)

246 - Purification of the RhopH complex for functional reconstitution of malaria nutrient channels Schureck, Marc A. (NIAID) Desai, Sanjay A. (National Institutes of Health)

247 - Study of reduced artemisinin susceptibility in West African Plasmodium falciparum isolates Sharma, Aabha I. (Harvard T. H. Chan School of Public Health) Demas, Allison R. (Ragon Institute of MIT and Harvard); Bopp, Selina; Volkman, Sarah K. (Harvard T. H. Chan School of Public Health); Hartl, Daniel L. (Harvard University); Wirth, Dyann F. (Harvard T. H. Chan School of Public Health)

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248- Extracellular Vesicles secreted by Entamoeba histolytica parasites contain small RNAs targeting specific genes Sharma, Manu (Stanford University); Morgado, Pedro; Zhang, Hanbang; Ehrenkaufer, Gretchen; Singh, Upinder (Stanford University)

249 - Maintenance of cholesterol homeostasis in sexual stages of P. falciparum Shukla, Anurag Morrisey, Joanne of; Vaidya, Akhil B. (Drexel University College of Medicine)

250 - The impact of PfK13 C580Y on global transcription through the intraerythrocytic developmental cycle of Plasmodium falciparum Sievert, Mackenzie AC (Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame); Checkley, Lisa A.; Davis, Sage Z.; , Gabriel J. (Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame); Nosten, Francois H. (Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University); Nair, Shalini (Texas Biomedical Research Institute); Khoo, Sok Kean (Department of Cell and Molecular Biology, Grand Valley State University); Anderson, Timothy JC (Texas Biomedical Research Institute); Ferdig, Michael T. (Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame)

251 - Plasmodium enolase and GAPDH as plasminogen and tPA receptors Silva, Thiago (National institute of health);

252 - Using Nanoparticles as a vehicle for the slow release of Artemisinin in the malaria parasite, Plasmodium falciparum Simantov, Karina (Hebrew University); Zemmour, Chalom; Stern, Tal; Benny, Ofra; Dzikoswki, Ron (Hebrew University)

253 - Novel haplotypes of PfCRT in Plasmodium falciparum from the Yunnan Province, China confer resistance to the first- line antimalarial piperaquine Small-Saunders, Jennifer L. (Columbia University Medical Center) Hagenah, Laura M.; Dhingra, Satish K.; Fidock, David A. (Columbia University Medical Center)

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254 - Toxoplasma gondii Apical Cap 9 is essential to anchor the conoid to the apical polar ring of mature parasites Soldati-Favre, Dominique (University of Geneva); Tosetti, Nicolo; Dos Santos Pacheco, Nicolas; Maco, Bohumil (University of Geneva)

255 - Is calcium the gear shift? The role of calcium-mediated phosphorylation of TgMyoA Stadler, Rachel V. (Department of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine); Rould, Mark A. (Macromolecular Consulting LLC); Vella, Stephen A. (Department of Microbiology Center for Tropical and Emerging Global Diseases, University of Georgia); Kelsen, Anne (Department of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine); Moreno, Silvia N J (Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia); Ward, Gary E. (Department of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine)

256 - Transcriptome-wide in vivo mRNA target identification of RNA-binding proteins essential for P. falciparum sexual development Stasic, Andrew J. (US Food and Drug Administration); Painter, Heather J. (US Food and Drug Administration)

257 - High-throughput screen to identify specific Inhibitors of the Plasmodium Protease, PfClpP Stephens, Dylon (The University of Georgia); Florentin, Anat; Muralidharan, Vasant (The University of Georgia)

258 - TcAMPK: identification and characterization of a cellular energy homeostasis hub regulator in Trypanosoma cruzi Sternlieb, Tamara (Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" – CONICET) Schoijet, Alejandra C.; Genta, Patricio D.; Alonso, Guillermo D. (Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" - CONICET, Ciudad Autónoma de Buenos Aires.)

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259 - Plasmodium-selective peptide vinyl sulfone proteasome inhibitors are potent inhibitors of multidrug-resistant malaria parasites and exhibit a low potential for generating resistance in vitro Stokes, Barbara H. (Columbia University Irving Medical Center Department of Microbiology); Yoo, Euna (Stanford University Medical Center Department of Pathology); Murithi, James M. (Columbia University Irving Medical Center Department of Microbiology); Luth, Madeline R. (UCSD School of Medicine Division of Host-Microbe Systems & Therapeutics); Afanasyev, Pavel; da Fonseca, Paula C.A. (MRC Laboratory of Molecular Biology); Winzeler, Elizabeth A. (UCSD School of Medicine Division of Host- Microbe Systems & Therapeutics); Ng, Caroline L. (University of Nebraska Medical Center Department of Pathology and Microbiology); Fidock, David A. (Columbia University Irving Medical Center Department of Microbiology)

260 - Human immune response differences between protected and not-protected participants vaccinated with radiation- attenuated Plasmodium falciparum sporozoites. Stuart, Ken (Seattle Children's Research Institute); Du, Ying (Seattle Children's Research Institute); Hertoghs, Nina; DeRosa, Stephen (Fred Hutchinson Cancer Research Center); Schwedhelm, Katharine (Fred Hutchison Cancer Research Center); Carnes, Jason; Duffy, Fergal; Neal, Maxwell (Seattle Children's Research Institute); McDermott, Suzanne (Seattle Children's Reearch Institute); Daubenberger, Claudia (Swiss TPH); Epstein, Judith (Navy Medical Research Center); Richie, Thomas; Hoffman, Stephen (Sanaria); Aitchison, John (Seattle Children's Research Institute); Gottardo, Raphael; McElrath, Julie (Fred Hutchinson Cancer Research Center)

261 - Dissecting Pathogenesis of Central Nervous System (CNS)-parasites by Electron Cryo-tomography (Cryo-ET) Sun, Stella Y. (Stanford University); Chen, Muyuan (baylor college of medicine); Kaelber, Jason T. (Rutgers University); Dong, Xiaoduo (National University of Singapore); Shi, Jian; Nematbakhsh, Yasaman; Lim, Chwee Teck (National University of Singapore); Schmid, Michael F.; Zarko, Li-av S.; Boothroyd, John C. (Stanford University); He, Cynthia Y. (National University of Singapore); Chiu, Wah (Stanford University)

262 - Vesicular mechanisms provide phenotypic assays of artemisinin resistance in Plasmodium falciparum malaria Suresh, Niraja (University of Notre Dame); Khair nima, Maisha (University of Notre Dame); Coppens, Isabelle (Johns Hopkins University); Bhattacharjee, Souvik (Jawaharlal Nehru University); Ghorbal, Mehdi; Haldar, Kasturi (University of Notre Dame)

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263 - The disulfide bonds as functional motifs of the Myb3 transcription factor in Trichomonas vaginalis Tai, Junghsiang (Institute of Biomedical Sciences); Chen, Shu-Hui (National Chen- Kung University); Tai, Junhg-Hsiang (National Taiwan University); Huang, Jessica (National Chen-Kung University)

264 - Pathogen Box Chemical Library as an antimalarial toolbox for phenotypic and chemogenomic studies on Plasmodium falciparum. Tanala Patra, Alok (Singapore University of Technology and Design)

265 - Lifecycle progression and sexual development of the apicomplexan parasite Cryptosporidium parvum Tandel, Jayesh (University of Pennsylvania); English, Elizabeth; Sateriale, Adam; Gullicksrud, Jodi; Beiting, Daniel (University of Pennslyvania); Pinkston, Brittain (University of Georgia); Striepen, Boris (University of Pennslyvania)

266 - The use of oxidative stressors and inducible targets for assessing the viability of foodborne Temesgen, Tamirat Tefera (Norwegian University of Life sciences); Tysnes, Kristoffer; Robertson, Lucy (Norwegian University of Life sciences)

267 - Ribozyme-mediated, multiplex CRISPR gene editing and CRISPR interference (CRISPRi) in rodent-infectious Plasmodium yoelii Walker, Michael P. (Penn State University); Lindner, Scott E. (Pennsylvania State University)

268 - Regulation of PIP39 expression by mitochondrial protein translocase, Tim50, and its role in Trypanosoma brucei infectivity TRIPATHI, ANUJ (MEHARRY MEDICAL COLLEGE); SINGHA, UJJAL K.; Chaudhuri, Minu (Meharry Medical College)

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269 - In vitro and in vivo evidence that GDV1 regulates sexual differentiation upstream of ap2-g Usui, Miho (Uniformed Services University of the Health Sciences); Prajapati, Surendra K. (Uniformed Services University of the Health Sciences); Ayanful-Torgby, Ruth; Acquah, Festus K.; Cudjoe, Elizabeth; Kakaney, Courage; Amponsah, Jones A. (Noguchi Memorial Institute for Medical Research); Obboh, (School of Medical Sciences, University of Cape Coast); Reddy, Deepti K.; Barbeau, Michelle C. (Uniformed Services University of the Health Sciences); Simons, Lacy M. (Loyola University Chicago); Czesny, Beata (Loyola University Chicago); Raiciulescu, Sorana; Olsen, Cara (Uniformed Services University of the Health Sciences); Abuaku, Benjamin K.; Amoah, Linda E. (Noguchi Memorial Institute for Medical Research); Williamson, Kim C. (Uniformed Services University of the Health Sciences)

270 - Functional characterization of a DNA sequence motif associated with Plasmodium falciparum gametocyte- associated transcripts Van Biljon, Riëtte (Pennsylvania) Russell, Timothy J.; Llinás, Manuel (Pennsylvania State University)

271 - Development of an ookinete and zygote inhibition assay for prescreening of transmission-blocking candidates Varadharajan Suresh, Ragavan (National Institute of Health); Miura, Kazutoyo; Deng, Bingbing; Zhou, Luwen (National Institute of Health); Tachibana, Mayumi (Ehime University); Long, Carole A. (National Institute of Health)

272 - High Molecular Weight Chitinase Invasion Complex Secreted by Plasmodium Ookinetes Enables Mosquito Midgut Invasion Vinetz, Joseph M. (Yale School of Medicine); Patra, Kailash P. (UCSD); Prieto, Helena (Western Connecticut State University); Yates, John R. (Scripps Research Institute); Kaur, Hargobinder (Yale School of Medicine)

273 - Molecular mechansims of host translational control during Toxoplasma gondii infection Louis-Philippe Leroux1, Julie Lorent2, Tyson E. Graber3, Visnu Chaparro1, Laia Masvidal2, Tommy Alain3, Ola Larsson2, Maritza Jaramillo1 1Institut National de la Recherche Scientifique, Quebec, Canada 2Karolinska Institutet, Stockholm, Sweden 3University of Ottawa, Ontario, Canada

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Session I: I’m In Love with the Shape of You: From Structure to Function

Chairs: Alexis Kaushanky and Chris de Graffenried

Sunday, September 15th, 2019

7:00pm – 9:00pm

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1 - Nanoscale Elucidation of the Invasion Apparatus of Apicomplexan Parasites

Segev Zarko, Li-av (Stanford University); Sun, Stella; Dahlberg, Peter (Stanford University); Pelt, Danile (University of California Berkeley); Chen, Jian-Hua (Lawrence Berkeley National Laboratory); Schmid, Michael; Galaz Montoya, Jesus; Moerner, W.E. (Stanford University); Larabell, Carolyn (University of California San-Francisco); Sethian, James (University of California Berkeley); Chiu, Wah; Boothroyd, John (Stanford University)

The phylum Apicomplexa includes several of the most important and prevalent eukaryotic human parasites, such as the malaria-causing Plasmodium spp. and Toxoplasma gondii that can cause severe neurological disease in the developing fetus. These intracellular parasites enter a host cell by deploying a remarkable machine at their anterior end known as the apical complex (AC), for which the phylum is named. At the start of invasion, the AC, including a unique spiral of -based fibrils called the conoid, is protruded and secretion events occur from two types of distinct secretory organelles, capsule-shaped micronemes and club shaped rhoptries. The exact means by which these various components of the AC coordinate invasion has been largely a mystery due, in part, to a lack of tools capable of resolving the structure of this extraordinary apparatus in its natural context. To address this need, we are developing a pipeline to image the complex and dynamic structure of the AC to high resolution in parasites devoid of any chemical fixation or staining. This pipeline starts with soft x-ray tomography of plunge frozen parasites to reveal the whole 3D shape of extracellular parasites and the organization of large subcellular organelles inside it. Next, cryo-fluorescence super resolution microscopy and cryo-electron tomography (cryo-ET) are used to precisely localize known apical proteins to the AC subcellular organelles through fusion with a photoactivable fluorescent protein, PAmKate, to record fluorescence originating from single molecules. Finally, nano-scale resolution images of these same parasite’s AC are acquired using cryo-electron tomography (cryo-ET) and annotation of the reconstructed tomograms is performed using mixed scale convolutional neural network analysis. Aligning the data from the three imaging techniques should provide the precise location of the protein while displaying the 3D organization of all the subcellular organelles and uncovering possible interactions between them. To start, we have used cryo-ET generated images of unprecedented resolution of the apical rings, a component of the AC thought to be conserved among all apicomplexan parasites whose structure and function is unknown. Our images reveal that the apical rings exhibit a highly organized structure of repeated units, allowing us to apply techniques of subtomogram averaging to resolve its structure at nano-scale resolution. To validate our pipeline, we have endogenously tagged SAS6-L, a protein that was previously shown to localize at the apical tip of the conoid and are analyzing this line through the methods described above.

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2 - An ORFeome-based Gain-of-Function library for Trypanosoma brucei suggests mitochondria as a key player melarsoprol killing and resistance

Carter, McKenzie R. (The George Washington University); Kim, Hee-Sok (Rutgers NJ Medical School); Gomez, Stephanie; Gritz, Sam (The George Washington University); Schulz, Danae (Harvey Mudd College); Hovel-Miner, Galadriel (The George Washington University)

Melarsoprol remains an essential treatment of second stage Human (HAT) for both human infectious sub-species (T.b. gambiense & rhodesiense). Treatment with melarsoprol is associated with high incidence of toxic side effects, cases of drug resistance, and increasing melarsoprol treatment failures. The mode-of-action and mechanisms of resistance to melarsoprol remain largely unknown, their elucidation could promote the identification of new drug targets against African trypanosomes and other Kinetoplastida parasites. Previous, RNAi-based genetic screening enabled the identification of key transporters and kinases in the melarsoprol pathway, but these approaches are not expected to identify drug targets or essential genes. In contrast to loss-of-function (RNAi-based) forward genetics approaches, Gain- of-Function studies (overexpression-based) can be advantageous in the identification of regulatory pathways and drug targets in a manner that does not exclude essential genes. Aspects of Trypanosome gene expression regulation preclude the use of traditional approaches for generating overexpression libraries (namely cDNA-based) and existing overexpression libraries, based on whole-genome fragmentation, lack specificity. To overcome these challenges, we produced a T. brucei whole-genome ORFeome, in which each targeted gene was PCR amplified and cloned for regulated inducible gene expression. The next-generation sequencing validated ORFeome was used to create a T. brucei Gain-of-Function (GoF) library containing 90% of the targeted genes with approximately 20-copies. To elucidate the mode-of-action of melarsoprol and discover the basis of reported treatment failures, we conducted a GoF genetic screen to identify genes that promote melarsoprol resistance. Induction of the GoF library in the presence of melarsoprol (17 nM) permitted the isolation of drug resistant cell populations. Following next-generation sequence analysis and statistical validation, 30 genes were significantly overrepresented in the melarsoprol resistant populations compared to GoF library input. Notably, the list of 30 hits included an anticipated target of melarsoprol (gamma- glutamylcysteine synthetase, Tb927.10.12370), yet most of the remaining genes have no known function and are annotated as conserved hypothetical. Upon closer examination, we found that approximately 1/3 of the proteins encoded by these genes are associated with the mitochondria or kinetoplast (by localization or proteomics), including one known protein of mitochondrial fatty acid biosynthesis, suggesting a new target organelle for melarsoprol’s mode-of-action. Further analysis of the genes arising from this study could identify targets for the generation of novel anti-trypanosomal drugs that are safer to administer than melarsoprol. This study also introduces the T. brucei ORFeome and GoF libraries as powerful new genetic tools in the field.

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3 - Bottom-up structural proteomics: cryoEM of protein complexes enriched from malaria parasites

Ho, Chi-Min (UCLA Dept. of Microbiology, Immunology & Molecular Genetics); Li, Xiaorun (California NanoSystems Institute); Lai, Mason (UCLA Dept. of Microbiology, Immunology & Molecular Genetics); Terwilliger, Thomas C. (Los Alamos National Laboratory and the New Mexico Consortium); Beck, Josh R. (Iowa State University Dept. of Biomedical Sciences); Wohlschlegel, James A. (UCLA Dept. of Biological Chemistry); Goldberg, Daniel E. (Washington University School of Medicine in St. Louis Depts. of Medicine and Microbiology); Fitzpatrick, Anthony WP (Zuckerman Institute, Columbia University Medical School); Zhou, Z. Hong (UCLA Dept. of Microbiology, Immunology & Molecular Genetics)

X-ray crystallography and recombinant protein production have enabled an exponential increase in atomic structures, but often require overexpression in recombinant systems and non-native constructs involving mutations or truncations, and are additionally challenged by membrane proteins and large multi-component complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic resolution cryoEM maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria parasite Plasmodium falciparum, an organism that has resisted traditional structural biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach opens the door to atomic structures of previously intractable biological systems such as P. falciparum, which has proven recalcitrant to structural study at atomic resolution prior to the recent resolution revolution in cryoEM.

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4. - Going “forward” with Leishmania classical genetics

Alves-Ferreira, Eliza (NIH); Inbar, Ehud (NIH, Bethesda.); Ferreira, Tiago (NIH); Khan, Asis (NIH); Lack, Justin; Sacks, David; E. Grigg, Michael (NIH)

Classical forward genetic studies have not been considered feasible within the genus Leishmania until very recently. Hence, the field has primarily taken reverse genetic approaches to study specific genes or gene families that impact Leishmania biology and pathogenesis. Now that it is possible to perform natural genetic crosses in the laboratory, we took a classic forward genetics approach to identify parasite genes that affect lesion size and vacuole formation. To do this, we performed an inter-species cross between L. amazonensis and L. major in permissive phlebotomine sand flies. Twelve independent hybrids were successfully recovered. Each hybrid possessed a unique karyotype profile and DNA content, according to PFGE and flow cytometry analyses. Seven hybrids were diploid, like both parents, whereas five were triploid. Whole genome sequencing established that each isolate was a full genomic hybrid. Consistent with previous crosses, different signatures of aneuploidy, CNV and gene dosage were identified. All F1 hybrids were screened for parasite-vacuole (PV) size that develops during a 72 hours macrophage infection. The two parental lines possess strong phenotypic differences in this trait: L. major develops a small PV with a single parasite per vacuole whereas L. amazonensis develops a large PV with multiple parasites per vacuole. Vacuole size was not an autosomal dominant trait, as 3 different phenotypes could be resolved. F1 progeny possessed either an L. major parental, L. amazonensis parental, or an intermediate phenotype (big vacuole with only 1 parasite) indicating that PV size is a multigenic trait. We are currently performing more F1 crosses and backcrosses to generated sufficient progeny to map genes associated with each phenotype by QTL.

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5t - New toolkit for high throughput imaging and image analysis of the chronic stage of Toxoplasma gondii

Winiger, Rahel R. (Laboratory of Molecular Parasitology); Fisch, Daniel (Host-Toxoplasma Interaction Laboratory); Yakimovich, Artur (MRC-Laboratory for Molecular Cell Biology); Hammoudi, Pierre-Mehdi (Department of Microbiology and Molecular Medicine); Ramakrishnan, Chandra (Laboratory of Molecular Parasitology); Soldati-Favre, Dominique (Department of Microbiology and Molecular Medicine); Hehl, Adrian B. (Laboratory of Molecular Parasitology)

The chronic stage of Toxoplasma gondii is poorly documented. How tissue cysts are established, maintained and how bradyzoites within these cysts interact with the host are only a few of the unanswered questions. Pivotal to the bradyzoite survival is the secretion of cyst wall (CW) material and the CW biogenesis. Only a few CW proteins are characterized and no extensive analysis of CW components has been performed. To address some of these questions, we developed a workflow consisting of a forward genetics approach, high content microscopy of mutant parasites and a machine learning-based image analysis pipeline to quantify phenotypic features. Targeted knockouts of bradyzoite specific proteins with a potential role in building and maintaining the structural integrity of the CW were generated using CRISPR/Cas9. A standardized protocol for the in vitro differentiation of knockout parasites into cyst-forming bradyzoites was established using a 96 well plate format for subsequent labeling and high-throughput fluorescent imaging. Imaged cysts are evaluated for cyst/CW phenotypes using the newly developed beta-version of HRMan (Host Response to Microbe Analysis), an image analysis pipeline, integrating machine learning algorithms and deep neural networks. This analytical approach, together with our established experimental workflow, allows for a straightforward high-throughput, high- content characterization of cysts/CW and has therefore the potential to become an essential new tool for a fast and unbiased phenotype evaluation of stage-specific loss of function (LOF) T. gondii. The identification of cyst/CW LOF phenotypes as well as the biological regulators behind the formation and maintenance of the CW will help to understand the range of functions of this barrier and eventually lead to novel intervention strategies against T. gondii.

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6t - Three-dimensional ultrastructure of Plasmodium falciparum throughout cytokinesis

Rudlaff, Rachel M. (Harvard Medical School); Kraemer, Stephan (Harvard University); Marshman, Jeffrey (Carl Zeiss Microscopy); Dvorin, Jeffrey D. (Harvard Medical School)

New volume electron microscopy techniques are being increasingly utilized to answer cell biologic questions. Here, we present an updated view of the ultrastructure of Plasmodium falciparum parasites throughout cell division using micrographs obtained with one such technique, focused ion beam – scanning electron microscopy (FIB- SEM). We imaged and rendered wild type schizonts at early segmentation, mid-segmentation, PVM rupture, and post-PVM rupture to show their 3D structure through cytokinesis. In early segmentation, we show a budding schizont with 2n and 4n nuclei, apical buds that contain one mature and one immature rhoptry, and a branched apicoplast and mitochondrion. In mid-segmentation, we capture a schizont with more pronounced buds. At this stage, nuclei are 2n or 1n, rhoptries are mature, and apicoplasts are divided while the mitochondrion remains connected. By PVM rupture, each daughter cell or bud contains a full set of organelles. These data illustrate the dynamic and complicated process of schizogony, but also raise several outstanding questions about Plasmodium development, including: 1) How does the parasite decide when to stop replicating and begin dividing? 2) How are organelles properly partitioned to individual daughter cells? 3) What proteins or protein complexes control these processes? We hope these illustrations spur discussion on these unusual aspects of Plasmodium cell biology.

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7t - VSG mosaics: when the color of your shoes determines the shape of your hat.

Gkeka, Anastasia (Deutsches Krebsforschungzentrum); Erben, Esteban; Zeelen, Johan P.; Stebbins, Erec C.; Papavasiliou, Nina F. (Deutsches Krebsforschungzentrum)

Trypanosoma brucei is a flagellated unicellar parasite, which causes African sleeping sickness and is transmitted via tsetse flies. This pathogen is highly adapted for life in the host’s environment, since it evades immune responses by periodic changing of its dense coat of Variant Surface Glycoproteins. VSGs can be novel distinct variants encoded in the genome, or mosaics arising later in infection from segmental gene conversion of donor VSG genes. They consist of a long N-terminal domain (NTD), exposed to the cell´s environment, and a shorter C-terminal domain (CTD). The CTD has been shown to be inaccessible to antibodies in live cells, making the NTD the most prominent target for antibodies. Our previous data regarding the structure of the variant VSG3 have revealed an O-linked carbohydrate (O-Glc) on the top of the NTD and that this sugar prevents immune recognition and pathogen clearance. Here we show that formation of mosaic VSG3 proteins, by retaining the VSG3 NTD and altering the CTD to the one from another VSG, results in loss of binding of the sugar-specific VSG3 monoclonal to the mosaic VSG covered parasites. However, the VSG3 polyclonal anti-sera can bind to all variants, hence suggesting either an alteration in post-translational modifications or a conformational change. Since it is known that mosaics are almost entirely CTD swaps (retaining the same NTD and altering the CTD), the question that arises is whether the CTD can determine the antigenicity of the NTD. Thus, more structural and mass spectrometry experiments are being performed to determine the nature of this change. In addition, repertoire analysis data will be produced to elucidate whether the parental VSG3 and the mosaics elicit distinct types of monoclonal antibodies, strengthening our above hypothesis.

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8t - Analysis of the Toxoplasma mitochondrial ATP synthase structure reveals how its shapes the unique cristae and highlights the role of phylum specific subunits

Sheiner, Lilach (University of Glasgow)

F1Fo-ATP synthase plays a crucial role in the maintenance of the membrane morphology and membrane potential essential for mitochondrial function. Through dimerization and assembly into dimer rows along the curved membrane region, ATP synthase shapes mitochondrial cristae. Cristae morphology varies widely between divergent mitochondrial lineages, which has been attributed to differing ATP synthase dimer structures and row geometries; however, the structural basis of the unique club-shaped cristae of Toxoplasmahas remained elusive.

Using electron cryo-tomography of whole Toxoplasmamitochondria we found that the apices of its club-shaped cristae are decorated by cyclic, supramolecular ATP synthase assemblies with a hexameric unit, which is unprecedented in any known ATP synthase.

We further utilized single particle analysis via cryo-EM, to determined the structures of this essential complex in its dimeric and hexameric form, allowing generation of a de-novoatomic model. The unique T. gondiiATP synthase dimer structure is dictated by 17 phylum-specific subunits. The structure identified boundary cardiolipin and revealed the interacting subunits that stabilize its higher oligomers to generate membrane curvature.

Thus, our results highlight T. gondiias a unique model system for investigating cristae biogenesis and pinpoint new targets for development of anti-apicomplexan drugs.

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Session II: Will the Circle be Unbroken: Cell Cycle - I

Chairs: Paul Sigala and Galadriel Hovel-Miner

Monday, September 16th, 2019

9:00am – 10:30am

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9 - A divergent cyclin/cyclin-dependent kinase complex controls progression through S- and M- phases during Plasmodium berghei gametogony

Balestra, Aurélia (Université de Genève); Rea, Edward; Zeeshan, Mohammad (University of Nottingham); Klages, Natacha (Université de Genève); Brusini, Lorenzo (University of Nottingham); Ferguson, David (Nuffield Department of Clinical Laboratory Science, University of Oxford.); Mourier, Tobias (Computational Bioscience Research Centre); Brady, Declan (University of Nottingham); Pain, Arnab (Computational Bioscience Research Centre); Tewari, Rita (University of Nottingham); Brochet, Mathieu (Université de Genève)

In most model organisms, major transitions of the cell cycle are triggered by variations in the activity of cyclin-dependent kinases (CDKs) bound to their primary regulator, the cyclin subunits. Cell division in the malaria parasite Plasmodium differs from canonical models in several ways. For example, CDKs and cyclins regulating DNA synthesis and mitosis in yeasts and are not clearly identified in Plasmodium. Instead, a number of divergent CDK-related kinases (CRKs) have been proposed to regulate cell cycle progression possibly without binding to a cyclin partner. Here, we show that the P. berghei CDK-related kinase 5 (CRK5) controls progression through both S- and M-phases during the three rounds of microgametocyte replication required to produce eight microgametes. CRK5 is additionally important for oocyst maturation, identifying it as an essential component at multiple stages of the parasite lifecycle during transmission through the mosquito. CRK5 activity requires binding to a divergent cyclin (SOC2) only found in Haemosporidia. The two proteins form a pre-existing complex in non-replicating gametocytes and remain bound over the first replication round suggesting that additional layers of regulation are necessary to activate CRK5 upon SOC2 binding. Consistently, both proteins are dynamically phosphorylated upon initiation of replication by multiple protein kinases including the Calcium-Dependent Protein Kinase 4, a master regulator of gametogony. Both proteins also show a high turnover throughout the two last rounds of microgametocyte replication, involving rapid protein translation followed by proteasome- dependent degradation. Our results present the first evidence that, despite the divergent nature of cell division in Plasmodium, cell cycle control relies on interactions between cyclins and CDKs with additional levels of regulation by post-translational modifications.

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10 - Deciphering the spatiotemporal organization of DNA replication during Plasmodium falciparum schizogony

Ganter, Markus (Center for Infectious Disease, Parasitology, Heidelberg University); Klaus, Severina; Machado, Marta; Klaschka, Darius; Guizetti, Julien (Center for Infectious Disease, Parasitology, Heidelberg University)

Cells typically multiply by duplicating their genome and subsequently dividing into two daughter cells. Plasmodium falciparum displays a remarkably different mode of replication and multiplies through schizogony: consecutive rounds of DNA replication and nuclear division occur, forming a multinucleated cell prior to cytokinesis. During schizogony nuclei divide asynchronously despite sharing the same cytoplasm, but the spatiotemporal organization of DNA replication and how it relates to asynchronous nuclear division are unknown. To investigate this, we generated a reporter parasite where nuclei are marked with mCherry and a component of the DNA replication fork is marked with eGFP. Using live cell imaging, we found that a nuclear eGFP signal coincided with increasing DNA content and that only a subset of nuclei showed an eGFP signal at a given time. These data indicate that DNA replication occurs asynchronously in P. falciparum. Next, we analyzed the timing of DNA replication events during early schizogony. Although asynchronous, DNA replication events followed a distinct temporal pattern, e.g. the time needed for the first round of replication was conserved across multiple parasites. Our results also indicate that the last round of DNA replication occurs approximately three hours prior to egress, suggesting that DNA replication and merozoite formation do not happen simultaneously. Together, our data indicate that DNA replication occurs asynchronously in P. falciparum. This surprising finding suggests that P. falciparum has likely evolved unique molecular mechanisms to control its unusual spatiotemporal organization of DNA replication, as it has for nuclear division during schizogony.

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11 - Calcium Influx through a Transient Receptor Potential Channel and its impact in the Toxoplasma lytic cycle

Márquez-Nogueras, Karla (University of Georgia); Kuo, Ivana (Loyola University Chicago); Moreno, Silvia (University of Georgia)

Ca2+ signaling impacts nearly every aspect of cellular life. Ca2+ signals result from the release of intracellular stores or from Ca2+ influx from the extracellular milieu. Plasma membrane channels control Ca2+ influx and respond to stimuli like membrane depolarization, specific agonists and intracellular store depletion. We studied Ca2+ entry in Toxoplasma and its downstream impact on its biology. Experimental evidence supports the presence of two calcium influx pathways with different affinities for Ca2+ and distinct downstream roles. We characterized a Toxoplasma gene annotated as Transient Receptor Potential (TRP) channel (TgGT1_310560, TgTRP2) and its potential role in Ca2+ influx. C-Terminal tagging of TgTRP2 showed localization to the plasma membrane. Disruption of the TgTRP2 gene with CRISPR-Cas9 resulted in mutant parasites defective in its Ca2+ influx with the residual influx activity being resistant to TRP Channel inhibitors. These parasites grew at a lower rate and were unable to invade host cells. Our data supported the involvement of TgTRP2 in calcium influx in Toxoplasma, important for its lytic cycle. We further characterized TgTRP2 and cloned the gene for expression in HEK cells genetically modified to not express the 3 isoforms of the IP3 Receptor. As TgTRP2 localized to the ER of HEK cells, we isolated nuclear membranes and characterized TgTRP2 by conducting electrophysiological measurements (single channel patch clamp recordings) of the ER-nuclear membranes. Using a variety of internal and external solutions with high and low Ca2+ concentration, we determined that TgTRP2 permeates Ca2+. Currents were significantly reduced in the presence of TRP Channel inhibitors. Altogether, our data suggest that TgTRP2 is a low affinity channel that likely mediates Calcium Influx in T. gondii.

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12 - A Trypanosoma brucei orphan kinesin uses an ATP- dependent sorting mechanism to re-organize microtubules into a parallel bundle to form the new posterior end during cytokinesis

Sladewski, Thomas Campbell, Paul; Sinclair, Amy; de Graffenried, Christopher (Brown University)

Microtubule crosslinking molecular motors are instrumental in organizing the cytoskeleton and establishing cell morphology. The protozoan parasite Trypanosoma brucei (T. brucei) is a notable system for studying microtubule organization because its cytoskeleton is made up almost entirely of microtubules. T. brucei has evolved a highly polarized cell body that is maintained throughout the cell cycle and is supported by an array of highly crosslinked microtubules that underlies the cell membrane. This unique cytoskeletal organization required the parasite to devise a distinct microtubule-based mechanism for cytokinesis involving microtubule re- modeling that generates a division fold and nascent posterior end within the existing array. To understand the mechanistic details of this process, we performed a proteomic screen to identify novel proteins involved in cleavage furrow ingression and uncovered an orphan kinesin, which we named KLIF (Kinesin Localized to the Ingressing Furrow), that localizes to the cleavage furrow of dividing cells and is essential for the late stages of cleavage furrow ingression. Using negative stain electron microscopy and immunofluorescence, we find that cells depleted for KLIF do not complete cytokinesis because they fail to form a new posterior end. To gain further mechanistic detail into how KLIF might function to form the new posterior, we performed in vitro biophysical assays on the purified motor. We show that KLIF interacts with microtubules to form parallel microtubule bundles. These bundles become polarized through an ATP-dependent sorting mechanism where antiparallel-overlapped microtubules are translocated and dissociated from the bundle, whereas parallel-overlapped microtubules become statically crosslinked. In addition, KLIF uses an alternative binding mode to tether the plus-ends of microtubules. These molecular features are likely important for bringing together a bundle of microtubule plus-ends that defines the shape of the new posterior and drive the completion of cleavage furrow ingression.

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13t - Mapping of origins of replication in Plasmodium falciparum

Gomes, Ana Rita (DIMNP, Université de Montpellier) CHU de Montpellier); Méchali, Marcel (Institute de Génétique Humaine); Lopez-Rubio, Jose-Juan (DIMNP, Université de Montpellier)

Understanding the principles governing DNA replication is critical for decoding the mechanisms that maintain genomic stability. In most studied DNA synthesis is restricted to once per cell cycle. Remarkably, at different stages in their lifecyle, malaria parasites undergo several rounds of DNA replication, after which newly formed nuclei are segregated into daughter cells. The factors driving this original cell cycle and re-replication events are not yet fully elucidated. DNA replication is initiated from specific sites scattered across the genome, termed replication origins but their number, structure and usage dynamics are not conserved across organisms. In Plasmodium their structure and usage dynamics are unclear. Using a population- based approach we captured and sequenced newly synthesized DNA strands produced during the first replicative cycle of schizogony and determined the nature of origins of replication in Plasmodium falciparum parasites. We estimated 761 putative origins, corresponding to a ~32Kb inter origin distance – a number on par with what was reported in yeast. However, unlike yeast, Plasmodium origins are strikingly GC rich. Furthermore, while studying their distribution throughout the genome we detected an association with a genomic feature termed G- quadruplex (G4). These are secondary structures of GC rich DNA, that arise from non-canonical pairing and have the ability to exclude nucleosomes. Additionally, G4s have been implicated in origin specification in mammalian cells. Interestingly, in P. falciparum parasites, the median distance from a given origin to the nearest G4 was of approximately -25bp, thus suggesting a functional and/or structure link between the two. This study comprises the first genome-wide description of origins of replication in malaria parasites and provides novel insight into their specification and regulatory cues. 88

14t - Single-cell transcriptomes of Plasmodium vivax parasites reveal extensive but continuous changes in gene expression during their intraerythrocytic cycle

Serre, David (University of Maryland School of Medicine); Cannon, Matthew (University of Maryland School of Medicine); Caleon, Ramon; Wellems, Thomas; Sa, Juliana (Laboratory of Malaria and Vector Research)

Studies of gene expression in Plasmodium are complicated by the continuous developmental changes of the parasites. As a consequence, most studies of blood stage parasites have relied on synchronized in vitro cultures of P. falciparum. Since P. vivax cannot be continuously propagated in vitro, this parasite remains poorly studied and our understanding of its biology primarily derives from P. falciparum, without accounting for their divergence and differences (e.g., gametocytogenesis, red blood cell (RBC) preference and sequestration). Recently, single-cell RNA-seq has been applied to P. falciparum and rodent malaria parasites but viability requirements have precluded such studies in other Plasmodium species, including P. vivax. Here, we describe the gene expression profiles of 9,215 individual P. vivax parasites obtained from infections of 10 splenectomized non-human primates with four P. vivax strains. Our data provide, on average, information on 19,282 mRNA molecules per individual parasite, allowing evaluations of even weakly expressed genes. Our analyses confirm that multiple stages, including gametocytes, are circulating in each infection, though their relative proportions vary greatly among infections. Our study reveals genes associated with gametocytogenesis and gametocyte differentiation, including some transcripts specifically expressed in the elusive P. vivax male gametocytes. Our findings also highlight the highly specific and tightly regulated expression of most genes during the intraerythrocytic cycle (IEC): many genes are expressed only during a short developmental period and very few genes, if any, are expressed consistently. Stage-specific gene expression regulation is also noticeable for AP2 genes, raising the intriguing possibility that successive expression of these master controllers of transcription could regulate the entire IEC. Finally, our analyses revealed that late schizonts expressed a significantly higher proportion of genes without P. falciparum orthologs, highlighting the differences in RBC invasion in these species and the need to specifically study P. vivax.

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Session III: Under Pressure: Host-Parasite Metabolis

Chairs: Galadriel Hovel-Miner and Paul Sigala

Monday, September 16th, 2019

10:30am – 11:30pm

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15 - LIPIN: a pivotal nexus in Toxoplasma lipid metabolism, channelling host-derived FAs to storage, preventing Lipotoxicity

Dass, Sheena (Apicolipid Team, IAB, CNRS UMR5309) Berry, Laurence (UNIVERSITÉ DE MONTPELLIER UMR 5235 (DIMNP)); Katris, Nicholas; Yamaryo- Botté, Yoshiki; Botté, Cyrille Y. (Apicolipid Team, IAB, CNRS UMR5309)

Lipid synthesis in apicomplexan parasites requires an essential combination of fatty acids synthesized de novo and scavenged directly from the host. The molecular mechanism by which the parasites combine and regulate fatty acid flux from these two sources, remains largely unknown. In this study, we identified and characterized a phosphatidic acid phosphatase, LIPIN, as the focal point of the parasite lipid homeostasis in Toxoplasma gondii. Toxoplasma tachyzoites are unable to survive a knockdown of LIPIN due to severe replication defect amongst several other membrane anomalies. This cytosolic enzyme catalyses the synthesis of diacylglycerol from phosphatidic acid, a central lipid precursor and signal transducer, as confirmed by heterologous complementation and lipidomics approaches. The disruption of LIPIN induces loss of parasite lipid storage concomitantly with an increase of the total lipid content (fatty acids), leading to rapid parasite death by ‘lipotoxicity’. With the help of 13C glucose-based fluxometrics using GC-MS, we identified that the source of these toxic fatty acids (FA) is the host (esp C18:1) whilst the FASII activity of the parasite is reduced. Electron microscopy reveals that TgLIPIN indirectly regulates nuclear membrane morphology and also the inner membrane complex biogenesis, suggesting the importance of lipid balance during the growth of parasite. Overall, we suggest that TgLIPIN protects parasite against FA-induced toxicity allowing a normal replication cycle within its host by regulating the critical levels of phosphatidic acid. The intracellular replication and survival of apicomplexan parasites within their host is thus governed by a tight regulation of lipid flux between cellular storage and membrane biogenesis.

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16 - Impact of Leishmania donovani infection on host macrophage mitochondrial metabolism, integrity and function

Acevedo Ospina, Hamlet (INRS-Armand-Frappier Santé Biotechnologie); Descoteaux, Albert (INRS-Armand-Frappier Santé Biotechnologie)

During Leishmania infection, metacyclic promastigotes are internalized by macrophages, create a specialized parasitophorous vacuole, and differentiate into amastigotes. To achieve this, promastigotes employ a panoply of virulence effectors including lipophosphoglycan (LPG) and the metalloprotease GP63. These parasite effectors are essential during parasitophorous vacuole modulation and to attenuate macrophage activation. Interestingly, Leishmania was shown to modulate AMPK and PI3K/AKT signalling axes, involved in host-mitochondrial regulation. Nevertheless, scarce attention has been paid to the characterization of mitochondrial metabolism during Leishmania infection, and to the effectors involved therein, that could be fulfilling a crucial role during pathogenesis. We hypothesize that, in macrophages, Leishmania modulate host- mitochondrial metabolism in an LPG- dependent manner. In infected BMM, host cell nuclear and mitochondrial genes that control mitochondrial biogenesis was assessed by real-time PCR, mitophagy was assessed by evaluating recruitment of Parkin to mitochondria via immunofluorescence. Host mitochondrial metabolic flux was assessed using Seahorse technology. We demonstrated that host cell nuclear and mitochondrial genes that control mitochondrial biogenesis are upregulated in an LPG-dependent manner. Importantly, we observed that the mtDNA/nDNA ratio increase during infection, indicating that LPG promotes mitochondrial biogenesis. Additionally, confocal analysis did not show differences in recruitment of mitophagy markers to host mitochondria during Leishmania infection. Metabolic flux analyzes showed that OCR/ECAR ratio is decreasing at 8- hour post-infection. Importantly, we observed that IRG-1, the enzyme that synthetize itaconate is 300 fold-times induced during infection in an LPG- dependent manner. This results suggest that LPG could modulate an energetic reprograming from oxidative phosphorylation to lactate production. This research will provide novel insights into the mechanisms by which Leishmania rewires host cell mitochondria metabolism to promote its own survival.

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17 - Mitochondrial acetyl-coA biosynthesis is essential during the red blood stages of human malaria parasite Plasmodium falciparum.

Nair, Sethu C. (Johns Hopkins University); Munro, Justin; Llinas, Manuel (Penn State University); Prigge, Sean T. (Johns Hopkins University)

Acetyl-coA is a central molecule in carbon metabolism. Malaria parasites have three major pathways to make acetyl-coA that are located different subcellular compartments: the apicoplast, mitochondrion and cytosol. Both the apicoplast and mitochondrial pathways use the pyruvate derived from glycolysis and enzymatically generate acetyl-coA using an organelle-specific pyruvate dehydrogenase enzyme. The cytosolic pathway relies on millimolar levels of exogenous acetate and its conversion to acetyl-coA using a cytosolic acetyl-coA synthetase enzyme. The apicoplast pathway is not essential during the blood stages, leaving the mitochondrial source of acetyl-coA a potentially important and vital source. In the present study, we generated a series of deletion mutants of important enzymes in the mitochondrial acetyl-coA biosynthetic pathway and showed that mitochondrial acetyl-coA biosynthesis is essential for the survival of the parasite. Unexpectedly, we found that two mitochondrial enzymes are capable of making acetyl-coA (Pyruvate Dehydrogenase and alpha- Ketoglutarate Dehydrogenase); deletion of both enzymes is required to kill blood stage parasites unless exogenous acetate is provided. Both enzymes rely on lipoic acid as a cofactor and we were able to simultaneously block the activity of both enzymes, and the production of acetyl-coA by genetically targeting lipoic acid metabolism. We studied the production of acetyl-coA in wild type and gene-knockout parasites using 13C-labeled glucose, acetate or glutamine and showed how these different carbon sources are metabolized in different metabolic scenarios. These results provide novel insights into parasite metabolism and identify an essential product of the mitochondrion.

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18 - Divergent Acyl Carrier Protein Coordinates Mitochondrial Fe-S Cluster Metabolism in Malaria Parasites

Falekun, Seyi P. (Department of Biochemistry, University of Utah); Jami, Yasi (Department of Biological Chemistry, University of California Los Angeles); Park, Hahnbeom (Institute for Protein Design, University of Washington); Wohlschlegel, James A. (Department of Biological Chemistry, University of California Los Angeles); Sigala, Paul A. (Department of Biochemistry, University of Utah)

Plasmodium malaria parasites harbor two endosymbiotic organelles, the apicoplast and mitochondrion, that are essential for blood-stage parasites and serve as antimalarial drug targets. The apicoplast retains a complete prokaryotic-like fatty acid biosynthesis (FASII) pathway that is dispensable for blood-stage parasites. The mitochondrion, despite lacking FASII enzymes, curiously retains a divergent acyl carrier protein (ACP). Unlike the apicoplast ACP, which features the conserved Ser that tethers the acylated phosphopantetheine group during FASII activity, the mitochondrial ACP (mACP) has replaced this Ser with a Phe that defies a canonical role as an acyl carrier protein. We have used CRISPR/Cas9 to tag mACP for conditional expression with the aptamer/TetR-DOZI system. Knockdown of mACP expression is lethal to parasites, indicating an essential role independent of FASII. This lethal phenotype can be rescued by decylubiquinone, suggesting a dominant dysfunction of the mitochondrial electron transport chain (ETC). Immunoprecipitation studies reveal that mACP interacts with the Isd11-Nfs1 complex, which is the core mitochondrial biogenesis machinery that produces the Fe-S cluster used by the Rieske protein for essential function in Complex III of the ETC. In yeast and humans, the acylated phosphopantetheine group of mACP is a key feature of its essential stabilization of the Isd11-Nfs1 complex. Plasmodium mACP lacks this prosthetic group and thus provides a new molecular paradigm for association with Fe-S cluster biogenesis machinery, stabilized by compensatory mutations in Isd11. This work defines a novel essential function for ACP in the Plasmodium mitochondrion that is likely to be conserved in other Apicomplexan parasites such as Toxoplasma and Babesia, which also retain an mACP homolog lacking the conserved Ser.

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19t - Trypanosoma congolense cytoadheres to the brain vasculature

Silva Pereira, Sara (Instituto de Medicina Molecular - João Lobo Antunes); De Niz, Mariana; Figueiredo, Luisa M. (Instituto de Medicina Molecular - João Lobo Antunes)

Trypanosoma congolense causes nagana in livestock, which is a major contributor to poverty in sub-Saharan Africa. Clinical outcome is unpredictable, ranging from a wasting, chronic disease to a rapidly fatal illness. Its sister species and the cause of human sleeping sickness, T. brucei, shows strong tropism to the brain and the adipose tissue, which directly relate to disease progression and pathology. T. congolense stays mainly in the circulating blood and rarely invades tissues, but whether it displays tropism to specific tissues remains unknown. Here, we have investigated tissue tropism of two strains of T. congolense in mice, representing both sides of the clinical spectrum (IL3000 and 1/148). Histology and fluorescence imaging showed that, unlike T. brucei, T. congolense parasites do not invade the cerebral parenchyma, but they accumulate in the brain vasculature through cytoadhesion to endothelial cells. Parasite cytoadhesion is detected early in infection and independently of strain virulence. By intravital microscopy, we describe the dynamic of cytoadhesion up to the first peak of parasitaemia. To investigate whether the brain-adhered parasites are different from the free-swimming parasites in circulation, we are comparing their transcriptomes using trypanosome-targeted RNA sequencing. Understanding the changes in gene expression in cytoadhered parasites and the potential mediators of cytoadhesion will reveal the molecular players involved in this important host-parasite interaction and potentially identify novel clinical targets.

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20t - Functional study of two Trypanosoma cruzi receptors (TcIP3R and TczAC) involved in signal transduction and important for parasite survival

Lander, Noelia (University of Georgia); Chiurillo, Miguel A.; Docampo, Roberto (University of Georgia)

Trypanosoma cruzi exhibits unique characteristics that make it an attractive model to study host-parasite interaction. We are interested in investigating cellular signaling cascades that are essential for T. cruzi survival throughout its life cycle. Calcium ion (Ca2+) is an important second messenger that regulates a vast repertoire of cellular processes in trypanosomatids, including host cell invasion, differentiation, motility, osmoregulation and cell bioenergetics. Recent work has established the presence and essentiality of an inositol 1,4,5-trisphosphate receptor (IP3R) in trypanosomes. This protein is responsible for Ca2+ release from intracellular stores. Adenylyl cyclases catalyze the synthesis of cyclic AMP (cAMP), another important second messenger involved in differentiation and motility of trypanosomes. These enzymes share little homology with their mammalian counterparts and have a completely different structure. In this work we are studying two proteins involved in different T. cruzi signaling pathways: TcIP3R and a calcium-sensitive receptor-type adenylyl cyclase (TczAC). We first investigated their cellular localization by either endogenous or overexpression tagging. Immunofluorescence analysis indicates that TcIP3R localizes to acidocalcisomes, while TczAC localizes to the flagellum. To investigate the role of these receptors in cell signaling and parasite survival we generated knockout cell lines of both genes by CRISPR/Cas9. TcIP3R-KO epimastigotes exhibit a lower growth rate compared to controls, whereas trypomastigotes show a defect in host cell invasion. These parasites also exhibit reduced mitochondrial Ca2+ uptake and oxygen consumption rates, increased pyruvate dehydrogenase (PDH) phosphorylation and decreased citrate synthase activity, highlighting the importance of IP3R-mediated calcium release for mitochondrial function and cell bioenergetics. Phenotypic characterization of TczAC-KO parasites is currently in progress, as well as calcium sensitivity of recombinant TczAC under physiological conditions.

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Session IV: Another One Bites the Dust: Drug Targets

Chairs: Vicki Jeffers and Adam Sateriale

Monday, September 16th, 2019

1:00pm – 2:15pm

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21 - Finding novel therapies against amebic parasites by screening the ReFRAME library

Marcolino, Monica Kangussu (Stanford University); Ehrenkaufer, Gretchen (Stanford University); Chen, Emily (Calibr Institute); Debnath, Anjan (University of California, San Diego); Singh, Upinder (Stanford University)

The pathogenic amebae, Entamoeba histolytica, Naegleria fowleri, Acanthamoeba castellani, and Balamuthia mandrillaris are deadly human pathogens with limited therapeutic options. Entamoeba causes dysentery and liver abscesses with invasive disease in 50 million people annually. Naegleria, Acanthamoeba and Balamuthia are free- living ameba which can cause opportunistic CNS infections which are rare but with a mortality rate over 95% and no optimal treatment regimen. Despite the urgent need, these parasites are woefully understudied. In this work we performed a screen against E. histolytica using the ReFRAME library, a ~12K-compound set designed for Repurpose, Focused Rescue and Accelerated MEdChem. After confirmation, we selected 16 priority compounds to advance further testing for the treatment of amebic dysentery and liver abscess based on their use in advanced clinical studies and low toxicity against human cells. We are evaluating the killing kinetics, and activity against cyst and metronidazole resistant parasites. We have previously noted a high overlap of active compounds between Entamoeba and the free-living amebae. Thus, we used the hits from primary screen on Entamoeba (159 compounds) against the free-living ameba Naegleria, Acanthamoeba and Balamuthia and found 87 hits active against at least one parasite. After running confirmatory tests, we selected 18 high-value priority compounds for amebic meningoencephalitis: 8 received FDA approval or orphan-designation, 10 are likely CNS- penetrant and 9 have nanomolar potency against at least one parasite. We have evaluated killing kinetics for selected compounds and found molecules with rapid killing of Balamuthia and Naegleria. We also identified compounds able to delay recrudescence of Balamuthia trophozoites. Our efforts have identified several high-value, highly effective compounds that can change the therapeutic landscape for these diseases.

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22 - Pharmacological Disruption of an ApiAP2 Transcription Factor in the Human Malaria Parasite Plasmodium falciparum

Russell, Timothy J. (Department of Biochemistry and Molecular Biology and Center for Malaria Research (CMaR), Pennsylvania State University); De Silva, Erandi K. (Myonexus Therapeutics, Inc.); Crowley, Valerie M. (University Health Network); Shaw-Saliba, Kathryn; McClean, Kyle J. (Molecular Biology and Immunology, Johns Hopkins Bloomberg School of Public Health); Josling, Gabrielle (Department of Biochemistry and Molecular Biology and Center for Malaria Research (CMaR), Pennsylvania State University); Panagiotou, Gianni (Leibniz Institute for Natural Products Research and Infection Biology, Hans Knöll Institute); Jacobs-Lorena, Marcelo (Molecular Biology and Immunology, Johns Hopkins Bloomberg School of Public Health); Llinás, Manuel (Department of Biochemistry and Molecular Biology and Center for Malaria Research (CMaR), Department of Chemistry, Pennsylvania State University)

Plasmodium parasites use fine-tuned transcriptional control to regulate gene expression events that are necessary for proliferation and transmission. Remarkably, they largely do this using a single family of 27 sequence specific DNA binding proteins called ApiAP2 proteins. The ApiAP2s are unique to Apicomplexans and contain AP2 DNA binding domains. ApiAP2 proteins are attractive drug targets due to their origin and lack of homology to any known transcription factors in humans. Here, we report the in silico prediction and in vivo investigation of a small molecule that disrupts the DNA binding activity of the ApiAP2 transcription factor PF3D7_1466400 (PF14_0633). The molecule, termed ‘Compound G’, has specific activity against PF3D7_1466400 DNA binding as measured by in vitro inhibition of binding to its cognate DNA sequence in an electrophoretic mobility gel shift assay (EMSA). Treatment of blood stage Plasmodium falciparum with Compound G was found to cause a growth arrest at the trophozoite stage. Additionally, Compound G was shown to phenocopy genetic deletion of the PF3D7_1466400 rodent malaria orthologue PbAP2-Sp by blocking development of salivary gland sporozoites in Plasmodium berghei. Comparison of the parasite transcriptome at the onset of growth arrest to the genome occupancy of PF3D7_1466400 measured by chromatin immunoprecipitation (ChIP) followed by deep sequencing revealed that a large fraction of downregulated genes may be directly controlled by PF3D7_1466400. Decreased occupancy of PF3D7_1466400 at its target genomic loci following Compound G treatment was demonstrated by ChIP followed by quantitative PCR, providing evidence that disruption of PF3D7_1466400 DNA binding by Compound G takes place in vivo. Further genetic studies using conditional knockdown in the blood stages and biophysical work with the purified AP2 domain are underway to validate the interaction between Compound G and PF3D7_1466400. This study demonstrates that members of the ApiAP2 protein family may serve as a target for pharmacological disruption of malaria parasite development. 101

23 - From Phenotypic Screens to Mode of Action: A Metabolomics Approach to Guide the Development of Novel Anti-Trypanosomal Drugs

Srivastava, Anubhav (Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC 3052) Creek, Darren J. (Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC 3052, Australia, Melbourne)

Background Access to large phenotypic screens has enabled the discovery of novel anti-infective compounds. Translating these compounds into new drugs faces a number of challenges. Finding the mode-of-action (MoA) can help in focussing efforts to develop the most promising leads. Method Using a medium-throughput method, we performed untargeted metabolomics analyses of Trypanosoma brucei, a kinetoplastid parasite responsible for neglected tropical diseases nagana and sleeping sickness. This allowed us to investigate the mode of action of 18 potent trypanocidal compounds from the Pathogen Box (available from Medicines for Malaria Venture). Results Over 500 metabolite features were identified in this study and mapped to the predicted metabolic network. This approach identified the metabolic pathways targeted by the most potent compounds which spanned nucleotide metabolism, lipid metabolism, co-factor synthesis and redox metabolism. Lysophospholipid metabolism, glutamate metabolism, purine salvage and S-adenosyl-methionine and polyamine pathways were found to be specifically hit. Interestingly, multivariate analyses showed that compounds with similar chemical structures did not always have similar biochemical activities. Conclusions This study showed that a simple metabolomics assay can rapidly reveal the MoA of newly discovered anti-infective compounds. This information can be used for prioritising compounds in the optimization pipeline and help in designing combination therapies that target discrete pathways to overcome emerging drug-resistance.

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24 - Comparative chemical genomics in Babesia species identifies a novel apicomplexan drug target

Keroack, Caroline D. (Harvard T. H. Chan School of Public Health); Elsworth, Brendan; Tennessen, Jacob A.; moreira, Cristina K.; Paul, Aditya S.; Duraisingh, Manoj T. (Harvard T. H. Chan School of Public Health)

Babesiosis is an emerging human zoonosis, and a well-established and widely distributed veterinary infection caused by 100+ species of Babesia parasite. This enormous diversity poses a challenge in identifying broadly effective therapeutic interventions. However, there is also a unique opportunity to leverage the power of comparative analyses in multiple Babesia spp. to explore novel conserved biology, facilitated by the existence of in vitro culture systems. The diversity of Babesia parasites, coupled with the lack of potent inhibitors necessitates the discovery of novel conserved targets. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of novel and conserved targets. CCG relies on parallel in vitro evolution of resistance in parallel independent populations of evolutionarily- related Babesia spp. (B. bovis and B. divergens). After screening the MMV Malaria box against both species, we identified a potent anti-babesial inhibitor, MMV019266. This compound is also strongly active in Plasmodium and shows some efficacy in Toxoplasma. We were able to rapidly select for resistance to this compound in two species of Babesia in parallel, achieving 10-fold or greater resistance after six weeks of intermittent selection. After sequencing of multiple independently derived lines in the two species, we were able to identify mutations in a single conserved gene in both species. This gene encodes a predicted membrane-bound metallodependent phosphatase (putatively named phoDapi) which is homologous to alkaline phosphatase D in other apicomplexa. Identification of this gene target was greatly facilitated by the comparative approach of utilizing two species. In B. divergens (Bdiv_001570c) we have identified different mutations at the same amino acid residue in the two independent selections (A: C197F, B: C197W). In B. bovis (Bbov_I003300) we were able to identify a mutation (G527S) which is spatially proximal to those found in B. divergens. In both cases, the mutations were found in the phoD-like phosphatase domain, proximal to the ligand binding site. Interestingly, the gene is predicted to contain an apicoplast leader sequence, and the ortholog has been identified in the apicoplast proteome in Plasmodium falciparum, suggesting a potential role in the apicoplast. Strikingly, MMV019266 does not exhibit a delayed death phenotype in Plasmodium, killing in the first cycle. The ability to identify phoDapi in two species, and in multiple independent selections, validates the role this gene plays as the etiological driver of resistance to MMV019266 in Babesia spp. Together, these data show the power of CCG to identify not only novel pan-babesiacidal compounds, but also to identify novel conserved apicomplexan biology.

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25t - Differential Expression and Localization of Cytochrome b5 in Giardia intestinalis during Nitrosative Stress Response

Mowat, Kaitlyn (Trent Univeristy);

Giardia intestinalis is an anaerobic intestinal parasite that causes giardiasis, a severe and infectious form of diarrhea that can lead to mortality in immunocompromised individuals. Giardia possesses a reduced form of mitochondria known as the mitosome. As Giardia lacks functional mitochondria, oxidative phosphorylation and heme biosynthesis do not occur, yet five heme proteins, a flavohemoglobin and four isotypes of the cytochrome b5 family of electron transfer proteins are expressed in Giardia. While the flavohemoglobin has roles in protecting Giardia from nitric oxide produced in the host, the function of the cytochromes, as well as the identity of their redox partners are unknown. As the cytochromes are redox-active proteins, we decided to examine the response of three of the smallest cytochromes (gCYTB5-I, II and III) to nitrosative stress as an initial step in identifying their function. Protein levels of the cytochromes and their subcellular localization were examined at different time points during the exposure of Giardia cultures to three nitrosative stressors (nitrite, GSNO and DETA-NONOate) using isotype-specific antibodies. Our preliminary results show that the levels of all three cytochromes change during nitrosative stress, and that gCYTB5-I also changes its subcellular location under these conditions. It is important to study the nitrosative stress response as the host macrophages release nitric oxide into the lumen of the intestines to eliminate the parasite from the body and many therapeutics (i.e. metronidazole) also utilize nitrogen-based reactive molecules to treat Giardiasis.

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26t - A Lysophospholipase involved in malaria parasite development: Potential targets for drug development

Mohmmed, Asif (International Centre for Genetic Engineering and Biotechnology.); Kumar, Pradeep (International Centre For Genetic Engineering and Biotechnology New Delhi); Botté, Yoshiki Yamaryo (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes); Thakur, Vandana (International Centre for Genetic Engineering and Biotechnology.); Botté, Cyrille (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes)

Lipid metabolism is essential for development and survival of malaria parasite in the host cell. Many current evidences support that lipid synthesis and trafficking are pertinent drug targets. In apicomplexa, lipid metabolism relies on the combination of the parasite metabolic capacities and the active scavenging of host resources. Synthesis, catabolism and recycling/reshuffling of lipids are thus central metabolic functions for membrane biogenesis/remodelling and the acquisition of lipid biosynthetic precursors from the host during the rapid growth of the asexual intraerythrocytic malaria parasite. Lysophospholipases (LPLs) catalyze the hydrolysis of acyl chains from membrane phospholipids and therefore promote recycling of host lipids by the parasite. We have thus initiated a biochemical and functional characterization of a selected lysophospholipase (labelled as LPL3) in malaria parasite, Plasmodium falciparum. Using GFP-tagging approach, we showed that LPL3 localized to the parasitophorous vacuole and seems very important for parasite development in blood stages. Inducible knock-down studies showed that the protein plays role in the schizogony of the parasite. Detailed lipidomics analysis revealed that LPL3 is involved in neutral lipid synthesis. Further, for biochemical characterization, we have successfully expressed and purified recombinant LPL3 using E.coli expression system. We developed a fluorescent based high throughput assay and used this to screen “Malaria Box” compound library. We identified few inhibitors of LPL3 having IC50 in micromolar range, which could be potential leads compounds for new anti-malarial. Taken together, our data strongly suggest a crucial role of LPL3 during the blood stages development of P. falciparum. We identified few inhibitors of target LPL3, which could pave the way to drug development for novel anti-malarial strategies.

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Session V: Highway to Hell: Functional Analysis

Chairs: Adam Sateriale and Vicki Jeffers

Monday, September 16th, 2019

2:30pm – 3:30pm

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27 - Divide and conquer: using FACS and RNAi screening to identify novel factors for mtDNA replication/segregation in trypanosomes

Miskinyte, Migla (University of Edinburgh); Ivens, Alasdair; Waterfall, Martin; Schnaufer, Achim (University of Edinburgh)

Trypanosomatid parasites cause a wide spectrum of diseases in humans and livestock but share a unique biological feature, the organisation of mitochondrial DNA (mtDNA) into a complex, massive structure, named kinetoplast (or kDNA). Replication, segregation and expression of kinetoplast genes are extraordinary complex processes, but crucial for the survival of trypanosomatids and a target of existing therapies for Trypanosoma brucei. These processes involve an estimated ~300 proteins, including components of the tripartite attachment complex (TAC), a structure that physically links kDNA to the basal body. Only a minority of these proteins have been identified and characterised functionally. In addition, very little is known about how these processes are regulated and integrated into cell and life cycle of the parasites. In order to build a more comprehensive picture of kDNA maintenance and regulation we developed an unbiased genetic screen that is based on genome-wide RNA interference, a novel kDNA staining protocol, fluorescence-activated cell sorting (FACS), and next generation sequencing. Following FACS, we were able to enrich for cells that lost kDNA (kDNA0 cells) after induction of gene-specific RNAi from 1% up to 90%. Genomic DNA was extracted from kDNA0 and uninduced control cells, RNAi inserts PCR-amplified and candidate genes identified by deep sequencing. The top candidate genes include several known kDNA maintenance factors, such as a mtDNA polymerase, a primase and several TAC proteins, thus validating our approach. Other hits include a number of previously uncharacterised ‘hypothetical’ proteins as well as proteins conserved in evolution that localise to the mitochondrion or to kDNA (www.TrypTag.org). We will present results on the validation and characterisation of these putative novel kDNA maintenance factors.

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28 - A genome-scale reverse genetic screen to identify functional determinants controlling Toxoplasma gondii sexual cycle

Sardinha-Silva, Aline (Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH); Yanes, Olivia (Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH); Vavrušková, Zuzka; Lukeš, Julius (Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Ceské Budejovice (Budweis)); Grigg, Michael E. (Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH)

Toxoplasma gondii transmission in nature is highly dependent on the parasite’s sexual cycle, which occurs exclusively in felids. However, the molecular pathways promoting sexual stage development are unknown. Our previous RNA-Seq work identified merozoite transcripts that are stage specific, conserved across the Apicomplexa, and thought to facilitate sexual competency. These include the SRS/6-Cys, FamA- D proteins and AP2 transcriptional factors, each of them comprising several members whose individual role in contributing to oocyst formation remains unknown. We employed a genome-scale reverse genetic signature-tag mutagenesis screen, using CRISPR/Cas9, to generate a library of >200 Toxoplasmastrains that each possess a unique barcode and are each deficient in a single SRS, FamA or AP2 gene. Our aim is to perform an input/output screen to identify sexual stage-specific proteins that are critical for oocyst formation, which would allow us to rationalize ways to block T. gondiitransmission. We pooled together 14 strains deficient in SRS and AP2 factor genes expressed in asexually replicating parasites and infected either HFF cells or mice. Mi-Seq sequencing was used to identify strains that failed to expand. SRS15B-, SRS22E-, SRS26B-, SRS26J-, AP2VI-3, AP2IX- 1- and AP2IX-6-KO strains presented improved tachyzoite in vitrogrowth, whereas, PLP1-, SRS22G- and AP2IV-3-KO showed impaired tachyzoite growth, compared to input sample. In mice, PLP1- , SRS15A-, SRS26B- and AP2IV-3-KO parasites failed to migrate to the spleen, whereas they had no defect in expansion in vitro. With the methodology in place, mice are currently infected with a pool of 24 knockouts for a proof-of-principle cat challenge experiment. We will present parasite genes identified as critical for Toxoplasmasexual development in order to block parasite transmission by cats. 109

29 - Identification of the Plasmodium falciparum Acetyl-CoA Synthetase as an emerging antiplasmodial drug target

Summers, Robert L. (Harvard T.H. Chan School of Public Health); Vanaerschot, Manu; Murithi, James M. (Columbia University); Pasaje, Charisse FJ (Massachusetts Institute of Technology); Luth, Madeline R. (University of California, San Diego); Magistrado-Coxen, Pamela (Harvard T.H. Chan School of Public Health); Carpenter, Emma F. (Wellcome Sanger Institute); Rubiano, Kelly; Striepen, Josefine; Bath, Jade (Columbia University); Munro, Justin T. (Pennsylvania State University); Pisco, Joao P.; Punekar, Avinash S.; Baragaña, Beatriz; Gilbert, Ian H. (Drug Discovery Unit, University of Dundee); Llinás, Manuel (Pennsylvania State University); Niles, Jacquin C. (Massachusetts Institute of Technology); Ottilie, Sabine; Winzeler, Elizabeth A. (University of California, San Diego); Lee, Marcus CS (Wellcome Sanger Institute); Fidock, David A. (Columbia University); Lukens, Amanda K. (The Broad Institute); Wirth, Dyann F. (Harvard T. H. Chan School of Public Health)

In recent years phenotypic screening has identified hundreds of drug-like compounds with potent activity against multiple stages of the malaria parasite Plasmodium falciparum, however the development of many of these promising compounds into new antimalarial drugs has been constrained by a lack of knowledge of their mechanism of action. By conducting resistance selection experiments and using whole genome sequencing, we identified mutations in the parasite’s Acetyl-CoA Synthetase (PF3D7_0627800; PfAcAS) which confer resistance to two structurally distinct antimalarial compounds, MMV084978 and MMV019721. Orthologues of PfAcAS in eukaryotes catalyze the formation of the central metabolite acetyl-CoA from acetate, coenzyme A and ATP, and participate in a range of essential processes including nutrient acquisition and biosynthesis, post-translational modification, and epigenetic regulation. Homology modelling of PfAcAS revealed that resistance mutations cluster around the predicted enzyme active site, suggesting that inhibitors may act by competing with the substrates of PfAcAS. Such active-site mutations may impair the function of the protein and reduce parasite fitness, however in vitro competitive growth experiments between wildtype and drug-resistant parasites demonstrated that parasites bearing the T648M or A597V mutations incurred little to no fitness costs relative to wildtype parasites. Western blot analyses revealed that exposure of wild-type parasites to MMV019721 caused a dose- dependent decrease in the acetylation of histones, which was abrogated in parasites resistant to the compound, suggesting that inhibition of PfAcAS may disrupt the epigenetic regulation of parasite DNA replication and gene expression. These findings identify PfAcAS as the target of structurally distinct antiplasmodial compounds and support the development of inhibitors of PfAcAS as antimalarial compounds with a novel mode of action.

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30t - Developing a scalable pipeline for functional genetic analysis in the malaria parasite, Plasmodium falciparum

Esherick, Lisl Y. (Massachusetts Institute of Technology) McGuffie, Bryan (Boston Children's Hospital); Cardenas, Pablo; Dey, Sumanta; Nasamu, Sebastian; Pasaje, Charisse F. (MIT); Dvorin, Jeffrey D. (Boston Children's Hospital); Niles, Jacquin C. (MIT)

Given the continuing emergence of Plasmodium falciparum parasites resistant to frontline antimalarials, there is an urgent need to develop new antimalarial drugs, especially those with novel mechanisms of action. Systematic identification and phenotypic characterization of essential P. falciparum genes can guide drug discovery efforts towards targeting novel genes and/or pathways. However, the limited genetic tractability of P. falciparum has previously posed significant challenges to high-throughput analysis of essential gene function. The development of CRISPR-based tools for genetic manipulation has enabled a wide variety of gene disruption and tagging strategies in P. falciparum. Using these tools, we are developing a scalable CRISPR- based genetic platform to disrupt genes via knock-out and/or the TetR- DOZI system for conditional translational knockdown. By using a conditional knockdown system, we are able to generate lines targeting essential genes by maintaining parasites under permissive conditions. In creating this platform, we are developing methods for automated construct design, parallel assembly of donor vectors for homology- directed repair, and pooled transfection. In addition, we have created arrayed and pooled assays to determine the growth phenotypes of individual knockdown lines, which are measured by luciferase or next generation sequencing, respectively. In assays comparing parasite growth under knockdown and permissive conditions, we find strong concordance between results obtained from small-scale pools and individually assayed parasite lines. By scaling up this resource, we will create arrayed and pooled libraries of transgenic parasites that should serve as a useful starting point for drug-discovery efforts and promote detailed study of essential gene functions in P. falciparum.

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31t - Development of resistance in vitro reveals novel mechanisms of artemisinin tolerance in Toxoplasma gondii.

Rosenberg, Alex (Washington University in St. Louis); Luth, Madeline R.; Winzeler, Elizabeth A. (University of California, San Diego); Behnke, Michael S. (Louisiana State University); Sibley, L David (Washington University in St. Louis)

Combination therapies that include artemisinin (ART) are currently the frontline therapy for Plasmodium falciparum infections. Resistance to ART is evidenced by the recent emergence and rapid spread of delayed artemisinin (ART) clearance by mutant forms of the PfKelch13 protein in Southeast Asia. This development heightens the interest in understanding the mechanism of action and potential resistance mechanisms to ART. Toxoplasma gondii is susceptible to ART treatment in vitro and in vivo, although it is less potent and hence generally not used therapeutically to treat toxoplasmosis. To explore potential mechanisms of action, we carried out long-term in vitro selection of two parasite lines using stepwise increases in drug concentrations to derive Toxoplasma parasites with elevated ART tolerance. Whole-genome sequencing analysis identified eleven mutations in nine different genes. We chose to focus on the genes encoding DegP protease and serine/threonine protein kinase because mutations in these genes occurred in both independently selected lines. When DegP/Kinase mutations were introduced into the parental parasite line by CRISPR/Cas9-mediated gene editing, they were sufficient to increase ART tolerance, confirming the functional relevance of these mutations. Further analysis of the ART resistant lines genomes revealed specific amplification of the mitochondrial genome in proportion to the resistance level. The mitochondria has previously been implicated in the mechanism of ART inhibition of yeast and cancer cells, suggesting that amplification might contribute to resistance by increasing critical targets. These studies implicate novel genes and mitochondrial expansion in development of resistance to artemisinin and suggest potential mechanisms of action.

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32t - Genetic screens reveal a central role for heme biosynthesis in artemisinin sensitivity

Sidik, Saima (Whitehead Institute); Harding, Clare (Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, University of Glasgow); Petrova, Boryana; Markus, Benedikt; Lourido, Sebastian (Whitehead Institute)

Artemisinins are powerful antimalarials, but resistance threatens their efficacy. To understand this resistance, we performed saturated CRISPR screens in the genetically tractable apicomplexan parasite Toxoplasma gondii. The artemisinin derivative dihydroartemisinin (DHA) kills T. gondii efficiently, and a mutation homologous to the Kelch13C580Y polymorphism that reduces P. falciparum’s DHA sensitivity reduces this drug’s effect on T. gondii, suggesting that some resistance mechanisms are conserved. A CRISPR screen performed in the presence of a sub-lethal concentration of DHA revealed that mutations in the putative porphyrin transporter Tmem14C increase T. gondii’s DHA sensitivity with the largest effect 24 hours into its intracellular growth cycle. This provides an additional parallel with P. falciparum, which is most sensitive to DHA during the trophozoite stage of its cell cycle. Using a lethal concentration of DHA, we found that mutations in heme biosynthesis and TCA cycle enzymes decrease T. gondii’s sensitivity to DHA — findings that are consistent with previous work demonstrating that heme activates DHA, and that underscore the power of CRISPR screens for identifying drug resistance mechanisms. Chemical inhibitors of heme biosynthesis and the TCA cycle also render T. gondii less sensitive to DHA, providing additional evidence that heme levels dictate the response to this drug. Our screen also identified a homolog of the serine protease DegP (DegP2), disruption of which reduces DHA sensitivity. In keeping with our model, ΔDegP2’s decreased DHA sensitivity correlates with decreased soluble heme. DegP2 mutants are viable, leading us to conclude that genetic modifications can reduce heme levels enough to reduce DHA sensitivity without precluding heme’s critical roles in respiration and as an enzymatic cofactor

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33t - DNA uptake agonists improve transfection efficiency and provide insights into plasmid uptake mechanism in malaria parasites

Crater, Anna K. (NIAID, National Institutes of Health); Garriga, Meera; Desai, Sanjay A. (National Institutes of Health)

P. falciparum grows within human erythrocytes and can take up plasmid from its host cell, permitting DNA transfection for basic molecular research. The mechanisms responsible for plasmid uptake and their physiological role for the intracellular parasite are unknown and have proven refractory, especially as they must facilitate transport of large DNA molecules across at least three membranes. Here, we identified agonists that improve transfection efficiency and help define the molecular mechanisms. Using a sensitive NanoLuc luciferase reporter under a strong promoter, we optimized erythrocyte plasmid loading and NanoLuc detection in a 384-well microplate format. These efforts permitted robust measurement of uptake and expression after only 48 hours of cultivation. We then carried out a high-throughput screen for chemical modulators of plasmid uptake. One scaffold yielded consistent activity, with the NPU-7 derivative having the greatest effect. DNA transfection with drug selection revealed that NPU-7 accelerated parasite outgrowth and improved CRISPR efficacy by ~ 10-fold. We excluded upregulation of transcription or translation as NPU-7 did not affect luciferase expression or activity in stably transfected lines. Single cell bioluminescent microscopy indicated that NPU-7 increases the percentage of cells that take up plasmid without altering the signal in each positive cell. Similar improvements were observed in P. knowlesi transfections using rhesus monkey RBCs, implicating a conserved target. While extended cultivation with NPU-7 was not detrimental, it abolished the stimulatory effect on plasmid uptake; genetic and epigenetic changes in these resistant progeny will be described and should unveil the molecular basis of plasmid uptake. Our findings may also revolutionize DNA transfection of malaria parasites, enabling large-scale genome modifications.

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Session VI: We’ve Gotta in to Get Out: Host-Parasite Interactions

Chairs: Catherine Merrick and Dawn Wetzel

Tuesday, September 17th, 2019

9:00am – 10:15am

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34 - Toxoplasma Ferlin1 facilitates trafficking and exocytosis of micronemes

Tagoe, Daniel A. (Boston College) Drozda, Allison; Coleman, Bradley (Boston College); Coppens, Isabelle (Johns Hopkins Bloomberg School of Public Health); Gubbels, Marc-Jan (Boston College)

Toxoplasma gondii pathology is driven by the lytic cycle made up of replication, egress, and invasion. The sequential secretion of three organelles, micronemes, rhoptries, and dense granules is essential for the lytic cycle, wherein Ca2+ signaling plays a pivotal role. Proteins with Double C2 (DOC2) domains are known Ca2+ sensors mediating protein secretion across eukaryotes. Of the 4 DOC2 genes encoded in the Toxoplasma genome, we report here on the role of Ferlin family protein TgFER1 in the lytic cycle. Conditional overexpression of a dominant negative FER1 (DN-FER1) allele lacking the C-terminal transmembrane domain results in pile up of fully mature microneme proteins in an apical compartment. Detailed assessment indicates this compartment is a dynamic cluster of mislocalized microneme organelles. In contrast to most other known proteins with characterized roles in protein sorting and organelle biogenesis, the rhoptry organelles appear completely normal, which indicates FER1 specifically acts on micronemes. Not surprisingly, DN- FER1 parasites are non-viable as they are unable to effectively secrete microneme proteins which results in defective egress, motility and invasion. On the other hand, conditional overexpression of full length FER1 parasites in intracellular Endo buffer triggered the secretion of their micronemes. This results in premature egress, which disrupts the lytic cycle and is also lethal. Taken together, these data show that FER1 is essential for both the trafficking and exocytosis of micronemes.

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35 - The first rhoptry bulb protein identified in Cryptosporidium is secreted during invasion and localized to the host cortical actin

Guerin, Amandine (UPENN); Roy, Nathan (Children's Hospital of Philadelphia); Berry, Laurence (University of Montpellier); Burkhardt, Janis K. (Children's Hospital of Philadelphia); Striepen, Boris (UPENN)

Cryptosporidium infection is a leading cause of child mortality, no vaccine is available and the current drug treatment against this diarrheal pathogen is inefficient. The unique way by which Cryptosporidium invades and manipulates its host cell is particularly interesting but poorly understood. Indeed, the parasite develops intracellularly at the apical surface of intestinal epithelial cells surrounded by a parasitophorous vacuole (PV) and is physically sustained on the edge of the cell by a host actin pedestal. We were able to follow Cryptosporidium invasion in combination with the host actin and membrane dynamic using live microscopy. The high speed of host cell actin polymerization at the apical end of the parasite leads us to hypothesize that the parasite secretes an effector protein to hijack this machinery. Rhoptries are apical organelles known to be secreted early during invasion by other apicomplexans and would contain the perfect candidate. Based on bioinformatic analysis, we have identified the first 5 rhoptry bulb proteins of Cryptosporidium. They all localized to the PV but two are also found secreted into the host cell. One candidate is particularly interesting as we found it associated with the host cortical actin by both microscopy and biochemistry experiments. It is particularly interesting that the protein is restricted to the apical end of polarized cells, both in vitro and in vivo. We were also able to recapitulate the localization of the protein in infected cells by ectopically expressing the protein in epithelial cell, and we define the N-terminal sequence to be sufficient for its localization. More work is ongoing to define the function of this essential rhoptry protein.

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36 - Unraveling the mechanism of microsporidia polar tube firing

Bhabha, Gira (Skirball Institute, NYU School of Medicine); Jaroenlak, Pattana (Skirball Institute, NYU School of Medicine); Cammer, Michael (NYU School of Medicine); Ekiert, Damian (Skirball Institute, NYU School of Medicine)

In one model of infection, microsporidia parasites employ a harpoon- like organelle called the polar tube that is propelled out of the spore to pierce host cell membrane under suitable conditions. The polar tube serves as a conduit, allowing the translocation of infectious material into the host. The molecular events driving polar tube firing are still poorly understood. We have optimized high speed, wide-field, live-cell imaging to capture the events during the germination process for two microsporidian species, Anncaliia Algerae and Encephalitozoon Hellem. The timescale of the polar tube firing event is less than two seconds with differences in velocity and acceleration between the two species. To follow how the nucleus and other organelles are transferred through the polar tube, we are optimizing the use of fluorescent dyes for live cell imaging. For over a century it has been known that the nucleus of the parasite must be translocated through the polar tube. However, this presents a paradox, as the tube diameter is more than 10-fold smaller than that of the nucleus, raising the question of how the nucleus may fit through the tube. We stained the parasite nuclei with fluorescent dye and performed live-cell imaging. The results revealed that the nucleus is translocated through the tube slightly after the tube is fired, and undergoes a remarkable degree of reorganization in order to fit through the tube, which retains approximately constant diameter throughout the process. These studies begin to provide insights into the initial stages of microsporidia polar tube firing and infection.

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37 - Human CD55 mediates parasite entry during the process of erythrocyte invasion by Plasmodium falciparum

Egan, Elizabeth - Shakya, Bikash (Stanford University) Patel, Saurabh D. (Zuckerman Mind Brain Behavior Institute, Columbia University); Tani, Yoshihiko (Japanese Red Cross Kinki Block Blood Center); Egan, Elizabeth S. (Stanford University)

Both host and parasite factors mediate attachment and internalization of Plasmodium falciparum during erythrocyte invasion. Previously, an RNAi screen for host determinants of P. falciparum invasion identified CD55 as essential for invasion. However, the molecular function of CD55 during invasion and how it interfaces with other events and factors known to mediate this complex process are unknown. We have investigated the function of CD55 using reverse genetics, antibody- based inhibition, immunofluorescence and live cell imaging. We generated isogenic CD55-null and control erythrocytes (cRBCs) from primary hematopoietic stem cells using CRISPR/Cas9 and ex-vivo erythropoiesis. P. falciparum invasion was markedly reduced in the CD55-null cRBCs as compared to isogenic controls, confirming the requirement for CD55 during invasion. Similarly, P. falciparum invasion was significantly inhibited in the presence of a polyclonal antibody targeting CD55. We used this inhibitory antibody to study the function of CD55 in real time using live cell microscopy. While several identifiable steps of the invasion process such as the pre-invasion time, frequency of attachment and cellular deformability were indistinguishable between anti-CD55 and control antibody conditions, parasite internalization was impaired in the presence of anti-CD55 antibody. Similar results were observed using CD55-null erythrocytes from a rare donor. Attached merozoites stimulated erythrocyte echinocytosis efficiently in the presence of anti-CD55 or control antibody, indicating that blocking CD55 does not impact rhoptry release and suggesting that CD55 functions downstream of this step. Co- localization experiments to visualize components of the moving junction point to a role forCD55 in progression of the moving junction during parasite internalization. These results suggest that CD55 plays a unique role relative to other receptors during the multi-step invasion process.

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38t - An ER CREC family protein regulates the egress proteolytic cascade in malaria parasites

Fierro, Manuel A. (The University of Georgia); Asady, Beejan; Brooks, Carrie F.; Cobb, David W.; Villegas, Alejandra; Moreno, Silvia N. J.; Muralidharan, Vasant (The University of Georgia)

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be the calcium (Ca2+) signaling hub and required for biogenesis of egress- related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum Endoplasmic Reticulum-resident Calcium-binding protein (PfERC), a member of the CREC family. Knockdown of PfERC shows that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythocytic lifecycle revealed that PfERC is essential for parasite egress but not required for protein trafficking or Ca2+ storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease, SUB1. Using double mutant parasites, we show that PfERC is required for the proteolytic maturation of the essential aspartic protease, Plasmepsin X, which cleaves SUB1. Further, we show that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish the ER- resident CREC family protein, PfERC, as a key early regulator of the egress proteolytic cascade of malaria parasites.

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39t - An unconventional myosin, TgMyoF is an organizer of the endosome-like compartments in Toxoplasma gondii

Heaslip, Aoife (University of Connecticut); Carmeille, Romain; Schiano, Irio (University of Connecticut)

Sequential release of proteins from the secretory organelles (micronemes and rhoptries) is critical for Toxoplasma gondii host cell invasion. Microneme and rhoptry proteins are synthesized in the ER and must be sequentially trafficked through a highly polarized endomembrane system, in order to reach their final destination in the mature secretory organelles. The strict spatial positioning of endosomal compartments is thought to optimize the trafficking of proteins this pathway. However, little is known about how the parasite cytoskeleton controls the positioning of these organelles or the transport of vesicles between these compartments. Our data demonstrate that an unconventional myosin, TgMyoF, and actin control the apical positioning of the endosome-like compartments (ELCs). In control parasites NeonFP-Rab5, EmeraldFP-Rab6, Neon-Rab7, DrpB- GFP and GFP-syntaxin-6 all localize, as expected, to distinct post- Golgi compartments. Live cell imaging demonstrates that the Rab5 and Rab6 compartments are highly dynamic and vesicles can be visualized budding from and fusing with these compartment. In addition, Rab5- and Rab6- positive vesicles exhibit directed motor-driven motion with velocities of 1.54±0.03mm/s and 1.01±0.02mm/s respectively. Upon TgMyoF knockdown (KD) or actin depolymerization with cytochalasin D, the ELCs became fragmented, static and distributed through the cytosol. TgMyoF-KD resulted in a 50% decrease in the number of directed transport events, while actin depolymerization resulted in an almost complete inhibition (>95%) of directed transport. TgMyoF KD results in a 50% decrease in parasite invasion and we hypothesize that this invasion defect is due to the suboptimal trafficking of proteins through the endosomal pathway in the absence of TgMyoF.

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Session VII: Dancing with the Devil: Host-Parasite Interactions

Chairs: Dawn Wetzel and Catherine Merric

Tuesday, September 17th, 2019

10:30am – 11:30am

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125

40 - Cell death mediated by the human trypanolytic serum protein Apolipoprotein L-1 is preceded by an accumulation of cytoplasmic calcium

Verdi, Joey (Hunter College, City University of New York); Giovinazzo, Joseph; Khalizova, Nailya; Weiselberg, Jessica; Thomson, Russell (Hunter College, City University of New York); Schreiner, Ryan (Weill Cornell Medical College); Raper, Jayne (Hunter College)

Humans are immune to infection by Trypanosoma brucei brucei. This immunity is mediated by the primate-specific cation channel forming serum protein Apolipoprotein L-1 (APOL1). APOL1 forms trypanolytic pH-gated cation channels in membranes at acidic pH, although the channels are only permissible to cations at neutral pH. We interpret that the most physiologically relevant mechanism by which this activity would lead to trypanosome death is through activation of the channel at acidic pH after endocytosis, followed by endocytic recycling of the channels to the plasma membrane where the pH returns to neutral. At neutral pH, APOL1 is permissible to several cations including both calcium and sodium. Inhibiting plasma membrane sodium flux significantly delays trypanosome lysis, whereas the role of calcium in trypanosome death has not been investigated. We developed a T. b. brucei parasite expressing gCAMP6f, a genetically encoded calcium-sensing GFP derivative. Delivering APOL1 to these trypanosomes leads to a rapid accumulation of cytoplasmic calcium that precedes cell death, suggesting that plasma membrane calcium flux is triggered by APOL1. To then validate these results in a new model system, we took advantage of another APOL1-mediated field of research. Two APOL1 genetic variants, called genotype 1 (G1) and G2, are associated with the development of a series of kidney disorders. Disease manifests through APOL1-mediated death of the kidney cells that produce the toxic variants of the protein. As G1 and G2 are being secreted by the cell, they are acidified in the secretory vesicles and subsequently neutralized once reaching the plasma membrane. In an attempt to draw any potentially existing parallels between APOL1-mediated kidney cell death and APOL1-mediated trypanosome death, we again employed gCAMP6f. We observed that G1 and G2-expressing kidney cells also accumulate cytoplasmic calcium, and that this accumulation is dependent on APOL1 protein reaching the plasma membrane. Together, these data suggest that plasma membrane ion flux is a major component of cell death in two distinct models of APOL1-mediated cytotoxicity.

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41 - Modulation of host cell responses and virulence by T.gondii's ROP55

Ruivo, Margarida; Penarete-Vargas, Diana (UMR5235 Universite de Montpellier); Hamie, Maguy; El Hajj, Hiba (American University of Beirut); Lebrun, Maryse (UMR5235 Universite de Montpellier)

The ability of Toxoplasma to cause disease depends on the coordinated secretion of specialized secretory organelles‚ namely micronemes‚ rhoptries and dense granules. During invasion‚ rhoptry proteins are injected in the host cell where they hijack host functions crucial to establish and maintain infection. Several ROPs have been described to be key components for parasite virulence. Here we report the characterization of a new rhoptry protein, ROP55, located at the parasitophorous vacuole membrane, with a role in controlling virulence in type I parasites. Parasites lacking ROP55 show a defect in growth in human and mouse fibroblasts. The deletion of ROP55 does not affect parasite invasion‚ egress‚ or replication; however the number of vacuoles after one day of infection is significantly reduced. Remarkably, this reduction is concomitant with a decrease in the number of host cells, suggesting that ROP55 controls cell death. This hypothesis is supported by an increase of host cell death measured by LDH release and the up-regulation of IL-1β. Similar results were obtained in mouse macrophages in the absence of any immune stimulation. Finally, we show that ROP55 is an important virulence factor in mice. The replication of KO parasites is completely abrogated in activated macrophages, and only 50% of mice die when infected with 1 million KO-ROP55 tachyzoites. All these phenotypes are fully restored in a strain ectopically expressing ROP55. Our results suggest a gatekeeping role for a new rhoptry protein, ROP55, against host cell death, a strategy to avoid clearance of the parasite during infection.

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42 - Identification of a novel protein complex essential for erythrocyte invasion by human-infective malaria parasites

Knuepfer, Ellen (The Francis Crick Institute); Wright, Katherine E. (Imperial College London); Green, Judith L. (The Francis Crick Institute); Kumar Prajapati, Surendra (Uniformed Services University of Health Sciences); Howell, Steven A. (The Francis Crick Institute); Moon, Robert W. (London School of Hygiene and Tropical Medicine, London.); Draper, Simon J. (The Jenner Institute); Rosanas-Urgell, Anna (Institute of Tropical Medicine); Higgins, Matthew K. (Department of Biochemistry, Oxford University); Baum, Jake (Imperial College); Holder, Anthony A. (The Francis Crick Institute)

Malaria is caused by Plasmodium parasites, which invade and replicate within erythrocytes. Plasmodium falciparum causes the most severe form of malaria in humans. Plasmodium parasites use multiple receptor-ligand interactions to gain entry into the host erythrocyte. Most of these invasion pathways are dispensable and this redundancy guarantees successful parasite proliferation. However, this redundancy in invasion ligand usage, together with extensive sequence polymorphisms, has hindered the successful development of effective blood-stage vaccines against malaria. Conversely, the interaction of the trimeric RH5-RIPR-CyRPA complex with basigin on the host erythrocyte is an essential step in P. falciparum invasion and believed to be important for Ca2+ signalling and rhoptry secretion. All three proteins have been identified as promising candidates for a blood stage vaccine. However, whereas RH5 is restricted to the subgenus, RIPR and CyRPA are also expressed in other human-infecting Plasmodium species. Analysing the role of PkRIPR in P. knowlesi host cell invasion identified a novel trimeric protein complex of PkRIPR with a thrombospondin-repeat containing protein, PkPTRAMP and a novel, cysteine-rich protein, PkCSS. Disruption of the PkRIPR-PkPTRAMP-PkCSS complex by inducible gene deletions resulted in blocked erythrocyte invasion and parasite death. Antibodies to PkRIPR, like antibodies to PfRIPR, inhibited invasion. In P. falciparum PTRAMP and CSS form a dimeric protein complex which co-exists on the merozoite with the trimeric RH5-RIPR-CyRPA complex. We have studied the effect of ptramp and css gene deletions on the parasite’s ability to attach to, deform, and invade erythrocytes. We demonstrate here the central role of PTRAMP and CSS in erythrocyte invasion by two important human pathogens.

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43t - Transcriptional modification of host cells harbouring Toxoplasma gondii bradyzoites prevents IFNgamma-mediated cell death

Seizova, Simona

Toxoplasma gondii tachyzoites extensively manipulate their host cell by exporting a distinct repertoire of effector proteins across the newly- established parasite vacuole (PV). This process interferes with the host transcriptional program, and is thought to enable parasite persistence and dissemination in spite of the host immune response. Eventually, Toxoplasma establishes a chronic brain infection that becomes a reservoir for disease reactivation and is seemingly drug resistant. Little is known about how this chronic stage of infection persists post-cyst formation, or whether protein export and host manipulation play a role in latency. Our research shows that in vitro bradyzoite-cysts alter the host transcriptional program via effector protein export mediated by the dense granule PV membrane protein, MYR1. We identified tachyzoite effector TgIST, an inhibitor of host IFN-gamma signaling, was exported during bradyzoite stages, suggesting a role for TgIST in chronic infection. Furthermore, we demonstrate that protein export is critical for protecting bradyzoite infected host cells from undergoing IFN - mediated cell death, thus enabling cyst persistence. This work provides the first evidence of the mechanism used by Toxoplasma bradyzoites for their long-term survival and identifies MYR1 as a potential drug target for the clearance of chronic toxoplasmosis.

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44t - EXP1 is required for organization of the Plasmodium falciparum parasitophorous vacuole membrane

Nessel, Timothy (Iowa State University); Beck, John M. (Iowa State University); Goldberg, Daniel E. (Washington University School of Medicine); Beck, Josh R. (Iowa State University)

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane (PPM) and constitutes the principal barrier between parasite and host compartments. The PVM is the site of several key transport activities but the basis for organization of this membrane system to fulfill its essential functions is unknown. A group of charged, single-pass integral membrane proteins constitutes a major component of the PVM proteome but the function of these proteins remains unclear. Prompted by proteomic analysis of the PVM, we sought to query the function of the longest known member of this group, EXP1. We adapted a CRISPR/Cpf1 genome editing system to install the TetR- DOZI-apatmer system for conditional translational control at the exp1 locus. EXP1 knockdown results in a lethal defect but does not impact parasite effector protein delivery into the erythrocyte, revealing an essential function distinct from the protein export pathway. Previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity. However, a mutant version of EXP1 lacking a residue important for this activity in vitro still provides substantial rescue of endogenous exp1 knockdown, indicating this proposed activity does not fully account for EXP1 function in vivo. To better understand the consequences of EXP1 knockdown, we examined parasite ultrastructure. Parasites lacking EXP1 display profound changes in vacuole morphology characterized by increased PVM separation from the PPM and accumulation of abnormal vacuole-enclosed membrane structures that contain host cytosol. Additionally, live fluorescence assays show alteration in PVM protein distribution following loss of EXP1. Collectively, our results reveal a novel PVM defect that suggests a critical role for EXP1 in maintaining proper PVM organization.

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45t - Systematic identification of diversity-encompassing variants of a new malaria vaccine target

Niare, Karamoko1,2,3, Timo Chege2, James Tuju2, Bourema Kouriba3, Gordon Awandare1, Julian Rayner4, Faith Osier2. 1 West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, 2Kenya Medical Research Institute – Wellcome Trust Research Programme, 3Malaria Research and Training Center. 4Wellcome Sanger Institute.

Background: The genetic diversity of malaria parasites is a major challenge in developing effective vaccines. Detailed data on the genetic diversity of many novel vaccine candidates are rare, and strategies to overcome this obstacle for vaccine development are limited. Objective: Here we used innovative approaches to analyze the nature and impact of the genetic diversity of a Plasmodium falciparum surface antigen, PF3D7_1136200, a newly identified blood-stage vaccine target. Methods: We harnessed public P. falciparum genomic datasets to study 2,512 field isolates from sub-Saharan Africa and South-East Asia. We measured its diversity PF3D7_1136200 and selection pressure within Africa. We also analyzed the population structure of this gene between Africa and Asia. To prioritize protein variants for broadly effective vaccine development, we used sensitive population genetics-based algorithms to cluster haplotypes that are identical by descent and state. Finally, we expressed all the common antigen variants as recombinant C- terminal His-tagged full-length proteins using mammalian expression system for immunological studies. Results: We observed 149 alleles within the coding region of the gene in Africa of which 48 were synonymous (S) and 101 were non-synonymous (NS). The minor allele frequency (MAF) ratio MAFS/MAFNS was 1.5. The gene showed two polymorphic regions within the N- and C- terminal ends and a conserved central sequence. The nucleotide and haplotype diversities did not vary in Africa where we observed a strong directional selection. We identified two equally dominant haplotype families including 125 NS unique haplotypes. Our algorithms grouped the unique haplotypes into 9 - 19 clusters. Interestingly, the top 5 largest clusters cover 92.1% of the field isolates, which predicts a high efficacy rate for pentavalent vaccines with this antigen. We found 34 common protein variants and recombinantly expressed 27 of them after removal of signal peptide and phosphatidylinositol anchor sequences. The native and heat-denatured forms of the expressed proteins were equally immunogenic, which suggests the presence of linear epitopes within the PF3D7_1136200 protein. Conclusion: Overall, our study provides a detailed insight into the genetic diversity of the new vaccine candidate, PF3D7_1136200, that could be valuable for the design of broadly effective multivalent vaccines. Keywords: Malaria – Vaccine – Genetic diversity – Computational modelling – PF3D7_1136200.

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Session VIII: Born to Be Wild: Infection Dynamics and Reservoirs

Chairs: Sabrina Absalon and Aoife Heaslip

Tuesday, September 17th, 2019

1:00pm – 2:10pm

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133

46 - Plasmodium falciparum dry season reservoir

Portugal, Silvia (Heidelberg University)

The dry season represents a major challenge for Plasmodium falciparumin many regions of the globe, where water availability limits mosquitoes to only part of the year. In Mali, P. falciparum parasites remain within children during the 5- to 6-month dry season, while no clinical malaria cases occur, waiting for transmission in the ensuing wet season. RNAseq data of parasites collected form children in Kalifabougou, Mali shows that during the dry season P. falciparumpresents an altered transcriptional profile compared to that of P. falciparum parasites from clinical cases in the transmission season, and that this is at least in part promoted by an increased circulating time of infected erythrocytes before adhesion to the vascular endothelium in the dry season. We further demonstrate that the host immune response minimally affected by the dry season persistent subclinical infections, and that parasite replication seems not to be impaired. We find, however, that circulating for longer periods prior to adhesion in the dry season potenties a low replication index and thus maintains infections at low levels and below the immune radar. We propose that P. falciparumvirulence in seasonal transmission areas is regulated so that the parasite adapts to a vector-free environment for long periods, decreasing endothelial binding and immunogenicity as infection progresses, instead enabling higher splenic clearanceto maintain low parasitaemias which allow bridging two transmission seasons several months apart.

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47 - Malaria synchrony is not driven by the host

Rijo-Ferreira, Filipa (HHMI, UT Southwestern Medical Center); Acosta-Rodriguez, Victoria (UT Southwestern Medical Center); Abel, John (Massachussets Institute of Technology); Kornblum, Izabela (HHMI, UT Southwestern Medical Center); Bento, Ines (Instituto Medicina Molecular); Matthews, Krista; Kilaru, Gokhul (UT Southwestern Medical Center); Mota, Maria M. (Instituto Medicina Molecular); Takahashi, Joseph S. (HHMI, UT Southwestern Medical Center)

Rhythmic fevers are one of the hallmarks of malaria: a consequence of the bursting of red blood cells (RBCs) upon completion of the synchronous asexual cell cycle of the parasite. RBCs burst 24h, 48h or 72h after invasion, depending on the Plasmodium species. Host rhythms of activity and food intake can influence the timing of the parasite asexual cell cycle. However, the mechanism for parasite synchrony, which is central to the rhythmic fever phenomenon, remains unknown. We hypothesized that parasites are able to maintain their synchrony independent from the host. Here we show that parasite rhythms are plastic and can adapt to a host that has a longer circadian clock, with the parasite slowing down its asexual cell cycle and gene expression to match the host rhythms. Despite sensing and adapting to host rhythms, we show that host rhythmic food intake does not drive parasite synchrony, since it persists even when host food intake is spread throughout the day. Furthermore, parasite rhythms are not only due to the cell cycle, as the quiescent sporozoite stage also displays rhythms in gene expression, especially in genes related to the motility required for host invasion. Finally, we demonstrate that the parasite population remains synchronous even when infecting a circadian clock mutant (arrhythmic) host, suggesting that host cues are not required for synchrony. The fact that rhythms exist across multiple parasite stages suggests that parasite rhythms are an advantage for the parasite. Thus, we propose that parasite rhythms, although sensing and entraining to the host, are not driven by the host, but rather generated by the parasite in order to anticipate its changing environment.

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48 - Using Toxoplasma gondii to redefine the neuronal innate immune response

Sambamurthy, Chandrasekaran (University of Arizona); Kochanowsky, Joshua (University of Arizona); Burciu, Camelia (Barrow Neurological Institute); Sattler, Rita (Barrow Neurological Institute); Koshy, Anita (University of Arizona)

Toxoplasma gondii naturally causes a lifelong, asymptomatic central nervous system (CNS) infection in immunocompetent humans and rodents. This tropism for and persistence in the CNS underlies T. gondii’s ability to cause devastating neurologic disease, and even death, in the immunocompromised. Despite being able to invade any nucleated cell in vitro, T. gondii persists primarily in neurons in vivo. Prior in vitro work suggested that neurons are persistently infected because, unlike other CNS cells, they lack the ability to clear intracellular parasites even in the setting of IFN-γ stimulation. Using a novel mouse model that allows us to permanently mark and track CNS cells injected with T. gondii effector proteins, we found that T. gondii predominantly interacts with neurons and that up to 95% of these T. gondii-injected neurons are not actively infected. These data suggest that, contrary to existing dogma, neurons may clear intracellular parasites. To test this hypothesis, we exposed primary pure murine neurons to IFN-γ or media and monitored neuron IFN-γ responses. We found that IFN-γ stimulated neurons upregulate classical IFN-γ response genes (STAT1) and GTPases known to be involved in the clearance of T. gondii in non-neuronal murine cells. Collectively, these findings suggest that IFN-γ stimulated neurons should be able to use these IFN-γ regulated GTPases to clear intracellular parasites. To test this possibility, we infected IFN-γ stimulated and unstimulated neurons and quantified the percentage of infected neurons over time. We found that stimulated and unstimulated neurons show similar infection rates early on, but, by 24hpi, the IFN-γ stimulated neurons show up to 15% decrease in the percentage of infected cells compared to unstimulated neurons. Additionally, using a GTPase resistant T. gondii strain for infection abrogates the difference in infection rates between IFN-γ stimulation and unstimulated neurons. Current work is focused on using transgenic mouse-derived neurons in vitro and conditional knock out mice as well as a new reporter system in vivo to confirm and extend our mechanistic understanding of this neuronal clearance of T. gondii. In addition, to translate our findings to human neurons, we are working on performing parallel studies in iPSC-derived human neurons. This work will be the first to show that neurons can clear T. gondii and will add to the growing literature that neurons have intact cellular immune responses.

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49 - Into the MIST: Adaptation to facilitate persistent infection of splenic macrophages enables malaria infection through stress erythropoiesis transmigration (MIST) and represents a novel exo-erythrocytic stage of the Plasmodium life cycle.

Fraser, James W. (Penn State University); Sim, Derek G. (Penn State University); Dey, Adwitia (Harvard Medical School); Jones, Matthew; Hart, Kevin J.; Llinas, Manuel; Lindner, Scott E.; Read, Andrew F.; Paulson, Robert F. (Penn State University)

Malarial infections of the mammalian host have classically been categorized into two key stages– an asymptomatic liver stage and a symptomatic blood stage. Many studies have observed that splenic macrophages take up merozoites during the blood stage but concluded that these parasites are of little consequence. However, using a model of acute infection in mice, we have now identified a significant role for stress erythropoiesis and splenic macrophages in malaria pathology. During acute infections, we observed that stress erythropoiesis was activated in the spleen, that the spleen, not the bone marrow is the primary site of erythrocyte production, and that macrophages in the spleen internalize erythrocytes harboring parasites that are detectable throughout the course of infection. Adoptive transfer of parasitized splenic macrophages transmits infection to naïve hosts, and macrophage-resident parasites remained detectable after 100 days. Interrogation of infected macrophages by RNA-seq revealed alterations to mitotic, immunologic, and metabolic pathways indicating that the parasite is adapted to survive in and commandeer these cells. Moreover, we are able to detect parasites in nucleated early-stage stress erythroid progenitors and terminally differentiating pre-erythrocytes within the spleen throughout infection. We propose that rather than infect these splenic erythroid progenitors directly, the parasite gains entry to them through associated macrophages, and are subsequently disseminated into the blood passively in the terminally differentiated erythrocytes. We have termed this process Malaria Infection through Stress Erythropoiesis Transmigration (MIST), and propose that these infections of an immune- privileged microenvironment within the spleen constitute a novel exo- erythrocytic stage of the Plasmodium life cycle that may provide a reservoir for long-term gametocytogenesis and chronic or relapsed disease.

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50t - Whole genome sequence comparative analyses of North African Leishmania infantum may reveal genetic and molecular determinants of the parasite tissue tropism.

Chakroun, Ahmed S. (Institut Pasteur de Tunis, Université Tunis El Manar); Guerbouj, Souheila (Institut Pasteur de Tunis, Université Tunis El Manar, Tunis - Belvédère); Leprohon, Philippe (Centre de Recherche du CHU de Québec, Université Laval); Harigua, Emna (Institut Pasteur de Tunis, Université Tunis El Manar, Tunis - Belvédère); Fathallah Mili, Akila (Université de Sousse); Ouellette, Marc (Centre de Recherche du CHU de Québec, Université Laval); Guizani, Ikram (Institut Pasteur de Tunis, Université Tunis El Manar, Tunis - Belvédère)

Leishmania parasites cause a wide spectrum of diseases that range from self healing skin lesions (CL) to visceral sickness (VL) which is lethal if not treated. These heteroxenous parasites belonging to the genus Leishmania constitute a public health scourge. Interestingly, the species L. infantum can be the etiological agent of both clinical affections in endemic countries. Whole genome sequencing (WGS) using Illumina platform, was carried out for 25 North African L. infantum strains causing VL (N = 14) or CL (N = 11) in Tunisia and Algeria. We used the most up to date L. infantum JPCM5 genome recently released on TriTrypDB as reference in the analyses. Read depth-based prediction of chromosome somy has highlighted the already established plasticity of the karyotypes. Nevertheless it did not show any consistency neither with the clinical form nor with the geographical origin since all the strains were interleaved. SNP variations detection and annotation using features including transcriptomic data gathered from “Leish-ESP - CBM” and locally curated using python scripts, illustrated mutations affecting CDS, UTRs and intergenic sequences. PCA (Principal Component Analysis) using the R package SNPRelate, performed on more than 170 K variable loci in linkage equilibrium, among which ~112 K newly discovered sites, produced clusters correlated to clinical manifestations. This supports the existence of different parasite populations associated to the clinical manifestations and the natural occurrence of genetic exchange.

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139

Session IX: Changes, Turn and Face the Strange: Developmental Stages

Chairs: Aoife Heaslip and Sabrina Absalon

Tuesday, September 17th, 2019

2:30pm – 3:30pm

140

141

51 - IPS cell technology: A great tool to study hypnozoites and pave the road towards malaria elimination

Pellisson, Melanie Anne-Marie Zeeman4, Thierry Doll3, Annemarie Voorberg-van der Wel4, Lucy Kirchhofer-Allan5, Sven Schuierer3, Guglielmo Roma3, Erika Flannery5, Sebastian Mikolajczak5, Clemens H. M. Kocken4, Pascal Mäser1,2, Matthias Rottmann1,2, Matthias Müller3 1Swiss Tropical and Public Health Institute (SwissTPH) 2University of Basel 3Novartis Institutes for Biomedical Research, Novartis Pharma AG 4Biomedical Primate Research Centre, Department of Parasitology 5Novartis Institute for Tropical Diseases, Novartis Pharma AG

One of the major challenges in eliminating malaria is the presence of hypnozoites in the human parasites P. vivax and P. ovale. Up to date, very little is known about this quiescent liver stage of the parasite, which hampers the search for compounds with hypnocytocidal activity. Here, we present the development of a P. cynomolgi in vitro model suitable to study the mechanisms underlying liver stage dormancy and ultimately allows screening of compounds for efficacy against hypnozoites. To be species compatible and enable cell production scale-up, we generated hepatocytes using induced pluripotent stem cells (iPS) derived from cynomolgus monkey. Generating simian hepatocytes from pluripotent cells is a promising alternative to the gold standard procedure using rhesus monkey primary hepatocytes. We established iPS cells from fibroblasts by over-expression of the reprogramming factors Oct4, Sox2, Klf4, and c-Myc. The produced cells were then differentiated into hepatocytes via an inducible approach using key transcription factors (HNF4α and FOXA3) for hepatic lineage formation. Characterization of the resulting hepatic cells displayed specific marker expression and function. These iPS- derived hepatocytes were subsequently infected with P. cynomolgi sporozoites. Immunofluorescence analysis with antibodies against Hsp70 and UIS4 revealed the presence of uninucleated parasites at 2 days post-infection (dpi). As in primary simian hepatocytes, both dividing and non-dividing forms were distinguished at 4 dpi and their respective development were followed until 12 dpi. At this time point, both persistent hypnozoites and asynchronously developed schizonts were identified. This infection model using cynomolgus monkey iPS- derived hepatocytes is a promising tool to study hypnozoite biology and may facilitate drug discovery against these quiescent parasites. 142

52 - Multiple aneuploidies are not the norm in natural, uncultured canine Leishmania infantum, but can be induced by in vitro culture-associated stresses.

Dobson, Deborah (Washington University); Shaik, Jahangheer (Washington University); Petersen, Christine (University of Iowa); Beverley, Stephen M. (Washington University)

Leishmania typically exhibit aneuploidy involving multiple chromosomes. However, the strains/species studied had been adapted to culture in vitro, raising the possibility that aneuploidy might be induced artificially by axenization-associated stresses. We examined L. infantum parasites from five naturally infected Missouri foxhounds. Aneuploidy was assessed by deep-sequencing and mapping to the L. infantum genome, yielding a ‘somy’ profile. We first isolated DNA from spleen biopsies bearing both dog and parasite material. Other than chromosome 31 (typically tetrasomic), the somy profile was ‘flat’ with a 2n somy profile. When placed into culture in 'rich' medium (Schneider's + serum), this ‘flat’ profile was maintained for at least 15 passages (~90 cell doublings), and was not altered by cryopreservation and revival. By 30 passages somy remained flat but with increasing variability, suggestive of low level aneuploidy. Single cell DNA sequencing explorations yielded uninformatively noisy profiles, so we generated clonal lines by limiting dilution from early isolates. 16 of 21 (80%) clones examined displayed a ‘flat’ somy profile, while the remainder showed unique aneuploidies involving a few (1-5) chromosomes. When ‘rich medium’ parasites were transferred to a standard medium (M199 + serum), growth was reduced for 5-6 passages. Concomitantly, somy profiles revealed multiple aneuploidies (5-12 chromosomes), whose patterns differed amongst isolates and grew increasingly variable with continued 'standard medium' culture. These results are concordant with a model where aneuploidy is absent naturally in canine L. infantum, but induced by stresses associated with axenization, media and/or single cell cloning in vitro. The seeming absence of natural aneuploidy may have important consequences to the parasites’ ability to adapt during the infectious cycle.

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53 - Plasmodium falciparum immature gametocytes development in human primary erythroblasts

Neveu, Gaëlle (INSERM, Cochin Institute); Richard, Cyrielle; Volpe, Fiona; Dupuy, Florian (INSERM, Cochin Institute); Annamalai, Pradeep (UPMC Faculté de Medecine); Naissant, Bernina (INSERM, Cochin Institute); Martins, Raphael M (CNRS5290, IRD224, University Montpellier 1 & 2, MIVEGEC); Vallin, Patrice; Andrieu, Muriel (INSERM, Cochin Institute.); Lopez-Rubio, José-Juan (CNRS5290, IRD224, University Montpellier 1 & 2, MIVEGEC); Mazier, Dominique (UPMC Faculté de Medecine); Verdier, Frédérique; Lavazec, Catherine (INSERM, Cochin Institute)

Plasmodium falciparum gametocytes, the only stages responsible for malaria parasites transmission from human to mosquitoes, have been shown to sequester and develop in the human bone marrow parenchyma, where they are enriched at erythroblastic islands. These specialized niches, where occurs the terminal erythroid differentiation, consist of a macrophage surrounded by differentiating erythroblasts. The presence of gametocytes in this niche may reflect either adhesion to bone marrow parenchyma cells or gametocyte development in erythroblasts. These mechanisms may contribute to immature gametocytes sequestration in the bone marrow parenchyma. Here, we have set up an infection protocol to produce gametocytes in a culture of human primary erythroblasts. By using colorimetric, fluorescent and electronic microscopy we have characterized the complete gametocyte development in these erythroid precursors. Although only late stages erythroblasts support P. falciparum infection, a delay in the erythroid maturation process upon infection allows gametocytes to complete their development until stage IV in nucleated cells. We have shown that extracellular vesicles produced by parasites are at least one of the factors inducing this delay in the rate of enucleation. Altogether, these results support the hypothesis that immature gametocytes may sequester in the bone marrow parenchyma by infecting late stage erythroblasts and that release of infected reticulocytes in circulation after erythroblasts enucleation may allow mature gametocytes to reach the peripheral blood.

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54t - A novel Babesia bovis secreted protein responsible for binding of infected erythrocyte to endothelial cells

Hakimi, Hassan (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University) Sakaguchi, Miako (Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University); Yamagishi, Junya (Research Center for Zoonosis Control, Hokkaido University); Yahata, Kazuhide; Kaneko, Osamu; Asada, Masahito (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University)

Babesia bovis causes the most pathogenic form of babesiosis in cattle, which results in high mortality in naive adults. B. bovis parasites invade host erythrocytes where they multiply and cause clinical symptoms. The parasite extensively modifies the structural and mechanical properties of host erythrocyte via the secretion of numerous proteins. These modifications mediate cytoadherence of infected erythrocytes in deep tissues and avoidance of clearance in the spleen. Despite their crucial role in virulence, pathogenesis, and as targets of host immunity, such proteins, have not been comprehensively characterized in B. bovis. We performed surface biotinylation of infected erythrocytes, followed by proteomic analysis. The approach was validated by the identification of a known surface exposed protein, variant erythrocyte surface antigen 1 (VESA1). We confirmed the export of several candidates using immuno fluorescence microscopy. Of these candidates the deletion of a putative membrane protein, the product of BBOV_III004280, using CRISPR/Cas9 system was unsuccessful indicating the essentiality of this gene for the parasite. Induced knock down using the glmS riboswitch system resulted in decreased growth rate, reduced ridge numbers on the erythrocyte surface, mislocalized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that this protein is a novel virulence factor for B. bovis. This advance in characterizing the B. bovis proteins secreted to the infected erythrocytes will aid in the development of new therapeutic strategies and novel vaccine candidates to combat bovine babesiosis.

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55t - Targeting Theileria effector proteins at the schizont membrane responsible for host cell transformation

Brühlmann, Francis (University of Bern); Woods, Kerry; Olias, Philipp (University of Bern)

Theileria is a genus of tick-borne parasites and a close relative to other apicomplexans such as Plasmodium. The two most virulent species, Theileria annulata andTheileria parva, cause Tropical Theileriosis and East Coast Fever (ECF) in cattle, respectively. T. annulata and T. parva are equipped with the unique ability among eukaryotic microorganisms to convert their mammalian host cell into a transformed immortalized and invasive stage. A striking difference between Theileria-transformed cells and cancer cells is that the malignant phenotype seems not to be based on genome instability or mutations. This implies that an armory of parasite proteins must be altering host signaling pathways in parasite-infected leucocytes. Only very few such effector proteins have been identified so far, although several, including subtelomeric SVSPs and the secreted TashATs, have been hypothesized to interfere with host cell signaling pathways. To investigate potential Theileria effector proteins passing the schizont membrane and to identify candidate proteins constituting a so far elusive export machinery, the TurboID proximity labeling technique was adapted to in-vitro cultivated T. annulata cell lines. For this, we utilized the promiscuous biotin ligase BirA* and fused it to known host proteins hijacked by the parasite to the membrane surface. Affinity purification and liquid chromatography- tandem mass spectrometry (LC-MS/MS) coupled with a bioinformatic approach utilizing whole genome sequencing data, identified multiple novel Theileria proteins, as well as several TashAT-like and SVSP proteins. Results of this approach will be presented, with a focus on novel candidate proteins that might help to explain the transformative nature ofTheileria parasite.

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56t - Epigenetic regulation of gametocytogenesis in P. berghei; the roles of HAT1, HDA1, and differential histone PTMs.

Power, B. Joanne J. (Pennsylvania State University); Burchmore, Richard (Glasgow Polyomics); Waters, Andrew P. (Wellcome Centre for Integrative Parasitology)

From invasion of the sporozoite, through Plasmodium development in the host liver, to intraerythrocytic parasite growth, all these stages of the malaria parasite life-cycle undergo asexual replication. Though these asexual stages are responsible for morbidity and mortality in the Plasmodium host, it is only with sexual development of the parasite, a process known as gametocytogenesis, that transmission from a host to a mosquito vector can occur, therefore ensuring the spread of the malaria parasite from one host to another. Using reverse genetics approaches, in combination with fluorescence- assisted cell sorting, a previously undetermined role for histone acetyltransferase 1 (HAT1) in P. berghei intraerythrocytic growth and gametocytogenesis was determined. In a similar manner, histone deacetylase 1 (HDA1), a protein previously shown to be transcribed alongside proteins AP2-G and LSD2 prior to schizont egress, was determined to be essential to optimal asexual blood-stage growth of P. berghei. In addition, acid extraction of histones and tandem mass spectrometry were used to determine the array of histone post-translational modifications (PTMs) that define sexual-stage gametocytes from mature asexual schizonts in P. berghei. Rapamycin-induced over-expression of pbap2-g was used to ensure sexual development of P. berghei parasites before isolation of mature gametocytes from asexual parasites. This analysis identified 430 histone PTMs from both P. berghei schizonts and gametocytes, 26 of which were repeatedly shown to be gametocyte- specific and 28 of which were schizont-specific. H3.3K18 trimethylation was enriched in all P. berghei schizont samples, with H2BK19ac, H2B.ZS9ph, and R95me1 enriched only at mature gametocyte stages. Many differential histone PTMs were located at nucleosome DNA entry/exit points, particularly, the histone 3 N-terminal tail and the unique aN helix.

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Session X: And the Seasons They Go Round and Round: Life-Cycle Differentiation

Chairs: Laura Kirkman and Diego Huet

Wednesday, September 18th, 2019

9:00am – 10:15am

148

149

57 - Functional characterizacion of Trypanosoma cruzi surface mucins in the infection of the invertebrate host camara, maria de los Milagros; Balouz, Virginia (IIB UNSAM, Buenos Aires.); Centeno Camean, Camila (IIB UNSAM); Cori, Carmen (CHIDECAR UBA); Lobo, Maite Mabel (IIB UNSAM); Lederkremer, Rosa (CHIDECAR); Gallo-Rodriguez, Carola (CHIDECAR UBA); Buscaglia, Carlos Andres (IIB UNSAM)

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a life- long and debilitating neglected illness of major significance to Latin America public health, for which no vaccine or adequate drugs are yet available. In this scenario, identification of novel drug targets and/or strategies aimed at controlling parasite transmission are urgently needed. In this work using a genetic and a biochemical approach we functionally characterised the role of T. cruzi surface mucins in the infection of the invertebrate host. By using ex vivo binding assays together with different biochemical and genetic approaches, we herein show that Gp35/50 kDa mucins, the major T. cruzi epimastigote surface glycoproteins, specifically adhere to the internal cuticle of the rectal ampoule of the triatomine vector ( T. infestans and R. prolixus), a critical step leading to their differentiation into mammal-infective metacyclic forms. Ex vivo binding assays in the presence of chemically synthesized analogs allowed the identification of a solvent-exposed peptide and a branched, galactofuranose (Galf)-containing trisaccharide (Galfβ1-4[Galpβ1-6]GlcNAcα) from their O-linked glycans as Gp35/50 kDa mucins adhesion determinants. Furthermore in vivo infection assays in R. prolixus revealed that parasites overexpressing Gp35/50 KDa mucins presented a higher infectivity in the insect host which was also correlated with a higher number of metacyclic forms in the rectal ampoule in comparison with control lines. Overall, these results provide novel insights into the mechanisms underlying the complex T. cruzi triatomine interplay. Most importantly, and taking into account that Galf residues are not found in mammals, we propose Gp35/50 kDa mucins and/or Galf biosynthesis as appealing and novel targets for the development of T. cruzi transmission-blocking strategies.

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58 - Transcription in Trypanosoma brucei: daring to be different

Florini, Francesca (IZB, University of Bern); Naguleswaran, Arunasalam (IZB, University of Bern); Heller, Manfred (DBMR, University of Bern); Roditi, Isabel (IZB, University of Bern)

The protein-coding genes of kinetoplastids, includingTrypanosoma brucei, are organised in polycistronic units reminiscent of bacterial operons. Messenger RNAs are transcribed as polycistronic precursors that are co- transcriptionally processed into monocistronic transcripts by trans-splicing and polyadenylation. Since kinetoplastidslack recognisable orthologues of many eukaryotic transcription factors, transcription in these organisms may have evolved to be fundamentally different. One example of this divergence is the large multigene family of retrotransposon hotspot (RHS) proteins, which is unique to trypanosomes. We recently showed that three RHS sub-families are involved in mRNA synthesis by RNA polymerase II (RNAPII). To further characterise proteins associated with RNAPII, we performed pulldowns with antisera directed against its largest subunit (RPB1). The RNAPII complex contained RHS proteins, nucleoporins from the inner ring/central channel and nuclear transport factors. Confocal microscopy confirmed that RPB1 colocalised with the nuclear envelope antigen NUP-1 at the nuclear rim. When cells were treated with Actinomycin D, which stalls the polymerase, anti-RPB1 antisera pulled down RHS4, but the remaining RHS, nucleoporins and the majority of transport factors were reduced ≥3-fold or absent. Active and stalled RNAPII also exhibited differences in phosphorylation of the C- terminal domain of RPB1. Furthermore, when transcription was blocked, poly(A) RNA accumulated in the nucleus and RPB1 no longer colocalised with NUP-1. Finally, we monitored nascent RNA synthesis and performed global run-on sequencing. The latter revealed that nascent transcripts are not uniformly derived from different chromosomes or even from a single transcription unit. These findings imply that nuclear architecture, regulated RNA export and/or nuclear RNA decay might all influence the steady state levels of mRNAs derived from the same polycistronic unit.

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59 - Identification of metabolic pathways that regulate sexual differentiation in P. falciparum.

Sollelis, lauriane (WCIP); Achcar, Fiona; Barrett, Michael; Marti, Matthias (WCIP, Glasgow)

Malaria remains a burden in endemic countries, where the disease continues to cause over 400, 000 deaths each year. During asexual replication of the parasite in red blood cells, a subset of parasites commits to a sexual differentiation pathway and become gametocytes, the only form transmissible to mosquitoes. Key to sexual commitment is the epigenetic activation of a transcriptional master switch, Pfap2-g, in the parasite. We have recently demonstrated that Pfap2-g can be activated by limiting the serum phospholipid lysophosphatidylcholine (LysoPC). Using transcriptional and lipidomic analyses we were able to define the signature of sexual commitment upon LysoPC restriction. These data demonstrated compensatory upregulation of ethanolamine kinase (Pfek) and phosphoethanolamine-methyl- transferase (Pfpmt) involved in de novo phospholipid biosynthesis through the Kennedy pathway. Notably, pmt is absent in the rodent malaria parasite and this lineage is also insensitive to LysoPC restriction. PfPMT is a highly expressed protein and requires three methyl groups in order to produce phosphocholine from phosphoethanolamine. Many critical processes in biology are regulated by methylation, mainly via the methyl donor S-Adenosyl-Methionine (SAM). Both epigenetics (via histone methylation) and lipid metabolism (i.e., the Kennedy pathway) are known sinks of methyl groups in cells. Here we hypothesized that alterations in methyl group abundance may affect growth and sexual commitment. To test this hypothesis, we recently generated a pmt knock-out parasite line. These parasites have a severe growth defect in absence of LysoPC but, surprisingly, no altered rate of commitment. However, sexual commitment can be blocked by adding SAM to minimal medium, both in WT and pmt KO parasites. Indeed, metabolomic and lipidomic analysis suggests alterations in the SAM biosynthesis and polyamine pathways under restrictive conditions. Data will be presented from our ongoing experiments following the fate of methyl groups during commitment using labelled precursors in WT and KO parasites, by proteomics, and parallel RNAseq analysis. Our data suggest that LysoPC availability and the subsequent regulation of PMT activity alter SAM levels, which ultimately affect the epigenetic regulation of ap2-g.

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60t - Characterization of a Novel Sensing Mechanism Governing Antigenic Variation and Immune Evasion in P. falciparum

Schneider, Victoria (The Rockefeller University); Ben Mamoun, Choukri (Yale University); Rhee, Kyu; Harris, Chantal; Kafsack, Bjorn; Deitsch, Kirk (Weill Cornell Medical College)

Plasmodium falciparum expresses a multi-copy gene family called var in the intraerythroyctic stages of its life cycle in a mutually exclusive manner. var genes encode the chief antigenic and virulence determinant of P. falciparum, PfEMP1, and switching between active genes results in antigenic variation, allowing the parasite to evade the human immune system and cause chronic infections. The molecular mechanisms that control activation and silencing of individual var genes, as well as coordination of the switching process, remain undefined, but it has been shown that changes in the transcription state of a var gene are controlled epigenetically. The methylation state of histone marks distributed at active and silent var genes, deposited by histone methyltransferases (HMTs), play prominent roles in var gene regulation. Previously, Ukaegbu et al., 2015 showed that manipulating deposition of these marks had a significant impact on var gene expression. Metabolism and epigenetic control of gene expression are linked, as HMT activity is dependent on the intracellular concentrations of methyl donors, which can fluctuate based on nutrient availability. Various studies in other organisms have shown that there is direct link between the level of intracellular S-adenosylmethionine (SAM), the principle methyl donor in biological methylation modifications, and histone methylation. To test the connection between metabolism and var gene expression, P. falciparum was cultured in media with altered concentrations of nutrients involved in SAM metabolism. Over time, conditions linked to changes in lipid metabolism were found to induce changes in var gene expression. This observation directly challenges the paradigm that antigenic variation in this organism follows an intrinsic, programed switching rate, which operates independently of any external stimuli, by instead suggesting it possesses the ability to sense environmental changes and respond to them by modifying gene expression. Here, we present results suggesting the novel possibility that P. falciparum does possess cellular machinery capable of sensing changes in the environment of its host and responding by altering antigen presentation. 153

61t - Examining trans-regulators of the Leishmania lifecycle

Walrad, Pegine B. (York Biomedical Research Institute, Univ. of York); R. Ferreira, Tiago; Pablos, Luis; Dowle, Adam; Forrester, Sarah; Parry, Ewan; V. C. Alves-Ferreira, Eliza; Newling, Katherine; Kolokousi, Foteini; Larson, Tony; Plevin, Michael (University of York, University of York.); K.Cruz, Angela (University São Paulo)

Constitutive transcription in Leishmania parasites is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few trans-regulator proteins are known. mRBPomes control gene regulation throughout the Leishmania lifecycle and LmjPRMT7 is a monomethylating enzyme implicit in pathogenesis. We independently and quantitatively isolated, identified and analysed both the comprehensive mRNA binding proteome and the protein targets of PRMT7 monomethylation in Leishmania. Utilizing optimised crosslinking and in-depth, quantified mass spectrometry, we present a comprehensive analysis of over 1,400 mRNA binding proteins (mRBPs) from the three main Leishmania lifecycle stages. We find a low correlation between transcript abundance and corresponding protein expression as well as stage-specific variation in protein expression versus RNA binding potential and target mRNA selection. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 17 target RNA-binding proteins (RBPs) hypomethylated in PRMT7-null mutants. PRMT7 can modify RBPs Alba3 and RBP16 as direct substrates in vitro and PRMT7 knockout reduces both RBP16 protein half- life and Alba3 mRNA-binding capacity in vivo. RNA immunoprecipitation (RIP) analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with target transcripts and thereby controls stability of delta-amastin surface antigen. Our study is the first to quantitatively identify the mRBPome and monomethylome of Leishmania parasites. Our results indicate that in L.mexicana parasites, RNA levels are not a strong predictor of whole cell protein expression or RNA binding potential. In addition, RBPs bind distinct transcriptome pools with stage-specific affinity. The novel role for PRMT7 arginine methylation of RBPs, suggests a post- translationally-directed regulatory pathway controlling both gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with novel mechanistic insight into the dynamic Leishmania trans-regulatory mRNA:Protein (mRNP) complexes that drive lifecycle progression to human infectious stages. 154

62t - Redox metabolomics and gametocytogenesis in the malaria parasite

Beri, Divya (Indian Institute of Science) Tatu, Utpal (Indian Institute of Science)

Plasmodium falciparum, the protozoan parasite causing cerebral malaria in humans, is one of the most successful human pathogens. Metabolic pathways of obligate, intracellular pathogens are highly intertwined with their host’s pathways to support pathogen growth, whilst the infected host struggles to maintain overall homeostasis. Inside the erythrocytes, the parasite multiplies via its asexual cycle consisting of merozoites, rings, trophozoites and schizonts. A fraction of these parasites exits this cycle and commit to form the transmissible forms called the gametocytes. Few years back our group demonstrated that ER stress caused by an external agent DTT triggers gametocytogenesis in the parasite. Intrigued by the presence of such stress-adaptive plasticity in the parasite, we decided to explore its redox metabolic pathways. We chose to study the transsulphuration (TS) pathway which is known to connect the redox molecule homocysteine’s catabolism to glutathione biosynthesis. Bioinformatically we observed that the parasite lacks two enzymes of the TS pathway which convert homocysteine to cysteine via cystathionine. Using targeted metabolomics approach, we confirmed this observation and demonstrated that the parasite effluxes homocysteine into the erythrocyte and culture medium. This was further supported by the clinical observation of hyper-homocysteinemia in malaria patients. Furthermore, our analysis revealed critical perturbations in the methionine cycle and glutathione homeostasis in the parasite. To explain the physiological effect of this redox perturbation, we challenged in vitro and in vivo parasites with homocysteine and observed physiologically relevant induction of gametocytogenesis. Our results indicate that self-generated redox stress, dearth of mechanisms to cope up with loss of redox homeostasis and ability of the accumulated redox metabolites to trigger gametocytogenesis, together provide for an autocrine/paracrine regulation of commitment in the malaria parasite. 155

Session XI: Express Yourself: Molecular Biology

Chairs: Diego Huet and Laura Kirkman

Wednesday, September 18th, 2019

10:30am – 11:30am

156

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63 - A Novel PNUTS-PP1 Phosphatase Complex Orchestrates RNA Pol II Transcription Termination in T. brucei

Kieft, Rudo (University of Georgia); Zhang, Yang; Marand, Alexandre P.; Moran, Jose Dagoberto; Bridger, Robert; Wells, Lance; Schmitz, Robert J.; Sabatini, Robert (University of Georgia)

O-linked glucosylation of thymine in DNA (base J) represents an epigenetic mark, regulating Pol II transcription termination within polycistronic gene clusters in Leishmania and T. brucei. Loss of base J leads to altered transcription termination and read-through transcription. The mechanism of termination in kinetoplastids, including the role of base J, is unknown. However, in humans and yeast, the transition from transcription elongation to termination involves dephosphorylation of the carboxy-terminal domain of Pol II by PP1 protein phosphatase as part of a multimeric complex composed of regulatory subunits PNUTS, Tox4, Wdr82 and PP1. In L. tarantolae we have identified a similar complex, including PP1, potential homologues of PNUTS and Wdr82, and a novel J-DNA binding protein (JBP3), presumably a functional homologue of the Tox4 DNA binding protein. In T. brucei we have verified the individual interactions of the multimeric complex, as well as the association of PNUTS and JBP3 with chromatin. Similar to the effects from the loss of J and H3.V, ablation of PNUTS, Wdr82 and JBP3 by RNAi leads to read-through transcription at Pol II termination sites and de-repression of Pol I transcribed VSG expression sites. We also detect increased transcription at regions between divergent Pol II promoters, presumably resulting from bi-directional promoter activity where non- productive RNA synthesis (antisense) is regulated by early termination. Similar increased antisense transcription at promoters is seen upon depletion of Wdr82 and PNUTS in human cells. Our data suggests a model where base J (via JBP3) recruits the PP1 complex to transcription termination sites at promoters and 3’ end of gene clusters.

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64 - Identification of a master regulator of differentiation in Toxoplasma

Waldman, Benjamin S. (Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology); Schwarz, Dominic (Whitehead Institute for Biomedical Research and Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg); Wadsworth II, Marc H. (Institute for Medical Engineering & Science (IMES), Department of Chemistry, and Koch Institute for Integrative Cancer Research, MIT); Saeij, Jeroen P. (Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis); Shalek, Alex K. (Institute for Medical Engineering & Science (IMES), Department of Chemistry, and Koch Institute for Integrative Cancer Research, MIT); Lourido, Sebastian S. (Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology)

Toxoplasma gondii chronically infects a quarter of the world’s population, and can result in life-threatening disease in immunocompromised patients. Acute symptoms of infection result from the rapid proliferation and lysis of host cells by Toxoplasma tachyzoites. Chronic infection is established by slower-growing bradyzoites, which form intracellular cysts resistant to current therapeutics and adaptive immunity. While this dramatic change in lifestyle is accompanied by widespread morphological, metabolic and transcriptional shifts, the mechanisms underlying and driving these differences have remained elusive. Differentiation of tachyzoites into bradyzoites can be induced in vitro through a variety of stress conditions, which has allowed intensive examination of transcriptional differences between these two stages. The inability to isolate Toxoplasma mutants displaying a complete block in differentiation has led to the view that no master transcriptional regulator of this process exists. Through a combination of Cas9-mediated genetic screening and single-cell transcriptional profiling, we have identified and characterized a single putative transcription factor (BFD1, bradyzoite formation deficient 1) as necessary and sufficient for differentiation. Translation of BFD1 appears to be stress dependent, and transient overexpression of BFD1 induces differentiation in the absence of stress. As a Myb-like factor, BFD1 provides a counterpoint to the ApiAP2 factors dominating our current view of parasite gene regulation, and represents a family of transcription factors previously overlooked as potential regulators of parasite-specific biology. BFD1 provides a genetic switch to study and control Toxoplasma differentiation, and will inform prevention and treatment of chronic infection.

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65 - Regulation of membrane trafficking by Giardia lamblia's sole RhoGTPase, GlRac

Thomas, Elizabeth B. (University of Washington)

Differentiation into environmentally resistant cysts is required for the transmission of the intestinal parasite Giardia lamblia. Previously, we showed that Giardia’s sole small RhoGTPase, GlRac, is required for the proper trafficking of Cyst Wall Proteins (CWPs) in Encystation Specific Vesicles (ESVs) - delay compartments where cyst wall protein is processed and held until secretion. To gain a mechanistic understanding of how CWP1 trafficking is regulated by GlRac, a proteomics screen was carried out to identify GlRac interacting proteins in encysting and non-encysting cells. We focused on characterizing proteins that were enriched in encysting cells and showed homology to known membrane trafficking proteins. Giardia lacks Golgi, endosomes and lysosomes and in the absence of canonical endomembrane organization the specific roles of these candidates are unclear. Remarkably, co-localization studies have implicated every protein examined so far in different stages of ESV trafficking. Of particular interest, we identified components of the Homotypic Fusion and Vacuolar Protein Sorting (HOPS) tethering complex, which is normally associated with lysosomal trafficking. Giardia lacks key components normally associated with this complex raising questions about how it functions in the absence of recognizable co-factors and whether it has been re-purposed for the regulation of secretion.

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66t - Functional and biochemical characterization of SPY- catalysed nucleocytosolic O-fucosylation in Toxoplasma gondii

Bandini, Giulia (Boston University); Agop-Nersesian, Carolina (Boston University); van der Wel, Henke; West, Christopher M. (University of Georgia); Samuelson, John (Boston University)

Protein glycosylation is one of the most abundant and widespread post- translational modifications. Glycosyltransferases acting on nucleocytosolic proteins are present in almost all eukaryotes, with O-GlcNAc transferase (OGT) being the most studied example. Modification of Ser/Thr residues by metazoan OGT affects the activity, localization or stability of many host proteins involved in transcription, translation, and signalling. We have previously shown that the human pathogen Toxoplasma gondii modifies with O-Fucose Ser/Thr residues on at least 70 proteins involved in mRNA processing, nuclear transport, and signalling. Recently, we identified the nucleocytosolic O-fucosyltransferase responsible as TgSPY, a paralog of OGT. Arabidopsis thaliana has also been shown to encode for an active SPY O-fucosyltransferase and homologs are predicted in other , including Cryptosporidium parvum. CRISPR/Cas9-mediated disruption of TgSPY causes loss of binding by the fucose-specific lectin Aleuria aurantia (AAL) and a minor deficit in parasite growth. At a molecular level we observe a decrease in steady state levels of reporter proteins that are fucosylated in parent cells, suggesting O-Fuc might be involved in protein stabilization. AAL binding can be rescued by complementation with the endogenous enzyme, but not Arabidopsis or C. parvum SPYs that are both proteolytically cleaved by tachyzoites. T. gondii spy was also disrupted in Cz1 Δku80 parasites expressing luciferase. Parental strain and Cz1 Δspy parasites display similar infection kinetics, although Δspy generates higher luminescence signals at the beginning of the chronic stage. Finally, we studied the biochemistry of TgSPY using a combination of in vitro and in vivo assays. Recombinantly expressed TgSPY hydrolyses only GDP-fucose when tested against a panel of GDP- and UDP-sugars. Complementation with catalytically impaired mutants and truncated versions of TgSPY has provided information on the conserved residues important for catalysis in both SPYs and OGTs.

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67t - The novel EhHSTF7 transcription factor binds to HSE and regulates the multidrug resistant Ehpgp5 gene expression in Entamoeba histolytica.

Bello, Fabiola (Centro de Investigación y Estudios Avanzados, IPN); Orozco, Esther (Centro de Investigación y Estudios Avanzados, IPN); Pérez, Guillermo; Gómez, Consuelo (Escuela Nacional de Medicina y Homeopatía, IPN)

Entamoeba histolytica parasite has the largest family of heat shock factors (HSF) among the eukaryotes conformed by seven members. Preliminary results suggest that EhHSTF7 factor could play an important role in Ehpgp5 gene regulation under emetine stress. However, there are no studies analyzing the role of any HSF in E. histolytica in response to emetine stress. Here, we analyzed the interaction of EhHSTF7 factor with the Heat Shock Element (HSE) of the Ehpgp5 gene, as well as the role of EhHSTF7 factor in Ehpgp5 expression. We performed electrophoretic mobility shift assays (EMSA), competition assays, and silencing by siRNAs to analyze EhHSTF7 factor interaction with HSE sequence. EMSA assays were carried out with EhHSTF7 recombinant protein and HSE-biotin sequence; we identified a complex formed by rEhHSTF7-HSE. Other greater complexes were observed with higher rEhHSTF7 concentration, suggesting an important role for protein concentration in the DNA-protein interaction. To analyze the importance of HSE nucleotides, we evaluate through competition assays the 5'-GAA-3' nucleotides of the HSE sequence, we identified that the 5'-GAA-3' nucleotides of the complementary strand of HSE have an important role in the binding of rEhHSTF7 factor. EhHSTF7 was silenced by siRNAs in trophozoites grown with emetine stress and without stress, then we evaluated the expression of Ehpgp5 using RT-PCR and western blot; we observed the higher silencing of EhHSTF7 factor with the siRNA467 and we also observed a decrease in EhPgp5 expression in trophozoites grown with siRNA467. These findings suggested that EhHSTF7 factor could play an important role in regulating EhPgp5 expression in E. histolytica trophozoites grown in emetine stress.

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Session XII: Mamma Mia, Here We Go Again: Cell-Cycle II

Chairs: Ellen Yeh and Rob Moon

Thursday, September 19th, 2019

9:00am – 10:00am

164

165

68 - The Plasmodium falciparum RNA-binding protein plasmei2 plays an essential role in the completion of liver stage development

Goswami, Debashree (Seattle Children's Research Institute); Betz, William; Locham, Navinder K. (Seattle Childrens Research Institute); Murphy, Sean C. (Department of Microbiology); Vaughan, Ashley M.; Kappe, Stefan H.I. (Seattle Childrens Research Institute)

The Plasmodium parasite life cycle is characterized by a highly specific expression of groups of proteins unique to each stage of parasite development. The parasite relies on post-transcriptional mechanisms to strictly maintain the specificity of this stage-dependent protein profile. Eukaryotic cells employ various RNA binding proteins (RBPs) for post transcriptional regulation of gene expression. A search for RBPs in Plasmodium expressed during the liver stage of the life cycle yielded the Plasmodium ortholog of the eukaryotic RBP, Meiosis inhibited 2 (Mei2) known to be essential for commitment to meiosis in the fission yeast Schizosaccharomyces pombe. We have previously shown that Plasmodium Mei2 (plasmei2) is conserved across Plasmodium species and exclusively transcribed in liver stages. Herein, we explore the role of Plasmodium falciparum (Pf) plasmei2 in the liver stage of parasite development by gene deletion. We found that Pf Dplasmei2 is dispensable for asexual blood stage growth, for gametocyte formation and the generation of either mosquito salivary gland sporozoites. Infection of human liver chimeric FRG huHep mice with Pf Dplasmei2 sporozoites showed normal invasion and trophozoite formation. The Pf Dplasmei2 liver stage parasite grows significantly in size and reaches its maximum development around day 6 post infection, following which it undergoes developmental arrest, in contrast to the wildtype parasite that continually grows until day 7 before its egress from the liver. Analysis of organellar development by immunofluorescent analysis of infected liver stages indicate that the apicoplast, mitochondria and endoplasmic reticulum remain branched and tubular in Pf Dplasmei2 liver stages and do not distribute evenly inside daughter merozoites, which is evident in WT day 7 parasites. The Pf Dplasmei2 liver stage does undergo schizogony but with fewer nuclei as compared to WT parasites on day 6, with an additional defect in DNA segregation. There is a lack of membrane invagination typical of cytomere formation in Pf Dplasmei2 mature liver stages, resulting in a failure to form exo-erythrocytic merozoites. When FRG huHep mice infected with Pf Dplasmei2 sporozoites were infused with human red blood cells (RBCs) on day 7 after infection, they did not become blood stage patent, in contrast to all mice infected with wildtype sporozoites. These results indicate that Pf plasmei2 is indispensable for liver stage development of the parasite with a possible role in regulating the translation of proteins required for segregation of DNA and organelles into exo-erythrocytic merozoites. These results also strongly qualify the Pf Dplasmei2 as a candidate for a genetically attenuated whole sporozoite vaccine.

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69 - Toxoplasma F-box Protein 1 Is Critically Required for Daughter Cell Scaffold Function During Parasite Replication

Baptista, Carlos G. (University at Buffalo); Lis, Agnieszka (University at Buffalo, Buffalo, NY.); Gas-Pascual, Elisabet (University of Georgia); Dittmar, Ashley; Sigurdson, Wade (University at Buffalo); West, Christopher M. (University of Georgia); Blader, Ira J. (Universty at Buffalo)

Toxoplasma gondii is an intracellular parasite responsible for one of the most common parasitic infections in humans. Toxoplasma replicates by a unique process named endodyogeny in which two daughter parasites develop within the mother. Early in endodyogeny, Toxoplasma forms a structure called Daughter Cell Scaffold (DCS) from where the daughter cell Inner Membrane Complex (IMC) will form. Despite its central role in parasite replication, the DCS is a poorly characterized structure. Here, we report that a component of the Skp1, Cullin, F-box containing (SCF-E3) ubiquitin ligase complex, Toxoplasma F-box Protein 1 (TgFBXO1) is a critical component of the DCS. First, we demonstrated that in interphase parasites that TgFBXO1 is an IMC-associated protein. Then, TgFBXO1 is recruited to the DCS immediately following centrosome duplication. Interestingly, TgFBXO1 recruitment to the DCS is dependent on its predicted N- myristoylation site and also on TgRab11b which is a small GTPase required for IMC biogenesis. While conventional microscopy shows that TgRab11b and TgFBXO1 colocalize at the DCS, super resolution microscopy reveals that the two proteins do not overlap but instead appeared to localize on distinct regions within the DCS. These data suggest that the DCS is composed of microdomains and that TgFBXO1 and TgRab11b are distinct markers for each region. Most significantly, we reported that knockdown of TgFBXO1 expression severally impairs Toxoplasma replication due to a defect in the organization and development of the daughter cell IMC. In summary, TgFBXO1 is the first protein identified to serve as an effector of the DCS.

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70 - ESCRTing material to the invader: Mechanisms underlying Toxoplasma gondii endocytosis of host cytosolic protein

Rivera-Cuevas, Yolanda (University of Michigan in Ann Arbor); McGovern, Olivia L.; Lawrence, Anna-Lisa; Carruthers, Vern B. (University of Michigan)

Parasites, by definition, depend on their hosts for resources; nonetheless, the mechanisms by which Toxoplasma gondii acquires nutrients to sustain its fast-replicate stage are poorly understood. Our group has discovered that the parasite can actively ingest host cytosolic proteins. However, the molecular mechanisms by which macromolecules traverse the parasitophorous vacuole membrane (PVM) remain unknown. We discovered a potential role for host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in parasite ingestion, based on observing 40% less ingested material after disruption of host ESCRT function by expressing a dominant negative mutant of Vps4A. Furthermore, we identified the parasite secretory protein GRA14 as a candidate for recruiting host ESCRT to the PVM based on it having several late-domain motifs, first described in retroviral proteins, that interact with the host ESCRT machinery to mediate viral budding. Interestingly, GRA14-deficient parasites had markedly lower percentage of ingested host-derived material compared to wildtype, providing the first evidence for a parasite protein involved in this process. Also, results from proximity ligation assays suggest that GRA14 is proximal to the host ESCRT protein ALIX. Thus, as a working model we propose that the parasite actively recruits host components to its PVM in a GRA14-dependent manner to trigger the formation of vesicles containing cytosolic material that further bud into the parasitophorous vacuole lumen, comparable to the mechanisms of ESCRT-mediated multivesicular body formation. Future studies are expected to provide insight on a novel mechanism of how T. gondii acquires host-derived resources to support its replication and development of infection.

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71 - A genetic trap: Resistance towards pyrazoleamide PA21A092 imparts hypersensitivity to the spiroindolone KAE609

Bhatnagar, Suyash (Drexel University College of Medicine); Morrisey, Joanne M.; Bergman, Lawrence W. (Drexel University College of Medicine); Painter, Heather J.; Llinas, Manuel (Penn State University); Fidock, David A. (Columbia University); Vaidya, Akhil B. (Drexel University College of Medicine)

Resistance towards several divergent chemotypes targeting the parasite Na+ homeostasis arise from mutations in the Na+/H+ ATPase, PfATP4. More than 20 different mutations in PfATP4 have been identified with varying degrees of sensitivity and resistance towards the other PfATP4 inhibitors. Our previous report (Vaidya et al. PMID: 25422853) showed that resistance to the pyrazoleamide C2-1 required simultaneous mutations in PfATP4 and PfCDPK5. We generated parasites resistant to another pyrazoleamide, PA21A092. Whole genome sequencing and analysis of PA21A092 resistant parasites also identified two mutated genes, PfATP4 (A211V) and PF3D7_0412000 (P576L). Interestingly, resistance to PA21A092 was accompanied by hypersensitivity to the spiroindolone KAE609, which was not observed in the C2-1 resistant parasites. Using CRISPR/Cas9 based genetic editing, we show that just the A211V mutation in PfATP4 was sufficient to recapitulate the PA21A092 resistance and KAE609 hypersensitivity. Furthermore, we observed that resistant parasites experienced elevated Na+ influx under treatment with KAE609 in comparison to PA21A092. We also show that the malaria parasites, unlike their mammalian hosts, cannot compensate for elevated Na+ influx and therefore succumb easily to Na+ homeostasis disruptions. Based on these observations, we conclude that the hypersensitivity of PA21A092 resistant parasites to KAE609 results from the lack of an effective compensatory mechanism to cope with the elevated Na+ influx caused by KAE609 treatment. Thus, the PA21A092 resistant parasites reach saturating [Na+] faster than their wild-type counterparts when treated with KAE609. These findings suggest the possibility that combining two different chemotypes targeting PfATP4 could force the parasites into a genetic trap where resistance to one would lead to hypersensitivity to the partner compound.

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Session XIII: Save the Best for Last: Host-Parasite Metabolism II

Chairs: Rob Moon and Ellen Yeh

Thursday, September 19th, 2019

10:30am – 11:30am

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72 - Trypanosoma cruzi intracellular amastigote glutamine metabolism and its impact on susceptibility to ergosterol biosynthesis inhibitors

Dumoulin, Peter C. (Harvard T.H. Chan School of Public Health) Vollrath, Joshua; Pagura, Lucas; Burleigh, Barbara (Harvard T.H. Chan School of Public Health)

Chagas disease is the clinical manifestation of infection with the kinetoplastid parasite Trypanosoma cruzi. In its mammalian host, T. cruzi only replicates intracellularly as an amastigote. Persistent tissue parasitism is the central determinant of disease and therefore drug discovery/development efforts are aimed at eliminating intracellular amastigotes. Recently a class of antifungal azole derivatives (ergosterol biosynthesis inhibitors – EBIs) performed well in pre-clinical assays, yet in clinical trials these compounds suppressed but failed to clear infection. The nature of the persisting parasite population during treatment remains unknown. One hypothesis is that surviving parasites are in a metabolically protected niche and are consequently refractory to EBIs. Consistent with this possibility, we have found that restriction of exogenous glutamine during intracellular T. cruzi amastigote replication in cell culture protects the parasites from the cytotoxic effects of EBIs. Using clonal outgrowth following exposure to EBIs we have determined that this protection is not the result of selection of a dormant sub-population. The antifungal/antiparasitic properties of the EBIs are derived from their inhibition of lanosterol 14α-demethylase (CYP51), a cytochrome P450 enzyme in the ergosterol biosynthesis pathway. Inhibition of CYP51 results in a block in ergosterol and accumulation of toxic sterol synthesis intermediates. We hypothesized that a reduced flux through the ergosterol biosynthesis pathway during glutamine starvation results in a concomitant reduction in toxic sterol intermediates. In support of this hypothesis, addition of farnesyl pyrophosphate (FPP), a precursor of ergosterol biosynthesis is able to re-sensitive amastigotes to EBIs in the absence of glutamine. Metabolic labeling experiments are currently underway track the carbons from glutamine into the sterol pools of the intracellular amastigote. These data underscore the potential for the immediate host metabolic environment to modulate the efficacy of anti-trypanosomal compounds that target parasite metabolism.

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73 - Defining the metabolic network of the malaria parasite reveals one-carbon metabolism is necessary for normal mitochondrial function

Cobbold, Simon A. (University of Melbourne); Marapana, Danushka S. (Walter and Eliza Hall Institute); Ralph, Stuart A. (University of Melbourne); Cowman, Alan F. (Walter and Eliza Hall Institute); Tilley, Leann; McConville, Malcolm J. (University of Melbourne)

Asexual development of the malaria parasite is associated with major restructuring of both parasite and host erythrocyte metabolism. As most current antimalarials target metabolic processes it is important to understand the parasite’s total metabolic capacity and identify new metabolic enzymes that are essential for intraerythrocytic development. Here we describe a new global stable isotope labelling strategy for comprehensively defining the metabolic changes that occur in human erythrocytes following malaria infection. Using untargeted LC-MS we traced the fate of ten major 13C-labeled carbon sources in both infected and uninfected erythrocytes. Differential analyses of all mass/charge features between infected and uninfected cells has led to a draft metabolome of the malaria parasite (942 metabolites). The draft metabolome constitutes 43% of the expected parasite metabolome, while an additional 112 metabolites were identified that were not predicted from genomic reconstructions. To further explore this ‘dark’ metabolome and validate the accuracy of the draft metabolome we investigated potential enzymes with no defined function which could potentially contribute to the dark metabolome. CRISPR/Cas9 was used to generate a dual DiCre/Ribozyme inducible knock-down system for 15 genes encoding uncharacterized metabolic enzymes, including the mitochondrial Serine HydroxyMethylTransferase (SHMT-M). Inducible disruption of SHMT-M impairs parasite development and perturbs mitochondrial metabolism. We determined that SHMT-M is necessary for providing one-carbon units for mitochondrial maintenance; demonstrating the utility of this approach for functional characterization of the parasite genome and search for novel antimalarial targets.

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74 - Trypanosoma cruzi mitochondrial pyruvate carrier (MPC) subunits do not form homodimers and are essential for pyruvate-driven respiration

Negreiros, Raquel S. (State University of Campinas); Vercesi, Anibal E. (State University of Campinas); Docampo, Roberto (University of Georgia)

Glycolysis generates pyruvate as its final product. Inside the mitochondria pyruvate can be converted into acetyl-CoA, which is used as substrate for the tricarboxylic acid cycle. Pyruvate metabolism is important to balance glycolysis and oxidative phosphorylation. Pyruvate availability in mitochondria depends on its active transport by the mitochondrial pyruvate carrier (MPC), which is formed by two subunits, MPC1 and MPC2, assembled as a hetero-dimer. In this work, we used the CRISPR/Cas9 technique for gene knockout and endogenous C-terminal tagging of Trypanosoma cruzi MPC1 and MPC2 subunits. Endogenous C-terminal tagging of TcMPC1 and TcMPC2 genes with 3xc-Myc showed that both proteins co-localize with MitoTracker to the mitochondria of epimastigote forms. Individual knockout of TcMPC1 and TcMPC2 genes was confirmed by PCR and Southern blot analyses. TcMPC1-KO or TcMPC2-KO cell lines did not show reduced growth rate in LIT culture medium as compared to the control cell line transfected with scrambled sgRNA. Digitonin- permeabilized TcMPC1-KO or TcMPC2-KO epimastigotes showed impaired oxygen consumption rates when pyruvate, but not succinate, was used as mitochondrial substrate as compared to the control cell line. The results suggest that, in contrast to human MPC subunits (Nagampalli et al., 2018), they do not form homo-dimers in the absence of each other. The monocarboxylate transporter inhibitor UK-5099 (50 μM) was also able to inhibit cellular respiration of control cell lines using pyruvate, but not succinate, as substrate. Thus, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, but not essential for growth of epimastigotes in rich medium. Acknowledgements: FAPESP grants 2013/50624-0 and 2017/25084-3

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75 - Membrane contact sites mediate lipid exchange at the Plasmodium – red blood cell interface

Garten, Matthias (NIH/NICHD); Beck, Josh R. (Iowa State University); Roth, Robyn (Washington University); Bleck, Christopher KE (NIH / NHLBI); Heuser, John; Tenkova- Heuser, Tatyana (NIH / NICHD); Goldberg, Daniel E. (Washington University); Zimmerberg, Joshua (NIH / NICHD)

To survive inside its host red blood cell, the malaria parasite must acquire all nutrients through an interface: the parasitophorous vacuole (PV). Small water-soluble molecules can cross the PV membrane exploiting channels formed by EXP2, while larger molecules like hemoglobin cross via an endocytosis-like mechanism. So far, no mechanism has been proposed that would explain the uptake of lipidic material across the PV lumen. Given the absence of evidence for PV lumen transport vesicles in electron micrographs and inspired by the close proximity of the parasite plasma membrane (PPM) to the PV membrane (less than 20 nm in many places), we hypothesized that the exchange of lipids is facilitated at membrane contact sites. Moreover, the distribution of the nutrient-permeable channel EXP2 and markers of the PV lumen had been described previously as patchy (‘beads-on-a-string’), suggesting domains for solute transport while also indicating sub-compartmentalization of the PV lumen. To understand how those observations are related to lipid and solute transport for Plasmodium falciparum, we correlated the spatial distribution of EXP2- mNeonGreen/mRuby3 and a PV lumen-targeted mRuby3 (PV-mRuby3) with a GFP tagged version of a known PPM lipid transporter, the Nieman- Pick related protein, PfNCR1. Using confocal microscopy, we found that EXP2 and the availability of PV lumen correlate to each other, while PfNCR1-GFP can be found in domains devoid of EXP2 and PV lumen. This finding was also confirmed using correlative light electron microscopy, showing PV-mRuby3 in areas of larger PV membrane – PPM distance only. The set of experiments presented here suggest that regions of close apposition of the PPM and the PV membrane are sites of lipid exchange. Functionally and structurally, this is the definition of a membrane contact site. Conceptually, this divides the PV into two distinct domains for the exchange of water soluble and lipidic material, helping to unravel the functions of this unique organelle for the parasite.

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Poster Session A

Monday, September 16, 2019

7:00 pm – 9:00 pm

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76 - New toolkit for high throughput imaging and image analysis of the chronic stage of Toxoplasma gondii Winiger, Rahel R. (Laboratory of Molecular Parasitology); Fisch, Daniel (Host- Toxoplasma Interaction Laboratory, London.); Yakimovich, Artur (MRC-Laboratory for Molecular Cell Biology); Hammoudi, Pierre-Mehdi (Department of Microbiology and Molecular Medicine, Geneva); Ramakrishnan, Chandra (Laboratory of Molecular Parasitology, Zürich); Soldati-Favre, Dominique (Department of Microbiology and Molecular Medicine, Geneva); Hehl, Adrian B. (Laboratory of Molecular Parasitology) For Abstract See Session I, 5t

77 - Three-dimensional ultrastructure of Plasmodium falciparum throughout cytokinesis Rudlaff, Rachel M. (Harvard Medical School); Kraemer, Stephan (Harvard University); Marshman, Jeffrey (Carl Zeiss Microscopy, Thornwood, NY.); Dvorin, Jeffrey D. (Harvard Medical School) For Abstract See Session I, 6t

78 - VSG mosaics: when the color of your shoes determines the shape of your hat. Gkeka, Anastasia (Deutsches Krebsforschungzentrum); Erben, Esteban; Zeelen, Johan P.; Stebbins, Erec C.; Papavasiliou, Nina F. (Deutsches Krebsforschungzentrum) For Abstract See Session I, 7t

79 - Analysis of the Toxoplasma mitochondrial ATP synthase structure reveals how its shapes the unique cristae and highlights the role of phylum specific subunits Sheiner, Lilach (University of Glasgow) For Abstract See Session I, 8t

80 - Mapping of origins of replication in Plasmodium falciparum Gomes, Ana Rita (DIMNP, Université de Montpellier) CHU de Montpellier); Méchali, Marcel (Institute de Génétique Humaine); Lopez-Rubio, Jose-Juan (DIMNP, Université de Montpellier) For Abstract See Session II, 13t

81 - Single-cell transcriptomes of Plasmodium vivax parasites reveal extensive but continuous changes in gene expression during their intraerythrocytic cycle Serre, David (University of Maryland School of Medicine); Cannon, Matthew (University of Maryland School of Medicine); Caleon, Ramon; Wellems, Thomas; Sa, Juliana (Laboratory of Malaria and Vector Research) For Abstract See Session II, 14t 178

82 - Divergent Acyl Carrier Protein Coordinates Mitochondrial Fe-S Cluster Metabolism in Malaria Parasites Falekun, Seyi P. (Department of Biochemistry, University of Utah); Jami, Yasi (Department of Biological Chemistry, University of California Los Angeles); Park, Hahnbeom (Institute for Protein Design, University of Washington); Wohlschlegel, James A. (Department of Biological Chemistry, University of California Los Angeles); Sigala, Paul A. (Department of Biochemistry, University of Utah) For Abstract See Session III, 18t

83 - Trypanosoma congolense cytoadheres to the brain vasculature Silva Pereira, Sara (Instituto de Medicina Molecular - João Lobo Antunes); De Niz, Mariana; Figueiredo, Luisa M. (Instituto de Medicina Molecular - João Lobo Antunes) For Abstract See Session III, 19t

84 - Functional study of two Trypanosoma cruzi receptors (TcIP3R and TczAC) involved in signal transduction and important for parasite survival Lander, Noelia (University of Georgia); Chiurillo, Miguel A.; Docampo, Roberto (University of Georgia) For Abstract See Session III, 20t

85 - Differential Expression and Localization of Cytochrome b5 in Giardia intestinalis during Nitrosative Stress Response Mowat, Kaitlyn (Trent Univeristy); For Abstract See Session IV, 25t

86 - A Lysophospholipase involved in malaria parasite development: Potential targets for drug development Mohmmed, Asif (International Centre for Genetic Engineering and Biotechnology.); Kumar, Pradeep (International Centre For Genetic Engineering and Biotechnology New Delhi); Botté, Yoshiki Yamaryo (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes); Thakur, Vandana (International Centre for Genetic Engineering and Biotechnology.); Botté, Cyrille (Institute for Advanced Biosciences, CNRS UMR5309, University Grenoble Alpes) For Abstract See Session IV, 26t

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87 - Developing a scalable pipeline for functional genetic analysis in the malaria parasite, Plasmodium falciparum Esherick, Lisl Y. (Massachusetts Institute of Technology) McGuffie, Bryan (Boston Children's Hospital); Cardenas, Pablo; Dey, Sumanta; Nasamu, Sebastian; Pasaje, Charisse F. (MIT); Dvorin, Jeffrey D. (Boston Children's Hospital); Niles, Jacquin C. (MIT) For Abstract See Session V, 30t

88 - Development of resistance in vitro reveals novel mechanisms of artemisinin tolerance in Toxoplasma gondii. Rosenberg, Alex (Washington University in St. Louis); Luth, Madeline R.; Winzeler, Elizabeth A. (University of California, San Diego); Behnke, Michael S. (Louisiana State University); Sibley, L David (Washington University in St. Louis) For Abstract See Session V, 31t

89 - Genetic screens reveal a central role for heme biosynthesis in artemisinin sensitivity Sidik, Saima (Whitehead Institute); Harding, Clare (Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, University of Glasgow); Petrova, Boryana; Markus, Benedikt; Lourido, Sebastian (Whitehead Institute) For Abstract See Session V, 32t

90 - DNA uptake agonists improve transfection efficiency and provide insights into plasmid uptake mechanism in malaria parasites Crater, Anna K. (NIAID, National Institutes of Health); Garriga, Meera; Desai, Sanjay A. (National Institutes of Health) For Abstract See Session V, 33t

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91 - Selection and biochemical characterization of novel leishmanicidal molecules by virtual and biochemical screenings targeting Leishmania eukaryotic translation initiation factor 4A

ABDELKRIM ÉP. GUEDICHE, Yosser zina (Institut Pasteur de Tunis); Harigua, Emna; Bassoumi, Imen (Institut Pasteur de Tunis); Zakraoui, Ons (Children's Hospital Oakland Research Institute); Banroques, Josette (Institut de Biologie Physico-chimique de Paris); Essafi-Benkhadir, Khadija; Barhoumi, Mourad (Institut Pasteur de Tunis); Munier-Lehmann, He lène; Nilges, Michael; Blondel, Arnaud (Institut Pasteur de Paris); Tanner, N. Kyle (Institut de Biologie Physico-chimique de Paris); Guizani, Ikram (Institut Pasteur de Tunis)

The identification of novel small and active molecules for the treatment of leishmaniases constitutes a research priority. Here, we expand on previous in silico investigations and virtual screens of small molecules targeting the Leishmania infantum initiation factor 4A (LieIF) as a potential drug target. LieIF belongs to the DEAD-box family of RNA helicases. DEAD-box proteins contain a highly conserved core structure with a dumbbell shape containing two, linked domains with structural homology to that of recombinant protein A (RecA). This core structure confers an ATP-dependent RNA-binding affinity, an RNA-dependent ATPase activity and an ATP-dependent RNA unwinding activity. We used the ATPase activity to establish a colorimetric assay in microtiter plates to screen for molecules that inhibit LieIF. We screened hundreds molecules previously identified by virtual screenings for their ability to bind to sites on LieIF that were important for the enzymatic activity. We discovered an interesting inhibitor: 6-α/β- aminocholestanol with an IC50 value of 150 ± 15 µM for 1 µM of LieIF. This compound also inhibits the RNA helicase activity of LieIF. The helicase assays and the ATPase competition experiments with the individual RecA-like domains and other proteins indicate that there are multiple binding sites on LieIF, and that the primary binding site is on domain 1 involving conserved RNA-binding motifs. Two out of ten identified chemical analogues of 6-α/β-aminocholestanol (6-α- aminocholestanol and 6-ketocholestanol) showed inhibitor effects on the ATPase activity of LieIF. Similar inhibitor effects were observed with mammalian eIF4A, but with different reaction profiles. All three molecules showed an anti-leishmanial activity against the promastigotes and the amastigotes of L. infantum parasites, and they showed non-significant toxicity toward macrophages. This study constitutes a first step towards the validation of LieIF as a drug target. It demonstrates biochemical differences between the Leishmania and mammalian eIF4A proteins, most notably in ATPase assays that show that rocaglamide affects the two proteins differently. To conclude, this work delivers a promising leishmanicidal molecule: 6-aminocholestanol with IC50 value lower than 1 μM on intracellular amastigotes, with little toxicity and with a selectivity index higher than 20. The 6-aminocholestanol constitutes a promising anti-Leishmania molecule that deserves further investigation. 181

92 - When a protein changes a job: novel noncanonical functions of Atg8 in malaria parasites

Walczak, Marta M. (Stanford University); Ganesan, Suresh M.; Niles, Jacquin C. (MIT); Yeh, Ellen (Stanford University)

Plasmodium spp, which cause malaria, and related apicomplexan parasites possess a unique non-photosynthetic plastid essential for parasites survival called the apicoplast. The apicoplast was acquired by secondary endosymbiosis; it is surrounded by 4 membranes, has its own genome and hosts several metabolic pathways. During blood stage development, the Plasmodium apicoplast forms complicated branched structures which ultimately divide and are segregated to daughter cells. However, despite its essentiality for parasite survival, the mechanisms behind apicoplast biogenesis remain largely unknown.We have recently showed that Atg8, a highly conserved protein required for autophagy in model eukaryotes, has a novel essential function in malaria parasites. Our data suggest that this function is in the division and/or segregation of the apicoplast to daughter parasites. Atg8 depletion leads to defects in apicoplast inheritance, and, as a result, parasites’ death.Here we further explore the mechanism of Atg8 during apicoplast biogenesis. We use co- immunoprecipitation and mass spectrometry to identify interaction partners of Atg8. For this purpose, we N-terminally tagged P. falciparum Atg8 with GFP in its genomic locus. In parallel, we use a functional complementation of Atg8 knockdown to map features of Atg8 that allow it to perform a non-canonical apicoplast-related function in apicomplexan parasites. Our results will provide insights into the novel mechanism of organelle biogenesis, which is critical to malaria parasites’ pathogenesis.

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93 - Depletion of cholesterol from the trophozoite stage of P. falciparum leads to bizarre consequences: Exploration with novel antimalarials.

Ahiya, Avantika I.(Drexel university, College of Medicine); Bhatnagar, Suyash; Morrisey, Joanne M. (Drexel University, College of Medicine); Beck, Josh R. (Department of Biomedical Sciences); Vaidya, Akhil B. (Drexel University, College of Medicine)

Previous studies from our laboratory have shown that short-term exposure to new antimalarial compounds causes rapid accumulation of cholesterol in the plasma membrane of the parasite. This effect appears to be a common consequence of inhibition of PfATP4 (Das et al., PMID:27227970) and PfNCR1 (Istvan et al., PMID:30888318). Previously it was shown (Lauer et al., PMID: 10899110) that cholesterol depletion by methyl-β-cyclodextrin (MBCD) causes extrusion of the trophozoite stages of the parasite out of the red blood cells (RBCs). This bizarre observation has remained unexplained. We have confirmed this observation by using a transgenic parasite line with fluorescently tagged parasitophorous vacuolar membrane (PVM). Upon MBCD treatment, ~60% of the trophozoites emerged out with PVM no longer surrounded by RBC membrane, but still tethered to intact RBCs. Our working hypothesis is that there is an active movement of cholesterol between the PVM and the RBC membrane, which is disrupted by MBCD treatment, resulting in parasite extrusion. In agreement with this hypothesis, we have observed that disruption of cholesterol homeostasis by treatment with either PfATP4 or PfNCR1 inhibitors prevented MBCD-mediated extrusion of trophozoites. Drugs that do not disrupt cholesterol homeostasis (e.g. chloroquine) did not affect MBCD mediated extrusion of trophozoites. These results suggest the existence of an active process of cholesterol exchange between PVM and the RBC membrane that is susceptible to PfATP4 and PfNCR1 inhibitors. We propose this process to be a part of the export of parasite-encoded proteins targeted to the RBC membrane, disruption of which could result in parasite demise.

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94 - Consequence of Babesia microti infection on susceptible mice in the absence or presence of Lyme spirochetes

Parveen, Nikhat (Rutgers-New Jersey Medical School)

Investigations of pathogenesis of Babesia microti and host response to infection using susceptible models have started relatively recently. We used young C3H mice to examine the effect of infection by this tick-borne Apicompexan parasite because this strain of mice is susceptible to B. microti infection and develops high parasitemia similar tot hat observed in some patients, and thus could provide answers to questions raised for human babesiosis. Babesia infected immunocompetent individuals often remain asymptomatic, such that blood donated by these people leads to transfusion transmitted babesiosis (TTB). TTB has become a major health concern now. The spleen plays an important role in B. microti clearance because splenectomized patients show high morbidity and even mortality, and because we observed pronounced splenomegaly in the infected mice. Agreeing with this premise, higher morbidity and mortality due to babesiosis are also reported in the elderly patients. We examined parasitemia of B. microti and assessed its impact on hemoglobin levels in blood, on spleen pathology and overall immune response in C3H mice. Peak parasitemia of >40% was immediately followed by diminished hemoglobin level while parasitemia was barely detectable by microscopy at 21 days of infection albeit we detected high B. microti DNA copies using our sensitive qPCR assay. We hypothesize that qPCR detects DNA released from recently lysed parasites or that associated with extracellular B. microti in blood, which are not easily detected in Giemsa-stained blood smears and might be the cause of underdiagnosis of babesiosis in patients.At the acute stage of infection, a significantly higher number of CD4+ T cells were present in spleens of mice and they produced TNF-a, IFN-?, IL-4 and IL-21, demonstrating high proliferation of these cells in response to B. microti infection. Overall, Th1 polarization of CD4+ splenic cells could help in the resolution of infection in mice while high levels of anti- inflammatory IL-10 cytokine producing CD4+ T cells and a high Tregs/Th17 ratio may prevent fatality due to excessive inflammation. At day 21 post-infection, significant splenic B and T cells depletion and increase in macrophages levels were observed in B. microti infected mice suggesting a role of macrophage in babesiosis resolution. Significant increase in IL-6 that promotes differentiation of Th17 cells was also observed but it resulted in only moderate changes in IL-17A, IL-17F, IL-21, and IL-22 levels, all secreted by Th17 cells. Therefore, a more thorough investigation is needed to determine the immune mechanisms involved in resolution of parasitemia or establishment of a carrier state for this parasite in the hosts. Simultaneous infections with B. microti and Lyme disease spirochete, Borrelia burgdorferi are now the most common tick-transmitted coinfections in the U.S.A. They are serious health problem because coinfected patients show more intense and persisting disease symptoms. B. burgdorferi is an extracellular spirochete and exhibits Lyme arthritis and carditis in C3H mice similar to that observed in humans. We found that B. burgdorferi infection attenuates parasitemia in mice while B. microti subverts the splenic inducible immune response, resulting in a marked decrease in splenic B and T cells, reduction in antibody levels and diminished functional humoral immunity. Suppression of adaptive immune response by B. microti in coinfected mice showed an association with Lyme disease manifestations exacerbation. We will carry out studies in the future to improve understanding of the molecular mechanisms that contribute to reciprocal effect of these two pathogens on each other and their overall impact on the infected hosts.

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95 - Comparative mapping of Plasmodium proteomes provides new insights into erythrocyte remodeling

Preiser, Peter (Nanyang Technological University); Anthony Siau1, Regina Hoo1*, Omar Sheriff1*, Donald Tay1*, Xue Yan Yam1, Han Ping Loh1, Marek Mutwil1, Meng Wei1, Siu Kwan Sze 1, Peter Preiser11Nanyang Technological University, School of Biological Sciences Singapore,2 Both authors contributed equally to this work

After invasion, Plasmodium extensively modifies the host-erythrocyte by secreting remodeling proteins into the parasitophorus-vacuole, the parasitophorus-vacuole-membrane (PVM), the host-cell cytoplasm and the erythrocyte membrane. However, without a common motif or structure available to identify these proteins, the Plasmodium remodelome remains elusive. Here, we mapped the proteome of five Plasmodium species. For this, infected red blood cell of P. yoelii, P. berghei, P. chabaudi, P. knowlesi and P. falciparum were fractionated and analyzed using quantitative TMT LC MS/MS revealing a proteome of ~2K to ~3K proteins per species. Differential analysis of the relative abundance of these proteins across the subcellular compartments allowed us to map their locations, independently of their predicted features. The proteome maps were further refined using gene orthology information and validated using genetic approaches. The analysis revealed that remodeling proteins encode for 8% to 17% of the parasite proteome and showed that while parasite proteins targeted to the periphery are mostly conserved across the genus, those exported beyond the PVM show significant lineage specificity. Reflecting the known differences between P. falciparum and rodent surrogate models, the distinct remodeling features noted for P. falciparum and rodent parasite support that these two lineages evolved differently. P. knowlesi shares both exported protein families with rodent parasites as well as some clusters of PEXEL protein families with P. falciparum suggesting that other plasmodium species have evolved a mixed approach. Our approach identified a significant number of remodeling proteins across the plasmodium genus likely to represent key functions used by the parasite to promote survival and virulence. The presence of different remodeling features in the plasmodium genus has important implications for the understanding of both common mechanisms and lineage-specific differences in the host-parasite interactions and could be crucial for the development of novel strategies against malaria.

185

96 - Charting the Basis for Toxoplasma gondii Virulence Traits Through Lab Adaptation

Primo, Vincent (Boston College); Rezvani, Yasaman (Department of Mathematics, University of Mass); Farrell, Andrew; Marth, Gabor T. (Department of Human Genetics and USTAR Center for Genetic Discovery, Eccles Institute of Human Genetics, University of Utah School of Medicine); Zarringhalam, Kourosh (Department of Mathematics, University of Massachusetts); Gubbels, Marc- Jan (Boston College)

The two Type I genotype Toxoplasma stains RH, a lab-adapted strain, and GT1, a non-lab adapted strain, have a genetic difference of only 0.002% (~1000 SNPs), but show remarkable phenotypic differences in vitro, including plaquing capacity and extracellular survival capability outside a host cell. These phenotypes are considered host-independent virulence factors for which the genetic basis remains largely unknown, despite efforts testing these genetic differences. We reasoned that epitstatic interactions could be responsible, which are hard to validate. Instead we subject the non-lab adapted strain, GT1, to prolonged in vitro cultivation to generate a lab adapted strain, thereby evolving host-independent virulence traits. By simultaneously tracking phenotypic and genetic changes over time we sought to identify alleles, expression patterns, and epistatic relationships to map the molecular basis of host-independent virulence traits in T. gondii. We lab adapted GT1 strain tachyzoites by standard serial passaging for >200 passages (~550 generations, ~2 years) and assessed the evolution of several phenotypes over time. We observed a steady increase in plaque size (2.85 fold), an increase in reinvasion efficiency (2.63 fold), and an increase in extracellular survival (2.15 fold). Comparative genomics using Whole Genome Shotgun Sequencing (WGSS) during this lab adaptation revealed an accumulation of mutations occurring relatively early during adaptation. Many of these mutations, however, occur in non- coding regions with only 1 non-synonymous mutation (L270R) within a phospholipid flippase protein. Since these mutations did not coincide with progressing lab adaptive traits, we next investigated changes in gene expression. We performed mRNA-seq on both intracellular and 6-hour extracellular parasites. This identified an abundance of differentially expressed (DE) genes, many of which belonging to the SRS surface protein and AP2 DNA binding gene families. Notably, extracellular parasites show a far greater number of DE genes trending up/down over time. Regression analysis of these trending genes identified 319 genes correlating (R2>0.70) with our observed changes in phenotype. Furthermore, gene set enrichment analysis (GSEA) revealed pathways shared with bradyzoite differentiation are involved. Validation efforts of our top candidate genes will be presented. In conclusion, having identified phenotypic, genomic, and transcriptomic changes, lab adaptation of the GT1 strain likely originates in changes of gene expression programs and is a promising approach for identifying the basis for host-independent virulence traits in the tachyzoite.

186

97 - Functional investigation of a nuclear binding partner of the bromodomain protein PfBDP1

Quinn, Jennifer (Institute of Microbiolgy - University Clinic Erlangen); Petter, Michaela (Institute of Microbiology - University Clinic Erlangen)

A critical mechanism of gene regulation in the malaria parasite P. falciparum is the dynamic modification of chromatin through posttranslational histone modifications. The combinatorial pattern of modifications such as acetylation and methylation confers a functional epigenetic code. Bromodomain proteins are so called “reader”-proteins, which bind to acetylated lysine residues present in histones. The P. falciparum bromodomain protein PfBDP1 was shown to bind to chromatin at promoters of invasion related genes and to coordinate their transcription. However, the mechanisms behind transcription control via PfBDP1 remain unclear.In other eukaryotic organisms bromodomain proteins regulate gene expression by recruiting transcription factors to gene promoters and by interacting with components of the Mediator complex. This multi-subunit complex is critical for several steps in transcriptional initiation by RNA Polymerase II. Co-immunoprecipitation followed by mass spectrometry showed that PfBDP1 interacts with a putative Mediator component, PF3D7_1124300, showing faint homology to the Dictyostelium discoideum subunit MED24. A combination of methods, including immunofluorescence and co-immunoprecipitation assays, as well as cellular fractionation, confirmed PF3D7_1124300 to be a nuclear protein directly binding to PfBDP1. To further investigate the function of PF3D7_1124300, we generated a parasite line in which PF3D7_1124300 can be conditionally depleted by utilizing the GlmS-ribozyme. Knock down of PF3D7_1124300 had no effect on parasite growth, but lead to reduced expression of small variant surface antigens, indicating that PF3D7_1124300 may play a role in regulating gene expression of subtelomeric genes. Currently, we are evaluating global transcriptional changes upon PF3D7_1124300 depletion by RNA-seq. We further aim to identify PF3D7_1124300 target sites by chromatin immunoprecipitation and sequencing (ChIP-seq). We will present preliminary data regarding the functional characterization of PF3D7_1124300 with respect to binding sites and its interaction with PfBDP1.

187

98 - Reversible glucan phosphorylation reveals a function for amylopectin in Toxoplasma gondii central carbon metabolism

Murphy, Robert D. (University of Kentucky); Dhara, Animesh; Watts, Elizabeth; Brizzee, Corey; Sun, Ramon; Sinai, Anthony; Gentry, Matthew (University of Kentucky)

The asexual stage of T. gondii is defined by rapidly dividing tachyzoites during the acute infection, and bradyzoites within a tissue cyst during the chronic infection. A distinguishing characteristic of bradyzoites is their ability to accumulate starch-like glucose polymers known as amylopectin granules (AGs), which are notably absent in tachyzoites. Importantly, the T. gondii genome encodes the activities needed for AG (starch) utilization. These include a glucan-water dikinase (“TgGWD”: TgME49_214260), a glucan phosphatase (“TgLaforin”: TgME49_205290), and multiple amylases, all of which comprise a cycle of reversible phosphorylation required for starch degradation. To examine their respective functions, expression of TgLaforin and TgGWD necessitated the use of an Sf9 insect cell expression system. This allowed us to further characterize TgLaforin and TgGWD using multiple biochemical and biophysical techniques. In addition to standard glucan kinase/phosphatase activities, we used gas chromatography-mass spectrometry and capillary electrophoresis to demonstrate that both enzymes perform as predicted in cell-free assays. Biophysical techniques including multi-angle light scattering and differential scanning fluorimetry have revealed oligomeric states for these proteins, and provided insights into protein organization, stability, and substrate specificities. A CRISPR-Cas9 knockout of TgLaforin in tachyzoites results in a slow developing, transient AG accumulation phenotype. These potential compensatory changes in the TgLaforin- KO, as revealed by metabolomics analyses, suggest a rewiring of central carbon metabolism. We are now investigating this population’s ability to convert from tachyzoite to bradyzoite and vice versa, and are currently working to determine the phenotypic effects of a TgGWD knockout. These findings suggest that AG metabolism may play roles not only in bradyzoites, but also within tachyzoites as well.

188

99 - Investigating paralogous ApiAP2 proteins with similar DNA binding specificities in Plasmodium falciparum

Bonnell, Victoria A. (The Pennsylvania State University) Josling, Gabrielle A. (The Pennsylvania State University); Gordân, Raluca (Duke University); Painter, Heather J. (Food and Drug Administration); Llinás, Manuel (The Pennsylvania State University)

Gene expression during the P. falciparum intraerythrocytic developmental cycle (IDC) is highly coordinated and is largely regulated by the Apicomplexan AP2 (ApiAP2) family of sequence- specific DNA binding proteins. In this work, we focus on the functional characterization of four ApiAP2 proteins (PF3D7_0420300, PF3D7_0802100, PF3D7_1305200, PF3D7_1456000) that bind a similar CACACA motif in vitro. These four proteins are expressed at different times during the IDC and contain paralogous AP2 DNA binding domains. Although these proteins all bind the CACACA motif, we predict that they regulate distinct subsets of target genes. Using both in vitro and in vivo approaches, we will define how these gene target preferences are established. To determine the comprehensive genome-wide targets for these ApiAP2 proteins, we are using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). As a complementary in vitro approach, we have measured the differential binding specificities against all intergenic instances of the CACACA motif across the entire P. falciparum genome using a recently developed genomic context protein binding microarray (gcPBM). These in vitro experiments test the relevance of the sequence context in which CACACA motifs are found across the genome, which likely impart differential target specificities. Our ChIP-seq results demonstrate that PF3D7_0802100 plays a role in activation of late stage genes, while gcPBM results suggest that paralogous AP2 DNA binding domains interact with diverse flanking regions surrounding the CACACA motif. To identify potential interacting partners we are using immunoprecipitation coupled with mass spectrometry (IP-MS). Further work into identifying gene targets and binding preferences will not only shed light on the mechanisms underlying P. falciparum gene regulation but will also identify specific DNA sequence elements that govern differential binding of paralogous transcription factors. 189

100 - Division and adaptation to host nutritional environment of apicomplexan parasites depend on apicoplast lipid metabolic plasticity and host organelles remodeling

Botté, Cyrille Y. (Apicolipid Team, Institue for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes) Katris, Nicholas J.; Amiar, Souad (Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes); Berry, Laurence (Dynamique des interactions Membranaires normales et pathologiques, UMR5235, Université Montpellier); Dass, Sheena (Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes); Shears, Melanie J. (McFadden Lab, School of Biosciences, University of Melbourne); Touquet, Bastien (Team Cell and membrane dynamics of parasite-host interaction, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR5309, Université Grenoble Alpes); Hakimi, Mohamed-Ali (Team Host-pathogen interactions and immunity to infections, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR5309, Université Grenoble Alpes); McFadden, Geoffrey I. (McFadden Laboratory, School of Biosciences, University of Melbourne); Yamaryo-Botté, Yoshiki (Apicolipid Team, Institute for adavanced Bioscience, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes)

Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases including malaria and toxoplasmosis. Apicomplexan parasites must obtain and combine lipids both from host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the actual role for each lipid source or how these are regulated upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase TgATS2, shows that the apicoplast provides local (lyso)phosphatidic acid balance, which is required for the recruitment of a novel dynamin (TgDrpC) critical during parasite cytokinesis. Disruption of TgATS2 led parasites to shift metabolic lipid acquisition from de novo synthesis towards host scavenging. Importantly, both lipid scavenging and de novo synthesis pathways exhibit major metabolic and cellular plasticity upon sensing host lipid- deprived environments through concomitant (i) up-regulation of de novo fatty acid synthesis capacities in the apicoplast, and (ii) parasite- driven host cell remodelling to generate multi-membrane-bound structures from host organelles that are imported towards the parasite.

190

101 - Toxoplasma gondii RON13 is a rhoptry neck kinase critical for rhoptry discharge and invasion

Ben Chaabene, Rouaa (UNIGE); Lentini, Gaelle; Mukherjee, Budhaditya; SOLDATI- FAVRE, Dominique (UNIGE)

As a member of the Apicomplexa, Toxoplasma gondii possesses two highly specialized, apical secretory organelles called rhoptries and micronemes that critically participate in invasion and egress from infected cell. The rhoptry neck (RONs) and bulb (ROPs) proteins play a pivotal role in invasion and subversion of host cellular functions, respectively. The aspartyl protease 3 (ASP3) is an essential maturase involved in the pre-exocytosis processing of several microneme and rhoptry proteins (1). Conditional depletion of ASP3 led to a severe defect in rhoptry discharge however the unprocessed substrate of ASP3 responsible for the phenotype is not known. Among the ASP3 substrates identified by terminal amine isotopic labeling of substrates (TAILS) analysis, we focused on TGME49_321650. This gene codes for RON13, a rhoptry neck protein that possesses a transmembrane spanning segment and a predicted Serine/Threonine kinase domain. We mapped the ASP3-specific site of cleavage on RON13 to a region between the transmembrane and the kinase domain and showed that maturation by ASP3 renders RON13 more soluble in the rhoptries. Parasites depleted in RON13 are severely impaired in invasion due to a defect in rhoptry discharge, resulting in the absence of moving junction formation. Using bioinformatic, we identified the putative motifs responsible for the kinase activity of RON13 and confirmed its activity by in vitro assay, making it the first active RON kinase identified so far. Functional complementation experiments established that the kinase activity is central to the observed phenotype. While the identification of RON13 substrates is underway, our preliminary data suggests that RON13-dependent phosphorylation appears to be implicated in the stability of the RONs complex.

1. Dogga SK, Mukherjee B, Jacot D, Kockmann T, Molino L, Hammoudi PM, et al. A druggable secretory protein maturase of Toxoplasma essential for invasion and egress. Elife. 2017;6.

191

102 - SLOPE: A two-part enrichment method effective on ring stage Plasmodium parasites

Brown, Audrey C. (University of Virginia) Guler, Jennifer L. (University of Virginia)

Plasmodium falciparum undergo rounds of asexual replication inside human red blood cells (RBCs), progressing from ring stage, to trophozoites and schizonts, before egress. Given the discovery of ring- specific artemisinin resistance and quiescence in P. falciparum, there is great urgency to better understand ring stage biology. However, lack of an effective enrichment method for this parasite stage has limited progress due to considerable host-contributed noise. Here, for the first time, we present a method for separating ring-infected RBCs from uninfected RBCs. This simple enrichment step effectively reduces contaminating host material, increasing sensitivity of downstream analyses such as metabolomics, proteomics, and genome sequencing. Our method takes advantage of the cholesterol-dependent lytic agent, streptolysin-O (SLO), to preferentially lyse uninfected RBCs as previously shown in 2007 by Jackson, et al. Following lytic treatment, Percoll gradient centrifugation removes lysed RBCs, leaving an intact cell population enriched for infected RBCs. We demonstrate that our SLO-Percoll (SLOPE) method is capable of enriching infected RBCs by over 20-fold and is successful in an array of in vitro conditions, including various parasite lines and species, media formulations, and artemisinin-induced quiescence. The alteration of external cholesterol levels modulates SLOPE effectiveness, confirming the role of RBC membrane cholesterol in lytic discrimination. Importantly, we show that enrichment does not impact parasite viability, which demonstrates the non-toxic nature of SLOPE. Targeted metabolomics of SLOPE- enriched ring stage samples demonstrates decreased noise due to host-contributed variation (i.e. blood or serum batch) compared to non- enriched ring stage samples. SLOPE is a scalable, rapid (30-40min), and non-toxic enrichment method ideal for use upstream of a variety of sensitive analyses.

192

103 - Unravelling the role of Pf113; novel PTEX accessory protein?

Bullen, Hayley (Burnet Institute); Palmer, Catherine (Bio21); Sanders, Paul; Jonsdottir, Thorey (Burnet Institute); Riglar, David (Harvard Medical School); Dans, Madeleine (Burnet Institute); Charnaud, Sarah (The Walter and Eliza Hall Institute, Parkville); Baum, Jacob (Imperial College London); Cowman, Alan (The Walter and Eliza Hall Institute); de Koning-Ward, Tania (Deakin University); Gilson, Paul; Crabb, Brendan (Burnet Institute)

Underpinning the survival of Plasmodium parasites in the human host is the translocon of exported proteins (PTEX); a parasite derived complex within the parasitophorous vacuole membrane that serves to export more than 400 proteins into the infected erythrocyte cytosol to mediate parasite survival. Whilst the core five components of the PTEX complex have been defined, there remain many questions as to how PTEX discriminates exported cargo proteins from resident parasitophorous vacuole (PV) proteins and how cargo proteins which vary tremendously in size and complexity are exported. Here we report on the characterisation of P. falciparum protein Pf113, which we believe is an auxiliary member of PTEX. We have investigated the localisation, expression and role of Pf113 and contend that Pf113 is carried into the erythrocyte by the invading parasite whereupon it resides fully within the PV and interacts with both PTEX and EPIC (exported protein- interacting complex). Conditional knockown of Pf113 results in morphological anomalies of the PVM which were exacerbated when protein cargo is inducibly trapped in PTEX. Pf113 knockdown parasites invaded erythrocytes normally suggesting the protein’s major role occurs within the PV after invasion. Here we contend that Pf113 serves to link PV-associated complexes such as EPIC, and specific subsets of exported proteins, with PTEX components to enable efficient export. At this stage it is not clear whether Pf113 helps prepare specific cargo types for export or if it removes aggregated or misfolded cargoes from PTEX to prevent clogging of PTEX.

193

104 - A Putative calcium proton antiporter of Toxoplasma gondii

Calixto, Abigail; Dykes, Eric J.; Moreno, Silvia NJ (University of Georgia)

Calcium (Ca2+) signaling is an evolutionary conserved process among all eukaryotes. Cytosolic Ca2+ increases result from Ca2+ influx from the extracellular environment through plasma membrane channels or from release of Ca2+ from intracellular stores. The cytosolic Ca2+ concentration is highly regulated and cells express a number of mechanisms to maintain low levels. These mechanisms consist of Ca2+-pumps at the plasma membrane and intracellular organelles like the endoplasmic reticulum and acidic compartments, Ca2+ exchangers and Ca2+ binding proteins that bind cytosolic calcium. In Toxoplasma gondii, an obligate intracellular parasite, a rise in intracellular Ca2+ impacts biological processes such as secretion of adhesive proteins, motility and invasion of and egress from host cells. These lytic cycle facets cause rapid and constant tissue disruption, supporting a role for Ca2+ signaling in pathogenesis. We characterized a putative calcium proton antiporter (Ca2+/H+-antiporter), Tggt1_319550, to study its role in T. gondii Ca2+ signaling. Ca2+/H+-antiporters use a proton gradient to sequester calcium into acidic stores. Tggt1_319550 belongs to a well-conserved family of transmembrane proteins predicted to be calcium transporters. The human and yeast orthologs localize to the golgi apparatus, whereas the Arabidopsis ortholog localizes to the . Using Crispr-Cas9, we introduced a multiple HA tag at the C-terminus of the Tggt1_319550 to study its localization. IFAs with anti- HA and co-localization with organellar markers showed ER and Golgi localization. We used Crispr-Cas9 to knockout the gene and isolated clones for phenotypic characterization. We did not observe a growth difference of the mutants and the cytosolic Ca2+ levels appeared normal. The uptake of Ca2+ by the ER calcium appeared to be affected, so we will present data on the potential role of this Ca2+/H+-antiporter in Toxoplasma ER Ca2+.

194

105 - Targeting the Human Malaria Parasite P. falciparum with Kalihinol Analogues

Chahine, Zeinab M. (UC Riverside); Prudhomme, Jacques M. (UC Riverside); Daub, Mary E. (UC Irvine); Ben Mamoun, Choukri (Yale School of Medicine); Vanderwal, Chris M. (UC Irvine); Le Roch, Karine M. (UC Riverside)

Plasmodium falciparum resistance to all current drug treatments threatens global malaria control eradication programs. Therapeutics with novel modes of action and low risk of generating resistance are urgently needed to combat drug-resistance in P. falciparum. Kalihinol is a novel natural compound that belongs to the Isocyanoterpene family (ICTs) and has shown exceptional potency against drug-sensitive and drug-resistant malaria parasites. This initial result suggests that these compounds may target a novel metabolic pathway from all current drug treatment. Here we explore the efficacy and mechanism of action of a Kalihinol analogue, MED6-189 on different stages of the human parasite life cycle, P. falciparum. Preliminary findings show highest potency of MED6-189 in Trophozoite and Schizont stages in the second erythrocytic cycle after incubation with the drug, indicating a ‘delayed death’ phenotype characteristic of a compound targeting the parasite apicoplast. Partial colocalization of the RFP-labeled molecules with GFP labeled apicoplast in D10_Acp_GFP transgenic lines were observed through immunofluorescence assays (IFAs). Preliminary data using Affinity chromatography followed by mass spectrometry suggest a potential role in apicoplast and trafficking. Results obtained using gene expression and metabolomics profiling under drug treatment will be further discussed and integrated with our initial result to better describe the mechanisms by which the parasite population collapses when treated with the drug. Understanding the mode of action of this compound will provide great promise in an effort to treat drug-resistant parasites.

195

106 - Crystal structure of a metacyclic Variant Surface Glycoprotein (mVSG) from Trypanosoma brucei

Chandra, Monica (German Cancer Research Center (DKFZ)); Branco, Francisco A.; Papavasiliou, Nina; Stebbins, Erec (German Cancer Research Center (DKFZ))

Trypanosoma brucei (T. brucei) causes African sleeping sickness in sub-Saharan Africa. It is transmitted from one mammalian host to another by the tsetse fly (Glossina spp.). The parasite’s surface is densely covered by the GPI-anchored Variant Surface Glycoproteins (VSGs). There are approximately 2,000 different VSG genes present in the T. brucei’s genomic repertoire. This vast number of VSG genes enables the parasite to switch an expressed VSG with an antigenically distinct VSG, a process known as antigenic variation. The parasite can thus continuously evade the host’s immune system. VSG is expressed in different life stages of T. brucei, the bloodstream and metacyclic stages. The former is present in the bloodstream of the mammalian host, and the metacyclic form inhabits the salivary gland of the tsetse fly and is transmitted to the mammalian host through a bite, initiating infection. A specific subset of five different VSGs is expressed in the metacyclic form (mVSGs). This study focuses on investigating the protein structures of mVSGs using X-ray crystallography. We have determined the first structure of one of the mVSGs, VSG531. The structure shows a similar fold when compared to the bloodstream VSG1 and VSG2, despite sharing low sequence identity. While maintaining similar fold, VSG531 has different surface properties compared to VSG1 and VSG2. Hence, it will elicit a different immune response from the host. Studying the protein structures of different mVSGs will give us a better understanding of the function of VSG in different life stages, as well as antigenic variation in T. brucei.

196

107 - First Insights into the Proteome and Endocytic Mechanism of the Cytostome/Cytopharynx Complex of Trypanosoma cruzi

Chasen, Nathan (Center for Tropical and Emerging Global Diseases, University of Georgia) Tarleton, Rick; Etheridge, Drew (Center for Tropical and Emerging Global Diseases, University of Georgia)

Trypanosoma cruzi, the causal agent of Chagas’ disease, spends the pathogenic portion of its lifecycle in mammalian host cells as actively replicating amastigotes. Unlike most intracellular pathogens, which reside inside a parasitophorous vacuole, T. cruzi amastigotes replicate directly in the cytosol, a compartment lacking in the simple nutrients required to support rapid parasite growth. As a result, T. cruzi must directly digest host cellular material to support its high metabolic rate. To accomplish this, the parasite employs an ancient endocytic organelle of protozoal origin, which has since been lost in other pathogenic kinetoplastids. This organelle, known as the cytostome/cytopharynx complex (CSP), starts at a pore on the parasite surface which leads to a long tubular invagination of the parasite membrane. Endocytosed cargo travels through the CSP, before ultimately fusing with parasite lysosomes for digestion. With the goal of elucidating the mechanistic underpinnings of this poorly understood feeding structure, we utilized a combination of immunoprecipitation and bioinformatic analyses to identify a number of novel CSP targeted proteins. These findings have provided insight into the mechanism of CSP uptake by identifying key machinery responsible for driving endocytosis. We hypothesize that T. cruzi is uniquely reliant on direct endocytosis of the host cell cytosol to satisfy its nutritional requirements and we expect that this work will lead to the identification of novel strategies for combatting this important pathogen.

197

108 - PfBTP3 reveals a novel role for the basal complex in sexual-stage malaria parasites

Clements, Rebecca L. (Harvard University); Streva, Vincent; Dvorin, Jeffrey D. (Boston Childrens Hospital)

Human malaria, which is caused by Plasmodium parasites, remains an important cause of global morbidity and mortality. Rapidly emerging resistance to first-line antimalarials imposes an urgent need for novel therapeutics. To successfully generate new antimalarials, we must gain a better understanding of the basic cell biology of Plasmodium falciparum, the parasite responsible for the deadliest cases of malaria. Segmentation, the process by which daughter parasites are formed within the common cytoplasm of the multi-nucleated mother, is a crucial process for asexual parasite replication in human red blood cells, and therefore, pathogenicity. Here we describe the discovery of a novel basal complex protein, PfBTP3 (Basal complex Transmembrane Protein 3, PF3D7_0704300). Although PfBTP3 is not essential for asexual replication in vitro, we find that knockdown of PfBTP3 increases gametocyte conversion. Using super-resolution microscopy, we demonstrate expression and assess localization PfBTP3 throughout gametocytogenesis. This study sheds light on the critical but poorly understood process of segmentation in P. falciparum and reveals a novel role for basal complex proteins in sexual-stage parasites.

198

109 - Plasmodium falciparum MCMBP depletion unveils the lack of spindle assembly checkpoint and the critical role of the DNA replicative machinery for nuclear division during schizogony.

Absalon, Sabrina (Indiana University School of Medicine) Dvorin, Jeffrey, Daniel (Boston Children's Hospital)

During the asexual stage of the life cycle, Plasmodium falciparum replicates via schizogony, a division mode that can be divided into a replicative phase and a budding phase. During the replicative phase, the parasite undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division with intact nuclear envelope. The budding phase occurs when the multi-nucleated syncytium is subjected to a synchronous round of karyokinesis, coinciding with segmentation of dozens of daughter cells known as merozoites. The molecular replicative machinery utilized by the Plasmodium parasite, as well as whether the parasite uses local and global checkpoints to control the timing of DNA replication and nuclear division, remain a major gap of knowledge in the biology of the parasite. We identified Pf3D7_1412100 as the P. falciparum Mini Chromosome Maintenance Binding Protein (MCMBP) orthologue. By co- immunoprecipitation, we confirmed that PfMCMBP interacts with the MCM complex, a multi-subunit complex with helicase function that unwinds DNA and gives rise to the replication fork. We successfully obtained a conditional knockdown of PfMCMBP using the destabilization domain system and proved its essentiality for parasite growth. Following depletion of PfMCMBP, schizont-stage parasites display an abnormal nuclear morphology (scattered DNA) and MTOC-like (microtubule organizing center) ectopic foci with extended spindle microtubules in multipolar conformation. Despite the severe and incomplete segregation of chromosomes, PfMCMBP-depleted schizonts segment and form non-invasive aneuploid merozoites with varied nuclear and cellular body size. Interestingly, our results closely resemble those in human cells where interference with MCMBP expression leads to centrosome amplification and nuclear abnormalities. Importantly, our data demonstrate that P. falciparum does not have a spindle assembly checkpoint to prevent unequal segregation of chromosomes during schizogony.

199

110 - Combined genetic and chemical approaches to uncover the essential ER redox network of the human malaria parasite Plasmodium falciparum

Cobb, David W. (University of Georgia) Kudyba, Heather M. (NIH); Hoopmann, Michael (Institute for Systems Biology); Bruton, Baylee W.; Krakowiak, Michelle W. (University of Georgia); Moritz, Robert (Institute for Systems Biology); Muralidharan, Vasant W. (University of Georgia)

Malaria remains a major health burden across the globe, with the most severe form of the disease caused by the eukaryotic parasite Plasmodium falciparum. Inside of the host red blood cell, P. falciparum maintains a complicated secretory pathway that originates in the endoplasmic reticulum (ER) and traffics proteins not only to the parasite plasma membrane, but also into the host cell and into unique organelles required for the parasitic lifecycle. This trafficking network is predicated upon the folding of diverse, newly synthesized proteins within the ER, which requires the correct formation of intra- and intermolecular disulfide bonds (oxidative folding). In addition to its role in the parasite’s complex trafficking requirements, the ER is essential for other critical biological functions, such as signaling events and lipid biogenesis, which are also likely influenced by the ER redox environment. In other eukaryotes, the Protein Disulfide Isomerase (PDI) family of enzymes has been identified as mediators of the redox environment of the ER, but little has been done to characterize these enzymes in P. falciparum. As a putative ER-resident chaperone with a thioredoxin domain, PfJ2 is a potential member of the P. falciparum PDI family. Using CRISPR/Cas9 editing of the parasite genome, we have confirmed localization of PfJ2 to the ER and shown through conditional knockdown that the protein is required for parasite survival in the RBC. Treatment of parasites with a crosslinker specific for redox-active cysteines traps clients to the PfJ2 thioredoxin domain. Potential substrates for this domain include other PDI family members and proteins localized throughout the secretory pathway of P. falciparum. The oxidative folding that occurs within the ER is essential for the parasite lifecycle, and therefore may be exploitable in the efforts to combat malaria, but the proteins that contribute to this process remain largely unexplored. Using a combination of CRISPR/Cas9 genetic editing and a redox- active crosslinker, we have begun to tease apart the pathway that leads to correct oxidative folding in the malaria parasite ER.

200

111 - Novel Antiplasmodial Compounds From Fungi

Collins, Jennifer (University of Central Florida) Cai, Shengxin; King, Jarrod; Wendt, Karen; Lee, Jinwoo; Cichewicz, Robert (University of Oklahoma); Chakrabarti, Debopam (University of Central Florida)

In our pursuit to discover the next generation of antimalarials from novel areas of chemical space we are screening a large library of diverse fungi to identify secondary metabolites with activity against the malarial parasite. Filamentous fungi are rich source of bioactive compound and are capable of synthesizing a wide range of novel pharmacophores. The Cichewicz fungal collection from the University of Oklahoma contains tens of thousands of novel fungal isolates secured from diverse habitats and ecological niches across the United States. We hypothesize that these fungal secondary metabolites, which are underexplored for antimalarial drug discovery, will provide us with a unique opportunity to explore medicinally relevant, but untapped chemical space for the discovery of essential malarial therapeutics. In this effort, we have screened libraries of 750 pure compounds, and 4,500 extracts obtained from diverse fungal sources for their ability to inhibit intraerythrocytic growth of P. falciparum Dd2 strain using a SYBR Green I-based phenotypic fluorescence assay. As a counter screen, we evaluated the cytotoxicity of the top hits in HepG2 cells using the MTS ((3-(4,5 dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) cell proliferation assay. Our hit rate, with an initial screening at concentrations of 1 µM for pure compounds or 2.5 µg/mL for crude extracts is approximately 10%. This screening has identified selective antiplasmodial compounds with picomolar EC50 values, including histone deacetylase inhibitors. These unique pharmacophores from wide areas of chemical space can provide chemical starting points to develop lead compounds against diverse cellular targets.

201

112 - Epigenetic control of Plasmodium falciparum gametocyte development through unique repressive markers of gene expression

Connacher, Jessica I. (University of Pretoria) Josling, Gabrielle A.; Llinas, Manuel (Pennsylvania State University); Birkholtz, Lyn-Marie (University of Pretoria)

Gene expression in the Plasmodium parasite is regulated by a variety of control mechanisms and the interplay between them including epigenetic control through the post-translational modification (PTM) of histones. The dynamic nature of the parasite’s histone PTM landscape, combined with subsets of highly abundant histone PTMs distinguishing each stage of the parasite’s life cycle, suggests this mechanism performs an essential role in driving asexual proliferation and sexual through the control of gene expression. Of note, we recently showed H3K27me2, H3K27me3, H3K36me2, H3K36me3 are highly abundant in stage II gametocytes. These four modifications are well characterised epigenetic regulators of differentiation in model eukaryotic cells, largely associated with repressive functions however, their relevance in malaria parasites is unknown. We therefore set out to determine whether these histone PTMs have a functional role in controlling gene expression, particularly during the differentiation of P. falciparum gametocyte development. ChIP-seq was used to generate a map of the genome-wide enrichment sites of H3K27me2, H3K27me3, H3K36me2, H3K36me3 in three distinct gametocyte populations. The results revealed highly specific patterns of enrichment for these histone PTMs within and upstream of gametocyte relevant genes. Combining the enrichment patterns with gene expression profiles allowed us to confirm that the histone PTMs investigated do indeed regulate the expression of genes critical for gametocyte development. We use ChIP-qPCR, western blotting and fluorescence microscopy to validate the results. Collectively, our data show that H3K27me2, H3K27me3, H3K36me2 and H3K36me3 perform vital and distinctive regulatory roles in stage II gametocytes, particularly through the repression of genes to allow for the processes of early gametocyte development unfold.

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113 - FLIM and Imaging Analysis of Plasmodium falciparum Exposed to Artemisinin

Connelly, Sean (Laboratory of Malaria and Vector Research, NIAID); Levine, Zoë (Laboratory of Malaria and Vector Research, NIAID); Manzella- Lapeira, Javier; Brzostowski, Joe; Krymskaya, Ludmila (Laboratory of Immunogenetics, NIAID); Rahman, Rifat; Sá, Juliana; Wellems, Thomas (Laboratory of Malaria and Vector Research, NIAID)

Plasmodium falciparum recrudescence after 3 – 7 days artemisinin monotherapy is well known. Recrudescence is also observed in vitro when early ring stage parasites are exposed to the drug. Parasites after recrudescence are just as sensitive to artemisinin as before the initial drug treatment, indicating no change of drug response phenotype. Microscopic observation of a delay in the intraerythrocytic cycle with arrested development of the ring-stage parasites suggested cell dormancy may provide a means to escape artemisinin toxicity. Here, we apply Fluorescence Activated Cell Sorting (FACS), Fluorescence Lifetime Imaging (FLIM), and super-resolution AiryScan confocal microscopy to compare ring-stage parasites exposed to dihydroartemisinin (DHA) or to control medium with dimethylsulfoxide (DMSO, DHA vehicle). Cell populations were isolated by FACS in each sample through staining with a DNA dye, SYBR Green, and a mitochondrial potential marker, MitoTracker Deep Red FM. The putative dormant parasite fraction was verified by re-culturing experiments. FLIM of intrinsic reduced nicotinamide adenine dinucleotide (NADH) was used to quantify the metabolic state and AiryScan confocal microscopy was used to visualize parasite structure. DHA-treated parasites showed a collapse of the nucleus and mitochondria as well as a lower mean lifetime, whereas parasites in control medium showed these two organelles apart from one other. After standardizing this method, other antimalarials will be tested to see if these changes are uniquely associated with DHA exposure. Further characterization of this phenomenon may provide insights on prevention of clinical recrudescence.

203

114 - Defining the functional secretory proteome during host invasion by the obligate intracellular parasite Toxoplasma gondii

Costa, Elizabeth A. (Whitehead Institute); Shortt, Emily; Lourido, Sebastian (Whitehead Institute)

Apicomplexans, including Toxoplasma gondii, are obligate intracellular parasites that rely on active invasion of host cells for their survival and replication. Secretion of proteins from specialized organelles called micronemes is essential for parasite motility, attachment to, and invasion of host cells, yet most characterized microneme proteins are dispensable for growth in human fibroblasts, suggesting extensive redundancy and/or context-specific functions. Recent identification of several microneme proteins with novel domains indicates that our current understanding of micronemes is incomplete. To define and characterize their contents, we affinity purified whole micronemes and performed quantitative mass spectroscopy. We could identify most known microneme proteins within the enriched fractions, validating our approach. We additionally identified many uncharacterized proteins with novel domain architectures, and we are currently employing biochemical and genetic approaches to characterize their roles in host cell recognition and invasion. To systematically dissect epistatic relationships between known and novel microneme proteins, we have developed a system to probe all pair-wise deletions using CRISPR- based screens. This genetic interaction map will enable us to define functionally related proteins and allow us to probe the phenotypes of functionally redundant components. These results will elucidate fundamental principles of host-parasite interactions during the crucial step of invasion.

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115 - The Acute Challenge Model: Sensitive moderate- throughput assessment of malaria T cell vaccine antigens in mice

Cruz Talavera, Irene (University of Washington); Stone, Brad; Murphy, Sean C. (University of Washington)

Identification of novel protective Plasmodium antigens is thought to be crucial for developing a vaccine that can induce a broad immune response and attain durable protection against this intracellular parasite. Pre-erythrocytic vaccine development for P. falciparum is faced with the complex problem of selecting genuinely protective T cell antigens out thousands of candidates. There are no proven methods for efficiently testing and selecting protective antigens in vivo. Furthermore, there are many antigens that are highly immunogenic but ultimately non-protective. To define protective antigens, we developed an “Acute Challenge” model that rapidly and sensitively detects protective T cell responses. In this model, multiple DNA vaccines targeting individual P. yoelii antigens are built in parallel using codon- optimized, 500 bp synthetic fragments. DNA vaccines are delivered twice by gene gun to induce CD8+ T cell responses in BALB/c mice. At the peak of the boosted immune response, we challenge the mice with 2-5x104 wild-type luciferase-expressing P. yoelii sporozoites and measure parasite burden using IVIS imaging 40-44 hours later. The Acute Challenge model gives rapid and sensitive results at a moderate throughput (35 days from priming to liver burden endpoint). Our focus is to assess P. yoelii candidate antigens of unknown protective potential that are known or putatively involved in hepatocyte invasion or exported from the parasitophorous vacuole into the host cell cytoplasm. Protective antigens are also assessed for their intracellular localization. Data from multiple Acute Challenge screens will be presented. This protection-forward approach contrasts with prior efforts at immunogenicity screening that have often led to identification of immunogenic but non-protective T cell responses.

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116 - Genetic inferences from sequencing of Pvdbp gene in Indian isolates of Plasmodium vivax

Kar, Sonalika (ICMR-National Institute of Malaria Research) Sinha, Abhinav (ICMR-National Institute of Malaria Research)

India exhibits varying Plasmodium vivax endemicity across the country. As reports on P. vivax drug resistance have been evidenced in various parts of the world, it is of prime importance to develop an effective P. vivax vaccine that will provide protection and prevent its transmission. The only leading P. vivax vaccine candidate, Duffy- binding protein (PvDBP II), interacts with Duffy Antigen Receptor for Chemokines to invade human reticulocytes, has been successfully evaluated under clinical trial Phase 1. The cysteine-rich region II of PvDBP contains critical binding motifs for reticulocyte invasion and has the highest rate of allelic polymorphisms among parasite isolates. Also, this region of Pvdbp gene is reported to be under positive selection. Hence, it is essential to consider and incorporate the outcomes of genetic diversity prior to developing a vaccine based on PvDBP II.Blood samples were collected from patients infected with P. vivax from three different geographic regions in India with reference to their endemicity. The 3762 bp Pvdbp gene was PCR-amplified and sequenced. The polymorphic characters and diversity of Pvdbp gene were analyzed using DnaSP5 and MEGA6 programs.A high rate of non-synonymous polymorphisms in region II of PvDBP was observed in comparison to rest of the Pvdbp gene. High genetic diversity in Pvdbp gene was observed in samples collected from northern India, while comparatively lesser diversity was observed in samples from north-eastern and western India.Region-specific genetic diversity as observed in the study should be taken into account when designing vaccine aimed at eliciting antibodies to inhibit reticulocyte invasion in a large country like India. Further statistical analyses are to be undertaken to prove the same

206

117 - Functional role of the HSP40 Protein in gametocytogenesis of Malaria parasite Plasmodium berghei.

Kashif, Mohammad (National Institute of Immunology); Singh, Agam P. (National Institute of Immunology)

Mohammad Kashif1 and Agam P Singh11 National Institute of immunology. 110067During its transitions to various stages, the Plasmodium parasite is subjected to various stresses that may lead to protein misfolding and aggregation. The chaperones help buffer against cellular stress by promoting productive folding of cellular proteins and preventing their misfolding and aggregation. They are considered future therapeutic targets in various cellular systems including Plasmodium. We endeavored to study a P. berghei HspJ protein of 62 kDa containing the PEXEL motif which we refer as PbHspJ62. Our results show that the protein is exported to host cytoplasm both at liver and blood stages. We checked PbHspJ62 protein expression in sporozoites, liver stages, and blood stages. Immuno Fluorescence Assay using anti-PbHspJ62 polyclonal antibody confirmed that protein has cytosolic expression in all the parasitic stages. In order to investigate the role of protein in Plasmodium, we generated a PbHspJ62 gene disrupted line of P. berghei. The targeted disruption of PbHspJ62 locus had no impact on parasite replication in erythrocyte but resulted in aberrant development of gametes. The knockout parasites were unable to form subsequent sexual stages because of aberrant gametogenesis. The result indicates a possible transcriptional or translational regulation of sexual stage gene at the asexual stage. In order to analyze the effect of gene knockout on parasite, we performed a whole transcriptome analysis on wild type versus PbHspJ62 knock out strain using RNA obtained from same. By performing technical and biological replicates we were able to quantify the transcriptional biological variation among these strains. We analyzed the host genes affected during PbHspJ62 knock out and observed genes involved in stage traversal from asexual to sexual stages had downregulated while asexual stage specific genes had upregulated. The results were also validated by performing qPCR. Our findings highlight the critical role that PbHspJ62 plays in facilitating the transition from asexual blood stage to transmissible gametocytes.

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118 - Conoid function in Apicomplexa is linked to a hybrid P- Type ATPase/Guanylate cyclase that controls lipid signalling required for activation of motility

Katris, Nicholas (Apicolipid Team, IAB, CNRS UMR5309); Yamaryo-Botte, Yoshiki (ApicoLipid Team, IAB, Universite Grenoble Alpes); Janouskovec, Jan (Department of Genetics, Evolution and Environment, University College London); Arnold, Christophe S. (ApicoLipid Team, IAB, Universite Grenoble Alpes); Yang, Annie SP; Sauerwein, Robert (Radboud University Medical Centre); McFadden, Geoff I. (School of Biosciences, University of Melbourne); Tonkin, Christopher J. (Walter and Eliza Hall Institute); Waller, Ross F. (Dept. of Biochemistry, University of Cambridge); Botte, Cyrille Y. (Apicolipid Team, IAB, CNRS UMR5309)

The phylum Apicomplexa is characterized by a unique structure known as the apical complex which is the site of secretion of invasion factors which facilitate a parasitic lifestyle. Additionally, some Apicomplexans such as Toxoplasma have a retractable conoid, a tight-knit, microtubular structure that protrudes and retracts from within the apical ring. The conoid, however, is absent in Plasmodium species, and the exact purpose of the conoid is somewhat mysterious. Here we find that the inhibition of a Toxoplasma conoid protein TgRNG2 prevents proper conoid extrusion which impacts the ability of an apical P-Type ATPase/Guanylate Cyclase (TgGC) to activate downstream activation of lipid seconds messengers Phosphatidylinositol (PI), Diacylglycerol (DAG) and Phosphatidic acid (PA) to facilitate intracellular calcium release via the Protein Kinase G (PKG) signalling pathway regulating motility. This implicates the highly conserved Apicomplexan PKG signalling network in Coccidian-specific conoid funcion. The speed with which lipid second messengers are turned over is spectacularly quick and we have tracked lipidomic fluxes of parasite DAG and PA in under 5 seconds. We infer that the conoid protein TgRNG2 has no clear homologue in Plasmodium for the reason that Plasmodium cannot egress prematurely instead undergoing a morphological change at the end of each life cycle. We propose that the conoid has evolved to coordinate egress of the asexual parasitic life stage in the absence of a major morphological change as in Plasmodium. This evolution is centred around a cGMP/PKG based signalling system that uses the P- type-ATPase phospholipid transporter domain of TgGC to sense the state of the external host environment with the conoid as the switch to turn motility on/off. 208

119 - Gain of function of human mechanoreceptor PIEZO1 inhibits replication of Plasmodium falciparum in dehydrated erythrocytes

Kegawa, Yuto (NIH); Waters, Hang; Bezrukov, Ludmila (NIH); Beck, Josh (Iowa State University); Glushakova, Svetlana; Zimmerberg, Joshua (NIH)

Gain of function (GoF) mutations in mechanoreceptor PIEZO1, a non- selective cation channel, were implicated in impaired malaria parasite propagation. Suggested cellular mechanism is based on the disturbed ion homeostasis in erythrocytes due to increased Ca2+ influx through the PIEZO1, activation of Ca2+-dependent K+ channel, efflux of K+ and erythrocyte dehydration. This mimics the parasite’s extracellular environment in sickle cells. We aimed to elucidate the pathophysiology of P. falciparum replication in parasite culture using blood of three African-American donors with the GoF-PIEZO1 mutation E756del. We quantified parasite replication of fluorescently labeled NF54 in donors’ blood and explored parasite egress and invasion using fluorescence microscopy. Blood of E756del carriers replicated parasites with equivalent efficiency compared to replication in non-carriers blood of either African or Caucasian donors. However, some schizonts in carriers’ blood had distorted morphology and defects in both parasite egress and invasion. Isolated dense erythrocytes from E756del carriers showed impaired replication, and defects in both egress and invasion as we previously described for sickle cells. Furthermore, activation of PIEZO1 in dehydrated erythrocytes by the drug Yoda1 exacerbated the observed defects.In summary, we showed that i) dehydrated erythrocytes with GoF PIEZO1 E756del are defective in supporting P. falciparum replication in vitro; ii) dehydrated sickle and GoF-PIEZO1 erythrocytes have a similar negative effect on the parasite cycle of replication. We propose that the similarity in replicative phenotype is due to a similarity in cellular mechanism of a parasite trying to replicate in a now hostile RBC cytoplasm, due to dehydration. Further investigation on the protective mechanism of erythrocyte defects on malaria parasite biology is warranted.

209

120 - Identification of Secretory Pathway Proteins within Toxoplasma gondii using RNA-Seq and CRISPR/Cas9

Kellermeier, Jacob A. (University of Connecticut) Carmeille, Romain; Heaslip, Aoife T. (University of Connecticut)

Toxoplasma gondii is an obligate intracellular parasite that needs to invade and replicate within cells of its host in order to survive. Host cell invasion critically depends on the parasite’s apical secretory organelles known as the micronemes and rhoptries which contain distinct complements of cargo proteins. Proteins destined for each organelle are synthesized in the endoplasmic reticulum (ER) and must traverse a highly polarized endomembrane system, consisting of multiple intermediate compartments (Golgi and the endosome-like compartments) before delivery to their final destination. Movement of proteins between organelles occurs via membrane-bound transport vesicles that bud from donor compartments and fuse with acceptor compartments, although the molecular mechanisms which control this process are not well understood. Our unpublished work has demonstrated that loss of the unconventional myosin, TgMyoF, results in defects in the positioning and integrity of the Golgi and endosome- like compartments. We hypothesize that the severe perturbation of endomembrane trafficking resulting from TgMyoF depletion will result in the aberrant expression of other genes in the secretory pathway, providing a means to identify novel proteins that are part of this important cellular pathway. To this end, we performed RNA-Seq to analyze the transcriptome of parental and TgMyoF-knockdown parasites. We identified over 100 genes that showed consistent differential expression in the absence of TgMyoF. We have begun determining the subcellular localization of 10 high priority candidate proteins by tagging the endogenous genes with a V5 epitope. Elucidating the localization and function of these proteins will provide vital new insight into the mechanisms of endomembrane trafficking in T. gondii.

210

121 - Deciphering the role of CD44 as a host factor for Plasmodium falciparum invasion

Kim, Chi Yong (Stanford University); Baro, Bàrbara (Stanford University); Salinas, Nichole (National Institutes of Health); Doerig, Christian D. (Monash University); Tolia, Niraj H. (National Institutes of Health); Egan, Elizabeth S. (Stanford University)

Erythrocyte invasion by P. falciparum requires interactions between several parasite ligands and host receptors. In previous work, we identified CD44 as a novel host factor necessary for efficient invasion, but its function is unknown. To investigate the role of CD44 during P. falciparum invasion, we generated CD44-null cultured red blood cells (cRBCs) using CRISPR/Cas9 in primary hematopoietic stem cells followed by ex-vivo erythropoiesis. P. falciparum invasion was significantly reduced in CD44-null cRBCs compared to isogenic controls, confirming that CD44 is required for efficient invasion. Recombinant CD44 was sufficient for binding free merozoites, suggesting that CD44 may serve as a receptor. To identify potential parasite ligands for CD44, we performed pull-down experiments followed by mass spectrometry. Two established invasion ligands, erythrocyte binding antigen-175 (EBA-175) and erythrocyte binding antigen-140 (EBA-140), were found to interact directly with CD44. Since EBA-175 and EBA-140 are known to interact with host cell glycophorins A (GYPA) and C (GYPC), respectively, we constructed cRBC mutants in GYPA, GYPC and/or CD44 to assess their relative contributions to erythrocyte binding by recombinant EBA proteins. While deletion of GYPA and GYPC led to reduced erythrocyte binding, deletion of CD44 had no effect, suggesting it may act as a co-receptor. As CD44 promotes intracellular signaling in non-erythroid cells, we sought to determine if EBA-175-induced changes in erythrocyte protein phosphorylation are CD44-dependent. Using phospho-specific antibody microarrays and 2D-DIGE we found evidence for CD44- dependent signaling pathways in cRBCs. In ongoing work we are exploring how these pathways facilitate efficient invasion and whether they can be manipulated using small molecules. Our findings on the molecular function of CD44 may lead to strategies for novel interventions against P. falciparum.

211

122 - The highly-unusual yet evolutionarily conserved fragmented mitochondrial genome of the coccidian, Toxoplasma gondii

Kissinger, Jessica C. (University of Georgia); Namasiviyam, Ranjani; Xiao, Wenyuan; Baptista, Rodrigo P.; Hall, Erica M. (University of Georgia); Troell, Karin (National Veterinary Institute); Doggett, Joseph S. (Oregon Health Sciences University)

The mitochondrial genome sequence, mtDNA, of examined apicomplexan parasites is highly-reduced‚ ~6 kb in length and consists of only three protein-encoding genes and highly-fragmented large and small subunit ribosomal genes. Despite apparent size and gene content conservation‚ the structures of apicomplexan mtDNA identified to date have been variable‚ consisting mostly of linear or linear-concatemer arrangements. Toxoplasma gondii and the related parasites Neospora caninum and Hammondi do not follow this paradigm. Three fundamental differences are observed. First, the mtDNA of Toxoplasma and Neospora are physically constructed of 21 distinct sequence blocks, none of which encodes a full gene. Second, the architecture (order, orientation and number) of the 21 sequence blocks is highly-variable but not random. Third, some arrangements of sequence blocks yield contiguous sequence capable of encoding one or more of the cytochrome genes, cob, coxI and coxIII. The three protein encoding genes are observed to be non-redundantly fragmented onto 13 of the 21 sequence blocks in a situation similar to what has been observed in the . The sequence blocks appear to be joined without the addition or removal of sequence. Nearly all mtDNA molecules sequenced thus far with single-molecule Oxford Nanopore technology are unique with respect to block number, order and orientation. The mtDNA molecules range in length from 2-23 kb. Full cytochrome transcripts are detected. Nanopore sequence analysis of an ENU cob point mutation revealed that homoplasmy is maintained. The tertiary structure of the genome sequence(s) and mechanism of replication remain elusive.

212

123 - Functional annotation of Plasmodium falciparum serine hydrolases through chemoproteomics

Klemba, Michael (Virginia Tech) Elahi, Rubayet; Ray, Keith; Dapper, Christie; Liu, Jiapeng; Helm, Rich (Virginia Tech)

Enzymes of the serine hydrolase superfamily are widely distributed and highly versatile catalysts that employ a nucleophilic serine sidechain. There are at least 42 serine hydrolase superfamily homologs encoded in the genome of the human malaria parasite Plasmodium falciparum and only ~8 of these has been characterized in any detail. We hypothesize that many of the as-yet uncharacterized serine hydrolases are involved in critical metabolic reactions in the parasite. Here, we describe a proteome-wide functional annotation of members of the serine hydrolase superfamily expressed in the asexual erythrocytic stage. A biotinylated, serine hydrolase-directed activity-based probe was employed to affinity purify serine hydrolases from soluble and membrane fractions of saponin-isolated asexual parasites, which were then identified by on-bead trypsin digestion and peptide LC-MS. To annotate serine hydrolases with potential lipolytic activity, competition experiments were conducted with the serine hydrolase-specific pan- lipase inhibitor isopropyldodecylfluorophosphonate. To further define hydrolase specificity, a focused library of subclass-specific serine hydrolase inhibitors was employed in competitive activity-based protein profiling. We will describe how our findings reveal the diversity of serine hydrolase expression in asexual parasites and enable the generation of testable hypotheses on the biological roles of individual serine hydrolases.

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124 - CD8 T cell IFN? responses are modulated by multiple T. gondii effectors.

Kongsomboonvech, Angel K. (UC Merced)

Host survival to Toxoplasma gondii infections is dependent upon protective CD8 T cell responses. These cytotoxic CD8 T cells are stimulated in recognition of cytosolic-derived antigen(s) that are presented on the surface of infected cells by MHC I, and then respond by secreting interferon gamma (IFN?). Since the manipulation of CD8 T cells may influence T. gondii’s ability to achieve chronic infection, we asked whether the parasite modulates activation of this cell type to promote transmission between hosts. To address this, we analyzed naïve CD8 T cell IFN? responses to the endogenous, vacuolar resident antigen, “TGD057”. Naïve TGD057 antigen-specific CD8 T cells were isolated from transnuclear mice and assayed for their ability to secrete IFN? to T. gondii•-infected bone marrow-derived macrophages. Here we report that the CD8 T cell response to TGD057 is independent of T. gondii’s protein export machinery, MYR1 and ASP5. Furthermore, a unique phenotypic pattern emerged where CD8 T cells responded vigorously to all T. gondii strains, except those from clade A, suggesting the presence of a Regulator Of CD8 T cell Response (ROCTR). Genetic mapping implicates multiple ROCTR candidates are at play, including two encoded on chromosomes X and XII, and a third encoded on chromosome VIIb that specifically modulates the early IFN? response of CD8 T cells. Moreover, this antigen-specific CD8 T cell IFN? response is dependent on the host inflammasome pathway, as well as the host’s Immunity-Related GTPases. Altogether, we hypothesize a two-step mechanism in which 1) a ROCTR(s) modulates the host’s ability to acquire vacuolar antigen, and 2) another ROCTR(s) intersects the host’s inflammasome pathway to influence the CD8 T cell IFN? production.

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125 - Functional analysis of putative circumsporozoite protein of Plasmodium vivax

Kumari, Sanju (National Institute of Malaria Research) Dash, Manoswini; Sinha, Abhinav (National Institute of Malaria Research)

Plasmodium falciparum infection results in more severe symptoms whereas Plasmodium vivax is most widely distributed. P. vivax has been neglected from several decades mostly because of lack of continuous in vitro cultivation system. In addition, P. vivax merozoites invade mostly immature reticulocytes, which is hard to procure regularly. These are some of the major obstacles which prevents P. vivax study at cellular and molecular level. Nevertheless, the complex life cycle and dearth of knowledge have hampered vaccine development in P. vivax. Circumsporozoite protein (CSP) is one of the antigen demonstrated as an effective vaccine candidate in P. falciparum, however in P. vivax, it is still under research. Interestingly, P. vivax encodes another protein, called the Putative CSP (puCSP). The genomic region harbouring puCSP had reduced genetic diversity in the flanking neutral loci. It indicates the possibilities of natural selection, as per the hitchhiking model of molecular evolution. Since the puCSP is entirely unexplored the present study is aimed at understanding the function of puCSP using molecular biology approach. A knockout of puCSP orthologue gene will be created using double crossover homologous recombination in Plasmodium berghei. The flanking sequences of puCSP orthologue gene have been cloned in Plasmodium berghei knockout vector. Plasmodium berghei schizonts will be transfected using knockout construct and knockout will be screened using drug pyrimethamine. Further the cellular localization of puCSP protein will be examined using endogenous puCSP antibody in P. vivax blood stages. Knockout study and biochemical characterization of puCSP would elucidate its role in P. vivax life cycle in detail.Plasmodium falciparum infection results in more severe symptoms whereas Plasmodium vivax is most widely distributed. P. vivax has been neglected from several decades mostly because of lack of continuous in vitro cultivation system. In addition, P. vivax merozoites invade mostly immature reticulocytes, which is hard to procure regularly. These are some of the major obstacles which prevents P. vivax study at cellular and molecular level. Nevertheless, the complex life cycle and dearth of knowledge have hampered vaccine development in P. vivax. Circumsporozoite protein (CSP) is one of the antigen demonstrated as an effective vaccine candidate in P. falciparum, however in P. vivax, it is still under research. Interestingly, P. vivax encodes another protein, called the Putative CSP (puCSP). The genomic region harbouring puCSP had reduced genetic diversity in the flanking neutral loci. It indicates the possibilities of natural selection, as per the hitchhiking model of molecular evolution. Since the puCSP is entirely unexplored the present study is aimed at understanding the function of puCSP using molecular biology approach. A knockout of puCSP orthologue gene will be created using double crossover homologous recombination in Plasmodium berghei. The flanking sequences of puCSP orthologue gene have been cloned in Plasmodium berghei knockout vector. Plasmodium berghei schizonts will be transfected using knockout construct and knockout will be screened using drug pyrimethamine. Further the cellular localization of puCSP protein will be examined using endogenous puCSP antibody in P. vivax blood stages. Knockout study and biochemical characterization of puCSP would elucidate its role in P. vivax life cycle in detail. 215

126 - A mutagenesis screen to identify genes involved in T. gondii autophagy during chronic infection

Wang, Fengrong; Carruthers, Vern B. (University of Michigan)

Toxoplasma gondii persists in humans by converting from active- replicating tachyzoites to slow-growing bradyzoites, which are encased in latent intracellular tissue cysts. Bradyzoites are impervious to existing treatments for toxoplasmosis, rendering chronically infected individuals at risk of progressive loss of vision or severe neural or pulmonary disease due to reactivated infection. Previously, our lab discovered that disrupting proteolysis within the parasite lysosome-like VAC/PLV led to accumulation of undigested autophagosomes and subsequently the death of bradyzoites in culture and in mice. Although this suggested an important role for autophagy during chronic infection, the autophagic pathway is poorly understood in T. gondii. It also lacks several core components of autophagy found in model systems. To identify novel autophagic players, we performed a forward genetic chemical mutagenesis screen with type II Pru strain expressing tdTomato-ATG8 as a marker of autophagic flux and bradyzoite specific GFP. After in vitro conversion to bradyzoites, we used FACS to capture the >95th percentile tdTomato-Atg8 of GFP positive bradyzoites. Four rounds of repeated differentiation and sorting resulted in an enriched population of mutant parasites that showed significantly higher tdTomato-ATG8 in bradyzoites than control parasites. Individual mutant clones were isolated and validated for abnormally elevated tdTomato-ATG8 fluorescence. Results from whole genome sequencing and identification of candidate autophagy genes will be presented. Novel players that are unique to protozoa will be prioritized for functional interrogation toward the prospect of impacting chronic Toxoplasma infection.

216

127 - APPL (Apple) and EEF (Eve) : Early endosome protein in liver stage malaria

Lahree, Aparajita (Instituto de Medicina Molecular- Joao Lobo Antunes);

The obligatory development of Plasmodium spp. in hepatocytes is hallmark of mammalian Malaria, which bestows mammoth numbers to the parasite to advance the erythrocytic infection and hitherto remains enshrouded in intriguing host-parasite biology. Metabolically privileged, hepatocytes serve the perfect host where the parasite siphons several biomolecules, interacts with host organelles and hijacks host signalling pathways. Of crucial interest here is the hepatocyte endocytic system that coordinates the endocytosis, sorting, signalling and recycling of key molecules in the cell thereby establishing its unique polarity and metabolism. In this apparently lucrative hub, the exo-erythrocytic form (EEF) of Plasmodium so far was only observed to interact with late endocytic compartments of the host, either for nutrient extraction or amidst host driven autophagy.We report here novelly, the association of the EEF with a host early endosomal protein: ‘adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1’ (APPL1) in hepatocytes. APPL1 is a multi-domain scaffolding protein and Rab5 effector, that senses endocytosed cargo for activation of signalling players such as Akt and AMPK, eventually leading to increased glucose uptake, cytoprotection and increased fatty acid oxidation among others. The EEF accumulates APPL1 in a host Rab5 independent fashion throughout its development via parasite factors. While replication arrest does not inhibit association with APPL1, only replicating and healthy parasites can accumulate ascending levels of this host protein. The curious absence of other classical host early endosome markers begs the question as to the purpose for such an interaction? Whether this is an attempt to amputate the host endocytic sensing mechanisms or to disguise the parasite as a large endosomal hub for redirection of host resources without triggering hostile host responses, remains presently under investigation.

217

128 - Exploring the content of noncoding RNAs, putative regulatory elements, in the protozoan parasite Leishmania

Cruz, Angela K. (University of Sao Paulo); Monteiro-Teles, Natalia M. (University of York); Dias, Leandro; Freitas-Castro, Felipe; Diniz, Juliana A. (University of Sao Paulo); Lorenzon, Lucas (University of Sao Paulo); Magalhaes, Rubens (University of Sao Paulo); Vasconcelos, Elton (University of Leeds); Myler, Peter (bCenter for Infectious Disease Research); Ruy, Patricia (University of Sao Paulo)

Several classes of noncoding RNAs (ncRNAs) have been revealed in recent years. ncRNAs are involved in a variety of regulatory processes in a wide range of organisms. Our laboratory is focused on understanding some of the layers at which regulation of gene expression occurs in Leishmania. Serendipitously, studying a group of short unannotated and polyadenylated transcripts from Leishmania major, we identified and partially characterized one of them, ODD3, arising from the 3’UTR of one of the two copies of a ribosomal protein gene (RPS16). ODD3 is detected as a ~150 nucleotide-long transcript. We extended the search for similar UTR-born to Leishmania donovani exploring the non- polysomal ribonucleoprotein fraction. Subsequently, an in-depth study on the modulation of gene expression across the life cycle stages of Leishmania braziliensis covering coding and noncoding RNAs (ncRNAs) was conducted. Analyses of differentially expressed (DE) genes revealed that most prominent differences were observed between the transcriptomes of insect and mammalian proliferative forms (6,576 genes). A computational pipeline and five ncRNA predictors allowed the identification of 11,372 putative ncRNAs. Most of the DE ncRNAs were found between the transcriptomes of insect and mammalian proliferative stages (38%). Of the DE ncRNAs, 295 were DE in all three stages and displayed a wide range of lengths, chromosomal distributions, and locations; many of them had a distinct expression profile compared to that of their protein-coding neighbors. A small group of the predicted transcripts was confirmed by northern blotting analysis. Knockout (KO) and aptamer tagging of 8 transcripts were obtained using CRISPR/Cas9 editing machinery, and the parasites’ phenotypes are under analysis. Modification of macrophage in vitro infection profile was witnessed for one of the evaluated ncRNA KO, and pulldown assays were conducted to identify ncRNA binding proteins. Among the novel putative ncRNAs disclosed in L. braziliensis there might be regulatory elements and are under investigation.Financial support: FAPESP (2013/50219-9), CNPq and CAPES

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129 - Challenges and Opportunities in Isolating Mitochondrial Ribosomes from Plasmodium falciparum

Dass, Swati (Drexel University College of Medicine); Mulaka, Maruthi; Ling, Liqin (Drexel University); Gutierrez-Vargas, Christina (2Department of Biochemistry and Molecular Biophysics and Department of Biological Sciences, Columbia University); Mather, Michael (Drexel University); Dvorin, Jeffery (Division of Infectious Diseases, Boston Children’s Hospital); Vaidya, Akhil (Drexel University); Frank, Joachim (Columbia University); Ke, Hangjun (Drexel University)

The mitochondrion of malaria parasites (Plasmodium spp.) is essential throughout the parasite’s developmental cycle and has been validated as an antimalarial drug target. In Plasmodium, mitochondrial protein translation is carried out by a mitochondrial ribosome (mitoribosome) that is composed of ribosomal proteins encoded on the nuclear genome and rRNAs encoded on the mitochondrial DNA (mtDNA). Unlike long and continuous rRNA genes present in mtDNA of most eukaryotes, the rRNAs of Plasmodium mitoribosome are highly fragmented and reduced in length of 20-195 bp, which are scrambled on both strands of the mtDNA. The list annotated mitoribosomal proteins in PlasmoDB is currently incomplete, suggesting many mitoribosomal proteins are highly divergent. It has remained unknown how the mitoribosome in Plasmodium works as protein translation machinery. We aim to develop a protocol to isolate mitoribosomes from Plasmodium falciparum to explore the unique compositional and structural features using various tools including mass spectrometry and cryo-electron microscopy. We have endogenously tagged the mitoribosome with a 3HA tag at a conserved mitoribosomal large subunit protein, PfmRPL23 (PF3D7_1239100). Using this parasite line, we have expanded our understanding of the challenges associated with isolating Plasmodium mitoribosomes. Here we discuss those challenges and advances leading to establishing a possible workflow for isolating mitoribosomes from Plasmodium falciparum. This will help us to understand the longstanding question in the field regarding biogenesis of Plasmodium mitoribosomes and will likely provide new avenues for drug interventions targeting the unique mitochondrial protein translation apparatus.

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130 - The microtubule-associated protein PAVE1 regulates the dynamics of the subpellicular microtubule array in Trypanosoma brucei de Graffenried, Christopher L. (Brown University); Sinclair, Amy N.; Sladewski, Thomas (Brown University)

The subpellicular microtubule array of the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly organized microtubule structure. The microtubules in the array are unusually stable and are crosslinked to each other and to the plasma membrane throughout the cell cycle. The array maintains the parasite’s asymmetric cell shape, which is required for its unique motility and pathogenicity. Despite its prominence in the cellular architecture of T. brucei, little is known about how the array is duplicated and subsequently partitioned during cell division. We have recently identified a protein termed PAVE1 that localizes to a subset of the inter-microtubule crosslinks present at the posterior end of the subpellicular array. PAVE1 depletion causes repositioning of the kinetoplast towards the posterior end of the cell. Closer inspection showed that the repositioning was due to a truncation of the posterior portion of the array, producing cells with frayed microtubule ends instead of a tapered posterior. Cells lacking PAVE1 rapidly cease to divide, suggesting that PAVE1 is necessary for the formation of a new posterior end during cell division. Pulse-chase experiments show that PAVE1 is added to the array in an ordered fashion and suggests that it plays an essential role in stabilizing the microtubules at the cell posterior. These results suggest that the subpellicular array is more dynamic than previously thought and that the microtubule crosslinks, which are present throughout the array, may be uniquely configured in different parts of the cell to perform distinct functions. Defining how the array is built, maintained, and segregated in T. brucei has broad implications in understanding the biogenesis of complex microtubule structures throughout biological systems.

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131 - Interspecies interaction in Plasmodium: opportunities and threats

Deora, Nimita (ICMR-National Institute of Malaria Research); Sinha, Abhinav (ICMR-National Institute of Malaria Research, Dwarka sector- 8)

P. falciparum-vivax mixed-infections present many challenges: low density P. vivax infection is difficult to detect by microscopy in P. falciparum-vivax mixed-infections as the former almost always dominates leading to false negative microscopy for P. vivax making molecular diagnosis more reliable. Further, P. falciparum-vivax mixed- infections are often therapeutically regarded as P. falciparum and treated with artemisinin-based combination therapy although P. vivax can adequately be treated with chloroquine. Thirdly, it is not known how individual species behave during P. falciparum-vivax mixed-infections with respect to asexual and sexual growth and response to antimalarials.P. falciparum-vivax mixed-infections might also lead to their co-transmission to mosquitoes with a possibility of sexual interactions in mosquito. Research is reported of understanding the inter-species sexual interactions in rodent malaria but no evidences exist of such studies for P. falciparum-vivax mixed-infections, particularly the mating behavior in mosquitoes.The present study is therefore being done to develop a protocol/method to separate the two species from mixed infection and to explore the possibility of hetero- specific fertilization between P. falciparum and P. vivax.The separation of two species from mixed infections will be attempted through FACS using forward/side scatter properties. This will add a tool to better understand the clinical biology of such mixed infections. Hetero-specific fertilization and zygote/ookinete formation will be examined by separating species specific male and female gametocytes using four different strategies and culturing cross-species opposite-sex gametocytes. This will open up a whole new paradigm as the resulting hybrid parasite will be an altogether new species and will be a possible threat to existing malaria elimination efforts.

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132 - Cryptosporidium parvum exports proteins into the cytoplasm of the epithelial host cell

Dumaine, Jennifer E (University of Pennslyvania); Sateriale, Adam; Gibson, Alexis (University of Pennsylvania); Reddy, Amita (University of Georgia); Striepen, Boris (University of Pennsylvania)

Infection with the protozoan parasite Cryptosporidium is a leading cause of diarrheal disease worldwide. is self-limiting in immunocompetent individuals; however, in the context of malnourishment or immunodeficiency, the disease is attributed to high morbidity and mortality. Upon ingestion of oocysts, Cryptosporidium infects the small intestine, where it drastically remodels the epithelial cell cytoskeleton to establish an intracellular but extracytoplasmic localization. We hypothesize that effector proteins exported to the host cell play critical roles in the initial establishment and maintenance of infection by modulating interaction with the host cell and host immunity. We assembled a prioritized list of candidate effectors based on a variety of functional genomic and population genetic parameters. Using CRISPR/Cas9 driven homologous recombination, we epitope tagged the endogenous loci of candidate proteins, and identified Medle 2 as the first example of a host targeted protein in Cryptosporidium. Medle 2 localizes to the cytoplasm of infected cells both in tissue culture and in the intestinal epithelium of infected mice. The protein is not apparent in sporozoites, but is detectable in the host cell cytoplasm as early as 6 hours post infection, suggesting a rhoptry-independent delivery system assembled by the trophozoite only after invasion. Treatment with Brefeldin A ablates Medle 2 export and Western Blot shows Medle 2 is N terminally processed before being exported to the host cell, highlighting the potential for mechanistic export in Cryptosporidium for the first time. Through cell biological and transcriptional studies, our current work aims to investigate the role of Medle 2 in contributing to ER stress during C. parvum infection as a potential function of this protein.

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133 - “Comparing luciferase expression in transfected stages of Plasmodium falciparum and Plasmodium knowlesi"

Ellis, Angela C. (NIAID); Sa, Juliana M.; Mu, Jianbing (NIAID); Barros, Roberto M. (Universidade Federal de São Paulo); Wellems, Thomas E. (NIAID)

Transfection of malaria parasites provides an important means to investigate the roles of specific genes in parasite biology and pathogenesis. Plasmodium falciparum and Plasmodium knowlesi are two species responsible for extensive malaria morbidity and mortality; unlike other widespread human parasites including Plasmodium vivax, P. falciparum and P. knowlesi can be readily cultivated and transfected under laboratory in vitro conditions. Here we describe the efficiencies of direct-electroporation on both P. falciparum and P. knowlesi using a firefly luciferase reporter construct at different intraerythrocytic stages. Preliminary data show variations in luciferase expression among different P. falciparum stages: signals in these stages are higher from the reporter construct introduced by direct-electroporation of rings than by direct-electroporation of schizonts. In contrast, previous literature has reported that direct-electroporation of mature P. knowlesi schizont stages (“segmenters”) with luciferase plasmids yielded greater expression than did direct-electroporation of late trophozoites or young developing schizont stages. These differences may reflect a relative robustness of P. knowlesi relative to P. falciparum segmenters and merozoites, so that P. knowlesi segmenters with developed merozoites can withstand direct-electroporation better than corresponding stages of P. falciparum. Experiments to directly compare luciferase expression from electroporated late-schizont/segmenter stages of P. falciparum and P. knowlesi are underway.

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134 - Giardia lamblia: Increased expression of long-chain fatty acid elongase alters global ceramide and phosphatidylcholine levels in cysts

Ellis, Cameron (University of Texas El Paso) Enriquez, Vanessa; Pence, Breanna; Almeida, Igor C.; Das, Siddhartha (University of Texas at El Paso)

Giardia lamblia, a waterborne parasite, is responsible for the intestinal infection (giardiasis) in humans and mammals. The infection is transmitted via cysts through contaminated food and water. The cysts undergo excystation in the small intestine to release new and replicative trophozoites. During excystation, cyst membranes (present beneath the cyst wall) are reorganized and become plasma membranes of trophozoites. Because the long-chain fatty acids (LCFAs) participate in membrane-lipid remodeling, we cloned and overexpressed giardial fatty acid elongase-1 gene (gFAELO-1, GL50581_2228) in trophozoites and encysted them in culture. gFAELO-1 was found to be expressed mostly in cysts rather than the trophozoites. Cysts with overexpressed gFAELO-1 appeared to be larger with fluffy and extended (wide) walls as evaluated by a confocal microscopic analysis. Because the morphological changes and cellular differentiation alters the lipid metabolism and global lipid homeostasis in higher eukaryotes, we questioned whether gFAELO-1 could influence the lipid profiles in Giardia cysts. To achieve this, lipids were extracted (from cysts) and analyzed by LC-MS/MS (Q ExactiveTM Mass Spectrometer). We identified 20 different ceramide species of ceramide (major species: was Cer d18:1/16:0-Cer) and observed that their levels were elevated dramatically by gFALEO expression. We also identified 66 species of phosphatidylcholine (major species: PC18:0/20:0-PC) and unlike ceramide, the overall PC pool was drastically reduced by gFAELO. Interestingly, giardial elongase had no significant effects on other cellular lipids. Thus, gFAELO-1 alters the global ceramide and PC levels in cysts, which could be associated with the excystation, signaling, and remodeling of membranes by emerging trophozoites. We propose that gFAELO-1 is an important enzyme and could serve as a potential target for designing anti-giardial therapies. 224

135 - Babesia species parasites can acquire resistance to heparin through the use of alternative invasion pathways

Elsworth, Brendan (Harvard School of Public Health); Keroack, Caroline D.; Tennessen, Jacob A.; Duraisingh, Manoj T. (Harvard T. H. Chan School of Public Health)

Current treatments for Babesia spp. infection are limited by severe side effects or the development of rapid resistance. Heparin and heparin-like molecules (HLM) have been shown to inhibit the ability of many parasites to invade their hosts cells, including multiple species of Babesia, Plasmodium, Toxoplasma, Trypanosoma and Cryptosporidium. In vivo experiments with B. microti and P. falciparum have shown that heparin is effective at reducing the parasite burden, and one HLM (sevuparin) has recently exhibited promising results in a phase II clinical trial against P. falciparum. We found that the IC50 of heparin against B. divergens proliferation is lower than that of P. falciparum, suggesting that HLMs could be promising new therapeutics against Babesia. Previous work has indicated heparin interacts with multiple parasite proteins but the mechanism of action in any parasite has yet to be clearly shown. We found that heparin has no effect on intracellular replication of Babesia spp. and that the effect on proliferation is due to inhibition of invasion of new host cells. However, following ~3 weeks of selection on heparin we were able to generate B. divergens and B. bovis parasites that are highly resistant to heparin inhibition of invasion and proliferation. Heparin resistant B. divergens are cross resistant against some, but not all HLMs tested, suggesting HLMs likely have multiple different mechanisms of invasion inhibition in Babesia. We found significant differences in the IC50 of heparin between B. divergens and B. bovis. B. divergens grown in either human or bovine RBCs showed only minor differences in inhibition, suggesting the differential heparin sensitivity of Babesia spp. is intrinsic to the parasite and the likely target is a parasite protein. To further understand the mechanism of resistance we investigated the sensitivity of the resistant and wild type strains to different enzyme treatments of the host RBCs that remove subsets of host receptors needed for parasite invasion. The majority of treatments produced the same level of inhibition between the two strains, however, the heparin resistant line was more able to invade RBCs treated with trypsin and chymotrypsin. This suggests that the heparin resistant parasites are relying more heavily on an alternative invasion pathway that is less sensitive to heparin and utilizes a receptor that is resistant to trypsin and chymotrypsin. Genome sequencing approaches will facilitate the identification of the parasite target or resistance mechanism of heparin and inform the ability to use HLMs for anti-babesial therapeutics while also providing insights into the biology of Babesia invasion. 225

136 - Arginine enables the adaptive proline response to halofuginone in P. falciparum

Fagbami, Lola (Harvard University)

We have previously identified the cytoplasmic prolyl tRNA synthetase (cPRS) in Plasmodium falciparum as the functional target of the natural product febrifugine and its synthetic analogue halofuginone. HFG treatment triggers a novel mode of drug resistance wherein intracellular proline levels are increased by 30 fold prior to any alteration in the target cPRS gene. This metabolic adaptation, termed the Adaptive Proline Response (APR), persists after drug withdrawal and renders the parasite tolerant to HFG treatment.We investigated the molecular basis of the APR by identifying the source of the increased proline using a multiplexed high-resolution mass-spectrometry (HRMS) based assay. After culturing HFG-induced parasites in the presence of orthogonally labeled proline and proline precursor amino acids (15N proline, 13C-15N arginine, and 13C5 glutamine), we observed predominantly 13C-15N-labeled proline, indicating that arginine is a major contributor.We then probed the essentiality of the arginine biosynthesis of proline by using CRISPR/Cas9 technology to generate independent parasites lines that lack ornithine d-aminotransferase (PF3D7_ 608800), a key enzyme in this metabolic pathway. Functional analysis of the ornithine d-aminotransferase (Dd2 ?OAT) knockout parasites revealed a complete absence of the arginine derived 13C- 15N-labeled proline. Dd2 ?OAT parasites exposed to HFG remain sensitive to drug and do not increase their intracellular proline levels. This observation that ornithine d-aminotransferase knockout parasites do not activate the APR demonstrates that arginine metabolism is required for the adaptive proline response to halofuginone.

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137 - Generation of in vitro Leishmania hybrids

Ferreira, Tiago R. (National Institute of Allergy and Infectious Diseases, National Institutes of Health); Louradour, Isabelle; Sacks, David (National Institute of Allergy and Infectious Diseases, National Institutes of Health)

Leishmania parasites have been shown to mate as promastigotes in the sand fly vector midgut. Using an experimental model of sand fly co- infection, we have generated large numbers of intra- and interspecific hybrids for which whole genome sequencing has revealed meiosis-like sexual recombination. Sexual reproduction in these parasites appears to be restricted to the sand fly midgut, and it has so far not been possible to produce genetic crosses in vitro. We report here a protocol that permits recovery of Leishmania hybrids generated in culture without vector-derived components. Two parental L. tropica transfectants resistant either to geneticin (Neo) or hygromycin B (Hyg), expressing respectively, GFP or RFP integrated into the chromosomal rRNA locus, were co-cultured in vitro. Crosses between these two L. tropica strains generated double-drug-resistant and double-fluorescent hybrids, though at a low frequency of the co-culture wells. PCR and heterozygous SNP analyses of 24 different intraspecies hybrids obtained from four independent experiments confirmed heterozygosity on four different chromosomes, suggesting that they are full genomic hybrids. In addition, interspecies L. major x L. tropica hybrids were obtained in vitro showing that this mechanism is not restricted to a single species. In conclusion, the in vitro hybrids may facilitate experimental study of the mating biology of Leishmania and be exploited for forward genetic analysis.

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138 - Determining how Toxoplasma gondii bradyzoites co-opt their host cell

Ferrel, Abel; Branon, Tess C. (Dept. of Molecular and Cell Biology, University of California, Berkeley); Ting, Alice Y (Departments of Genetics, Biology, & by courtesy, Chemistry, Stanford University); Boothroyd, John C. (Department of Microbiology and Immunology, Stanford University)

Toxoplasma gondii is an obligate, intracellular parasite capable of causing severe infections of warm-blooded animals. During asexual reproduction in a host, this apicomplexan exists as a tachyzoite in the acute stage of infection and a bradyzoite during the chronic stage. Infection of a cell produces a unique niche for the parasite named the parasitophorous vacuole (PV) initially composed of host plasma membrane invaginated during invasion. Others have recently shown how tachyzoites are able to co-opt the host cell by translocating parasite “effectors” across the PV membrane (PVM) into the host cytosol. Our lab performed a genetic screen and identified Myc regulation 1 (MYR1), a protein found at the PVM, as necessary for effector protein translocation and consequent upregulation of host c- Myc expression. Although studies have demonstrated tachyzoite-host interactions, how bradyzoites alter host functions and if this is MYR1- dependent has not been carefully examined. I am addressing this gap by identifying proteins expressed during the bradyzoite stage which make it out of the PV and into the host cell. To do this, I have engineered a host cell line expressing a promiscuous biotin ligase, TurboID, localized to the host nucleus and will infect with wild type or ?myr1 parasites. This will allow for the biotinylation of all proteins, host and parasite, found within the nuclear compartment. Biotinylated proteins will be purified by affinity purification and submitted for mass spectrometry. Together, this will help identify proteins secreted by bradyzoites and allow us to begin understanding how an infected host cell is affected by T. gondii in the chronic stage of infection.

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139 - m6A methylation of the polyA-tail regulates VSG transcript stability in trypanosomes

Figueiredo, Luisa M. (Instituto de Medicina Molecular, University of Lisbon); Viegas, Idalio; Rodrigues, Joao; Macedo, Juan P. (Instituto de Medicina Molecular, University of Lisbon); Aresta-Branco, Francisco (German Cancer Research Center (DKFZ)); Jaffrey, Samie (Weill Medical College of Cornell University)

Trypanosoma brucei is covered by a dense coatof variant surface glycoproteins (VSG). The VSG transcript is very abundant, stable and has a half-life of around 4.5hr. The molecular basis for the stability of VSG mRNA is unknown. In other eukaryotes, N6-methyladenosine (m6A) has recently emerged as an RNA modification involved in post- transcriptional gene regulation. In this work, we hypothesized that m6A could be involved in stabilizing VSG mRNA. Using tandem-mass spectrometry, we first confirmed the presence of m6A in mRNA harvested from bloodstream and procyclic forms of trypanosomes, representing around 0,06% of adenosines, similar to other eukaryotes. Immunoblotting analysis revealed that m6A is enriched in the VSG transcript. RNAseH mapping showed that, unlike any other eukaryote, m6A is present in the polyA tail. Given that polyA tails protect transcripts form degradation, we hypothesized that m6A may be the reason why VSG transcripts are unusually stable and abundant. Using multiple assays to follow VSG degradation, we showed that polyA tail is deadenylated and VSG mRNA is degraded only when m6A has been removed from the polyA tail, strongly indicating that m6A protects VSG mRNA from degradation and thus contributes to its stability. We are currently characterizing the cis-acting elements that determine the specificity of VSG polyA methylation. This work identifies RNA modifications as a new mechanism to control the regulation of protein-coding genes in African trypanosomes.

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140 - Functional characterisation of the chromatin remodeling enzyme Snf2L in Plasmodium falciparum

Watzlowik, Maria (University of Regensburg)

The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific positions of nucleosomes that restrict the accessibility of DNA elements for sequence specific binding of regulatory factors. Cells have evolved ATP-dependent chromatin remodeling enzymes that can position nucleosomes, move and evict histone octamers from DNA and incorporate histone variants at specific genomic locations. These enzymes are essential regulators of all DNA dependent processes. The mechanisms of chromatin dynamics in Plasmodium falciparum (Pf)gene regulation and the functions of these enzymes are currently not known. A previous study showed that the biochemical properties of the histones reflect the cellular chromatin architecture of Pf,exhibiting reduced nucleosomal stability, shorter nucleosome repeat lengths and most importantly lacking the capability of sequence dependent nucleosome positioning. Now we address the function of the chromatin remodeling enzymes in Pf, which are highly divergent insequence when compared to their human counterparts. We initiated thefunctional characterization of PfSnf2L, an essential protein in Pfand a single member of the ISWI-family proteins. Interestingly, the enzyme shows distinct remodeling activity and regulation features when compared to the human homologue. The enzyme shows binding to DNA, nucleosomes and chromatin with a preference for canonical Pf nucleosomes. We could also substantiate ATP- dependent nucleosome mobilization activity, but PfSnf2L lacks the ISWI-characteristic dependency on the histone H4-tail. Intact nucleosomes are required for movement but not for the stimulation of the ATPase activity.The striking differences in the sequence and mechanism of the human and Pf remodeler suggest that epigenetic regulation differs from common rules, potentially presenting novel targets for antimalarial drugs.

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141 - Revising the karyotype of Toxoplasma gondii and synteny with Neospora caninum using single molecule sequencing

Xia, Jing (University of Pittsburgh); Bainbridge, Rachel (University of Pittsburgh); Venkat, Aarthi; Ay, Ferhat (La Jolla Institute for Allergy & Immunology); LeRoch, Karine (University of California Riverside); Boyle, Jon (University of Pittsburgh)

The Toxoplasma and Neospora caninum genomes still have multiple gaps due to repetitive and putatively unclonable sequences. Here we report direct, single molecule sequencing and de novo assembly of genomes for multiple Toxoplasma strains and Neospora caninum (Liverpool) using the MinION nanopore sequencer. We obtained between 200,000 and 650,000 reads per strain (up to ~7.4 Gb of sequence) and more than 90% of the reads could be mapped to the reference genome. The resulting assemblies improved T. gondii and N. caninum contiguity (N50 of ~6.6Mb) and increased the overall assembled sequence by ~2 Mb. For all de novo assemblies multiple complete chromosomes were fully assembled as evidenced by clear telomeric repeats on the end of each contig. Interestingly, for all of the Toxoplasma gondii strains that we sequenced (RH, CTG, II×III F1 progeny clones CL13, S27, S21, and S26), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome. This was due to a repeatable and consistent fusion of chromosomes VIIb and VIII. These data were further validated by mapping existing T. gondii ME49 Hi-C data (Bunnik et al., PNAS 116:3183-3192) to our assembly. Changes in genome architecture that occur during sexual recombination will also be discussed. Finally, when we compared the T. gondii and N. caninum assemblies we found some chromosomes that had nearly 100% synteny while others lacked clear synteny, suggesting that multiple large scale translocation events have occurred in T. gondii and N. caninum since their most recent common ancestry.

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142 - Characterizing insulinase-like proteases in Cryptosporidium parvum

Xu, Rui (Washington University in St. Louis; East China University of Science and Technology); Xiao, Lihua (South China Agricultural University); Feng, Yaoyu (South China Agricultural University; East China University of Science and Technology); Sibley, L. Daivd (Washington University in St. Louis)

Cryptosporidium spp. are apicomplexan parasites that are an important cause of diarrhea in humans and animals. Insulinase-like proteases (INS) belonging to the superfamily of M16 metalloproteases are one of the largest protein families within the small proteome of Cryptosporidium. Recent comparative genomic analysis indicated that INS proteases are common in Cryptosporidium spp., with 12~22 genes being identified in different species. Cryptosporidium parvum has 22 INS proteins, 13 of which are expressed at high levels during early stage of the life cycle. The INS proteins are comprised of both active sites and domains which lack the canonical residues required for catalysis. A number of them are also predicted to have signal peptides, suggesting that they may be secreted. In this study, we focused on INS1 (cgd1_1680), INS3 (cgd2_920), and INS4 (cgd2_930), which contain a zinc-coordinating motif HxxEH as well as homology to inactive catalytic domains found in insulin-like proteases. INS1 also contains a putative signal peptide suggesting it may be secreted. We tagged these proteases by inserting an epitope tag at the C-terminus of the protein using CRISPR/Cas9 genome editing. Transgenic parasites were expanded using an air-liquid transwell system for epithelial cell culture that provides continuous culture system to culture transgenic parasites. Based in immunofluorescence staining, INS1 protein was expressed in a punctate pattern throughout parasite, while INS3 and INS4 were associated with the nucleus. We are currently attempting to delete the INS encoding genes or use conditional degradation systems in C. parvum to elucidate their biological functions.

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Poster Session B

Tuesday, September 17, 2019

7:00 pm – 9:00 pm

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143 - An ER CREC family protein regulates the egress proteolytic cascade in malaria parasites Fierro, Manuel A. (The University of Georgia); Asady, Beejan; Brooks, Carrie F.; Cobb, David W.; Villegas, Alejandra; Moreno, Silvia N. J.; Muralidharan, Vasant (The University of Georgia) For Abstract See Session VI, 38t

144 - An unconventional myosin, TgMyoF is an organizer of the endosome-like compartments in Toxoplasma gondii Heaslip, Aoife (University of Connecticut); Carmeille, Romain; Schiano, Irio (University of Connecticut) For Abstract See Session VI, 39t

145 - Transcriptional modification of host cells harbouring Toxoplasma gondii bradyzoites prevents IFNgamma-mediated cell death Seizova, Simona For Abstract See Session VII, 43t

146 - EXP1 is required for organization of the Plasmodium falciparum parasitophorous vacuole membrane Nessel, Timothy (Iowa State University); Beck, John M. (Iowa State University); Goldberg, Daniel E. (Washington University School of Medicine); Beck, Josh R. (Iowa State University) For Abstract See Session VII, 44t

147 - Systematic identification of diversity-encompassing variants of a new malaria vaccine target Niare, Karamoko1,2,3, Timo Chege2, James Tuju2, Bourema Kouriba3, Gordon Awandare1, Julian Rayner4, Faith Osier2. 1 West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, 2Kenya Medical Research Institute – Wellcome Trust Research Programme, Kilifi, Kenya, 3Malaria Research and Training Center 4Wellcome Sanger Institute For Abstract See Session VII, 45t

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148 - Whole genome sequence comparative analyses of North African Leishmania infantum may reveal genetic and molecular determinants of the parasite tissue tropism. Chakroun, Ahmed S. (Institut Pasteur de Tunis, Université Tunis El Manar); Guerbouj, Souheila (Institut Pasteur de Tunis, Université Tunis El Manar, Tunis - Belvédère); Leprohon, Philippe (Centre de Recherche du CHU de Québec, Université Laval); Harigua, Emna (Institut Pasteur de Tunis, Université Tunis El Manar, Tunis - Belvédère); Fathallah Mili, Akila (Université de Sousse); Ouellette, Marc (Centre de Recherche du CHU de Québec, Université Laval); Guizani, Ikram (Institut Pasteur de Tunis, Université Tunis El Manar, Tunis - Belvédère) For Abstract See Session VIII, 50t

149 - A novel Babesia bovis secreted protein responsible for binding of infected erythrocyte to endothelial cells Hakimi, Hassan (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University) Sakaguchi, Miako (Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University); Yamagishi, Junya (Research Center for Zoonosis Control, Hokkaido University); Yahata, Kazuhide; Kaneko, Osamu; Asada, Masahito (Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University) For Abstract See Session IX, 54t

150 - Targeting Theileria effector proteins at the schizont membrane responsible for host cell transformation Brühlmann, Francis (University of Bern); Woods, Kerry; Olias, Philipp (University of Bern) For Abstract See Session IX, 55t

151 - Epigenetic regulation of gametocytogenesis in P. berghei; the roles of HAT1, HDA1, and differential histone PTMs. Power, B. Joanne J. (Pennsylvania State University); Burchmore, Richard (Glasgow Polyomics); Waters, Andrew P. (Wellcome Centre for Integrative Parasitology) For Abstract See Session IX, 56t

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152 - Copper metabolism in Naegleria spp.

Ženíšková, Katerina (Department of Parasitology, Faculty of Science, Charles University in Prague); Grechnikova, Maria; Mach, Jan; Šuták, Róbert (Department of Parasitology, Faculty of Science, Charles University in Prague)

Copper is an essential trace element for all organisms. Its presence is necessary in key biochemical reactions; as a cofactor of complex IV it conveys electron transfer in the respiratory chain, it has a function in detoxification reactions of superoxide radicals and it also participates in import of iron ions into the cell. On the other hand, copper ions also take part in reactions leading to generation of reactive oxygen species that damages the structure of lipids, proteins and DNA. Therefore, the homeostasis of this metal in the cell should be strictly regulated. Many studies also describe the importance of copper ions as a virulence factor of pathogens, where these ions induce the immune response of the host organism.Our project is focused on the copper uptake, detoxification and intracellular distribution within the free-living Naegleria gruberi and its related brain-eating amoeba Naegleria fowleri. N. gruberi is considered as a model organism for studying the pathogenic N. fowleri and our experiments including proteomic analysis and measuring the activity of respiration chain confirmed that N. gruberi is able to change the energy metabolism in relation to copper availability. Furthermore, the dependence of uptake of iron on copper was confirmed. We have also identified several N. fowleri copper transporters and proposed a role of a copper exporting ATPase in copper detoxification pathway. Taken together, we are uncovering elaborate pathways of copper homeostasis utilized by Naegleria amoebas.

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153 - High-throughput functionalization of the Toxoplasma gondii proteome

Smith, Tyler (Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology); Lopez-Perez, Gabriella (University of Puerto Rico at Mayaguez); Lourido, Sebastian (Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Biology)

Apicomplexans are some of nature’s most widespread parasites, including the causative agents of toxoplasmosis (Toxoplasma gondii), cryptosporidiosis (Cryptosporidium spp.), and malaria (Plasmodium spp.). Due to their divergence from model eukaryotes, apicomplexans have evolved an array of phylum-specific adaptations; however, most apicomplexan proteins remain uncharacterized both in their localization and function. The recent adaptation of CRISPR/Cas9 technology to T. gondii enabled the high-throughput characterization of the parasite genome. Such screens quantified the fitness cost associated with the disruption of each gene. These screens lack rapid temporal control and require follow-up studies to determine protein function and localization. To address these challenges, we developed a high-throughput (HiT) CRISPR-mediated tagging vector to rapidly functionalize the C-termini of target proteins with a payload. Utilizing the HiT vector, we tagged a library of genes with the mini auxin-inducible degron (mAID) linked to a fluorophore and epitope tag. This enabled rapid and reversible knock- down of the targeted proteins. We assayed the function of each tagged mutant using pooled screens in the presence or absence of auxin. We also identified novel localizations for several proteins by subcloning the tagged populations. This system extends the applications of genome- wide screens into complex cellular phenotypes, providing a new platform for the dissection of apicomplexan cell biology.

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154 - Identification of the F-actin capping protein in the regulation of morphological transition of Trichomonas vaginalis

Hsu, Hong Ming (The Division of Tropical Medicine and Parasitology, College of Medicine, National Taiwan University); Wang, Kai-Hsuan (The Division of Tropical Medicine and Parasitology, College of Medicine, National Taiwan University)

Trichomonas vaginalis colonized on the urogenital tract of human is the causative agent of world-spread trichomoniasis. In this parasite, the association of actin-based machinery in morphological transformation upon the process of adhesion and migration on host cells had been reported decades ago, but remains unclear molecular mechanism to date. In this study, the expression of an F-actin capping protein (TvFACP) and filamentous actin (F-actin) were detected to the level higher in those high-adherent clinical isolates and regulated by environmental iron in a dose-dependent manner. Also, iron simultaneously diminished the polymerization of F-actin and morphology transformation in this parasite progressing adhesion. By the transgenic system, TvFACP overexpression substantially impaired the efficiency of F-actin assembly, and also decreased the morphological transformation population in the high-adherent isolates. On the other hand, TvFACP directly interacted with actin to form the protein complexes when examined by GST pull-down assay and immunoprecipitation. All data together, we speculated that TvFACP may act as an attenuator in the dynamics of F-actin assembly in T. vaginalis. Iron presumably regulated expression of TvFACP with the activity binding to actin filaments, interfering with the efficiency of F- actin reorganization and morphological transformation, eventually leading to the reduction of parasite adhesion to host cells. The functional study of F-actin capping protein is a hot issue in the research of high eukaryotes, and our findings may bring new ideas in the investigation of actin-mediated pathogenesis in T. vaginalis.

240

155 - From chaos comes order: how an intrinsically disordered Toxoplasma effector subverts interferon signaling

HUANG, ZHOU (Washington University in St. Louis); Liu, Hejun; Amarasinghe, Gaya K.; Sibley, L. David (Washington University in St. Louis)

As a widespread and successful apicomplexan parasite, Toxoplasma gondii can infect most warm-blooded hosts. One pivotal reason for the success of Toxoplasma infection is its ability to subvert host signaling and escape from innate immune responses. Among the secreted effectors that alter host function are dense granule (GRA) proteins that are secreted outside the parasitophorous vacuole and which often traffic to the host nucleus. As a frontline in host defense, the IFN-g stimulated STAT1 pathway is repressed by various pathogens using different mechanisms. Previous work from our lab identified the intrinsically disordered dense granule effector TgIST (T. gondii inhibitor of STAT1 transcription (TgIST), which is responsible for blocking STAT1 signaling by all three major T. gondii clonal lineages. TgIST blocks type I and II interferon signaling both in rodent and human cells by directly binding to STAT1 in the host nucleus and recruiting a repressive complex called nucleosome remodeling and deacetylase (Mi-2/NuRD), which is thought to result in transcriptional repression. To reveal the molecular mechanism of TgIST-mediated transcription repression, the regions responsible for binding to STAT1 and Mi- 2/NuRD were defined by immunoprecipitation using Ty-tagged truncated versions of TgIST expressed in HEK293 cells and by limited proteolysis and MS/MS analysis. Three STAT1 binding and one Mi- 2/NuRD binding domains were validated by co-purification of recombinant proteins in E. coli. Furthermore, molecular docking and crystallography successfully revealed the structural basis for the interaction between TgIST and STAT1-phosphorylated homodimer. Defining the structural basis for TgIST-STAT1 binding may enable future identification of inhibitors that block binding and reverse the repression of IFN-g signaling, thus restoring host immunity.

241

156 - Characterizing the phylum-specific adaptations of the apicomplexan ATP synthase

Huet, Diego (University of Georgia and Whitehead Institute for Biomedical Research) Lourido, Sebastian (Whitehead Institute for Biomedical Research, MIT Department of Biology)

Apicomplexans are able to survive in a wide range of cell types, demonstrating their metabolic plasticity and ability to access nutrients from diverse and changing environments. At the mitochondrial inner membrane, the ATP synthase plays a key role in this metabolic plasticity. Our work has previously identified ICAP2 as an essential component of the ATP synthase complex, partially homologous to the b subunit of the mammalian ATP synthase stator. Interestingly, the protein also harbors domains absent from stator subunits in other model organisms. ICAP2 possesses a C-terminal extension and a putative EF-hand–like domain conserved only in apicomplexans, suggesting that calcium may regulate its function. To understand more about the role of these apicomplexan-specific domains, we generated three ICAP2 conditional knockdown strains expressing different tagged versions of the protein to functionally test for their ability to complement the loss of the wildtype allele. The first strain expresses a full-length version of ICAP2 while the second expresses a protein lacking the C- terminal extension. The third strain possess a mutated version of the putative calcium-binding domain of ICAP2, where the residues predicted to coordinate calcium have been mutated. Preliminary observations suggest that only the full-length version of ICAP2 is able to complement the loss of the endogenous protein, suggesting that both the C-terminal extension and calcium-binding domain of ICAP2 are required for parasite fitness. Further characterization of the domain is currently undergoing, including its recombinant expression and analysis of the ATP synthase complex composition in the presence or absence of calcium. Studies of the ATP synthase in other organisms have uncovered links to mitochondrial morphology and calcium signaling. The characterization of the apicomplexan-specific domains of ICAP2 will likely uncover similar links to diverse cellular processes in these parasites.

242

157 - Towards characterization of protein export machinery and effectors throughout Plasmodium development

Hussain, Tahir (Iowa State University); Beck, John M. (Iowa state university); Beck, Josh R. (Iowa State University)

In the vertebrate stage of infection, obligate intracellular malaria parasites undergo development in both hepatocyte and erythrocyte host cells. Successful establishment of a niche for replication in these remarkably diverse environments is achieved through delivery of an arsenal of exported effector proteins into the host cell. This process is best characterized in the blood stage where hundreds of effector proteins are transported across the parasite vacuole (PV) membrane and into the host cell by the Plasmodium translocon of exported proteins (PTEX), facilitating key changes to erythrocyte permeability, rigidity and cytoadherence. The core PTEX machinery in the blood stage is comprised of the HSP101 unfoldase which is coupled to the EXP2 PV pore through a flange-like structure formed by PTEX150. In contrast, few exported proteins have been identified in the liver stage and the nature of the vacuolar export machinery remains largely unknown. Intriguingly, HSP101 is not expressed during much of liver stage development, suggesting key mechanistic differences for protein export into the hepatocyte. In an effort to identify novel exported liver stage effectors and machinery, we targeted the BioID2 proximity- labeling system to the Plasmodium berghei PV in multiple configurations, including fusion to EXP2. This facilitated comparative PV proteomic analysis between the blood and liver stages, identifying known exported and PV components as well as putative novel proteins in each stage. Ongoing work to characterize the function of PTEX components in the liver stage and identify novel effectors and PV machinery will be discussed.

243

158 - Coactosin phosphorylation controls Entamoeba histolytica cell membrane protrusions and cell motility

Huston, Christopher D. (University of Vermont); Teixeira, Jose E.; Hasan, Muhammad M.; Lam, Ying Wai (University of Vermont)

Entamoeba histolytica’s ability to balance adherence and cell migration presumably affects its ability to cause invasive colitis. We previously showed that silencing expression of the plasma membrane metalloprotease EhMSP-1 decreases amoebic motility while increasing adherence. We now report results from a phosphoproteomics study done to clarify the mechanism of these phenotypic changes. Comparison of phosphoproteins in wild-type and EhMSP-1-silenced trophozoites revealed altered phosphorylation of several cytoskeletal proteins, specifically increased EhCoactosin phosphorylation at Ser147 and decreased unconventional myosin-IB phosphorylation at Ser757.Follow up focused on EhCoactosin, which is reportedly required for amoebic erythrophagocytosis. Coactosin is a member of the actin depolymerizing factor-homology (ADF-H) protein family; it binds to both monomeric and polymerized actin, and stabilizes F-actin filaments. Coactosin is not known to be phosphoregulated, but the related ADF-H family member Cofilin is phosphoregulated in mammalian cells where dephosphorylation alters its actin severing and depolymerizing activity. To investigate EhCoactosin phosphoregulation, we compared control parasites to two EhCoactosin mutant strains: Coac-D (Ser147 replaced by aspartate (S147D))(phosphomimetic); and Coac-A (Ser147 replaced by alanine (S147A))(non- phosphomimetic).Coac-A mutants presented a lower number of and more coordinated cell membrane protrusions compared to Coac-D when quantified using time-lapse microscopy. Coac-A mutants also migrated longer distances in Matrigel. Both EhCoactosin mutant proteins colocalized with actin, and neither mutation significantly altered adherence or phagocytosis. Based on the observed differences in pseudopod formation and cell migration, we conclude that EhCoactosin is phosphoregulated, which we believe represents the first example of Coactosin phosphoregulation. Studies are underway to test the activity and actin binding capacity of recombinant mutant EhCoactosin proteins, and to identify the phosphatases and kinases that act on EhCoactosin.

244

159 - Fast-killing inhibitors of the malaria parasite, Plasmodium falciparum, that disrupt cytosolic and digestive vacuole pH

Imlay, Leah S. (UT Southwestern Medical Center); White, John (University of Washington); Hollibaugh, Ryan (UT Southwestern Medical Center); Palmer, Michael J. (Medicines for Malaria Venture); Rathod, Pradipsinh K. (University of Washington); De Brabander, Jef K.; Phillips, Margaret A. (UT Southwestern Medical Center)

As resistance to current therapies develops and spreads, new antimalarial treatments, especially those with novel targets, are urgently required. We have identified two compounds, DSM453/459, with potent antimalarial activity (10-20 nM EC50s) and a rate of kill approaching that of artemisinin and chloroquine. Generation of resistant parasites has proven difficult, suggesting that development of clinical resistance may also be very slow. Although the compounds were developed as part of a series of dihydroorotate dehydrogenase (PfDHODH) inhibitors, they are poor PfDHODH inhibitors. Additionally, their toxicity is not rescued by expression of yeast DHODH, as is the case for other PfDHODH inhibitors, suggesting that DSM453/459 act against another target. We describe efforts to identify their target(s) in order to facilitate development of improved compounds or allow screening for additional inhibitors. We find that these compounds cause dysregulation of pH maintenance systems, including acidification of the parasite cytosol and alkalization of the digestive vacuole, a lysosome-like compartment that is normally maintained at an acidic pH. Because many ion homeostasis mechanisms involve exchange or cotransport between H+ and other ions, it is likely that regulation of non- H+ ions may also be affected. We are also in the process of using chemoproteomic (click chemistry/pulldown) approaches to identify the protein target of these compounds.

245

160 - A role for the conserved var gene var2csa in coordinating antigenic variation by malaria parasites

Zhang, Xu (Weill Cornell Medical College); Deitsch, Kirk (Weill Cornell Medical College)

To maintain chronic infections, malaria parasites must continuously alter the proteins exposed to the host’s immune system, a process called antigenic variation. For Plasmodium falciparum, the major antigen on the surface of infected red blood cells is P. falciparum erythrocyte membrane protein-1 (PfEMP1), a protein encoded by the multi-copy var gene family. With about 60 family members, var genes are transcribed in a mutually exclusive manner and transcriptional switching between genes results in antigenic variation. var gene activation and silencing are regulated epigenetically, however the mechanisms that coordinate switching between active genes remain unknown. Previous mathematical modeling of var gene switching patterns, both from experimental infections and from long-term cultures, suggested that expression switching is not random, but rather displays properties of a structured network. Moreover, these models predicted the existence of a gene that serves as a “sink node” or “switching intermediate” within the network to coordinate switching events, however the nature of the putative node was not defined. Previously, when we destabilized epigenetic regulation of the var gene family using either genetic or chemical methods, we selectively activated a uniquely conserved gene called var2csa, suggesting that this gene may play a central role in coordinating switching, consistent with the previously predicted sink node. In this study, we investigated the hypothesis that var2csa plays an essential role in coordinating var gene expression by serving as a “switching intermediate” within the var gene network. Using CRISPR-CAS9 mediated genome editing, we modified the var2csa locus through promoter deletion, promoter replacement, or loss of the entire locus through truncation of the subtelomeric region containing the gene. These modifications had profound effects on var gene switching, leading to parasites that either cannot switch or that switch at an extremely high rate. These data define a novel function for the conserved var2csa locus in coordinating transcriptional switching events within the var gene family and thereby mediating antigenic variation.

246

161 - EuPathDB resources: tools for analysis, integration and discovery

Harb, Omar S.; Brunk, Brian (University of Pennsylvania); Warrenfeltz, Susanne; Kissinger, Jessica (University of Georgia); Roos, David (University of Pennsylvania)

The Eukaryotic Pathogen Databases (EuPathDB, http://eupathdb.org) provide free online access to data mining tools across a wide variety of datatypes. An advanced search strategy system enables interrogation of both preloaded datasets and user private datasets. Results of search strategies can be visualized or analyzed further (e.g. GO, metabolic pathway enrichment, etc…). In addition, data can be analyzed privately using a Galaxy workspace where all EuPathDB genomes have been preloaded. EuPathDB supports over 230 organisms within Amoebazoa, Apicomplexa, , Diplomadida, Trichomonadida, Kinetoplastida, , fungi and host organisms (e.g. mouse, human, etc…). For these organisms, EuPathDB integrates a wide range of data including genome sequence and annotation, transcriptomics, proteomics, phenomics, epigenomics, metabolomics, whole genome population and host-pathogen interaction data. Data are analyzed using standard workflows and an in-house genome analysis pipeline generates data including domain predictions, orthology profiles across all genomes and GO term associations. Search results can be combined into strategies that easily merge evidence from diverse data types and across organisms. Easily accessible tools enhance the search strategy system and include dynamic data visualization, comparative genome analysis, population genetics tools, and functional or pathway enrichment. This comprehensive resource empowers the scientific community to mine data and develop hypothesis driven research. Future work will focus on providing additional tools for private data analyses, data sharing and the expected merger with VectorBase (http://vectorbase.org). To learn more about what is new, ask us questions, contribute user comments or to provide suggestions visit our booth during all poster sessions.***presented on behalf of the entire EuPathDB team.

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162 - A combination of reverse genetics and live microscopy shows distinctive roles for the P. knowlesi Duffy binding protein and normocyte binding protein families during erythrocyte invasion.

Hart, Melissa (LSHTM); Mohring, Franziska; Charleston, James (LSHTM); Hall, Joanna; Almond, Neil (NIBSC); Saibil, Helen (Birkbeck, University of London); Moon, Robert (LSHTM)

The adaptation of P. knowlesi to continuous culture in human red blood cells has provided exciting opportunities to investigate erythrocyte invasion biology, both to compare with features identified within the traditionally studied P. falciparum, as well as features shared with the more closely related P. vivax. Two major protein families, the erythrocyte binding like proteins (EBLs/EBAs) and the reticulocyte binding like proteins (RBLs/RHs) have been studied extensively in P. falciparum and are hypothesized to have overlapping, but critical roles during the invasion process. P. knowlesi includes a much smaller repertoire of these two families, of which one EBL, Duffy binding protein alpha (DBPa) and one RBL, normocyte binding protein Xa, (NBPXa) are both essential for invasion of human red blood cells. Importantly, like P. vivax, P. knowlesi relies upon the interaction of DBPa with its RBC receptor, Duffy antigen receptor for Chemokines (DARC). By taking advantage of the unique biological features of P. knowlesi, which has merozoites double the size of P. falciparum that are longer lived, as well as newly adapted techniques for CRISPR-Cas9 genome editing, we have undertaken a comprehensive genetic analysis of the P. knowlesi EBL/RBL families and combined this with live microscopic analysis to unravel their roles during invasion. Using both conventional gene knockouts and a conditional DiCre approach in simultaneously tagged lines, we have demonstrated distinctive roles between both families, and show that the role of NBPXa precedes that of DBPa. Finally, we have demonstrated that P. knowlesi merozoites are able to undergo productive gliding motility prior to invasion, both on glass and RBC surfaces. This has allowed us to study motility as a distinct process to invasion for the first time in the merozoite, and thus begin to unravel additional steps leading to invasion.

248

163 - Probing Cryptosporidium sexual differentiation with small molecules

Hasan, Md Mahmudul (University of Vermont) Jumani, Rajiv S. (University of Vermont); Klopfer, Connor; Donnelly, Liam; Franco, Sebastian (University of Vermont); Love, Melissa S.; Mcnamara, Case W. (Calibr); Huston, Christopher D. (University of Vermont)

Apicomplexan Cryptosporidium parasites undergo both asexual and sexual development in a single host. Therefore, unlike Plasmodium species, inhibiting the sexual phase could be a strategy to treat cryptosporidiosis. Cryptosporidium infects the human colorectal carcinoma cell line HCT-8 in vitro. However, the culture population collapses upon parasite gamete formation (~72 hours post infection), likely due to inefficient mating. Inhibiting sexual differentiation could therefore also be a way of keeping the parasites in the asexual replication cycle to establish a simple continuous culture system. As a means to testing this hypothesis, we sought to identify small-molecule inhibitors of sexual differentiation. The parasite DNA Meiotic Recombinase 1 (DMC1) protein is exclusively expressed in Cryptosporidium gametogenesis and can be readily detected by immunostaining. Using DMC1 as a marker for sexual differentiation, we developed an assay to test the effects of small-molecule inhibitors on asexual and sexual development of the C. parvum Iowa strain. The ReFRAME library, a collection of ~13,000 approved drugs and clinical- stage candidates, was used to validate the assay. Our screen identified 11 compounds that specifically inhibit C. parvum sexual differentiation. Interestingly, there were temporal differences in their activity in the HCT-8 infection model; some inhibit differentiation when added as late as 34 hours post-infection, while some only work if added within 12 hours post-infection. These data suggest stepwise regulation of Cryptosporidium sexual differentiation, and that the inhibitors act at multiple checkpoints along the way. To better understand the pathway, we are currently performing mRNA-seq experiments to examine the effects of compounds on host and C. parvum gene expression during parasite sexual differentiation.

249

164 - Thioredoxin peroxidase-I and its nuclear role in Plasmodium falciparum

Heinberg, Adina (Hebrew University); Amit-Avraham, Inbar; Thompson, Emilie; Dzikowski, Ron (Hebrew University)

Plasmodium falciparum, the causative pathogen of malaria, resides in human red blood cells (RBC), a place abundant in oxygen and iron. To prevent damage generated in this pro-oxidative environment, Plasmodium has several redox proteins including peroxiredoxins. One such peroxiredoxin, thioredoxin peroxidase-1 (Trx-Px1) is known to be a cytoplasmic protein that plays a role in protecting the parasite from oxidative and heat stress. However, the role that Trx-Px1 plays in the nucleus and in affecting gene expression hasn't been addressed. Another key to P. falciparum’s survival is its ability to modify the iRBC surface with cytoadherent proteins. These proteins are encoded by the multi-copy gene family named var and they are expressed in a mutually exclusive manner, where only one gene is expressed at a time. We identified Trx-Px1 as a nuclear protein that was bound to the antisense long non-coding RNA that is associated with the active var gene. We generated an HA-tagged Trx-Px1 parasite line and found that Trx-Px1 localizes to a distinct region on the nuclear periphery early in the cell cycle when var gene transcription occurs. In addition, Trx-Px1 is upregulated in response to heat stress. We hypothesize that nuclear Trx-Px1 could play a role in transcriptional regulation in the face of environmental insults.

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165 - The role of RNA-binding proteins abundant in Apicomplexans (RAPs) in mitochondrial biogenesis of the human malaria parasite, P. falciparum

Hollin, Thomas (University of California, Riverside); Prudhomme, Jacques (University of California, Riverside, Riverside.); Pasaje, Charisse F.; Falla, Alejandra (Massachusetts Institute of Technology); Saraf, Anita; Florens, Laurence (Stowers Institute for Medical Research); Niles, Jacquin C. (Massachusetts Institute of Technology); Le Roch, Karine G. (University of California, Riverside)

In the malaria parasite, Plasmodium falciparum, gene regulation depends on mechanisms acting at the transcriptional, post- transcriptional and translational levels throughout its life cycle. Our understanding of these mechanisms is incomplete and represents a critical step to fight malaria. In eukaryotes, genes regulated at the post- transcriptional level require diverse pathways and involve RNA-Binding Proteins (RBPs) that are essential to RNA processing at multiple levels such as splicing, stability, localization and translational efficiency. While at least 18% of the Plasmodium genomes encode for potential RBPs, these proteins have been poorly characterized throughout the parasite life cycle. Comparative computational analysis recently identified an expanded RNA-binding protein family that seems to be unique to Apicomplexan parasites (RBPs abundant in Apicomplexans or RAPs). Twenty-two of these RAPs were identified in P. falciparum. To investigate the role of these RAPs in the development and survival of P. falciparum, we first selected two RAPs, PF3D7_0105200 and PF3D7_1470600 predicted to target the parasite mitochondrion. Using the CRISPR/Cas9 approach together with the tetR inducible system, we showed that both localize in the mitochondria and are essential to the parasite survival. Preliminary data using ribonucleoprotein interaction network, gene expression profiling and metabolomics analyses indicate their role in mitochondrial biogenesis. The validation of the essentiality of these atypical RAPs could facilitate our ability to design new drugs against malaria.

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166 - I-BET is an anti-Toxoplasma agent that targets TgBDP3, a bromodomain protein in the TFIID complex

Jeffers, Vicki (University of New Hampshire); Hanquier, Jocelyne; Sullivan Jr., William J. (Indiana University School of Medicine)

The acetylation of histone tails is a critical process for the regulation of gene expression in Toxoplasmagondii. The bromodomain is the recognition module for acetylated lysines and bromodomain-containing proteins are recruited to acetylated histone tails to coordinate transcriptional regulatory complexes. There are twelve bromodomain- containing proteins in Toxoplasma, only two of which (the GCN5 lysine acetyltransferases) have been characterized. We demonstrate that TgBDP3, which contains two tandem, N-terminal bromodomains, is an essential nuclear protein that is associated with the TFIID transcriptional initiation complex. Chromatin-immunoprecipitation (ChIP) studies have identified the genome-wide binding sites of TgBDP3, revealing the parasite gene networks that are subject to TgBDP3 regulation. We have previously reported that the bromodomain inhibitor I-BET151 has selective anti-parasitic activity against Toxoplasma. Using chemoproteomics approaches with a linkable I-BET analogue, we have determined that TgBDP3 is a target of I-BET151 and that the inhibitor binds the first (but not the second) bromodomain of TgBDP3. The impact of TgBDP3 inhibition by I- BET151 on TgBDP3 chromatin recruitment and parasite transcription is assessed by ChIP and quantitative RT-PCR, validating TgBDP3 as a target for I-BET151 parasite inhibition.

252

167 - PCR-HRM illustrates diverse malaria parasites circulating in urban-restricted non-human primates in Kenya

Jeneby, Maamun (International Centre of Insect Physiology and Ecology- icipe); Villinger, Jandouwe (International Centre of Insect Physiology and Ecology- icipe)

As members of diverse habitats, free-ranging nonhuman primates (NHPs) may serve as sentinels for surveillance of parasitic infectious diseases. The aim of this study was to utilise the robust polymerase chain reaction (PCR) coupled with high-resolution melting (HRM) analysis in investigating, for the first time, the prevalence and identity of enzoonotic malaria parasites infecting free-ranging common NHPs found within selected urban centres in Kenya, East Africa. A total of 126 Chlorocebus aethiops (African green monkeys - AGMs) and 65 Papio anubis anubis (olive baboons) were sampled in situ for 5 ml of blood and released. Exploratory PCR–HRM analysis designed to amplify malaria parasites, genus Plasmodium, identified diverse species of malaria parasites closely related to human Plasmodium falciparum, rodent’s Plasmodium berghei and chimpanzee’s Plasmodium reichnowi in African green monkeys. This result highlights the potential risk of enzootic smian malaria transmission to humans by peridomestic mosquitoes within urban centre with NHPs.

253

168 - GPI Anchored Micronemal Antigen is required across multiple stages of the Plasmodium life cycle

Jennison, Charlie Armstrong, Janna M.; Patel, Hardik (SCRI); Minkah, Nana (SCRI); Wilder, Brandon K. (SCRI); Sheikh, Amina; Vaughan, Ashley M.; Kappe, Stefan HI (SCRI)

GPI Anchored Micronemal Antigen (GAMA), also known as Putative Secreted Ookinete Protein 9 (PSOP9), expressed in multiple stages of the malaria life cycle is believed to play roles in both parasite invasion and egress. In Plasmodium berghei, GAMA appears to be required for robust infection of the midgut by the motile ookinete. Oocysts that do form reach full maturity but sporozoites fail to egress, halting the life cycle at this point. These midgut sporozoites are not infective to mice. In P. falciparum, where previous knock-out attempts were unsuccessful, GAMA has been shown to bind erythrocytes and play a role in the sialic acid independent invasion pathway, with GAMA specific antibodies showing growth inhibition activity. The mechanisms underlying these multiple roles across life cycle stages remain undescribed. We have reconfirmed the previously described defects in ?GAMAP. berghei parasites and found intact oocysts and sporozoite- free salivary glands up to day 40 post infection. Intriguingly, a chimeric P. berghei parasite expressing P. falciparum GAMA, complemented the function of GAMA in ookinetes, recovering normal infectivity to the mosquito midgut, but mature oocyst sporozoites were unable to egress and these midgut sporozoites remained minimally infective to mice.We are now undertaking a promoter-swap strategy to understand the role GAMA plays across the mosquito and pre-erythrocytic stages of the life cycle. The characterization of parasites expressing GAMA under the stage specific promoters CTRP (ookinete), CAP380 (oocyst), and TRAP (sporozoite), reveal unique roles for GAMA. In addition, epitope tagging is being undertaken to investigate the spatial and temporal localization of GAMA, alongside efforts to understand the role of P. falciparum GAMA beyond the erythrocytic stage.

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169 - Contractile vacuole and flagellar homeostasis are regulated by mechanosensitive channels.

Jimenez, Veronica (Center for Applied Biotechnology Studies and Department of Biological Science, College of Natural Sciences and Mathematics, California State Universi); Fonbuena, Joshua; Feldman, Marc (1Center for Applied Biotechnology Studies and Department of Biological Science, College of Natural Sciences and Mathematics, California State); Augusto, Ingrid; Miranda, Kildare (2Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro)

In all cell types, mechanosensation is responsible for sensing and responding to changes in pressure, osmolarity, and tension of the membrane. In bacteria, mechanosensation is associated with virulence-related traits such as biofilm formation and quorum sensing. Despite developing in a constantly changing environment, little is known about mechanosensing machinery and its physiological role in protozoan parasites. In T. cruzi, we have identified and characterized a mechanosensitive channel (TcMscS) that shares structural and functional features with the small conductance mechanosensitive channel, MscS of E. coli. Gene knockout by CRISPR-Cas9 severely reduced the growth of extracellular and intracellular parasites and decreased its ability to respond to osmotic stress. Global transcriptomic analysis by RNAseq showed hundreds of differentially expressed genes in TcMscS-KO epimastigotes. Interestingly, a number of genes encoding for flagellar proteins are significantly downregulated. We verified the expression at protein level and found that PFR2, FCaBP and other key flagellar components are reduced. Additionally, the parasites show abnormal morphology, shorter flagella and the presence of multiple basal bodies. This is consistent with motility and infectivity defects observed in the TcMscS-KOs.Our results indicate a connection between the contractile vacuole function, flagellar homeostasis and cell fitness that is regulated by mechanosensitive channels.The presence of TcMscS homologues in other protozoans suggests a conserved sensing mechanism that could unveil important aspects of the host-parasite interaction.

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170 - Phenotypic Characterization of an Essential Mitochondrial Protein with Unknown Function in Plasmodium falciparum

Lamb, ian M. (Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine) Morrisey, Joanne; Daly, Thomas (Drexel University College of Medicine); Jenkins, Bethany M.; Mather, Michael M. (Drexel University College of Medicine); Bergman, Lawrence (Drexel University College of Medicine); Vaidya, Akhil M. (Drexel University College of Medicine)

Mitochondrial functions are essential throughout the life cycle of malaria parasites and have been validated as targets of antimalarial drugs. We are exploring the possibility that additional mitochondrial functions may provide targets for the discovery of novel antimalarial drugs. Here, we seek to characterize a Plasmodium protein encoded by the gene PF3D7_0105500 with unknown function. This gene is highly conserved and limited only to the Myzozoan superphylum. Divergence of Apicomplexans and Dinoflagellates from was accompanied by a massive reduction in mtDNA open reading frames and genome size, highlighted by the complete loss of mtDNA in the Apicomplexan genus Cryptosporidium. Interestingly, PF3D7_0105500 orthologues are absent in Ciliates but conserved in most other apart from Cryptosporidium, suggesting it is a Myzozoan innovation important to maintaining mtDNA functions subsequent to a drastic reduction in its size. It also appears to be localized to the parasite mitochondrion as judged by a biotinylation-based proximity labeling screen of the P. falciparum mitochondrial proteome. Using a newly-developed system of conditional gene knockdown, we derived a transgenic parasite line in which the PF3D7_0105500 gene product is tagged in a manner that expression is dependent on the presence of anhydrotetracycline (aTc). Withdrawal of aTc caused parasite demise, showing essential function of this gene for parasite survival. We have successfully expressed the PF3D7_0105500 protein product recombinantly, and further characterization of this protein is ongoing.

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171 - High level resistance to P. falciparum cytochrome B inhibitors

Lane, Kristin D. (NIAID/NIH); Mu, Jianbing (NIAID/NIH.); Lu, Jinghua; Liu, Anna; Remcho, Parks (NIAID/NIH); Windle, Sean (University of Washington); Wellems, Thomas (NIAID/NIH)

The mitochondrial electron transport chain is essential in all life stages of Plasmodium falciparum parasites and remains an attractive target for antimalarial development. From the 106/1 lab line, drug pressure with the cytochrome B (CytB) inhibitor, atovaquone (ATQ), selected the clinically relevant CytB Y268S mutation. This clone was highly resistant to ATQ and the succinate dehydrogenase antagonist, plumbagin (PL), without observed cross-resistance to CytB inhibitors, CK-2-68 and RYL-552. Secondary drug pressure with either CK-2-68 or RYL-552 resulted in resistant mutants lacking additional CytB mutations detected by Sanger sequencing. In addition to high level ATQ resistance observed in the ATQ-selected clone, these new lines demonstrated new features of cross-resistance to CK-2-68 and RYL- 552, and increased susceptibility to PL.A previously selected CytB V259L mutant (RYL-552 pressure) displayed cross-resistance to both CK-2-68 and RYL-552, and only some ATQ tolerance compared to 106/1. Sequencing after secondary selections with CK-2-68 or ATQ revealed additional CytB changes. ATQ pressure resulted in a L144S, V259L mutant demonstrating mid-level ATQ resistance, and decreased susceptibility to antimycin A (AMA, a CytB inhibitor of the quinol reduction pocket), compared to the V259L parent. CK-2-68 selection resulted in a double Y126C, V259L mutant with high-level resistance to CK-2-68 and AMA. We docked the compounds into the wildtype and mutant CytB and calculated the estimated free energy of binding and estimated inhibitory constants. High-level ATQ resistance is underpinned by PfCytB Y268 mutations, in agreement with biological data. On the background of the PfCytB V259L mutant, the Y126C substitution may confer high-level CK-2-68 resistance.

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172 - Activation of erythrocyte permeability enhances drug uptake by malaria sexual parasites

Lavazec, Catherine (Inserm U1016/CNRS UMR8104); Barbieri, Daniela (Inserm); Bouyer, Guillaume (CNRS); Dupuy, Florian; Marteau, Anthony (Inserm); Sissoko, Abdoulaye (Université de Paris); Neveu, Gaelle; Bedault, Laurianne (Inserm); Roman, Diana (Université de Paris); Siciliano, Giulia; Alano, Pietro (Istituto Superiore di Sanita); Martins, Rafael; Lopez Rubio, Jose Juan (CNRS); Clain, Jérome; Duval, Romain (Université de Paris); Egée, Stéphane (CNRS)

Following malaria parasite invasion, the infected erythrocyte displays important alterations of its membrane properties. To ensure the transport of nutrients and waste products necessary for their replication and survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These New Permeation Pathways (NPPs) have been extensively characterized in asexual parasite stages, whereas the existence of NPPs has never been detected in gametocytes. Using a diversity of techniques such as isosmotic lysis, electrophysiology, fluorescence tracer uptake and viability assays, we show here that NPPs are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocytogenesis. Our results indicate that NPPs contribute to the uptake of artemisinin derivatives by immature stages and suggest that the slowdown of NPPs activity in mature stages may account for their refractoriness to these drugs. Importantly, our results show that NPPs activity is regulated by cyclic AMP signaling, and interfering with this pathway can reactivate erythrocyte permeability and restore uptake of artemisinin derivatives by mature gametocytes. These processes are predicted to play a key role in P. falciparum gametocytes susceptibility to antimalarials and are proposed to represent novel and unconventional route to target parasite transmission.

258

173 - In Search of Novel Antimalarials: Chemical Starting Points from a Natural Product Library Phenotypic Screen.

Lawong, Aloysus (UT Southwestern Medical Center); Margaret, Phillips (UT Southwestern Medical Center); Shingare, Rahul (UC Santa Cruz); MacMillan, John (UC Santa Cruz)

Emerging resistance of the Plasmodium parasite to current therapies underscores the need for new anti-malarial agents with broad ranging activity against multiple stages of the parasite. Overcoming emerging drug resistance requires new drugs with novel modes of action. With the aim of identifying new classes of anti-malarials our search began with a whole cell phenotypic screen of a marine natural product fraction library representing 30,000 natural products. We identified and validated an active fraction with nanomolar (Pf3D7 EC50 5.4 nM) plasmodicidal activity and some cytotoxicity (0.077 µM) against K562 mammalian leukemia cells. Sequence similarity match from genomic DNA extracts established the fraction as a secondary metabolite of the filamentous bacteria Streptomyces rochie. Bioassay-directed purification yielded a pure active moiety, and structural assignment by a combination of mass spectrometry and nuclear magnetic resonance spectroscopy revealed an 18-membered macrolide. The active moiety is the previously isolated threonyl tRNA synthetase inhibitor, borrelidin, reported in 1949 by Berger et al. Current efforts are geared towards compound isolation from other active fraction hits in the screen with micro-molar (Pf3D7 EC50 <1 µM) plasmodicidal activity and low or no mammalian cytotoxocity.

259

174 - Dynamic properties of the Leishmania cap-binding protein IF4E-1

Leger-Abraham, Melissa (Harvard Medical School);

Leishmaniasis disease is caused by protozoan parasites, Leishmania, which are spread to humans through the bite of infected sandflies. Regulation of their gene expression occurs mostly post- transcriptionally, and the different patterns of gene expression that are required throughout the parasites’ life cycle are primarily regulated at the level of translation. During the translation initiation step, Leishmania cap-binding proteins (LeishIF4Es) engage a hypermethylated cap structure found at the 5’-end of mRNAs, named cap-4, that is different from the m7GTP cap found in mammalian cells. While LeishIF4E-1 has emerged as a crucial cap-binding protein in the human stage, the molecular details of its interaction with cap-4 are poorly understood. In this study, we applied a combination of biophysical techniques (NMR spectroscopy and X-ray crystallography), and molecular biology approaches to study the structure/function of LeishIF4E-1.

260

175 - Distinct roles of small subunit proteins of the mitochondrial ribosome in Plasmodium falciparum

Ling, Liqin (1, Department of Microbiology and Immunology, Drexel University College of Medicine; 2, West China Hospital, Sichuan University) Mulaka, Maruthi; Dass, Swati; Mather, Michael (Center for Molecular Parasitology, Department of Microbiology and Immunology Drexel University College of Medicine); Riscoe, Michael (Portland VA Medical Center); Zhou, Jing (Department of Laboratory Medicine, West China Hospital, Sichuan University); Ke, Hangjun (Center for Molecular Parasitology, Department of Microbiology and Immunology Drexel University College of Medicine)

At a size of 6 kb, the mitochondrial DNA (mtDNA) in Plasmodium spp., the causative agent of malaria, is the smallest among all eukaryotes’ mitochondrial genomes. It only encodes three proteins of the mitochondrial electron transport chain (mtETC), cytochrome b, cytochrome c oxidase subunit I and III. Besides a minimal size, the Plasmodium mtDNA encodes many highly fragmented rRNA genes in length of just 20-195 bp. It has remained unknown if and how these fragmented rRNAs form a mitochondrial ribosome (mitoribosome) with ribosomal proteins encoded on the nuclear genome. The overall goal of our study is to explore mechanisms that assemble fragmented rRNAs with mitoribosomal proteins to constitute a functional ribozyme. Here, we have genetically characterized three annotated mitoribosomal proteins of the small subunit of Plasmodium falciparum mitoribosome, PfmRPS12, PfmRPS17, PfmRPS18, using a CRISPR/Cas9 based conditional knockdown approach, the TetR-DOZI-aptamer system. Immunofluorescence assays showed all three proteins are localized to the mitochondrion. Immunoelectron microscopy suggested PfmRPS18 is localized near the mitochondrial inner membrane. PfmRPS18 also forms a large molecular weight complex detected on blue native gel. Conditional knockdown of these proteins resulted in mild (PfmRPS12&PfmRPS18) to severe (PfmRPS17) growth arrest in the parasite. As readout of mitochondrial protein translation, the enzymatic activity the bc1 complex in these knockdown parasites decreases in varying degrees. The knockdown parasites also displayed distinct levels of hypersensitivity to atovaquone, ELQ-300 and other antimalarial inhibitors targeting the mtETC. Together, these data suggest loss of different mitoribosomal proteins of Plasmodium falciparum leads to deficiency in mitochondrial protein translation at distinct levels. We are currently characterizing how these mitoribosomal proteins directly interact with fragmented rRNAs. 261

176 - Evidence that transcription blockage is associated with signalling to the death in Trypanosoma cruzi

BERTOLDO, WILLIAN R. (UFMG); MACEDO, ANDREA M. (UFMG); FRANCO, GLORIA R. (UFMG); MACHADO, CARLOS R. (UFMG)

Transcriptional stress leading to RNA polymerase stalling can be caused by DNA damage. Stalled RNA polymerase is important to signal to initiate transcription- coupled repair (TC-NER). Our group have already showed that high doses of UVC kill the cells very faster. This death is signalized, since the use of ATM and ATR inhibitors can prevent death. One of the goals of this work is to verify whether transcription blockade after UVC damages is the main reason to the T. cruzi death. The pre-treatment with alpha-amanitin (60ug / ml), a selective inhibitor of RNA polymerase II, showed that these cells become resistant to UVC radiation, however, when this drug was removed and the transcription process restarted, cells become sensitive to UVC again. This data suggests that the death is dependent of an active transcription process. Previous results from our lab showed cells exposed to gamma irradiation (500Gy) abolished transcription process 24 and 48 hours after irradiation. So, we exposed cells to UVC in different hours after gamma irradiation. Just after the gamma irradiation, when the transcription process continuous to work, the gamma irradiated cells present similar UVC sensitivity when compared with non- gamma irradiated cells. However, the gamma irradiated cells are more resistant to UVC when the transcription process are absent (24 and 48 hours after gamma irradiation). This higher resistance persists until gamma irradiated cells return the normal growth. Furthermore, in T. cruzi, CSB is the key protein that recognizes RNA polymerase stalled .The overexpression of TcCSB increase cells sensitivity to UVC in an ATM/ATR dependent way. On the other hand, the single-knockout (CSB +/-) cells shows resistance. During the DNA damage response (DDR), deubiquitination activity demonstrated to be related to TC-NER. USP7 protein controls the CSB ubiquitination state during DDR, cells pre-treated with PR-619 (20 uM), a reversible USP7 inhibitor, become more resistant to UVC. However, in the same conditions CSB+/- cells were more sensitive than untreated cells (p < 0,002). So, the inhibition of deubiquitination influence in T. cruzi UVC response, possibly due to interference in TcCSB levels and the signalling. As our group have already observed that drugs that interfere in DNA replication is not lethal to T. cruzi, we verified if the sensitivity to UVC is different in cell cycle phases. For this study, cells were synchronized with hydroxyurea (20 mM), and the cell cycle determined by FACS. Cells were treated with 1500 J/m2 of UVC in different cell cycle phases. We observed synchronized T. cruzi cells (WT and CSB+/-) were more sensitive in G1 phase than the untreated cells (p <0.02). Interestingly, T. cruzi cells in G2/M were more resistant than the unsynchronized cells (p <0.003). We are looking for to know if these differences are due to difference in transcription process or due to differences in signalling process. So, all the data suggest that the blockage of transcription process is an important process in the signalling death in T. cruzi. 262

177 - Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome

Bhanot, Purnima (Rutgers - New Jersey Medical School) K. Govindasamy, R. Khan, M. Snyder, H. J. Lou, P. Du, Kudyba, Heather M. (NIH), V. Muralidharan, B. E. Turk, P. Bhanot

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

263

178 - Conserved heterodimeric structure & glycan specificity of C-type lectins Anopheles gambiae CTLMA2 and CTL4

BISHNOI, RITIKA

The C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCPC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 exhibit charge complementarity that promotes the formation of higher oligomeric states at physiological pH. Both lectins bind specific sugars, with an apparent preference for glycosaminoglycan motifs comprising β1-3/β1-4 linkages between glucose (Glc), galactose (Gal) and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is functional in vivo, reversing the enhancement of phenoloxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection

264

179 - A novel amino acid substitution in the AP2 domain of Pfap2-g is associated with a gametocyte-deficient phenotype

Prajapati, Surendra K. (Uniformed Services University of the Health Sciences); Morahan, Belinda J. (Monash University); Barbeau, Michelle C.; Hupalo, Daniel (Uniformed Services University of the Health Sciences); Wilkerson, Matthew; Dalgard, Clifton L. (Uniformed Services University of the Health Sciences); Williamson, Kim C. (Uniformed Services University of the Health Sciences)

An in depth understanding of the molecular mechanisms that regulate the switch between the asexual and sexual stages of the parasite is important for the design of effective anti-malaria transmission control measures. Currently two genes, gametocyte development protein 1 (gdv1) and an AP2 domain transcription factor apetella-2 (ap2-g), have been found toregulate P. falciparum gametocytogenesis.Gdv1 deletion or conditional-degradation results in a dramatic decrease in gametocyte formation, while ap2-g-knock out (KO) parasites do not form any gametocytes. In this sexual differentiation pathway, expression levels of ap2-gare positively regulated by GDV1 protein. To further interrogate the signaling pathway, we targeted a gene (PFB0665w) computationally annotated as aserine/threonine kinase (stpk) with an expression profile similar to gdv1. The resulting P. falciparum KO line (?STPK-3D7) showed a complete loss of gametocyte production, however this phenotype was not reproduced in another independent KO attempt. Whole genome sequencing of the gametocyte-deficient ?STPK-3D7 line revealed an intact gdv1 locus, however ap2-g had a SNP in the AP2 domain that results in a change from valine to leucine (V2163L). This region is free from SNPs in field isolates suggesting the V2163L substitution is not selected under field conditions. Transcriptome analysis of ?STPK-3D7 ring stage parasites revealed a RNA pattern consistent with reports from other ap2- g modified gametocyte deficient lines (?AP2-G-3D7 and F12), including down-regulation of ap2-g. We propose that the V2163L substitution is critical for AP2-G function. Further work is in progress to confirm that restoring the wildtype allele will restore gametocyte production.

265

180 - Plasticity of iron metabolism of brain eating amoeba

Mach, Jan (Charles University) Ženíšková, Katerina; Arbon, Dominik; Malych, Ronald; Grechnikova, Maria; Sutak, Robert (Charles University)

Iron plays a crucial role in many cellular pathways of all organisms. It is the most limiting factor for growth and/or virulence in different environments. Unlike the free-living organisms, parasites are solely dependent on the available host resources. This fact is widely misused by the host which tries to conceal essential resources, like iron, from parasites by elaborate methods. Naegleria fowleri is a free-living amoeba found in the warm freshwaters all around the world. In certain conditions, it can enter the human body through the nose and cause fatal disease Primary Amoebic Meningoencephalitis. It is a very severe and fast progressive disease which kills the patient within a few weeks. Efficient treatment is virtually nonexistent, and the fatality rate is above 97%. Although the condition is rare, the number of misdiagnosed cases is probably high since the symptoms often resemble other infections.Our work focuses on the impact of iron-rich and iron-poor conditions on N. fowleri metabolism. We show that iron is indeed vital and affects the growth of the cells in-vitro. Further, we performed proteomic and transcriptomic profiling and advanced metabolomic analysis to search for affected proteins and pathways in different iron conditions. We described the effect of iron-depleted condition on downregulation of nonessential iron containing pathways such as phenylalanine degradation. Measuring the overall respiration showed, that alternative oxidase is utilized in iron-deficiency, possibly compensating for the insufficient iron, which is necessary for the respiratory chain complexes. Another attribute affected by iron availability is the extent of phagocytosis. Altogether, our results show the plasticity of N. fowleri iron metabolism while facing different iron availability in the surrounding environment.

266

181 - Utilizing collateral sensitivity to dihydroorotate dehydrogenase inhibitors as a strategy to suppress pathways to resistance

Mandt, Rebecca E.K. (Harvard T.H. Chan School of Public Health); Lafuente-Monasterio, Maria Jose (Tres Cantos Medicines Development Campus); Luth, Madeline R. (University of California San Diego, School of Medicine); Reynolds, Matthew (Harvard College); Ottilie, Sabine; Winzeler, Elizabeth A. (University of California San Diego, School of Medicine); Gamo, F. Javier (Tres Cantos Medicines Development Campus); Wirth, Dyann F.; Lukens, Amanda K. (Harvard T.H. Chan School of Public Health)

Emerging resistance to frontline therapies necessitates the development of antimalarials with novel modes of action. Unfortunately, resistance sometimes arises rapidly even for novel drugs never-before introduced to parasite populations. During Phase 2 clinical trials with Plasmodium dihydroorotate dehydrogenase (DHODH) inhibitor DSM265, two patients developed resistant infections, with recrudescent parasites harboring C276Y or C276F mutations in DHODH. Our lab identified several point mutations in DHODH conferring between 10 to 1000-fold resistance to DSM265 using in vitro evolution. We find that many mutations do not impose a fitness cost in in vitro competitive growth assays. There are a variety of chemotypes that target Plasmodium DHODH, and we previously showed that resistance to one DHODH inhibitor can cause increased sensitivity to other classes of inhibitors, a phenomenon termed ‘collateral sensitivity’. Here, we characterize the cross-resistance and collateral sensitivity profiles of DSM265-resistant lines against a compound panel from our recently-published Tres Cantos Open Lab Screen. One compound, TCMDC-125334, is active against all mutant lines tested. Notably, the clinically-relevant C276Y line is highly-sensitive to this compound. In vitro selections with TCMDC-125334 yielded parasites with moderate (2 to 3-fold) resistance, which have copy number variation at the dhodh locus. We have begun selections with TCMDC-125334 and DSM265 in combination, and have thus far been unable to isolate resistant parasites. Interestingly, selections with the hyper-sensitive C276Y line selects for parasites with a dose-response phenotype similar to wildtype for TCMDC-125334, but which are highly resistant to DSM265 and other DHODH inhibitors. Overall, this work aims to characterize the emergence of resistance to novel antimalarials, and to ultimately identify compounds that can better suppress the emergence of resistant parasites.

267

182 - Guanabenz Reverses a Key Behavioral Change Caused by Latent Toxoplasmosis in Mice by Reducing Neuroinflammation

Martynowicz, Jennifer. (Indiana University School of Medicine); Augusto, Leonardo; Wek, Ronald C. (Indiana University School of Medicine); Boehm II, Stephen L. (Indiana University Purdue University); Sullivan Jr, William J. (Indiana University School of Medicine)

Toxoplasma gondii is an intracellular parasite that causes persistent, lifelong infections in one-third of humans worldwide. The latent tissue cyst form (bradyzoite) evades host immunity and is impervious to current drugs. While acute infection can be life threatening to immunosuppressed individuals, chronic infection has been linked to behavioral changes in rodents and neurological disease in humans. Notably, chronic infection in mice leads to hyperactivity in an open field assay. How Toxoplasma related behavioral changes occur, and whether they are due to parasite manipulation or the host response to infection, remains an outstanding question. We have previously shown that the anti-hypertension drug, guanabenz, reduces cyst burden in the brains of BALB/c mice up to 80%, providing a means to interrogate behavioral changes in mice with reduced parasite burden. We chose to use two different mouse strains due to differences in susceptibility to infection. We treated both strains of mice with guanabenz (5mg/kg/day ip for 3 weeks) after chronic infection with Toxoplasma was established, and then assessed locomotor activity. We observed the expected increase in locomotor activity in each chronically infected mouse strain. Remarkably, guanabenz reversed this behavior change in both BALB/c and C57BL/6, yet only decreased brain cyst burden in BALB/c mice. Examination of the brains showed that guanabenz decreased inflammation and perivascular cuffing in both strains. Our study shows that it is possible to reverse a key behavioral change associated with chronic Toxoplasma infection. Surprisingly, the rescue from parasite- induced hyperactivity correlates with a decrease in neuroinflammation instead of reduced cyst counts, suggesting that some behavioral changes arise from host responses to infection rather than a parasite driven process.

268

183 - Unveiling the role of UIS4 during malaria liver stage of infection

M'Bana, Viriato (Instituto de Medicina Molecular, João Loo Antunes, Lisboa); Lahree, Aparajita; Slavic, Ksenija; Mota, Maria (Instituto de Medicina Molecular, João Loo Antunes, Lisboa)

Hepatocyte infection by Plasmodium sporozoites is the first natural step towards the establishment of malaria disease. Inside hepatocytes, the parasite lives in a specialized compartment enveloped by a membrane called parasitophorous vacuole membrane (PVM). The PVM represents the interface between the parasite and the host cell, and most likely plays different roles that may range from protection and signalling to waste elimination. Nevertheless, little is known about the molecular players and interactions occurring at this interface.A subset of proteins encoded by the “Upregulated in Infective Sporozoites” (UIS) genes have been identified, and it has been shown that some of them are exported to the PVM. One such protein is UIS4, which is essential for parasite survival. Despite its crucial role for malaria parasites during the liver stage of the disease, the biological function of UIS4 remains to be elucidated.To uncover the role of UIS4, its potential interacting partners, were targeted by immunoprecipitated using an antibody against UIS4 both in cells infected with UIS4-KO parasite or wild type. By our surprise, actin was identified as a UIS4 interacting partner. Furthermore, in vitro assay of actin, polymerisation was performed using the recombinant soluble domain of UIS4, and the results reveal that UIS4 promotes actin polymerization. This interaction is sufficient to dramatically alter the host actin dynamics around the growing EEF leading to EEF death, as shown by live imaging.Altogether, this study revealed a previously unknown interaction between a critical parasite protein essential for the success of the liver stage of infection and host actin.

269

184 - Evolution of a new pathway of reserve carbohydrate biosynthesis in Leishmania parasites

McConville, Malcolm j. (University of Melbourne); Serne, Fleur (University of Melbourne); Ralton, Julie (University of Melbourne, Parkville); Nero, Tracy (University of Melbourne); Kloehn, Joachim; Cobbold, Simon; Hanssen, Eric; Parker, Michael; Williams, Spencer (University of Melbourne); Davies, Gideon (University of York)

Leishmania synthesize a novel intracellular carbohydrate reserve, composed of ß-1,2-mannan oligosaccharides, termed mannogen. Nothing is known about the enzymes involved in mannngen synthesis and degradation or the function of this pathway in mammalian-infective stages. Here we describe a new class of enzymes that catalyze the sugar nucleotide-dependent elongation of mannogen chains, as well as their phosphorolytic turnover. These dual activity enzymes, termed mannosyltransferases/phosphorylases (MTPs), are related to bacterial mannan phosphorylases, and are likely to have been acquired by horizontal transfer from gram-positive bacteria. The MTPs catalyze the constitutive turnover of mannogen in all parasite stages, suggesting that this cycle functions as a metabolic rheostat, regulating sugar phosphate and phosphate levels. Knock-out studies show that mannogen turnover protects obligate intracellular parasite stages from glucose toxicity in vivo and is essential for thermotolerance and infectivity in the mammalian host. Our studies suggest that the acquisition of this pathway played a key role in allowing Leishmania to establish an obligate intracellular life style in the mammalian host and that the MTPs are potential drug targets.

270

185 - Identification of antigens and antibodies important for protection conferred by radiation-attenuated Plasmodium falciparum sporozoite vaccination

McDermott, Suzanne M. (Seattle Children's Research Institute); Osman, Rahwa; Stuart, Ken (Seattle Children's Research Institute)

There are over 200 million cases and one million deaths caused by malaria worldwide each year. A highly-efficacious vaccine that prevents infection is an urgently needed tool to combat this situation. Immunization with liver-stage arresting, radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) confers sterilizing protection, and high anti-malaria antibody titers are important for protection. Together these results indicate that antigens from pre- erythrocytic (PE) stages of the P. falciparum life-cycle and the and adaptive immune responses induced during these stages are effective vaccine candidates. However, the specific antigens and antibodies behind this protection are still unknown. To identify these unknown antibodies and cognate PE antigens we are using state-of-the-art molecular immunological approaches to study samples from PfRAS vaccine trials in which protection was determined by Controlled Human Malaria Infection (CHMI). To identify protective antigens, we are preparing yeast antigen display libraries that comprehensively represent the P. falciparum proteome that will be screened with serum samples from non-protected and protected individuals following PfRAS immunization. In parallel, we are identifying and characterizing B-cell receptor repertoires that are associated with protection using next generation sequencing of single chains as well as natively paired heavy and light chains. We expect these studies will identify protective antibodies, and clonal diversity and frequency associated with protection following malaria vaccination. Thus, our work will inform much-needed improved vaccine design and expand our knowledge of protective malaria antigens and host antibodies, as well as providing important tools for the research community.

271

186 - A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Meister, Thomas R. (Stanford University); Tang, Yong (Stanford University); Walczak, Marta (Stanford Universtiy); Pulkoski-Gross, Michael J. (Stanford University); Hari, Sanjay B.; Sauer, Robert T. (Massachusetts Institute of Technology); Amberg-Johnson, Katherine (Stanford University); Yeh, Ellen (Stanford Universtiy)

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid—the apicoplast—which originated from a secondary (eukaryote–eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast, and a new fluorescent reporter for organelle loss. Thus far, we have validated 6 candidate genes (out of 12 identified) to be essential for apicoplast biogenesis. These include a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity, and a CaaX-like protease that has also likely gained a novel function. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. These newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.

272

187 - A novel putative telomere-binding protein in Plasmodium falciparum

Merrick, Catherine J. (Cambridge University); Edwards-Smallbone, James (Keele University); Jensen, Anders L.; Totanes, Francis I.G.; Roberts, Lydia E. (Cambridge University); Hart, Sarah R. (Keele University)

In the early-diverging parasite Plasmodium, only a few telomere-binding proteins have thus far been identified and several of these appear to be unique. Because of this, and because telomere maintenance is crucial for all rapidly-dividing single-celled organisms, the study of telomere biology in Plasmodium is of particular interest, and could reveal new, specific targets for antimalarial therapy.Here, we report the identification of the previously uncharacterised P. falciparum GBP2 protein as a putative telomeric factor. It was identified via an agnostic pulldown and mass- spectrometry approach targetting telomeric chromatin: ‘Proteomics of Isolated Chromatin fragments’ (‘PICh’). This was applied here for the first time in Plasmodium and is orthogonal to previously published methods for identifying telomeric factors. PfGBP2 is partially homologous to S. cerevisiae GBP2, which has both telomere-binding and mRNA-binding roles: ScGBP2 contains three RRM domains – an RNA/DNA-binding fold – whereas PfGBP2 is predicted to contain two. Using two of its RRM domains, ScGBP2 binds preferentially to G-rich RNA and DNA sequences, such as S. cerevisiae telomere repeats. Consistently, in EMSA assays, recombinant PfGBP2 also binds to G-rich DNA oligos.PfGBP2 is apparently essential in erythrocytic parasites (very deleterious knockout phenotypes were reported in the piggyBac and PlasmoGEM screens). We were also unable to express a tagged GBP2 gene episomally, suggesting that over-expression is likewise deleterious. The endogenous gene could, however, be tagged, allowing us to demonstrate that the protein appears by immunofluorescence as peri-nuclear foci (characteristic of telomeric factors), and by western blot in the nucleoplasm and chromatin-bound fractions, although some protein also appears in the cytoplasm. We are now conducting ChIP to establish genomic locations for PfGBP2 in vivo.In summary, we report a new, essential Plasmodium telomere-binding factor, expanding our understanding of telomere biology in this organism.

273

188 - Plasmodium histone deacetylase 1 (HDA1) plays important, dual, sex specific roles in gametocyte maturation and viability

Millar, Scott (University of Glasgow); Power, B. Joanne; Martin, Julie; Hughes, Katie; Waters, Andy (University of Glasgow)

In order for Plasmodium species to transmit to a mosquito vector they must transition from an asexual parasite of the erythrocytic cycle into a sexual male or female gametocyte. Epigenetically regulated expression of ap2-g, a member of the AP2 family of transcription factors, at crucial time points in asexual parasites was identified as orchestrating this developmental change, guiding the parasite through gametocytogenesis. Although some epigenetic actors controlling ap2- g expression and thus commitment have been identified the full extent of epigenetic regulation remains unclear. Histone deacetylase 1 (HDA1) has recently been implicated in gametocytogenesis as its transcript is upregulated in analyses of early gametocytes of P. falciparum. Using reverse genetic approaches, we deleted hda1 (pbhda1-, PBANKA_1335400) in P. berghei. The resulting KO exhibited a reduced asexual growth rate and gametocytes that were slower to mature with a resulting male to female gametocyte ratio that was skewed compared to controls due to reduced production of female gametocytes. These phenomena may be linked to reduced ability of pbhda1- parasites to deacetylate H3K122Ac, an epigenetic mark associated with chromatin accessibility and transcription. Gametocytes of pbhda1- could not generate ookinetes which genetic crossing revealed to be due to a defect in male gametocyte exflagellation and fertility. The exflagellation defect is potentially linked to hyperacetylation of cytoskeletal proteins mirroring the activity of huHDAC6, the closest human homologue of PbHDA1, which regulates cell motility via deacetylation of key cytoskeletal proteins.Our analyses indicate that HDA1 performs at least two functions influencing gene transcription through epigenetic regulation of H3K122Ac and male gamete flagellar formation in different sub-cellular locales at distinct points in the parasite life cycle.

274

189 - Investigation of transcriptional regulation in Plasmodium ookinetes

Modrzynska, Katarzyna K. (University of Glasgow) Morton, Rachel; Kirchner, Sebastian (University of Glasgow); Nyarko, Prince (West African Centre for Cell Biology of Infectious Pathogens (WACCBIP)); Waters, Andrew P. (University of Glasgow)

Transmission from the mammalian host to the mosquito vector is a main bottleneck in the life cycle of malaria parasites. During this process, the transmissible parasite forms (gametocytes) emerge, fertilise and, within 24h, form extracellular, motile forms called ookinetes. The transition from gametocytes to ookinetes requires a dramatic change in the parasite’s gene expression. The mechanisms controlling transcriptional regulation at this stage, however, are not fully understood.Previous studies identified six transcription factors from the apiAP2 family with potential function during the ookinete development. In this project, GFP tagging of these transcription factors in the rodent malaria P.berghei has revealed the spatial and temporal localisation of each TF through a 24hr time course sexual development. For three of them, which showed a distinct nuclear localisation in zygotes or ookinete, ChIP analysis was also performed, mapping their genomic occupancy and their putative target genes. In parallel we have also analysed the chromatin structure (using an ATAC-seq protocol) and selected epigenetic marks in the developing ookinetes.Analysis of these datasets revealed that at least two different transcription factors acting in concert are responsible for the regulation of expression in the developing ookinetes. Each of these factors binds to a distinctive sequence motif and taken together they explain the majority of open chromatin regions identified at this stage. These findings contribute to our understanding of the ookinete development and gene expression regulation in Plasmodium.

275

190 - Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research

Mohring, Franziska (London School of Hygiene and Tropical Medicine) Hart, Melissa N. (London School of Hygiene and Tropical Medicine); Rawlinson, Thomas A. (The Jenner Institute, University of Oxford); Henrici, Ryan; Charleston, James A.; Diez Benavente, Ernest; Patel, Avnish (London School of Hygiene and Tropical Medicine); Hall, Joanna (National Institute of Biological Standards and Control); Almond, Neil (National Institute for Biological Standards and Control); Campino, Susana; Clark, Taane G.; Sutherland, Colin J.; Baker, David A. (London School of Hygiene and Tropical Medicine); Draper, Simon J. (The Jenner Institute, University of Oxford); Moon, Robert W. (London School of hygiene and Tropical Medicine)

Due to their unique biology, relapsing Plasmodium vivax and zoonotic Plasmodiumknowlesi parasites represent a distinctive challenge for malaria eradication. To replicate, both parasites must invade red blood cells via interaction of Duffy binding protein (PvDBP or PkDBPa respectively) with human red blood cell Duffy antigen receptor for chemokines, DARC. PvDBP is a lead vaccine candidate, but due to the lack of a long-term in vitro culture system, vaccine studies were limited to ex-vivo and protein binding assays for characterisation of inhibitory antibodies. The DBP protein family has also been implicated in both host and cellular tropisms which have particular importance in unravelling key differences between the closely related species. Understanding the biology of both parasites was also constrained by the lack of robust molecular and genetic approaches.Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic P. knowlesi line in which the full length PkDBPa gene was replaced with PvDBP. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Further, the transgenic line is used to screen a panel of monoclonal antibodies from human volunteers immunised in a clinical trial of PvDBP. This identifies a broadly neutralising antibody which also inhibits invasion of all 13 tested clinical isolates of P. vivax. The hierarchy of mAb potency against the transgenic P. knowlesi model mirrored that observed in two parasite isolates isogenic for SalI PvDBP, suggesting this model is highly predictive of P. vivax neutralisation.

276

191 - Targeting parasite-produced MIF as an anti-virulence strategy to reduce tissue damage

Moonah, Shannon. (University of Virginia); Ghosh, Swagata; Padalia, Jay (University of Virginia)

INTRODUCTION: Treatment interventions for protozoan parasites are severely hindered by emerging drug resistance, drug toxicity, ineffectiveness of existing drugs and limited antimicrobial options. Therefore, new therapeutic strategies are urgently needed for the unrelenting protozoan parasite threat to global health. While substantial progress in antivirulence approaches has been made in bacteriology, virulence factor inhibition in parasitology remains significantly understudied. Many medically important protozoans secrete an inflammatory MIF cytokine homolog, which acts as a virulence factor and is linked to severe disease. STUDY OBJECTIVE and METHODS: Here, we used Entamoeba histolytica, a protozoan that causes inflammatory diarrhea (amebic colitis), as the model organism, and mouse model that mirrors severe human infection to investigate parasite-produced MIF as an antivirulence target. RESULTS and DISCUSSION: We found that intestinal inflammation and tissue damage was significantly reduced in mice treated with metronidazole combined with anti-E. histolytica MIF antibodies compared to metronidazole only. Thus, these findings suggest antibiotics combined with anti-parasite MIF blocking antibodies might improve outcomes in severe infections.

277

192 - Interactions between ribosomal proteins and fragmented rRNAs in the mitochondrial ribosome of Plasmodium falciparum

Mulaka, Maruthi (Drexel University College of Medicine); Das, Swati (Drexel University College of Medicine); Ling, Liqin (Drexel Univeristy College of Medicine); Ke, Hangjun (Drexel University College of Medicine)

The origin of mitochondria of all eukaryotes can be traced back to an alpha-bacterium. As decedents of a bacterium evolutionarily, mitochondria maintain a genome and a translation system. The parasitic lifestyle in apicomplexan parasites has apparently driven a reductive evolution in the mitochondrial genome. The mitochondrial DNA (mtDNA) of most apicomplexan parasites is small, encoding just three proteins of the electron transport chain, cytochrome b, cytochrome c oxidase subunits I&III. In malaria parasites, the 6 kb mtDNA is the smallest organellar genome among all eukaryotes. Besides a highly compacted size, the 6 kb mtDNA encodes more than 30 rRNA fragments in varying lengths of 20-195 bp. These rRNA fragments assemble into mitochondrial ribosome (mitoribosome) with mitoribosomal proteins imported from the cytosol. The mechanisms by which fragmented rRNAs assemble with mitoribosomal proteins to form a functional ribosome are largely unknown. In this study, we intend to assemble fragmented rRNAs with annotated mitoribosomal proteins using biochemical and computational approaches. We are currently expressing rRNA fragments using in vitro transcription and annotated mitoribosomal proteins using recombinant protein expression systems. We are performing experiments to define which rRNA fragment interacts with a corresponding protein partner. In parallel, using protein modeling tools, we are attempting to obtain a predicted 3D structure of each of the annotated mitoribosomal proteins in the database (~ 40 proteins). Using available high-resolution ribosome structures of model organisms as template, we intend to computationally superimpose the obtained individual structures of mitoribosomal proteins of Plasmodium falciparum to the known ribosomal structures. These computational 3D models will help us to understand how fragmented rRNAs interact with mitoribosomal proteins in a unique fashion.

278

193 - Using metabolomics to understand the role of acetyl-CoA in asexual Plasmodium falciparum parasites.

Munro, Justin (Pennsylvania State University); Allman, Erik (Pennsylvania State University); Nair, Sethu; Prigge, Sean (Bloomberg School of Public Health); Llinás, Manuel (Pennsylvania State University)

Emerging antimalarial drugs have displayed exceptional potency for perturbing coenzyme A (CoA) biosynthesis. To establish the significance of the mitochondrial generation of acetyl-CoA as a major function of coenzyme A, we are characterizing a set of knockouts in lipoate ligase 2 (LipL2) and branched chain keto-acid dehydrogenase (BCKDH). LipL2 is responsible for the lipoylation of mitochondrial keto- acid dehydrogenase E2 subunits, which are required for the dehydrogenation of the corresponding keto-acid substrate while BCKDH has been implicated as a substitute enzyme for pyruvate dehydrogenase with the role of generating acetyl-CoA in Plasmodium species. Interestingly, LipL2 can only be knocked out by adding a significant amount of exogenous acetate whereas the BCKDH knockouts were viable without acetate supplementation. Through a combination of metabolomics experiments using acetate supplementation and isotope labeling with these knockout parasite lines, we find that both the mitochondrial and cytosolic generation of acetyl-coA are required for asexual parasite development. In the mitochondrion, acetyl-CoA is generated by the mitochondrial PDH-like enzyme, BCKDH, while the cytosolic generation of acetyl-CoA is performed via acetyl-CoA synthetase. The reason for the essentiality of these processes is most likely for nuclear product acetylation, such as histones, in both asexual-stage parasites and gametocytes according to the downstream incorporation of acetyl-CoA into acetylated amino acids. In addition, the knockout of LipL2 resulted in a severe decrease in pyrimidine biosynthesis precursors, which suggests that pyrimidine biosynthesis and acetyl-CoA generation may be linked in Plasmodium species. This work demonstrates that enzymes that result in the generation of acetyl-CoA are viable, multi-stage drug targets, which will be beneficial to future resistance modeling and drug deployment studies.

279

194 - Targeting Aurora-like Kinases in Plasmodium falciparum

Murillo_Solano, Claribel; Vienneau-Hathaway, Jannelle (UCF); Murithi, James (Columbia University Medical Center.); Collins, Jennifer; Shaw, Justin (UCF); Buskes, Melissa; Ferrins, Lori (Northeastern University); Chakrabarti, Ratna (UCF); Pollastri, Michael (Northeastern University); Fidock, David A. (Columbia University Medical Center); Chakrabarti, Debopam (UCF)

The unique features of the Plasmodium cell cycle offer opportunities for the development of therapeutics directed against essential components that are involved in its regulation. Cell division kinases such as Aurora kinases are likely to play pivotal roles in both erythrocytic and pre- erythrocytic stages where during schizogony multiple rounds of nuclear division take place. Plasmodium Aurora-related kinases (Arks) 1 and 2 are homologous to Aurora A and B Ser/Thr kinases that are expected to be involved in cell division and have been shown to be essential. To better define the function of PfArks we have introduced the HA-tag at the C-terminus of PfArk1 and PfArk2 using a CRISPR/Cas9 knock-in approach. Integration at the correct locus was confirmed by PCR and sequencing. Additionally, to discover potent and selective small molecule inhibitors of PfArks, we screened a library of optimized mammalian Aurora kinase inhibitors that have evolved from a general pharmacophore model for Ser/Thr kinases. We identified novel potent inhibitors (EC50 < 1 µM) in phenotypic screening using SYBR Green I fluorescence-based assays. One of the inhibitors, DC02391, targets phosphorylation of the myelin basic protein by recombinant PfArk2 in a dose-dependent manner. This compound completely inhibited histone H3 phosphorylation, a signature event by human Aurora B, in parasite exposed to 5XEC50 (500 nM) of the compound. Studies are underway to assess bead-based native enzyme activity and critically analyze changes in subcellular distribution following inhibitor treatment using the HA-tagged PfArk1 and PfArk2 lines.

280

195 - Compound stage-specificity and metabolic profiling to advance malaria drug discovery

Murithi, James (CUIMC); Owen, Edward (Penn State University); Otillie, Sabine (UCSD); Chibale, Kelly (UCT); Winzeler, Elizabeth (UCSD); Llinás, Manuel (Penn State); Vanaerschot, Manu; Fidock, David (CUIMC)

Target identification plays a key role in antimalarial drug development. Resistance selections and other ‘omic studies are unfortunately not always able to identify a compound’s target. Insights into the stages at which antimalarial candidates are maximally active can provide important clues about their mode of action. Here we report on a medium-throughput in vitro asexual blood stage susceptibility assay that determines compound activity at five different developmental stages: early and late rings, early and late trophozoites, and schizonts. We categorized a set of 36 known antimalarials and candidate compounds based on the susceptibility profile across life stages and the timing of compound peak activity. This identified compounds with broad activity profiles as well as those with stage-specific activity. Subsequently, we assessed 33 of these compounds for their metabolic perturbations in drug-treated parasites in an effort to identify targeted pathways. Collectively these data helped us discriminate between modes of drug action and modes of drug resistance, as exemplified by the various compounds showing different profiles while all encountering resistance mediated by the ABC transporter PfABCI3. We also identified a specific biphasic dose-response at the late trophozoite stage for DSM265 and atovaquone, likely related to the dual importance of dihydroorotate dehydrogenase and cytochrome bc1 in maintaining the mitochondrial electron transport chain and pyrimidine biosynthesis. Ultimately, asexual blood stage drug specificity and metabolomics profiling helped prioritize compounds for further development and can provide useful information when considering optimal partner drug combinations.

281

196 - Discovery and Characterization of Antiplasmodial Compounds Targeting Protein Kinases

Bohmer, Monica (University of Central Florida) Barq, Alya (University of Central Florida); Sausman, William (University of Texas at San Antonio); Chakrabarti, Ratna (University of Central Florida); Hanson, Kirsten (University of Texas at San Antonio); Chakrabarti, Debopam (University of Central Florida)

Reversible phosphorylation of cellular proteins, through the action of protein kinases and protein phosphatases, are key regulatory switches of various cellular processes in eukaryotic cells, including Plasmodium. Phosphosignaling has been shown to be of importance at all stages of parasite maturation and the work done in our laboratory has established the dynamics of protein phosphorylation during intraerythrocytic life cycle of the parasite using quantitative phosphoproteomics. Therefore, in our pursuit to discover novel antimalarials acting on multiple stages of parasite growth, we have focused on inhibitors of Plasmodium protein kinases. In this effort we have screened a kinase-focused library of 5,280 compounds for their ability to inhibit the asexual growth of the parasite using SYBR Green I-based fluorescence assay. The utility of compounds in this library has been validated through in silico modeling and on-target profiling against mammalian kinases. We identified 10 hits exhibiting nanomolar EC50 values (Z’>0.7). As a counter screen, we evaluated the cytotoxicity of the active compounds in HepG2 cells using the MTS cell proliferation assay. Many of the active compounds were found to be selective. We tested asexual stage active compounds against liver stages using the P. berghei-HepG2 infection model which supports complete development through to hepatic merozoite formation, using a parasite line expressing luciferase and GFP. The compounds were tested at 0.5 and 5 micromolar concentrations with compounds present from 2-72 hours post-infection using a multimodal workflow developed in the Hanson lab. One of the compound, DC-03466 exhibited modest effects in the 48 hpi biomass assay, but block hepatic merozoite formation quite efficiently. Efforts are underway to elucidate the targets of the active compounds.

282

197 - Host adaptation in Toxoplasma gondii - What makes this parasite so “successful”?

Naor, Adit (Stanford); Boothroyd, John C. (Stanford)

Toxoplasma gondii is considered one of the most successful parasites in nature based on its worldwide distribution, high prevalence and extremely broad host range (almost any cell type in nearly all warm blooded animals can be infected). We know that Toxoplasma’s virulence is highly dependent upon allelic variation and hypothesize that these differences in virulence are driven by regional differences in the predominant hosts.To test this hypothesis, we have performed an in-lab evolution study wherein we serially pass Toxoplasma in tissue culture, in three different host cell lines: human foreskin fibroblasts, mouse 10T1/2 fibroblasts and a line of kidney-derived fibroblast-like cow cells known as Madin-Darby bovine kidney (MDBK) cells. Following approximately 1000 generations in these different host cell lines we assessed the growth, mouse-virulence and gene expression profile of the three adapted lines. Remarkably, parasites grown on the cow cells showed ~15-fold increased ability to attach to any of the three host cell lines compared to parasites passaged on the human cell lines. We were able to identify two other differences that could be the mechanisms underlying this phenotype: 1) higher expression levels of particular parasite surface proteins involved in attachment; and 2) a more rapid secretion of micronemal proteins. Using RNA-Seq on the three evolved lines, we observed reproducible differences in parasite gene-expression depending on the host cell line on which they were passed. Interestingly, we observed that many of these changes in parasite gene expression and phenotype arise within just a few passages on the different host cell lines. We have also tested the virulence of these adapted lines in mice and show that passage history of the parasite affects virulence in this model. Currently, we are testing the role of the differences in gene expression in the ability to grow on the different host cell lines and their impact on mouse-virulence.

283

198 - Tadalafil clears gametocytes from blood circulation in humanized mice

N'DRI, Marie-Esther (Inserm U1016, Institut Cochin, CNRS, UMR8104, Université Paris Descartes); BARBIERI, Daniela; GOMEZ, Lina; DUPUY, Florian (Inserm U1016, Institut Cochin, CNRS, UMR8104, Université Paris Descartes); FRANETICH, Jean François; MAZIER, Dominique (Sorbonne Université, INSERM, CNRS, Centre d'Immunologie et des Maladies Infectieuses, CIMI); MORENO-SABATER, Alicia (Sorbonne Université, Inserm U1135, CNRS ERL 8255, Centre d'Immunologie et des Maladies Infectieuses (Cimi-Paris); LAVAZEC, Catherine (Inserm U1016, Institut Cochin, CNRS, UMR8104, Université Paris Descartes)

A switch in mechanical properties of the infected erythrocyte between the immature and mature gametocyte stages has been reported as a marker of the final step of Plasmodium falciparum gametocytogenesis. This mechanism is controlled by intracellular levels of cAMP which determine the rigidity status of the infected erythrocyte. We have shown that phosphodiesterase (PDE) inhibitors increase the stiffness of mature gametocyte-infected erythrocytes (GIE) and impair their circulation in an in vitro model for splenic retention. These findings suggest that PDE inhibitors may promote gametocytes elimination by the spleen, however they have never been confirmed in vivo. Here, we show that the inhibition of cAMP degradation with tadalafil, an approved drug used to treat erectile disorder, significantly increases the stiffness of mature GIE in a dose-dependent manner. We also found that retention rates of mature GIE are comparable in the in vitro spleen mimic and in immunodeficient mice injected with purified GIE, thus indicating that these mice are a relevant in vivo model to validate our previous in vitro results. Using flow cytometry, qPCR and bioluminescence imaging, we observed that the kinetics of gametocytes clearance after a single GIE injection in immunodeficient mice are significantly faster when GIE are pre-incubated in vitro with tadalafil. In a model of humanized mice infected with P. falciparum, which allows the complete development of the erythrocytic cycle till mature gametocytes stages, tadalafil-treated mice cleared mature GIE at a higher rate than in untreated mice. Overall, these results indicate that tadalafil treatment clear mature gametocytes from blood circulation in vivo.

284

199 - Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites

Okada, Megan (University of Utah); Okada, Amanda L.; Maschek, John A. (University of Utah); Swift, Russell (Johns Hopkins); Prigge, Sean T. (John Hopkins); Sigala, Paul A. (University of Utah)

Plasmodium falciparummalaria parasites harbor a non-photosynthetic plastid organelle called the apicoplast that houses core metabolic pathways and is essential for parasite viability. Isoprenoid precursor biosynthesis of isopentenyl pyrophosphate (IPP), via the non- mevalonate/MEP pathway, is an essential apicoplast function required for crucial cellular processes, including protein prenylation for vesicular trafficking and biosynthesis of mitochondrial ubiquinone and heme a. Since IPP supplementation rescues lethal apicoplast loss, isoprenoid biosynthesis has been thought to only serve essential roles outside this organelle. Here we provide direct evidence that IPP synthesis is critical for apicoplast branching and segregation into daughter merozoites. Parasites treated with the MEP inhibitor fosmidomycin (FOS) fail to elongate and divide the apicoplast despite still reaching schizogeny. This elongation defect in FOS can be fully rescued with concomitant IPP treatment. However, delaying IPP supplementation until schizogeny rescues parasite viability without rescue of apicoplast inheritance, such that daughter parasites have lost the organelle. Thus, the critical role of IPP for apicoplast biogenesis precedes broader essentiality outside the organelle. We have localized a previously annotated polyprenyl synthase (PPS) to the apicoplast lumen. Conditional knockdown of this enzyme appears to block apicoplast biogenesis and inheritance by daughter merozoites, whose lethality can be rescued by IPP. We posit that this apicoplast PPS functions downstream of IPP synthesis to produce longer-chain isoprenoids that play a critical but undefined role in expanding the apicoplast membrane during organelle biogenesis. This work provides a new paradigm for isoprenoid utilization in P. falciparumparasites, identifies a novel essential branch of apicoplast isoprenoid metabolism that can serve as a new drug target, and exemplifies an organelle maintenance pathway whose inhibition defies delayed death to kill parasites in the first cycle.

285

200 - Fatty acid elongases are critical for maintaining membrane lipid homeostasis in Trypanosoma cruzi

Pagura, Lucas (Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health); Dumoulin, Peter C. (Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health); Mendes, María Tays; Ellis, Cameron C.; Almeida, Igor C. (Biological Sciences Department, University of Texas at El Paso); Burleigh, Barbara A. (Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health)

During its complex life cycle, Trypanosoma cruzi undergoes distinct morphological and physiological transitions. Conversion between developmental stages is accompanied by membrane lipid remodeling as the parasite adapts to life in an insect or mammalian host, however, the mechanisms by which these parasites regulate membrane lipid composition are currently unknown. Here, we focused on the modular fatty acid elongase (ELO) system as a candidate pathway for regulation of membrane lipid synthesis in T. cruzi. This pathway, first described in African trypanosomes1, consists of four enzymes (ELO1-4) that sequentially elongate short chain fatty acid (SCFA) substrates C4:0 to C10:0 (ELO1) and C10:0 to C14:0 (ELO2). Long-chain fatty acids (LCFA), C16:0 and C18:0, are generated by ELO3 and ELO4 generates very long-chain polyunsaturated fatty acids. To gain mechanistic insights into the role of fatty acid elongases in the regulation of membrane lipid composition in T. cruzi, we generated individual parasite lines with functional disruptions in elo1, elo2, or elo3 genes using CRISPR/Cas9 as confirmed by PCR and Southern blot. Analysis of phospholipids from T. cruzi epimastigotes by LC-MS/MS revealed a substantial shift in the fatty acyl moieties toward shorter chain fatty acids (C10:0, C12:0, C14:0, C14:1) as compared to the LCFAs (C16:0, C18:0, C18:1, C18:2) highly represented in WT parasites. These changes were most pronounced in the ?elo3 mutant which also displayed profound morphological defects in the epimastigote stage. All of the mutants were able to establish infection in mammalian cells, however intracellular growth of the ?elo2 and ?elo3 amastigotes was markedly reduced as compared to WT. Moreover, our data indicate that the expression, trafficking, and/or distribution of major surface glycoproteins may be dysregulated in ?elo3 mutant amastigotes as suggested by the uncharacteristic punctate labeling of the parasite surface and flagellum visualized by immunofluorescence microscopy. Combined, our data suggest that despite previous demonstrations of lipid and fatty acid scavenging by T. cruzi2, exogenously acquired fatty acids do not efficiently compensate for the loss of endogenous LCFA synthesis in the ?elo mutants, supporting the role for the ELO pathway in maintaining homeostatic control of membrane lipid composition in T. cruzi.1. Lee, S. H., Stephens, J. L., Paul, K. S. and Englund, P. T. (2006). Fatty acid synthesis by elongases in trypanosomes. Cell 126:691-699, doi:10.1016/j.cell.2006.06.0452. Gazos-Lopes, F., Martin, J. L., Dumoulin, P. C. and Burleigh, B. A. (2017). Host triacylglycerols shape the lipidome of intracellular trypanosomes and modulate their growth. PLoS Pathogens 13, e1006800, doi:10.1371/journal.ppat.1006800 286

201 - The Plasmodium Clp protease system controls plastid biogenesis via degradation of specific substrates

Florentin, Anat (University of Georgia); Stephens, Dylon; Anaguano, David; Brooks, Carrie; Muralidharan, Vasant (University of Georgia)

Like other apicomplexans, Plasmodium falciparum contains a plastid called the apicoplast, that functions to produce essential metabolites. Drugs that target the apicoplast are clinically effective, with most of them inhibiting the organelle protein synthesis machinery. However, the bulk of the apicoplast proteome is encoded by the nuclear genome and transported to the plastid, with little or no regulation by the organelle itself. Here, we propose that without the means to regulate most of its own protein synthesis, the apicoplast controls its proteome and biology through an organelle specific proteolytic machinery. Using a combination of genetic and biochemical approaches, we show that the Clp system regulates the apicoplast proteome, thus controlling the biology of the organelle. We have targeted the Clp protease (PfClpP), chaperone (PfClpC), inactive protease (PfClpR) and substrate adaptor molecule (PfClpS) and found that interfering with expression, activity or interactions between any of these genes, results in apicoplast loss. We have generated null mutant of the PfClpP protease (PfClpPKO), and discovered that it is essential due to its role in apicoplast biogenesis. A PfClpP conditional mutant (PfClpPapt) revealed the robustness of its activity. Expression of the PfClpC chaperone with a mutation in the protease binding site (PfClpCF1103W) revealed its role as an essential regulator of complex activity. A combination of affinity purification and proximity biotinylation was used to determine complex composition and identify interactors and regulators. Generating double mutants of PfClpPapt and PfClpPTRAP, a dead-protease variant, revealed the enzymatic properties of the complex and was used to isolate specific substrates. Furthermore, we have investigated the molecular and biological functions of the substrate adaptor molecule, PfClpS, and showed that the PfClpS conditional mutant (PfClpSapt) phenocopies the effects of other Clp mutants. Lastly, we used an in vivo reporter, to show that PfClpS mediates degradation by binding specific substrates and feeding them into the Clp complex. We concluded that the ClpR/P/C/S system regulates the biology of the apicoplast via protein degradation and has the potential to be an excellent drug target.

287

202 - Roles of calcium signaling and cyclic nucleotide signaling in control of invasion and egress of bradyzoites

Fu, Yong (Washington University School of Medicine); Brown, Kevin; Jones, Nathaniel; Sibley, L. David (Washington University School of Medicine)

Toxoplasma gondii is an obligate intracellular parasite that has evolved complex developmental stages important for pathogenesis. Immune pressure contributes to the differentiation of rapidly dividing tachyzoites into the relatively quiescent bradyzoites, which can establish lifetime infection in host muscle and CNS tissue in the form cysts. Previous studies have showed the invasion and egress of tachyzoites depend on microneme exocytosis controlled by protein kinase G and calcium-dependent kinases. These pathways are negatively regulated by cAMP signaling, thus promoting intracellular development. Although these pathways are well studied in tachyzoites, the coordination of calcium-dependent and cyclic nucleotide-dependent kinases in control of bradyzoite invasion and egress is poorly understood. To address this important question, we generated type II ME49 strain dual-reporter parasites that express mCherry driven by BAG1 promoter and calcium indicator GCaMP6f. We observed that bradyzoites induced in vitro (pH 8.2, 8 days culture) presented a much slower increase in the Ca2+ level than that of tachyzoites after agonist stimulation. In parallel, to investigate microneme secretion in bradyzoites, we constructed dual-indicator parasites expressing microneme protein MIC2 fused with Gaussia luciferase (GLuc) and BAG1 promoter-driving mCherry. We showed that BSA rather than ethanol independently triggered MIC2-Gluc secretion which could be enhanced by combined utilization of ethanol and BSA. Next, we aim to generate cAMP and cGMP fluorescent- indicator parasites in order to determine the response of in vitro induced bradyzoites to agonists of cyclic nucleotide stimulation. In parallel, we will perform conditional protein degradation of adenylate cyclases (ACs) in ME49-TIR1 parasites using auxin-inducible degron (AID) tagging system. Collectively, defining the roles of calcium and cyclic nucleotide signaling pathways in bradyzoites will lead to the better understanding of the mechanism of invasion and egress by T. gondii.

288

203 - Exploring COP9 signalosome as a novel therapeutic target for protozoan parasites

Ghosh, Swagata (University of Virginia) Moonah, Shannon (University of Virginia)

INTRODUCTION and HYPOTHESIS: Protein homeostasis is essential for life, thus, protein degradation pathways have emerged as promising drug targets for treating protozoan diseases. We recently discovered a parasite-encoded CSN5 like protein, the predicted catalytic subunit of the COP9 signalosome, which is conserved in many medically important protozoan parasites. Here, we hypothesize that COP9 signalosome regulates protein degradation and is therefore essential for parasite survival and a viable anti-parasitic target. METHODS: To test this hypothesis, we used Entamoeba histolytica as a model parasite and performed genetic knockdown studies along with tetracycline-induced overexpression to study the dominant negative effect of EhCSN5 mutants. Also, we used in-silico molecular docking for screening small molecule inhibitors of EhCSN5. RESULTS and DISCUSSION: Genetic alteration of EhCSN5 by two independent approaches revealed that EhCSN5 is essential for normal growth and viability of E. histolytica. Reduced EhCSN5 protein expression inhibited cell viability. Overexpression of an enzymatic mutant form of the protein showed dominant negative effects on the growth and viability of the parasite. Screening a pool of metalloproteinase inhibitors by molecular docking with EhCSN5 catalytic site, revealed a potential inhibitor that was capable of killing E. histolytica parasites using an in-vitro inhibition assay, with significantly higher efficacy compared to the traditional drug metronidazole. Therefore, our findings so far, support the notion that COP9 signalosome in E. histolytica is essential for the parasite’s viability and provides a promising avenue for anti-parasitic drug targeting. We are currently investigating the EhCSN5 inhibitor using a mouse model that mirrors human E. histolytica infection. Experiments to test the molecular effects of genetic and pharmacological disruption of EhCSN5 enzymatic activity, are also underway. Further knowledge of the EhCSN5 pathway opens up opportunities not only for E. histolytica, but other difficult-to-treat protozoans that produce their own CSN5. 289

204 - Genome Wide CRISPR/Cas9 Knockout Screen Identifies Host Genes Important for Cryptosporidium parvum Infection

Gibson, Alexis R. (University of Pennsylvania); Sateriale, Adam R.; Beiting, Daniel R.; Striepen, Boris R. (University of Pennsylvania)

Cryptosporidium is an obligate intracellular protozoan parasite, and the second leading cause of severe diarrhea and diarrheal-related death in children worldwide. There are currently no vaccines and the only FDA approved drug has low efficacy in the immunocompromised individuals who need it most. Despite this massive impact on public health, little is known about the host response to Cryptosporidium. We conducted a genome-wide CRISPR/Cas9 knockout screen to discover host genes necessary for Cryptosporidium parvum infection. Human intestinal epithelial cells, HCT-8s, stably expressing Cas9 were transfected with a lentiviral sgRNA library targeting every gene in the human genome. Selection for resistance to cell death was accomplished by three consecutive 72-hour C. parvum infections. After each challenge, cells were removed for gDNA extraction and sequenced to determine the abundance of each sgRNA. Of the top 25 candidates, the most highly enriched pathway, type III interferon (IFN?) signaling, is known to control viral infections in epithelial cells. RNAseq analysis of cells sorted using a fluorescent reporter parasite line indicates that IFN?, specifically IFNL1, is being produced by infected cells and not bystanders. CD151, a member of the tetraspanin superfamily, was also a top candidate. CD151 has been shown to activate Cdc42, a small GTPase, involved in actin remodeling required for successful C. parvum infection. Genes in pathways of host glycosaminoglycan synthesis and glycosylphosphatidylinositol (GPI) anchor synthesis were also highly enriched, likely responsible for facilitating parasite attachment and invasion. Further research is necessary to understand the importance of these pathways in host defense and the role parasite manipulation may play in these responses.

290

205 - Elucidating spatially-resolved changes in host signaling during Plasmodium liver-stage infection

Glennon, Elizabeth (Seattle Children's Research Institute); Pan, Liuliu; Liang, Yan (NanoString Technologies); Kaushansky, Alexis (Seattle Children's Research Institute)

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and travel to the liver where they infect a single hepatocyte. Current evidence suggests hepatocytes are significantly altered upon Plasmodium infection, creating opportunities for the development of host-targeted therapeutics that specifically target infected cells. However, the low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo, and existing studies have been unable to consider how protein and phosphoprotein differences might be altered at different spatial locations within the heterogeneous liver. We have applied digital spatial profiling to characterize host signaling within Plasmodium yoelii-infected BALB/c mouse livers. Digital spatial profiling uses antibodies that have been linked to a barcoded oligonucleotide with a photocleavable linker to measure levels of 50- 100 host total and phosphorylated proteins within defined regions of fixed tissue sections. This enables us to gather in vivo data with minimal, rapid processing of the tissue. We were able to reliably detect protein and phosphoprotein levels in single P. yoelii-infected hepatocytes, facilitating the evaluation of variation in host signaling among individual infected cells within the same host. Moreover, we identified alterations in host signaling within uninfected cells that neighbor infected hepatocytes. Identifying signaling that propagates from infected cells may provide insight into how the parasite creates a favorable microenvironment within the liver and how liver stage infection influences subsequent immunity. By identifying changes in phosphosignaling within and around infected cells, we hope to gain insight into the cellular niche that promotes liver stage parasite development, and to identify potential targets for host-directed therapies.

291

206 - Trypanosoma brucei and Trypanosoma congolense cathepsin L proteolyse human TLF and free recombinant APOL1

Gonzalez, Bernardo (Hunter College); Raper, Jayne (Hunter College); Verdi, Joe (Hunter College); Thomson, Russell; Sanchez, Alan (Hunter College)

Humans and some non-human primates are immune to infection by T.b.brucei due to trypanosome lytic Apolipoprotein L-1 (APOL1). ApoL1 is the active component of trypanosome lytic factors (TLF1 and TLF2) which are present in these primate sera. After TLF enters the parasite, APOL1 inserts into the endosome membrane, forming pH-dependent cation channels which leads to colloid-osmotic swelling and lysis of the parasites. Currently, the endocytic pathways of TLF1 and TLF2 as well as the factors associated with the transit of these complexes have yet to be elucidated. Studies utilizing a T. brucei RNAi library identified an array of proteins and factors possibly involved in intracellular TLF trafficking, including the lysosomal cathepsin L (CatL). We aim to evaluate the activity of the CatL of T. brucei (TbCatL) and T. congolense (TcCatL) on recombinant APOL1 and TLFs. To carry out this study, we constructed HIS-tagged TbCatL and TcCatL linked to human interleukin-2 (IL2) signal peptide to generate secreted versions of the proteases in cell culture. The cDNAs were cloned into a mammalian expression vector, transfected into Chinese hamster ovary cells, and purified by metal affinity and size exclusion chromatography. The enzymatic activity was assessed using a synthetic CatL substrate and a CatL-specific inhibitor. We observed that incubation of human APOL1 (hAPOL1) or baboon APOL1 (bApoL1) with CatL leads to complete proteolysis of both Apoliporoteins. CatL also proteolyzed human TLF1 (hTLF1) and human TLF2 (hTLF2). Why then can APOL1 and TLFs kill African trypanosomes? Further studies will aim to determine if TLFs or APOL1 are proteolyzed by CatL in vivo, or whether APOL1 attaches to the endolysosomal membrane prior to detection by CatL.

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207- The Canonical Histone H2A, of the Malaria Parasite Plasmodium falciparum is Phosphorylated and Recruited to Foci of Damaged DNA

Goyal, Manish (Hebrew University) Singh, Brajesh Kumar; Dzikowski, Ron (Hebrew University)

Plasmodium falciparum parasites proliferate within the circulating red blood cells and is responsible for the deadliest form of human malaria. These parasites are exposed to numerous intrinsic and external sources that could cause DNA damage; therefore, they have evolved efficient mechanisms to protect their genome integrity and allow them to proliferate in such conditions. In yeast and mammals, double strand breaks rapidly lead to phosphorylation of the core histone variant H2A.X which marks the site of damaged DNA. Here we show that in Plasmodium falciparum parasites, that lack the H2A.X variant, the canonical PfH2A is phosphorylated on serine 121 upon exposure to DNA damage. We further demonstrate that phosphorylated PfH2A is recruited to foci of damaged chromatin shortly after exposure to source of damage and that the phosphorylation is decreased over time as the parasite repairs its DNA. Altogether these findings present novel marker for DNA damage in Plasmodium that could be used as a useful tool to study DNA damage response in these deadly pathogens.

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208 - Full development of tsetse-transmitted trypanosomes in advanced human skin tissue models

Engstler, Markus (University of Wuerzburg)

African trypanosomes are transmitted by the tsetse fly through deposition of the infective metayclic stage parasites into the mammalian skin. Our knowledge about the early stages of human trypanosome infection at the site of theinsect’s bite remains limited. Therefore, we propose bioartificial human skin as versatile model system for the investigation of tsetse-borne trypanosome infections. We have developed novel primary human skin equivalents with improved mechanical properties. Our skin model resembles native human skin in its histological architecture and distinctive physiology. We have used tsetse flies to successfully simulate the natural infection process by direct transmission of trypanosomes to our artificial skin models. High-end 4D imaging of the injection site revealed an unexpected complexity of the skin lesions, with direct implications on the early dissemination of the trypanosomes within the host dermis. Most importantly, the parasites complete their natural developmental progamme within the human skin model. We found a very rapid transition from the transmitted quiescent metacyclic trypanosomes to proliferative slender bloodstream forms, and further differentiation to fly-transmissible forms. We have also documented the interaction of trypanosomes with dermal fibroblasts, and have conducted an extensive dual RNA-Seq analysis of trypanosome infections of primary human skin models.

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209 - Complexome profiling reveals Plasmodium falciparum mitochondrial protein dynamics (during sexual differentiation)

Evers, Felix (Radboud Institute for Molecular Life Sciences); Cabrera-Orefice, Alfredo; Brandt, Ulrich (Radboud University Medical Center); Kooij, Taco W.A. (Radboud Institute for Molecular Life Sciences)

The Plasmodium falciparum mitochondrion has hallmarks of an attractive drug target as it is essential and sufficiently distinct from host mitochondria. Currently, atovaquone is the only validated antimalarial targeting the mitochondrion. Identification of additional mitochondrial targets has been limited by the uncommon and poorly understood characteristics of this organelle in Plasmodium species. Among other peculiarities each parasite harbours only a single mitochondrion, which contains the smallest known mitochondrial genome and has limited significance for energy generation during blood-stage development. Rather, the organelle is critical in various anabolic pathways such as pyrimidine or Fe-S cluster biosynthesis. During gametocytogenesis the mitochondrion undergoes heavy expansion and de novo formation of cristae, which is accompanied by a shift of the parasite towards increased mitochondrial catabolism. Here, we used complexome profiling to obtain an unbiased view of the protein changes underlying this phenomenon. Complexome profiling is a technique that combines native gel electrophoresis and mass spectrometry to assess composition and relative abundance of protein complexes. We applied this technique to mitochondrial fractions of P. falciparum asexual and sexual blood stages. The presence of various previously characterized protein complexes validated our approach. We identified novel protein complex components, as well as poorly recognizable orthologues of subunits known from eukaryotic model systems. Furthermore, we revealed remarkable differences in abundance and presence of mitochondrial complexes between gametocytes and asexual blood stages. These findings allow us to derive new hypotheses about the changing role of the mitochondrion during sexual differentiation of the parasite and beyond. By deciphering the parasite’s mitochondrial complexome we expect to highlight divergent protein complexes as attractive potential drug targets not detectable by traditional proteomic approaches.

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Poster Session C

Wednesday, September 18, 2019

3:00 pm – 5:00 pm

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210 - Characterization of a Novel Sensing Mechanism Governing Antigenic Variation and Immune Evasion in P. falciparum Schneider, Victoria (The Rockefeller University); Ben Mamoun, Choukri (Yale University); Rhee, Kyu; Harris, Chantal; Kafsack, Bjorn; Deitsch, Kirk (Weill Cornell Medical College) For Abstract See Session X, 60t

211 - Examining trans-regulators of the Leishmania lifecycle Walrad, Pegine B. (York Biomedical Research Institute, Univ. of York); R. Ferreira, Tiago; Pablos, Luis; Dowle, Adam; Forrester, Sarah; Parry, Ewan; V. C. Alves-Ferreira, Eliza; Newling, Katherine; Kolokousi, Foteini; Larson, Tony; Plevin, Michael (University of York); K.Cruz, Angela (University São Paulo) For Abstract See Session X, 61t

212 - Redox metabolomics and gametocytogenesis in the malaria parasite Beri, Divya (Indian Institute of ScienceTatu, Utpal (Indian Institute of Science) For Abstract See Session X, 62t

213 - Functional and biochemical characterization of SPY- catalysed nucleocytosolic O-fucosylation in Toxoplasma gondii Bandini, Giulia (Boston University); Agop-Nersesian, Carolina (Boston University); van der Wel, Henke; West, Christopher M. (University of Georgia); Samuelson, John (Boston University) For Abstract See Session XI, 66t

214- The novel EhHSTF7 transcription factor binds to HSE and regulates the multidrug resistant Ehpgp5 gene expression in Entamoeba histolytica. Bello, Fabiola (Centro de Investigación y Estudios Avanzados, IPN); Orozco, Esther (Centro de Investigación y Estudios Avanzados, IPN); Pérez, Guillermo; Gómez, Consuelo (Escuela Nacional de Medicina y Homeopatía, IPN) For Abstract See Session XI, 67t

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215 - Decoding combinatorial patterns of histone post- translational modifications of Plasmodium falciparum. von Grüning, Hilde (University of Pretoria); Coradin, Mariel; Mendoza, Mariel G. (University of Pennsylvania); Sidoli, Simone (Albert Einstein College of Medicine); Garcia, Benjamin A. (University of Pennsylvania); Birkholtz, Lyn-Marie (University of Pretoria)

In eukaryotes, histone post-translational modifications (PTM) generate chromatin structures that are conducive to gene expression regulation. These covalent chemical motifs have been suggested to establish specific epigenetic states through a “histone code” that mediate cellular processes. Although the presence a few co-existing histone PTMs in Plasmodium falciparum parasites have been previously described, the global landscape and the functional contribution to gene regulation have not been elucidated. We aim to quantify and map all combinatorial histone PTMs in P. falciparum during both the asexual and gametocyte developmental stages and to provide evidence that these combinations of histone PTMs result in functionally distinct outcomes. Using advanced quantitative mass spectrometry, we present here the dynamic and complex patterns of histone PTM combinations occurring specifically in each developmental stage, suggesting an alternative higher order mode of gene regulation in the parasite. Proteomic chromatin profiling revealed the proteins that associate with genomic regions histone PTMs that co-exist in combination, including specific reader enzymes. Next, interrogation of the genomic distribution of these combinatorial histone PTMs through ChIP-sequencing will be performed. Ultimately, a clearer understanding of the full extent and functional complexity of the combinatorial histone code could contribute to our knowledge base of the unique developmental processes required for P. falciparum sexual differentiation and other biological processes.

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216 - The regulation of translational control during human-to- mosquito transmission of Plasmodium falciparum

Bennink, Sandra (Division of Cellular and Applied Infection Biology, RWTH Aachen University); von Bohl, Andreas; Ngwa, Che Julius; Pilch, Nicole (Division of Cellular and Applied Infection Biology, RWTH Aachen University); Minns, Allen; Orchard, Lindsey; Lindner, Scott; Llinás, Manuel (Department of Biochemistry and Molecular Biology, The Pennsylvania State University); Pradel, Gabriele (Division of Cellular and Applied Infection Biology, RWTH Aachen University)

The human malaria parasite Plasmodium falciparum exhibits a complex life cycle during which it has to adapt to changing environments. In order to pre-adapt to the transmission from the human host to the mosquito vector, the gametocytes store transcripts that encode proteins needed for parasite development in the mosquito in stress granules (SGs). We recently described a novel SG component of female gametocytes, the seven-helix protein 7-Helix-1, which represents a homolog of the human stress response regulator LanC- like 2. Assembly of SGs is usually initiated by phosphorylation of the translation initiation factor eIF2α. While eIF2α phosphorylation can be induced in Plasmodium gametocytes by treatment with the poison sodium arsenite, gametocytes lacking 7-Helix-1 show a decreased eIF2α phosphorylation, indicating impaired SG formation in the absence of 7-Helix-1. In SGs, 7-Helix-1 interacts with a variety of ribonucleoproteins, such as CITH, DOZI, and PABP1. The protein further forms a complex with the RNA-binding protein Puf2, a translational regulator of the female-specific antigen Pfs25, as well as with pfs25-coding mRNA. In accord, gametocytes deficient of 7-Helix- 1 exhibit impaired Pfs25 synthesis. Interestingly, we were not able to detect binding between 7-Helix-1 and the RNA-binding protein ALBA4, suggesting that ALBA4 acts independently of 7-Helix-1. However, the lack of 7-Helix-1 leads to an up-regulation of ALBA4 as well as a down- regulation of elongation factor eEF1β, pointing to an imbalance of translational control components in the absence of 7-Helix-1. Our combined data point to a key role of 7-Helix-1 in re-initiating protein synthesis of SG-stored mRNAs at the onset of P. falciparum gametogenesis.

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217 - Genetic mapping of strain specific differences in autophagy effector recruitment to the Toxoplasma gondii parasitophorous vacuole

Radke, Joshua (Washington University School of Medicine); Sibley, David (Washington University School of Medicine)

Autophagy (ATG) or autophagy related genes play a critical role in the innate immune response by targeting intracellular pathogens for elimination. Recently, our laboratory identified an innate, noncanonical autophagy pathway that regulates T. gondii infection in a strain- dependent manner in hIFN-? activated human cells. The core set of ATG16L1, ATG12 and ATG5 proteins are involved in recruitment of LC3 to the parasitophorous vacuole (PV), yet upstream activators (i.e. Beclin and Atg14) and downstream degradation functions (i.e. lysosomal fusion) are not required. In humans, PVs containing susceptible strains (type II and III) of T. gondii are ubiquitinated, followed by recruitment of the ATG adapters p62 and NDP52, and finally engulfed in multiple layers of host membrane resulting in the stunting of parasite growth. However, type I strains of T. gondii (type I) are largely resistant to ATG mediated growth restriction. Here we interrogated the genetic differences in autophagy effector recruitment between the resistant type I (GT1-SNF) strain and the susceptible type III (CTG-ARA) strain of T. gondii using 34 unique progeny isolated from a genetic cross (IxIII). Two quantitative trait loci (QTL) associated with autophagy effector recruitment phenotype were identified, one on Chr. II (LOD 3.35) and a second on Chr. VIII (LOD 4.05). We used a directed CRISPR screen to evaluate a priority set of genes identified within the Chr.VIII peak to determine their role in ATG recruitment in human cells. Our findings suggest a model whereby differential recognition of ASP5- dependent exported PV membrane protein(s) is responsible for strain- dependent recognition by the ATG pathway.

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218 - Cells in tandem: A novel essential “basal body” protein in Trypanosoma brucei

Ramanantsalama, Miharisoa Rijatiana MR (UMR5234 CNRS University of Bordeaux, MFP); Sahin, Annelise; Bonhivers, Mélanie; Robinson, Derrick DR; Dacheux, Denis (UMR5234 CNRS University of Bordeaux)

Trypanosoma brucei is a protozoan flagellate parasite responsible for Animal African Trypanosomiasis (Nagana) with related species being responsible for Human African Trypanosomiasis (sleeping sickness). Diagnosis and treatment of trypanosomiasis remain complex and need to be improved for eventual elimination. This parasite has several organelles such as a single mitochondrion, a motile flagellum nucleated from basal bodies and a specialized endo- exocytotic compartment called the flagellar pocket (FP). The FP is an invagination of the plasma membrane and the only site for endo- and exocytosis. A cytoskeletal structure called the flagellar pocket collar (FPC) is localized at the neck of the FP, at the exit site of the flagellum. In 2008 and 2017, our group identified and characterized the two first components of the FPC, TbBILBO1(2) and a partner, TbFPC4(3) respectively. TbBILBO1 is a cytoskeletal protein essential for FP and FPC biogenesis, endocytic activity, repositioning of the new flagellum and cell viability. Here, we demonstrate that an uncharacterized protein Tb1180, localized at the base of the flagellum, interacts with proteins of the FPC complex, including TbBILBO1. By performing in situ gene tagging, we located Tb1180 at the mature/maturing basal body and our analysis indicates that is part of an annular structure. Domain analysis by allele replacement, overexpression and Yeast-Two- Hybrid approaches indicate that the N-terminus domain is sufficient for targeting but also in self-interaction. The interaction with other partners involves the coiled-coils domain. RNA interference knockdown of this protein, in the bloodstream form blocks cytokinesis during the first cell cycle stage resulting cells that are attached in an unconventional manner that we call “cells in tandem effect”. This severe change of the cell morphology leads to cell death demonstrating the essentiality of Tb1180. According to these results and regarding the partial colocalization with TbRP2, a transitional fibres protein, we suggest that Tb1180 is a novel protein of the transition fibres as well as a novel marker of basal body maturation.(1) Bonhivers M, et al, PLoS Biol. 2008 May 6;6(5):e105.(2) Florimond C, et al, . PLoS Pathog. 2015 Apr 14;11(4):e1004844.(3) Albisetti A, et al, PLoS Pathog. 2017 Nov 1;13(11):e1006710. 302

219 - Oligomerization of Anti-malarial drug target PfATP4 is essential for its function

Ramanathan, Aarti A. (Drexel university); Morrisey, Joanne; Mather, Michael W.; Bergman, Lawrence W.; Vaidya, Akhil B (Drexel university)

Plasmodium falciparum P-type cation ATPase (PfATP4) is a Na+/H+ efflux pump crucial for maintaining low cytosolic Na+ concentration in malaria parasites during its intraerythrocytic lifecycle. In recent years, multiple studies have shown PfATP4 to be the target to a vast number of chemical scaffolds including candidate antimalarials Cipargamin (KAE609), SJ733 and PA21A092. By using blue-native gel electrophoresis we found that PfATP4 exists as a higher molecular weight complex. Immunopurification and proteomic studies revealed that the complex is homo-oligomeric in nature and that it is formed co- translationally. Phylogenetic analysis suggests that ATP4 from apicomplexans and chromerids form a distinct class of P-type ATPases having fewer transmembrane helices than in other related pumps. We hypothesized that this truncation of trans-membrane helices necessitates oligomerization for its function. Based on structural analysis, we predicted involvement of p-p interactions between aromatic amino acids within the transmembrane domain to be critical for oligomerization. To test this hypothesis, we mutated three such aromatic amino acids in the last transmembrane helix of PfATP4. The wildtype and the mutated PfATP4 genes were introduced into a P. falciparum line at the attB locus with its endogenous PfATP4 under conditional regulation. Whereas the wildtype PfATP4 expressed from the attB locus was able to form distinct oligomers, the mutant PfATP4 did not. Strikingly, unlike the wildtype, the mutant PfATP4 failed to functionally complement the knockdown of the endogenous gene, leading to parasite demise. These results strongly suggest that proper functioning of PfATP4 requires co-translational oligomerization of the protein, pointing to yet another potential means to interefere with parasite physiology.

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220 - Plasma membrane is repaired by acid sphingomyelinase in E. histolytica

Ramirez-Montiel, Fatima (Universidad de Guanajuato) Mendoza-Macias, Claudia; Andrade-Guillen, Sairy; Rangel-Serrano, Angeles; Paramo-Perez, Itzel; Rivera-Cuellar, Paris (Universidad de Guanajuato); España-Sanchez, Liliana (Centro de Investigación y Desarrollo en Electroquímica); Anaya-Velazquez, Fernando; Franco, Bernardo; Padilla- Vaca, Felipe (Universidad de Guanajuato)

The host-amoeba relationship is based on a series of interplays between host defense mechanisms and parasite survival strategies. While host cells elaborate diverse mechanisms for pathogen expulsion, E. histolytica trophozoites have also developed complex strategies to counteract host immune response and facilitate its own survival while confronting host defenses; which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Secreted aSMase activity detected was stimulated by Mg+2 and inhibited by Co+2. Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase in the secreted aSMase activity. Streptolysin O form pores in the plasma membrane of E. histolytica trophozoites that initiates the uncontrolled entry of Ca2+, recognized as the primary trigger for cell responses which favors the migration of the lysosomes to the periphery of the cell, fuses with the PM and release their content, including aSMase to the external side of the cell. The secreted aSMase favoring the internalization of the lesion for its degradation in phagolysosomes. During the early stages of PM damage, the pores are rapidly blocked by patch-like structures that prevent the lysis of the trophozoite and immediately begin internalizing the lesion. Overall, these novel findings show that the aSMase6 enzyme from E. histolytica promotes the repair of the PM damaged to prevent losing cell integrity that could act when encountered with the lytic defense systems of the host.

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221 - PfGCN5, a global regulator of stress responsive genes, modulates artemisinin resistance in Plasmodium falciparum

Rawat, Mukul (Indian Institute of Science Education and Research, Pune) Kanyal, Abhishek; Sahasrabudhe, Aishwarya (Indian Institute of Science Education and Research); Vembar, Shruthi S. (Institute for Bioinformatics and Applied Biotechnology); Lopez-Rubio, Jose-Juan (Dynamique des Interactions Membranaires Normales et Pathologiques); Karmodiya, Krishanpal (Indian Institute of Science Education and Research)

Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinins, which threatens malaria control and elimination strategies. Oxidative stress and protein damage responses have emerged as key players in the generation of artemisinin resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress responsive and multi-variant family genes in poised state and regulates their expression under stress conditions. We have also provided biochemical and cellular evidences that PfGCN5 regulates stress responsive genes by acetylation of PfAlba3. Furthermore, we show that upon artemisinin exposure, genome-wide binding sites for PfGCN5 are increased and it is directly associated with the genes implicated in artemisinin- resistance generation like BiP and TRiCchaperone. Moreover, inhibition of PfGCN5 in artemisinin-resistant parasites, Kelch13 mutant, K13I543T and K13C580Y (RSA~ 25% and 6%, respectively) reverses the sensitivity of the parasites to artemisinin treatment indicating its role in drug-resistance generation. Together, these findings elucidate the role of PfGCN5 as a global chromatin regulator of stress-responses with potential role in modulating artemisinin drug resistance, and identify PfGCN5 as an important target against artemisinin resistant parasites.

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222 - Evaluation of WR99210 from different manufacturers for selection of transfected Plasmodium falciparum

Remcho, T. Parks. Liu, Anna; Wellems, Thomas E.; Lane, Kristin D. (NIAID/NIH)

Prior to the 1997 report of its use for selection of P. falciparum transformants, WR99210 had entered pre-clinical trials as a potent antimalarial compound with nanomolar efficacy. Because of its specificity in targeting dihydrofolate reductase (DHFR) of P. falciparum and not the human enzyme, WR99210 was soon widely employed for the selection of parasite lines transformed with plasmid constructs expressing human DHFR. Early studies sourced WR99210 from Walter Reed Army Institute of Research directly, and subsequent studies obtained the compound from Jacobus Pharmaceuticals (JP) or, more recently, Sigma Aldrich (SA). Effective compound concentrations (EC50s) can vary depending on source. Indeed, following relatively ineffective selections of transformants with WR99210 from SA, we established that P. falciparum DD2 parasites survived constant drug pressure of 2nm WR99210 from SA, but not from JP. Further testing indicated greatly different EC50s of 0.32nM vs. 547nM for the JP- and SA-sourced WR99210, respectively. Potential causes of these discrepancies are being investigated. Assured and effective sources of WR99210 will be vital to the continued utility of plasmid constructs and transfection methods developed over the past 20 years.

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223 - RNA secondary structure determination of translationally repressed transcripts in P. falciparum gametocytes

Rios, Kelly T. (Pennsylvania State University) Lindner, Scott E. (Pennsylvania State University)

In Plasmodium falciparum female gametocytes, hundreds of transcripts are translationally repressed, including pfs25 and pfs28, and translational repression is mediated by RNA-binding proteins (RBPs), including PfPuf2. How specific transcripts are selected by RBPs for preservation in the female gametocyte is not well understood, and a conserved sequence motif has not been identified within those transcripts. PfPuf2 binds to a single-stranded, U-rich sequence called a Puf-binding element (PBE) that is present in ~20% of Plasmodium transcripts globally, but not all transcripts that contain PBEs are translationally repressed (e.g. housekeeping genes). Two PBEs are present in the 5’ UTR of pfs25, but only one of those elements is sufficient for translational repression in an in vitro reporter translation assay. We hypothesize that mRNA secondary structure contributes to the selectivity of RBPs for transcripts for translational repression, and that structure is involved in modulating the accessibility of PfPuf2 to its binding sites. To test this experimentally, we are determining the global, in vivo DMS reactivity profile of RNAs in wild-type and pfpuf2-parasites using Structure-seq, a method that chemically modifies on the Watson- Crick face of non-base paired adenosine and residues. To improve the quality of sequencing runs, we have also adapted an RNaseH-based method for ribosomal RNA depletion. This method, along with minimal free energy predictions, allows a comparison of functional and non-functional PBEs, like those in the 5’ UTR of pfs25, as well as the identification of putative, cis-regulatory RNA structures.

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224 - Quantifiable imaging-based physiological parameters to define chronic Toxoplasma infections in vivo

Watts, Elizabeth (University of Kenucky College of Medicine); Murphy, Robert; Dhara, Animesh (University of Kentucky College of Medicine); Patwardhan, Abhijit (University of Kentucky College of Enginnering)

Symptomatic toxoplasmosis associated with immune suppression is typically triggered by the reactivation of bradyzoites within tissue cysts. Despite their central role in pathogenesis, little is known about the basic biology of bradyzoites. Our work (Watts et al. mBio 2015) was the first to definitively demonstrate that bradyzoites within tissue cysts in vivo are dynamic replication competent entities. Our ability to quantify the intracyst bradyzoite burden (BradyCount 1.0), capture replicating parasites (TgIMC3 staining) and define the relative recency of replication (TgIMC3 intensity) establish that tissue cysts are heterogeneous as are the physiological properties of the bradyzoites they house. We further confirm that this heterogeneity is evident with regard to mitochondrial activity and the accumulation of amylopectin granules (AG). With this study, we aim to develop a new standard to assess the progression of the chronic Toxoplasma infections and to translate this approach to the assessment of existing and future drugs in vivo. Toward this end, we are developing BradyCount 2.0 to morphologically quantify nuclear profiles and bradyzoite TgIMC3 scaffolds (as a markers of active and past replication), mitochondrial profiles and the distribution of AG as reporters of energy and intermediary metabolism. By recording and integrating multiple parameters on individual encysted bradyzoites, we will establish a framework to define the chronic infection and the impact of drug intervention employing quantifiable physiological metrics. These inputs will be used to refine a Markov Chain-based data-driven computational model to understand this enigmatic phase, for which effective treatments are lacking. These studies will greatly improve the sensitivity of testing of both existing and future drugs while providing crucial mechanistic insights.

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225 - Characterization of a Novel Protein Phosphatase 1 Complex Involved in RNA Polymerase II Transcription Termination in Kinetoplastids

Zhang, Yang

O-linked glucosylation of thymine in DNA (base J) represents an epigenetic mark regulating Pol II transcription termination within polycistronic gene clusters in Leishmania and T. brucei. While the mechanism of Pol II transcription termination in kinetoplastids is unknown, the loss of base J led to read-through transcription at termination sites. To elucidate the role of base J in termination, we identified a base J binding protein (called JBP3) in a complex with PP1 protein phosphatase, a putative PP1 interactive-regulatory protein (PIP) and Wdr82 in Leishmania. Transcription termination in humans involves dephosphorylation of the CTD of Pol II by PP1 as part of a similar multimeric complex where the PIP homologue binds PP1 via a conserved RVxF motif, regulates PP1 activity and acts as a scaffold for the entire complex. This is consistent with our findings that ablation of PIP, Wdr82, or JBP3 by RNAi in T. brucei leads to read-though transcription at transcription termination sites. To understand the mechanism of termination in kinetoplastids, we are characterizing the nature of the PJW/PP1 complex. PIP deletion analysis indicates JBP3 and Wdr82 bind the C-terminal domain of PIP. While the purified complex from Leishmania has in vitro protein phosphatase activity, the T. brucei complex lacks phosphatase activity and PP1 by MS, suggesting possible differential PP1-PIP interactions in different organisms. This is supported by the observation that TbPIP is phosphorylated in vivo and presumably regulates its interaction with PP1. We are currently exploring the role of the RVxF motif as the PP1 interactive domain in PIP and the role of PP1 in termination. The results from these experiments will be discussed.

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226- Show me your ID and come in - Targeting of the lysosomal degradome in Trichomonas vaginalis

Zimmann, Nadine (BIOCEV); Rada, Petr; Hrdý, Ivan; Tachezy, Jan (BIOCEV)

Among sexually transmitted human pathogens, Trichomonas vaginalis is the most common non-viral agent. In the vagina, it feeds on the vaginal microbiota as well as on epithelial cells and erythrocytes through phagocytosis. Subsequent destruction occurs in lysosomes, eukaryotic organelles with a low pH.and a set of hydrolases that not only degrade phagocytosed material, but also cargos that enter the cell through secretory, endocytic, and autophagic pathways. Lumenal ‘degradome’ proteins possess one or more intrinsic targeting sequences (LTS) that ensure their lysosomal localization, out of which the motif Asn-X-Ser/Thr is the most common.Here we investigated the composition of T. vaginalis lysosomes and the lysosomal targeting of a selected Cathepsin L-like cysteine peptidase.Initially, we identified the gene for Rab7, a potential lysosomal marker, subcloned it and verified its localization through immunofluorescence (IF) microscopy. Next, lysosomes were isolated with two different approaches. The first approach was based on differential and 45% Percoll gradient centrifugation. The second approach made use of magnetic Dynabeads coated with FITC-labeled lactoferrin which led to phagocytosis of the beads. The phagocytosed beads were then separated from non-lysosomal cell compartments. The analyses revealed 49 putative lysosomal proteins. The best candidates were chosen to confirm their cellular localization by IF. Finally, a Cathepsin L-like cysteine peptidase that possesses two putative LTS was selected to further examine how it is targeted to the lysosomal compartment. We therefor substituted the asparagine and serine/threonine in both LTS to serine, alanine, glutamine, and lysine, respectively, and expressed the mutant protein with C-terminal hemagglutinin tag. Subsequent IF microscopy showed that the mutant protein no longer localizes to lysosomes after any of the substitutions, indicating the asparagine based motif to be essential for lysosomal localization. 310

227 - Dissecting the role of plasmepsin II and III in piperaquine resistant P. falciparum lines

Walsh, Breanna (Harvard T.H. Chan School of Public Health); Summers, Robert; Volkman, Sarah; Wirth, Dyann (Harvard T.H. Chan School of Public Health); Bopp, Selina (Harvard T.H.Chan School of Public Health)

Loss of efficacy of dihydroartemisinin (DHA)/piperaquine (PPQ) has been reported in Southeast Asia. We have described a bimodal growth response to increasing PPQ concentrations in PPQ resistant P. falciparum isolates from Cambodia. We chose the area under the curve (AUC) instead of the conventional half-maximal effective concentration (EC50) used in drug assays to quantify the bimodal response. Cloned parasites of a single Cambodian isolate varied only in their plasmepsin II and III copy numbers but not in other loci associated with PPQ resistance such as pfcrt or pfmdr1 copy numbers assessed by whole genome sequencing. To understand the influence of plasmepsins on the bimodal phenotype we generated plasmepsin II, plasmepsin III and plasmepsin II/III combination KO lines. Our data demonstrate that a reduction in either plasmepsin II or III decreases the AUC compared to the parental line. To understand if increased plasmepsin copy numbers could protect from hemoglobin catabolism disruption by PPQ we analyzed Fe fractions (hemoglobin, free heme and hemozoin) of parasite lines with different plasmepsin copy numbers. While there was a significant increase in freee heme upon PPQ treatment indicating inhibition of hemozoin formation by PPQ, there was no significant difference between parasites with different plasmepsin copy numbers. In contrast, increaseing extracellular pH or changing homeostasis of the food vacuole with pH modulators did reduce survival under high PPQ exposure. We demonstrate a functional role of plasmepsins in the response to PPQ and identify a contribution of pH homeostasis in the food vacuole.

311

228 - An intravenous preparation of the antimalarial OZ439 for nonhuman primate laboratory studies

Sa, Juliana M. (NIAID) Salzman, Rebecca E. (NIAID); Melendez- Muniz, Viviana A. (NIAID); Figan, Christine E.; Moraes-Barros, Roberto R. (NIAID); Burrows, Jeremy (Medicines for Malaria Venture); Walker, Larry (Campbell University); Wellems, Thomas E. (NIAID)

Malaria cases have been significantly reduced since widespread use of artemisinin-based combination therapies (ACTs). ACT combines the fast and strong antimalarial activity of an artemisinin derivative compound (ART), which typically has a short plasma half-life, with a long-lasting partner drug to eliminate residual parasites not killed by ART. OZ439 is a synthetic ozonide compound that shares with ART derivatives an endoperoxide moiety, but it was developed to have a longer plasma half-life than ART and to be delivered as a single dose, rather than three daily doses. OZ439, is highly hydrophobic and difficult to dissolve, with limited validated effective preparations, and its oral bioavailability to nonhuman primates can be unreliable. Here we describe a micelle-based delivery system for OZ439 intravenous (IV) administration to Aotus monkeys. Plasma levels measured in uninfected animals treated with a single 10 mg/kg dose of OZ439 were comparable to those reported from humans who received the drug by oral administration. P. falciparum infected monkeys presenting recrudescence after a 3-day 4 mg/kg AS IV treatment were given a single 10 mg/kg dose of OZ439. Parasite clearance half-lives (t1/2s) of the OZ439-treated recrudescences were similar to the primary AS treatment t1/2s, and parasites also recrudesced after this monotherapy treatment. Micelles may support effective single-dose malaria treatment by IV administration, which can now be further investigated in Aotus.

312

229 - A novel Plasmodium yoelii serine threonine kinase related to exflagellation center formation

Ishizaki, Takahiro (The Institute of Tropical Medicine (NEKKEN), Nagasaki University); Chaiyawong, Nattawat; Asada, Masahito; Yahata, Kazuhide; Culleton, Richard; Kaneko, Osamu (The Institute of Tropical Medicine (NEKKEN), Nagasaki University)

Malaria parasites proliferate by repeated invasion of and multiplication within erythrocytes in the vertebrate host. During asexual proliferation, some of the sexually committed parasites undergo sexual stage differentiation and gametocytes circulate in host peripheral blood until mosquito ingestion. Once gametocytes are fed by mosquito, male gametocytes are egressed from host erythrocytes and activated microgametes start exflagellation for fertilization. Some kinases were known as multi-functional regulator to control critical parasite life cycle, however un-characterized kinases are still remained. Here, we focus on a novel serine threonine kinase (STK) in Plasmodium yoelii that is highly expressed in schizonts and male gametocytes. First, we tagged endogenous STK with Myc epitopes or deleted this gene locus by CRISPR/Cas9 method (?STK parasites). Immunofluorescent assay for Myc epitope-tagged parasites revealed this STK was partially colocalized with AMA1 and MTIP in schizonts. ?STK parasites showed significant growth defect and infected mice survived significantly longer. Egress ability was not changed, whereas, invasion ability was significantly decreased ?STK parasites. We found this STK is also functional at gametocyte stage. There were no clear phenotypes in male and female gametocytemia and egress from erythrocytes after activation with xanthurenic acid. However, the number of exflagellation center and oocyst number in the mosquito midgut were significantly decreased in ?STK parasites. Together, we revealed that this STK is involved in parasite erythrocyte invasion and exflagellation of male gametocytes. These data indicate that this STK phosphorylate molecules involved in the upstream events of erythrocyte invasion and exflagellation.

313

230 - The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development

Devendra Kumar Gupta1, Laurent Dembele1, Annemarie Voorberg Vander- wel2, Stefan H. I. Kappe3, Erika L. Flannery1, Sebastian A. Mikolajczak1, Pablo Bifani1, Clemens H. M. Kocken2, Thierry T. Diagana1 1Novartis Institute for Tropical Diseases 2Biomedical Primate Research Centre 3Center for Infectious Disease Research

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Little is known about the regulation of dormancy and hypnozoite activation. Here, we characterize the Plasmodium Liver-Specific Protein 2 (LISP2) as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

314

231 - G-quadruplex interactome in Plasmodium falciparum

Gurung, Pratima (LPHI, University of Montpellier); Gazanion, Elodie (Université de Montpellier); Gomes, Ana-Rita; Guitard, Vincent (LPHI, University of Montpellier); Paeschke, Katrin (University Clinic Bonn Biomedical Center (BMZ)); Alberti, Patrizia (Nucleic Acid Structures, Telomeres and Evolution,MNHN,CNRS,UMR 7196); Urbach, Serge (BioCampus Montpellier, CNRS UMR 5203); Lopez-Rubio, Jose-Juan (LPHI, University of Montpellier)

Guanine-quadruplex (G4) are non-canonical DNA structures that can regulate biological processes such as transcription, replication and telomere maintenance. In the AT-rich genome of Plasmodium falciparum they are enriched in the telomeric and sub-telomeric regions. They are associated with recombination events in virulence genes and with telomere maintenance. However, there is a lack of experimental evidence of their biological role and their interacting proteins.We here provide the first snapshot of the G4 interactome obtained from the combination of two complementary approaches: yeast one hybrid and DNA pulldown, followed by LC-MS/MS. Using a validated stable G-quadruplex probe as bait, we identified ~61 putative G4 interacting proteins. In other organisms, the orthologs of most of our candidates were previously shown to interact with G4s, thus strengthening our results. We have chosen three candidate genes in order to explore how they contribute to G4-mediated regulatory processes: Fen1, GBP2 and DNAJ. These are predicted to be involved in maintaining genome stability. We are now studying the G4 binding affinities by EMSA of our candidates, and exploring their role in parasite gene regulation using a combined CRISPR/Cas9-DiCre conditional gene knockout approach. Overall, this study will shed light on this understudied G4 regulatory mechanism in malaria parasites.

315

232 - Translocation of dense granule effectors across the parasitophorous vacuole membrane in Toxoplasma-infected cells requires the activity of ROP17, a rhoptry protein kinase.

Panas, Michael W. (Stanford University); Ferrel, Abel; Naor, Adit (Stanford); Tenborg, Elizabeth (UC Davis); Lorenzi, Hernan (JCVI); Boothroyd, John (Stanford)

Toxoplasmagondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing 3 components, MYR1, MYR2 and MYR3. To determine if other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host-cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and, thereby, inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM, but that translocation can be rescued “in trans” by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17’s role in regulating GRA translocation is carried out on the host- cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction.

316

233 - Comparative transcriptomics of three lizard Plasmodium species

Pangburn, Sarah (CUNY Graduate Center); Borner, Janus; Perkins, Susan (American Museum of Natural History)

The malaria parasites (Plasmodium spp.) are a polyphyletic group within the haemosporidian genera, which infect a range of vertebrate hosts including humans, other mammals (bats, rodents, apes), and sauropsids (birds, lizards). Recent phylogeny work indicates that mammalian Plasmodium resulted from a divergence from a sauropsid malaria ancestor, which was followed by a switch back to the sauropsid hosts. Studying sauropsid Plasmodium parasites is advantageous in that it can tell us more about the biological method of infection in wild hosts, but still holds relevance to human infective malaria due to the evolutionary relationship between mammalian and sauropsid Plasmodium. , P. azurophlium, and P. leucocytica infect the lizard , which is endemic to Saba island in the eastern Caribbean. These three parasites are ideal study organisms due to their unique biological mechanisms and niche partitioning within the host. Plasmodium floridense infects red blood cells and produces hemozoin as a byproduct of hemoglobin digestion, both of which are typical features of Plasmodium species. Conversely, Plasmodium azurophlium infects red blood cells but does not produce hemozoin, and P. leucocytica infects white blood cells (and, therefore, does not produce hemozoin). Comparative transcriptomic analyses will help elucidate the metabolic pathways utilized by these parasites during infection. Broader implications of this study include baseline data about the molecular mechanisms of infection that may be applied to future studies of disease prevention in human-infective malaria.

317

234 - Cellular signaling in the regulation of Giardia cyst formation

Paredez, Alexander (University of Washington); Shih, Han-Wei; Alas, Germain (University of Washington)

Many eukaryotes, pathogenic and free-living alike, encyst during their life cycle. Major morphological changes occur as Giardia parasites transition from trophozoites to infectious cysts. Among these changes are flagellar retraction and adhesive ventral disc disassembly, which causes the parasites to release and be cleared from the host intestine. Therefore, the encystation pathway has the potential to be exploited for therapeutic development. We previously published that GlRac, Giardia’s sole Rho family GTPase, has a role in regulating encystation. Constitutive GlRac signaling reduced Cyst Wall Protein1 (CWP1) production, the authoritative marker for Giardia differentiation. This defect in encystation can be rescued by modulating cyclic adenosine monophosphate (cAMP) levels. Cyclic adenosine monophosphate (cAMP) is a second messengers that regulates differentiation in many protists and parasites. Our data implicates cAMP signaling as part of the signaling pathway that regulates Giardia differentiation and GlRac appears to be an upstream regulator. Current results will be presented.

318

235 - Characterization of a Plasmodium falciparum acetyl-CoA synthetase and targeted discovery of inhibitors using functional genomics

Pasaje, Charisse Flerida A. (Massachusetts Institute of Technology); Cárdenas Ramirez, Pablo (Massachusetts Institute of Technology); Hollin, Thomas; Abel, Steven (University of California, Riverside); Summers, Robert L. (Harvard T.H. Chan School of Public Health); Lukens, Amanda K. (Harvard T.H. Chan School of Public Health, Boston.); Vanaerschot, Manu; Murithi, James (Columbia University Irving Medical Center); Ruth, Madeline R.; Ottilie, Sabine (University of California, San Diego); Le Roch, Karine (University of California, Riverside.); Fidock, David A. (Columbia University Irving Medical Center, New York); Winzeler, Elizabeth A. (University of California, San Diego); Wirth, Dyann F. (Harvard T.H. Chan School of Public Health); Niles, Jacquin C. (Massachusetts Institute of Technology)

Acetyl-CoA is an essential metabolite at key junctions of cellular and metabolic processes including gene regulation through protein acetylation. One route to producing this cofactor in the human malaria pathogen, Plasmodium falciparum, is via direct synthesis from acetate catalyzed by the parasite’s acetyl-CoA synthetase, PfAcAS (PF3D7_0627800). To study PfAcAS, we used CRISPR-Cas9 to introduce a C-terminal epitope tag and a TetR-DOZI- RNA aptamer module to conditionally regulate PfAcAS expression levels. We determined that PfAcAS is essential for parasite growth, and its knockdown results in multiple defects across the intra-erythrocytic developmental cycle including: stalling of development at trophozoite stage; compromised daughter cell segmentation; and failure to egress from the host red blood cell. PfAcAS predominantly localizes to the nucleus, but redistributes to nuclear-associated punctate structures in late stages, suggesting a potential role in acetylation of nuclear proteins such as histones involved in gene regulation. Amongst other systems- level approaches, we are using developmental stage-dependent transcriptomic analyses to determine whether PfAcAS knockdown impacts global gene expression. Interestingly, evolution of resistance and whole-genome sequencing identified several compounds as potential inhibitors of PfAcAS. Using a phenotypic screening platform based on our conditional gene regulation technology facilitated further verification of these compounds as specific PfAcAS inhibitors. Altogether, these findings demonstrate that PfAcAS is critical for proper blood stage parasite development and a promising antimalarial target.

319

236 - Quantitative method for PK/PD studies of DHODH inhibitors in vivo

Pontikos, Michael; Leija, Christopher; Williams, Noelle S.; Phillips, Margaret A. (University of Texas Southwestern Medical Center)

Malaria remains a significant cause of morbidity and mortality in tropical and subtropical regions of the world, with the most severe consequences found in developing countries. Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the de novo biosynthesis pathway of pyrimidines, catalyzing the oxidation of dihydroorotate to orotate. DHODH inhibitors are clinically in use for treatment of rheumatoid and psoriatic arthritis and multiple sclerosis. Malaria lacks the salvage pathway for pyrimidines; thus, the parasite relies on the de novo pathway to meet its needs for pyrimidine synthesis. The DHODH inhibitor DSM265 was identified as a potent antimalarial compound by our group, validating DHODH as a target for the treatment of malaria. DSM265 has shown good efficacy against P. falciparum malaria in Phase II clinical trials in patients and also the potential to act as a chemopreventive agent in Phase I studies using a sporozoite challenge model. DSM265 has low nanomolar activity against Plasmodium DHODH and strong selectivity against the human enzyme. However, it inhibits rodent DHODH in the micromolar range, complicating the use of mouse and rat preclinical models. In order to assess species differences in on-target toxicity between mammalian hosts, we developed a liquid-chromatography/mass spectrometry method for monitoring the effects of DHODH inhibitors on endogenous DHODH. We first tested the approach in vitro against cell lines from different mammalian species. Dose- dependent increases in dihydroorotate were seen in both human and mouse cell lines when treated with the known mammalian DHODH inhibitor A77 1726 (the active metabolite of leflunomide). To obtain an in vivo proof of concept, mice were dosed with leflunomide or a vehicle control and blood and urine were collected over a period of four days. Dihydroorotate concentrations increased 100- and 6000-fold in blood and urine respectively in the leflunomide treated mice verses the vehicle treated mice. The advantages of using urine as the biomarker matrix cannot be understated, especially in the developing world: its collection is non-invasive, does not require specialized training or costly equipment, and is produced in large quantities. In this study, there is the additional advantage that due to the role of the kidney in maintaining blood homeostasis, larger differences in biomarker levels are seen in the urine than in the blood. This study validates the approach of using upstream metabolites of DHODH as biomarkers for DHODH inhibition. This study was funded by Medicines for Malaria Venture. 320

237 - The impact of organelle dynamics on mitochondrial structure and function in kinetoplastids

Povelones, Megan L. (Penn State Brandywine); DiMaio, John (Penn State Brandywine); Ruthel, Gordon (University of Pennsylvania); Malfara, Madeline F.; Iatsenko, Ekaterina; Pitts, Joseph; Jackson, Jasmine (Penn State Brandywine)

Kinetoplastids and apicomplexans have a single mitochondrion. The distinctive shape of this organelle dynamically changes during the cell and life cycle. In other eukaryotic cells, mitochondrial shape is regulated by fusion and fission events called mitochondrial dynamics. These processes are essential for organelle function as well as the faithful distribution of mitochondrial contents at cell division. We have quantitatively defined distinct mitochondrial shapes that correlate with the cell cycle of the mosquito kinetoplastid, Crithidia fasciculata. We have also used live-cell imaging to demonstrate that the mitochondrion is dynamic, including both fusion and fission of tubules. Using RNAseq, we have compared transcript abundance in cultured swimming and adherent C. fasciculata, and found a dramatic regulation of their transcriptome, indicating that these are distinct developmental stages. Genes associated with mitochondrial energy production are enriched in the swimming form, suggesting that, as in T. brucei, mitochondrial metabolism is developmentally regulated. We propose that mitochondrial dynamics maintains the distinct mitochondrial shape of kinetoplastid parasites, ensures equal partitioning of the mitochondrial network in dividing cells, and allows adaptation to different energetic environments. We have evidence that the mitochondrion is also dynamic in T. brucei, in which we can use genetic tools to evaluate the effect of predicted mitochondrial dynamics proteins on remodeling. To further explore mitochondrial dynamics in kinetoplastids, we are developing fluorescent markers for other compartments, including the outer mitochondrial membrane and the ER. Through these studies, we hope to provide insight into the conservation and evolution of mitochondrial dynamics in eukaryotes.

321

238 - Identification of a mechanism of resistance to itraconazole in Toxoplasma gondii

Alday, Phil H. (VA Portland Healthcare System); Zhicheng, Dou (Clemson University); Carruthers, Vern (University of Michigan Medical School); Doggett, Joseph S. (VA Portland Medical Center)

Itraconazole is a triazole antifungal in widespread clinical use that has a low-nanomolar IC50 against T. gondii in vitro and reduces brain cyst burden in a mouse model of toxoplasmosis. In both fungi and trypanosomes, itraconazole inhibits the enzyme lanosterol 14 alpha- demethylase. This cytochrome P450 catalyzes a key step in the synthesis of ergosterol, an important component of fungal and trypanosomal cell membranes. The target of itraconazole in T. gondii is unknown. We present evidence that the single cytochrome P450 encoded in the Toxoplasma genome is not the target of itraconazole, and that itraconazole interacts with a novel protein in T. gondii. We generated itraconzole-resistant clonal strains of T. gondii by chemical mutagenesis and clonal selection at 10x itraconazole IC50. Comparing whole-genome sequencing data from parental and resistant strains yielded 19 nonsynonymous single-nucleotide variants (SNVs) across 14 genes in the resistant strain. Only one of these genes, TGGT1_281440, has a CRISPR screen score indicating a significant contribution to fitness. We identified a V3414E mutation in this clone and subsequently confirmed this mutation in two other clones produced in separate mutagenesis experiments. Additionally, we discovered a nearby M3412K mutation in another resistant clone. TGGT1_281440 is predicted to encode a 390kD transmembrane protein of unknown function that localizes to the mitochondrion. Structural modeling with I- TASSER predicts the formation of a beta-barrel in the C-terminal region of the protein. A similar beta-barrel structure has been found in the human voltage-dependent anion channel 1 (VDAC1), an outer mitochondrial membrane protein that also binds itraconazole. These results indicate that itraconazole interacts with a novel target in T. gondii and have important implications for future optimization of triazoles as treatments for toxoplasmosis.

322

239 - Understanding how var2csa uORF signals for cellular localization in Plasmodium falciparum

Assaraf, Shany (Hebrew university) Fastman, Yair; Dzikowski, Ron (Hebrew university)

Pregnancy-associated malaria (PAM) caused by the protozoan parasite Plasmodium falciparum results from placental sequestration of iRBCS expressing a unique PfEMP1 that adhere to chondroitin sulfate A (CSA) and therefore named VAR2CSA. Among the var gene family, var2csa is the only gene that contains an upstream open reading frame (uORF) which was shown to be involved in the translation regulation of the downstream VAR2CSA. We previously found that the peptide encoded by the uORF signals for cellular localization of the downstream protein to the ER. To better understand the mechanisms by which the uORF signals for cellular localization we performed functional deletion analysis, using a uORF-GFP ectopic expression plasmid and identified an 8 aa sequence essential for its cellular localization. Preliminary Co-IP experiments of parasites overexpressing uORF-myc plasmid followed by mass spectrometry indicate that the uORF peptide interacts with many proteins which participate in the cellular trafficking machinery. We currently expand our study to investigate the role of RNA binding proteins in regulation of transcript containing uORFs.

323

240 - Elucidating the nutrient acquisition strategies of Cryptosporidium.

SOMEPALLI, MASTANBABU (University of Pennsylvania); Berry, Laurence (Université de Montpellier); Striepen, Boris (University of Pennsylvania)

The apicomplexan parasite Cryptosporidium is a major cause of diarrheal morbidity and mortality worldwide. Despite this enormous global burden, effective treatment options are lacking and the frontline drug nitazoxanide has limited efficacy in the most susceptible individuals. Thus, there is an urgent need for developing safe and efficacious treatments. The nutrient acquisition is a fundamental aspect of host-pathogen interactions and is a potential target for anticryptosporidial agents. Genome sequencing studies have revealed Cryptosporidium has minimal anabolic capabilities and relies on a repertoire of transporters (>100) to scavenge nutrients from the host cells. Several studies have demonstrated that the intracellular stages of Cryptosporidium possess a feeder organelle at the host-parasite interface, which is believed to be involved in nutrient uptake from the host cells. To understand the Cryptosporidium nutrient acquisition mechanisms, we identified a putative hexose transporter through BLAST searches. The CpHT1 (hexose transporter) belongs to the Major Facilitator Superfamily class of integral membrane proteins that mediates uptake of sugars. The CpHT1 encodes a 479 amino acid polypeptide with a signal peptide and 12 transmembrane domains. To investigate further, we generated transgenic C. parvum parasites expressing CpHT1-HA fusion protein using the CRISPR/Cas9 system. Subcellular localization studies using super-resolution microscopy and immuno-electron microscopy showed that CpHT1 is expressed through all the intracellular stages and is exclusively localized to the feeder organelle at the host-parasite interface. Attempts to knockout the cpht1gene were unsuccessful indicating it is likely essential for the parasite growth and development. These results provide evidence that the CpHT1 is located at the host-parasite interface during the course of intracellular development and may play a critical role in sugar uptake from the host cells. 324

241 - Ablation of the TSSA (Trypomastigote Small Surface Antigen) gene causes infection impairment in Trypanosoma cruzi trypomastigotes

Balouz, Virginia (Institute for Research in Biotechnology); Cámara, María de M; Rodriguez, Matías E. (Institute for Research in Biotechnology); Cruz-Bustos, Teresa (Center for Tropical and Emerging Global Diseases (CTEGD)); Masip, Yamil E. (Institute for Research in Biotechnology); Berná, Luisa (Institut Pasteur de Montevideo); Burasi, Florencia; Lobo, Maite; Centeno Camean, Camila (Institute for Research in Biotechnology); Robello, Carlos (Institut Pasteur de Montevideo); Docampo, Roberto (Center for Tropical and Emerging Global Diseases (CTEGD)); Tekiel, Valeria; Buscaglia, Carlos A. (Institute for Research in Biotechnology)

Trypanosoma cruzi is the etiological agent of Chagas disease. TSSA (Trypomastigote Small Surface Antigen) is encoded by a multicopy mucin-like gene showing polymorphisms among parasite isolates. These polymorphisms correlate with differential antibody responses in T. cruzi-infected humans and differential adhesion towards non-macrophagic cell monolayers. The TSSA variant present in TcII, TcV and TcVI DTUs has been characterized as a parasite adhesin, engaging surface receptor(s) and inducing signaling pathways on the host cell as a prerequisite for trypomastigote internalization. Most interestingly, trypomastigotes over-expressing TSSA displayed improved adhesion and infectivity towards non-macrophagic cell lines in vitro. To get further insights into the functional significance of TSSA, we applied CRISPR/Cas9 technique in the RA strain (TcVI) to obtain TSSA-KO parasites. After antibiotic selection, epimastigotes were cloned and genotypified by PCR and PacBIO WGS. Clones of interest, i.e. those bearing partial or complete tssa gene tandem ablation, were cycled in vitro and the expression of TSSA protein in trypomastigotes was assessed by Western blot, IFA and flow cytometry. TSSA-KO clones were significantly less infective in non-macrophagic cell monolayers and in a three-dimensional cell array as compared to control cell lines (wild-type and/or TSSA-partial KO) in in vitro infection assays. Interestingly, TSSA-KO parasites showed an attenuated phenotype in vivo evidenced by a decreased parasitemia and virulence in BALB/c mice as compared to control cell lines. Altogether, our results shed new light on the interaction between T. cruzi and the mammalian host. Elucidation of this interphase and the molecules and signals involved is essential for the discovery of novel targets of intervention in Chagas disease.

325

242 - Functional mapping of the ap2-g upstream sequence in Plasmodium falciparum

Basson, Travis (Swiss TPH, University of Basel) Voss, Till S. (Swiss TPH, University of Basel)

In Plasmodium falciparum, the switch from the asexual replicating cycle to differentiation into the non-proliferating blood stage forms known as gametocytes is essential for transmission between humans via the mosquito vector. The transcription factor PfAP2-G is the master regulator of sexual commitment. The ap2-g locus is epigenetically silenced by heterochromatin protein 1, which antagonizes sexual conversion in proliferating parasites. ap2-g can be activated by the upstream regulator gametocyte development protein 1, and this process is triggered by deprivation of the host-derived factor lysophosphatidylcholine. Parasites expressing PfAP2-G terminate cyclical asexual replication and undergo gametocyte differentiation instead. We reasoned that regulatory silencer and activation elements situated in the upstream sequence of ap2-g are presumably connected to these alternative states of gene transcription. Here, we used CRISPR/Cas9-based gene editing to generate a series of cell lines where a reporter cassette has been inserted into the dispensable cg6 locus on chromosome 7. These contain ap2-g upstream sequences controlling the expression of the drug-selectable marker blasticidin deaminase fused to green fluorescent protein (BSD-GFP), as well as Nano-Luciferase (N-LUC). In this context, the readouts of the BSD- GFP fusion and N-LUC reporters allow us to investigate ap2-g upstream sequence promoter activity at the population and single cell levels. Altogether, we aim to use this system for the identification of regions of the upstream sequence responsible for establishing the silenced or active states of ap2-g expression. This knowledge could lead to the identification of specific cis-acting elements and regulatory factors implicated in the epigenetic regulation of sexual commitment.

326

243 - Analysis of interacting partners of the nuclear Giardia intestinalis cytochrome b5

Batoff, William

Giardia intestinalis is a waterborne enteric parasite that lacks a fully functional mitochondria and the capacity for heme biosynthesis. Despite this, Giardia encodes a handful of heme proteins, including four cytochrome b5 isotypes (gCYTB5-I – IV) of unknown function. Using isotype-specific antibodies in immunofluorescence microscopy experiments we observed gCYTB5-I in the nucleolus, gCYTB5-II in endocytic vesicles in the cytoplasm, and gCYTB5-III in the nucleus. The nuclear location of gCYTB5-III was supported by immunoblot analysis of crude cytoplasmic and nuclear enriched fractions of Giardia trophozoites. To gain an understanding of the possible roles and partners of gCYTB5-III, immunoprecipitation experiments were performed on lysates from Giardia trophozoites pre-treated with a permeable crosslinker to stabilize protein interactions. While mass spectrometric analysis of the immunoprecipitate identified proteins expected to be in the nucleus (histones, histone modifying enzymes), it also identified proteins that are mitosomal markers (ferredoxin, mitochondrial HSP70) and proteins involved in iron-sulfur cluster assembly (IscS, IscA). Interestingly, GiOR-1, a flavoenzyme that has been shown to mediate electron transfer from NADPH to recombinant Giardia cytochromes [Pyrih, J. et al. Eukaryot. Cell 13, 231–239 (2014)] was also identified. Thus, our immunoprecipitation results provide evidence for GiOR-1/gCYTB5 interactions in vivo. Curiously, although GiOR-1 has been found to localize to the mitosomes [Pyrih, J. et al. Mol. Microbiol. 102, 701–714 (2016)], gCYTB5s were not detected in this organelle [Jedelský, P. L. et al. PLoS One 6, 15–21 (2011)]. These conflicting results suggest that at least one of the cytochromes (gCYTB5-III) is located in the mitosomes, or that some of the proteins associated with the mitosomes may also be present in the nucleus.

327

244 - Plasmodium falciparum Merozoite surface proteome and Post-Translational Modifications (PTMs)

Saini, Ekta (ICGEB,New Delhi) kaur, Inderjeet (ICGEB.); chauhan, charu; Sampathkumar, Gopalan (National Institute of Immunology); Malhotra, Pawan (ICGEB.)

Malaria accounts for enormous disease burden globally which is predominantly caused by Plasmodium falciparum. Currently, there is no malaria vaccine available. RTS,S, a sporozoite targeting vaccine, has completed three clinical trials in Africa with very moderate efficacy of 29- 36%. An efficacious malaria vaccine remains a long-term struggle. Asexual blood stages of the parasite are responsible for the clinical symptoms of the disease. Several merozoite antigens have been promoted as promising vaccine candidates.Merozoite surface architecture and complexity of the merozoite surface are not well understood. To understand the mechanism of pathogenesis, it is desirable to illustrate the complete process of merozoite invasion of human erythrocyte. The process of merozoite invasion is a complex, multi-step event involving numerous groups of antigens and multiple receptors. This complexity has proved challenging in the generation of an effective malaria vaccine.It is important that PTMs in plasmodium should be investigated as regulatory pathways to provide new insights in vaccine development. The major aim of this study is to illustrate the surface proteome of merozoite. At the onset, we did a complete proteomic analysis of the merozoite, after stripping the surface proteins from the intact merozoites. We next raised antibodies against this complex mixture and studied the potency of this antisera in invasion inhibition assay. We also analysed the reactivity of these antibodies to P. falciparum infected patient sera to identify immunodominant proteins. Further, using proteomics approach, we have identified large number of merozoite surface proteins undergoing Phosphorylation, methylation, acetylation and glycosylation. We evaluated some of these proteins with antibodies present in laboratory to show that RAP1, RAP2, RhopH3 and CLAG9 undergo phosphorylation and methylation. We also show magnitude of these PTMs on merozoite surface by immunofluorescence assay. Furthermore, we wish to investigate the role of PTMs in invasion of host RBC by the merozoite. Together, our results show that proteins of merozoite surface undergo extensive post-translation modifications and might play a role in merozoite invasion of host erythrocytes.

328

245 - Characterizing the Myosin F interactome in Toxoplasma gondii using BioID

Schiano, Irio Heaslip, Aoife T. (University of Connecticut)

Myosin F is an unconventional myosin motor conserved across the apicomplexan phylum. In Toxoplasma gondii, myosin F (TgMyoF) is required for the correct inheritance of the apicoplast, an essential organelle required for lipid synthesis and processing, as well as transport of dense granules, which are secretory organelles that modify the intracellular environment of the host cell. TgMyoF is a class 27 myosin with structural similarity to the canonical cargo transporter Myosin V. In other eukaryotes, this myosin directly interacts with its vesicular cargo through adapter proteins such as melanophilin and Rab GTPases. However, because of the C-terminus differences between TgMyoF and Myosin V, it is unclear how or if TgMyoF interacts with its vesicular cargo. Here we use close proximity biotin labeling (BioID) and LC-MS/MS to identify TgMyoF interacting partners. Endogenous TgMyoF was tagged at its C-terminus with BirA* or a mutant analog, TurboID. These biotin ligases label proteins within ten nanometers of TgMyoF, but at different rates. BirA* and TurboID achieve similar levels of protein biotinylation after 18 hours and 1 hour, respectively. Because of the rate differences between these two proteins, we predict we will identify dynamic interactions that occur on a time scale of minutes to hours using TurboID. Identifying TgMyoF- interacting proteins will provide new insight into the mechanisms of organelle and vesicle transport in Toxoplasma gondii.

329

246 - Purification of the RhopH complex for functional reconstitution of malaria nutrient channels

Schureck, Marc A. (NIAID) Desai, Sanjay A. (National Institutes of Health)

Intracellular malaria parasite development requires host cell remodeling and activation of a nutrient uptake channel on the erythrocyte membrane. This nutrient channel is essential and has been linked to the rhoptry high-molecular weight protein complex (RhopH), but how these proteins contribute to channel formation is unknown. Here, epitope-tagged RhopH members were generated by DNA transfection and used to optimize protein solubilization and purification. We screened detergents and buffers to identify conditions that yield maximal recovery of intact RhopH complexes, as evaluated with native PAGE. Limited proteolysis revealed a well-ordered complex and delineated protease-resistant domains. Reconstitution of the RhopH complex from parasite cultures into model membranes will be described. These studies may conclusively resolve the composition of the parasite nutrient channel, address possible roles of soluble modulators or accessory proteins, and facilitate drug discovery targeting a conserved nutrient uptake mechanism in malaria parasites.

330

247 - Study of reduced artemisinin susceptibility in West African Plasmodium falciparum isolates

Sharma, Aabha I. (Harvard T. H. Chan School of Public Health) Demas, Allison R. (Ragon Institute of MIT and Harvard); Bopp, Selina; Volkman, Sarah K. (Harvard T. H. Chan School of Public Health); Hartl, Daniel L. (Harvard University); Wirth, Dyann F. (Harvard T. H. Chan School of Public Health)

Current emergence of artemisinin resistance in South East Asia threatens the success of artemisinin combination therapies (ACTs) in battling malaria. We sought to investigate artemisinin resistance in the African context through thirteen rounds of dihydroartemisinin (DHA) pulses in West African Plasmodium falciparum resulting in two independent artemisinin resistant parasite lines Pikine_R and Thiès_R. Whole genome sequencing identified ten mutations in seven genes, of which only two loci (pfcoronin Pf3D7_1251200 and a conserved Plasmodium gene of unknown function Pf3D7_1433800) were shared by both the selected parasites. We previously knocked in pfcoronin WD-40 propeller domain mutations in West African parasites and 3D7 laboratory strain to establish their sufficiency in conferring artemisinin resistance measured by in vitro Ring-stage Survival Assay (RSA). To investigate the resistance contribution of Pf3D7_1433800, the other gene of unknown function, we reverted its mutation in Thiès_R (I575M) using CRISPR/Cas9 gene editing. We also reverted pfcoronin mutation in Thiès_R (G50E) for comparison. While reverting pfcoronin mutation significantly reduced RSA survival of Thiès_R, reverting Pf3D7_1433800 mutation did not. Reverting pfcoronin mutations in Pikine_R also significantly reduced its RSA survival, confirming PfCoronin to be both necessary and sufficient for artemisinin resistance in West African parasite background. Additionally, we tested potential resistance synergy between pfkelch13 and pfcoronin by generating double mutant parasites with PfCoronin R100K & E107V mutations and PfKelch13 C580Y in 3D7 laboratory strain. RSA Survival rates of double mutants were similar to PfKelch13 C580Y alone, suggesting no synergy between potentially independent pathways of resistance. Investigation of pfkelch13-independent biological mechanisms of artemisinin resistance will make significant contributions to our currently limited understanding of artemisinin resistance. 331

248 - Extracellular Vesicles secreted by Entamoeba histolytica parasites contain small RNAs targeting specific genes

Sharma, Manu (Stanford University); Morgado, Pedro; Zhang, Hanbang; Ehrenkaufer, Gretchen; Singh, Upinder (Stanford University)

Extracellular vesicles (EVs) are membrane bound vesicles secreted to the external environment by both eukaryotic and prokaryotic cells. EVs can contain lipids, proteins and nucleic acids and have an important role in cell-cell communication through transfer of their cargo. Here we show for the first time the presence of extracellular vesicles in axenic in vitro cultures of Entamoeba histolytica, a unicellular protozoan infecting the gastrointestinal tract. We found that amoebic conditioned medium contains particles consistent with expected size of exosomes (~100 nm). Electron microscopy of the EVs showed a morphology similar to EVs identified in other organisms. Immunoblotting showed the enrichment of amoebic proteins such as light chain of cell surface galactose-/N-acetylgalactosamine binding lectin (Lgl) and tetraspanin (an exosome marker protein) in the EVs. Interestingly, RNAi pathway Argonaute proteins (EhAGO2-2, EhAGO2-3), which bind amebic small RNAs (sRNAs), were also present in EVs. Characterization of the EV proteome by mass spectrometry showed an enrichment of proteins associated with vesicle formation, cell-signaling and metabolism, and cytoskeletal proteins. Analysis of the EV-RNA showed the presence of small RNA in the range of 18-32 nucleotides which were protected against RNase treatment. Previously we had identified endogenous sRNA populations involved in gene silencing in amoeba; sequencing of EV sRNAs showed that a subset of this anti-sense RNA population was also present inside EVs. In summary, our data shows that E. histolyticasecretes EVs that are similar in size and shape to previously characterized exosomes from other organisms and contain a distinct subset of amoebic proteins. The presence of RNAi pathway proteins together with anti-sense RNAs suggests a possible role of EVs in gene silencing mediated inter-cellular communication.

332

249 - Maintenance of cholesterol homeostasis in sexual stages of P. falciparum

Shukla, Anurag Morrisey, Joanne of; Vaidya, Akhil B. (Drexel University College of Medicine)

Previous studies from our laboratory have shown cholesterol homeostasis in asexual stages of Plasmodium falciparum to be critical for parasite survival. Short term exposure to antimalarial compounds that target either PfATP4 (Das et al; PMID: 27227970) or PfNCR1 (Istvan et al; PMID: 30888318) lead to cholesterol accumulation in plasma membrane as judged by saponin sensitivity. These studies also reveal that maintenance of cholesterol homeostasis is an active process mediated by parasite encoded molecular pathways. PfATP4 inhibitors have been shown to be active against sexual stages of P. falciparum therefore we were interested to examine status of cholesterol homeostasis in gametocytes. Gametocytes enriched from the NF54 strain of P. falciparum were exposed to 10X EC50 of PfATP4 inhibitors (PA21A092) or PfNCR1 inhibitor (MMV0009108) for two hours. Subsequent treatment with varying saponin concentration followed by western blotting for cytosolic protein aldolase permitted assessment of cholesterol content in gametocyte plasma membrane. Treatment with these compounds resulted in increased saponin sensitivity of gametocytes as judged by reduction of parasite associated aldolase. These results are consistent with the proposition that, as in asexual stages, gametocyte stages of parasites also maintain an active process of cholesterol exclusion from plasma membrane.

333

250 - The impact of PfK13 C580Y on global transcription through the intraerythrocytic developmental cycle of Plasmodium falciparum

Sievert, Mackenzie AC (Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame); Checkley, Lisa A.; Davis, Sage Z.; Foster, Gabriel J. (Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame); Nosten, Francois H. (Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University); Nair, Shalini (Texas Biomedical Research Institute); Khoo, Sok Kean (Department of Cell and Molecular Biology, Grand Valley State University); Anderson, Timothy JC (Texas Biomedical Research Institute); Ferdig, Michael T. (Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame)

Emerging artemisinin resistance (Art-R) in the malaria parasite, Plasmodium falciparum, is a complex phenotype, making it difficult to determine the precise mechanism(s) of resistance. SNPs in pfk13 are molecular markers for Art-R; however, the role of pfk13 in the mechanism of resistance remains unclear. It is imperative to uncover mechanisms of resistance to head off total failure of this drug. An interesting but understudied phenotype associated with Art-R is the ‘delayed ring’, characterized by an extended ring stage and a shortened trophozoite stage of the Intraerythrocytic Developmental Cycle (IDC). This pause in development has been proposed as a mechanism by which the parasite extends the least susceptible stage of the IDC and shortens the most susceptible stage. To examine how pfk13 C580Y affects the developmental cycle, we conducted a transcriptional timecourse over the IDC using CRISPR/Cas9 edited parasites generated by introducing C580Y into an artemisinin sensitive parent line isolated from the Thailand-Myanmar border. Transcripts were collected from highly synchronized cultures at 4h intervals from 0 to 32h post invasion (hpi) and every 8h from 32 to 56 hpi. We are analyzing these data for insights about the impact of pfk13 C580Y on the transcriptional profile as a readout of developmental patterns of the malaria parasite.

334

251 - Plasmodium enolase and GAPDH as plasminogen and tPA receptors

Silva, Thiago (National institute of health);

Plasmodium falciparum uses human plasmin bound to its surface for successful infection of the mosquito midgut. Plasmin is formed through the proteolytic cleavage of its zymogen, plasminogen, a reaction mediated by the plasminogen activators, tPA and uPA. Recently, we found that plasminogen activation on the surface of macrogametes and sporozoites requires the binding of tPA to the cell surface. Surface Pfenolase has been described as a receptor for human plasminogen; and surface PfGAPDH works as a receptor for plasminogen in various pathogens including bacteria, fungi and parasites. Some plasminogen receptors can also be co-receptors for the plasminogen activators, e.g. enolase. In Plasmodium, the cell surface receptor for tPA is currently unknown. We hypothesized that Pfenolase and PfGAPDH, previously reported to occur on the surface of P. falciparum, are receptors for plasminogen and tPA. We observed that both, Pfenolase and PfGAPDH, are detected on the surface of several developmental stages of the parasite, including micro- and macrogametes, zygotes, ookinetes, and sporozoites. Furthermore, we found that tPA and plasminogen bind non-competitively to both Pfenolase and PfGAPDH in vitro. This binding is mediated by the kringle domains of both tPA and plasminogen. We are currently testing the potential of Pfenolase and PfGAPDH as transmission-blocking targets, either individually or in combination. We are also testing the potential of these antibodies to preclude the recruitment of plasminogen and tPA to the parasite surface.

335

252 - Using Nanoparticles as a vehicle for the slow release of Artemisinin in the malaria parasite, Plasmodium falciparum

Simantov, Karina (Hebrew University); Zemmour, Chalom; Stern, Tal; Benny, Ofra; Dzikoswki, Ron (Hebrew University)

Plasmodium falciparum’s ability to develop drug resistance is a major barrier in malaria control and elimination. Artemisinin and its derivatives (ARTs) are the most effective drugs to treat malaria today, but due to recent emergence of parasites with decreased sensitivity to ARTs, the efficiency of artemisinin-based drug therapy is threatened. The decreased sensitivity to ARTs manifests as a longer clearance half-life when exposed to the drug, and has been associated with mutations in PfK13. It was shown that parasites carrying these mutations may decelerate cell cycle progression resulting in quiescence-associated reduced sensitivity to ART. We used nanoparticles (NPs) to encapsulate artesunate, a derivative of artemisinin, in an attempt to increase the drug’s half-life and overcome the long-clearance time of these parasites. The formulation of these NPs allows them to be stable and water soluble making it a good candidate in prolonging the release and half-life of ART. Our data shows that NPs that were added to the cultures were found in infected erythrocytes and were localized to the parasites. We show that these encapsulated artesunate NPs effectively kill the parasites in a short time and increase the parasite's sensitivity in ring stage survival assays. Additional experiments are carried on to test if this method of encapsulation could overcome the reduced susceptibility of P. falciparum to ARTs.

336

253 - Novel haplotypes of PfCRT in Plasmodium falciparum from the Yunnan Province, China confer resistance to the first- line antimalarial piperaquine

Small-Saunders, Jennifer L. (Columbia University Medical Center) Hagenah, Laura M.; Dhingra, Satish K.; Fidock, David A. (Columbia University Medical Center)

Malaria caused by Plasmodium falciparum (Pf) remains an important public health threat in border regions of the Yunnan Province, China due to their proximity to bordering malaria-endemic countries. Parasites in this region are exposed to varying, country- specific antimalarial regimens. Earlier reports indicated that piperaquine (PPQ) resistance was present in this region, a consequence of earlier PPQ monotherapy, and PPQ combined with dihydroartemisinin (DHA) has encountered widespread resistance across the Greater Mekong Subregion. To date, few studies have explored the Pf drug resistance landscape in this region, and rarely do these determine full-length pfcrt sequences. Here we have used zinc- finger nuclease-based gene editing to introduce novel pfcrt alleles, reported in Yunnan Province, into Dd2 parasites. Three lines were created: China E (GB4 + I371R), China B (China E +E75D/A144Y/S220A) and China C (China B + R371I reversion). These recombinant lines expressing the China B and C alleles demonstrated distended digestive vacuoles in trophozoites and schizonts, with the largest vacuoles noted in China C. China E, B and C lines were all sensitized to the former first-line drug chloroquine (CQ) , despite having the K76T mutation. China C demonstrated the largest changes in antimalarial susceptibility, with sensitization to CQ, monodesethyl-chloroquine, and quinine. Resistance to other antimalarials, including mefloquine, pyronaridine, ferroquine, md- amodiaquine, dihydroartemisinin, and lumefantrine, was not affected. China C demonstrated significant PPQ IC50 and IC90 increases. PPQ survival assays demonstrated significant resistance in the China C line. Ongoing fitness assays will reveal the interplay between parasite fitness and drug resistance in these lines. This work provides new insights into PfCRT-mediated drug resistance in China and identifies new PfCRT haplotypes that may be driving PPQ resistance in Yunnan Province. 337

254 - Toxoplasma gondii Apical Cap 9 is essential to anchor the conoid to the apical polar ring of mature parasites

Soldati-Favre, Dominique (University of Geneva); Tosetti, Nicolo; Dos Santos Pacheco, Nicolas; Maco, Bohumil (University of Geneva)

Toxoplasma gondii belongs to the coccidian sub-group of Apicomplexa that possesses an apical complex composed of a conoid, made of unique tubulin polymer fibers. This intriguing dynamic organelle extrudes in extracellular motile and invasive parasites and is associated to the anterior polar ring, a microtubule-organizing centre for 22 subpellicular microtubules (MTs). The cortical MTs are linked via small filaments composed of alveolins to the Inner Membrane Complex (IMC) at the pellicle.The Apical Cap 9 (AC9) is an alveolin network protein previously localized to the apical IMC plate (Chen et al. CMI 2017). Immunofluorescence analysis by super-resolution microscopy shows that AC9 signal is arranged in ~22 rows reminiscent of MTs organization. Conditional depletion of AC9 using the Auxin-induced Degron (AiD) system uncovers a very severe loss of fitness. Parasites lacking AC9 replicate normally but are defective in microneme secretion and hence fail to egress and invade. Remarkably, upon AC9 depletion, a series of conoid markers (MyoH, GAC, AKMT, FRM1, CPH1, ICMAP1 and RNG2) are not found anymore in the mature parasites although they are still present in the nascent daughter cells. Closer examination by electron microscopy on intracellular parasites and on extracellular deoxycholate-extracted parasites confirmed that the conoid is lost. Despite its localization at the proximity of the cortical MTs, AC9 is not binding to MTs in vitro. Given its limited solubility, we embarked on proximity labeling by the biotin ligase BirA approach to determine AC9 interacting partners. We are currently dissecting the components of the AC9 complex to identify the binding determinant to MTs and to elucidate how the conoid is anchored to the polar ring.

338

255 - Is calcium the gear shift? The role of calcium-mediated phosphorylation of TgMyoA

Stadler, Rachel V. (Department of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine); Rould, Mark A. (Macromolecular Consulting LLC); Vella, Stephen A. (Department of Microbiology Center for Tropical and Emerging Global Diseases, University of Georgia); Kelsen, Anne (Department of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine); Moreno, Silvia N J (Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia); Ward, Gary E. (Department of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine)

Toxoplasma gondii uses an actomyosin-based motile system to invade into and egress from host cells and to disseminate through host tissues. Because motility is essential for the in vivo virulence of T. gondii, the proteins that drive and regulate parasite motility are promising targets for the development of new drugs to treat infection. A central component of the parasite’s motility system is an unconventional class XIV single-headed myosin, TgMyoA. Calcium is known to activate motility, and a T. gondii-encoded calcium-dependent protein kinase, TgCDPK3, has been implicated in both TgMyoA phosphorylation and initiation of parasite motility in 2D. Using parasites expressing the fluorescent calcium reporter GCaMP6f, we show here that parasite intracellular calcium levels oscillate as the parasite moves in 3D through a model extracellular matrix. Intriguingly, parasite speed oscillates during 3D motility with a periodicity similar to that of the calcium oscillations. Furthermore, we show that recombinant TgMyoA containing phosphomimetic mutations at the sites of TgCDPK3 phosphorylation has enhanced motor activity. We therefore hypothesize that motor function and parasite motility are regulated by calcium- and TgCDPK3-mediated phosphorylation of TgMyoA. To test this hypothesis, we used our 3D motility assay and custom software to quantitatively show that parasites lacking functional TgCDPK3 have defects in 3D motility. We are currently exploring: (a) whether the motility defect of these parasites is due specifically to reduced TgMyoA phosphorylation, and (b) whether the regular pattern of acceleration/deceleration the parasite exhibits along its 3D trajectory is caused by the calcium oscillations. This work will broaden our understanding of the role played by calcium in the regulation of apicomplexan parasite motility. 339

256 - Transcriptome-wide in vivo mRNA target identification of RNA-binding proteins essential for P. falciparum sexual development

Stasic, Andrew J. (US Food and Drug Administration); Painter, Heather J. (US Food and Drug Administration)

Efforts to eradicate the global malaria health burden rely on the disruption of Plasmodium falciparum transmission. Understanding gene regulatory mechanisms that enable maturation of the transmissible sexual stages is essential to this effort. It is well established that both post-transcriptional regulation and translational repression play a major role in maturation and fertilization during sexual development. However, due to lack of effective methodologies, in vivo identification of the exact targets of RNA binding proteins (RNA-BPs) essential for sexual development remained elusive. Here, we aim to establish the use of photoactivated ribonucleoside- enhanced crosslinking and immunoprecipitation (PAR-CLIP) for the transcriptome-wide identification of mRNA-protein interactions. This method exploits the use of a transgenic P. falciparum expressing a yeast fusion of phosposribosyltransferase and cytosine deaminase (yFCU) which can incorporate thiol-modified uracil into mRNA (PMIDs: 29985403 & 28416533), enabling transcriptome-wide in vivo identification of the specific targets of any RNA-BP via photoactivated covalent crosslinking to thiolated transcripts. To validate PAR-CLIP in P. falciparum, we will investigate RNA-BPs that have previously demonstrated to play a role in sexual development, including PUF2, DOZI and CITH. These genes will be GFP-tagged in P. falciparum strains expressing yFCU and thiol-labeled mRNAs will be photo-crosslinked to the RNA-BP. The resulting RNA- protein complexes will be immunoprecipitated and the crosslinked mRNA sequenced for target identification (PMID 27871973). The exact RNA-BP binding sites will be identified by taking advantage of an induced nucleotide mismatch signature which is a result of crosslinking. Overall, this approach will provide a method for fully characterizing essential RNA-BPs and broaden our understanding of post-transcriptional regulation during parasite transmission for the identification of potential intervention strategies.

340

257 - High-throughput screen to identify specific Inhibitors of the Plasmodium Protease, PfClpP

Stephens, Dylon (The University of Georgia); Florentin, Anat; Muralidharan, Vasant (The University of Georgia)

Plasmodium falciparum, the causative agent of malaria, has a class of genes that have a prokaryotic origin known as the Clp family of chaperones and proteases. Several of them, including the PfClpP protease, localize to a unique metabolic organelle in the parasite known as the apicoplast. Not much is known about apicoplast biology, but it has been shown that drugs that target this non-photosynthetic plastid are clinically effective. Our data show that the PfClpP protease is essential for asexual growth of the parasite, and that it forms a proteolytic complex with other Clp proteins to regulate apicoplast biogenesis. We generated a conditional knockdown mutant of PfClpP and were able to reduce protein levels of ClpP in the parasite by over 95%. Nevertheless, residual PfClpP protein levels in the cell were enough to maintain its biological function and prevented any phenotypic effect despite its essentiality. Using the PfClpP conditional mutants as a tool, we screened for small molecules that act as specific PfClpP inhibitors and exhibit differential effect depending on cellular levels of PfClpP. As a proof of concept, we started with small chemical libraries such as the Pathogen Box, a small library of 400 compounds, developed by the Medicines for Malaria Venture, to screen against the parasites. Compounds from this library are known to be effective against multiple infectious diseases such as malaria, tuberculosis, and dengue fever, but many of their targets are unknown. Our working hypothesis is that under knockdown conditions, small molecules that are PfClpP specific will become more effective at killing the parasites due to a significant reduction of the target. Screening the pathogen box, we have optimized and validated this method for high throughput drug screening and are moving forward to screening of larger chemical libraries to identify drugs that inhibit this essential and unique protease in the parasite plastid.

341

258 - TcAMPK: identification and characterization of a cellular energy homeostasis hub regulator in Trypanosoma cruzi

Sternlieb, Tamara (Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" – CONICET) Schoijet, Alejandra C.; Genta, Patricio D.; Alonso, Guillermo D. (Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" - CONICET , Ciudad Autónoma de Buenos Aires.)

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved in maintaining energy homeostasis in response to different stresses in many organisms. During the transition between the mammalian host and the insect vector, Trypanosoma cruzi, the causative agent of Chagas disease, faces different types of environmental fluctuations, all of which prompt the parasite to remodel its metabolism to adapt, survive and differentiate into the next stages of its life cycle. Recently, it was shown that Trypanosoma brucei AMPK is involved in the differentiation from the bloodstream slender to stumpy stage and in surface protein expression changes in response to nutritional stress. This underscores the relevance of AMPK for parasite life cycle progression. We identified four candidate genes for the AMPK subunits of T. cruzi (a1 and a2 catalytic subunits, ß and ? regulatory subunits). The ß and ? subunits are largely conserved in their domain structure relative to the mammalian orthologs. However, the alpha subunits show significant sequence and structure differences from the human counterparts. The presence of these subunits in T. cruzi epimastigotes was confirmed by RT-PCR, Western blot using a phospho-AMPKa specific antibody, mass spectrometry and by kinase activity assays using the specific AMPK substrate SAMS. TcAMPKa1 over-expressing epimastigotes showed a lower growth rate in basal culture conditions compared to the control, while a2 over-expression had the opposite effect. We found there is upregulation of AMPK activity under epimastigote starvation, and that dorsomorphin, a specific AMPK inhibitor, also inhibits T. cruzi AMPK. Each of these subunits could complement the ‘glucose dependent’ phenotype of S. cerevisiae conditional mutants lacking the respective subunit of the AMPK ortholog SNF1. Starving assays with AMPKa over-expressing parasites also showed a possible role of AMPK in autophagy. Our results show, for the first time, the presence of a functional AMPK orthologue in Trypanosoma cruzi. 342

259 - Plasmodium-selective peptide vinyl sulfone proteasome inhibitors are potent inhibitors of multidrug-resistant malaria parasites and exhibit a low potential for generating resistance in vitro

Stokes, Barbara H. (Columbia University Irving Medical Center Department of Microbiology); Yoo, Euna (Stanford University Medical Center Department of Pathology); Murithi, James M. (Columbia University Irving Medical Center Department of Microbiology); Luth, Madeline R. (UCSD School of Medicine Division of Host-Microbe Systems & Therapeutics); Afanasyev, Pavel; da Fonseca, Paula C.A. (MRC Laboratory of Molecular Biology); Winzeler, Elizabeth A. (UCSD School of Medicine Division of Host-Microbe Systems & Therapeutics); Ng, Caroline L. (University of Nebraska Medical Center Department of Pathology and Microbiology); Fidock, David A. (Columbia University Irving Medical Center Department of Microbiology)

The spread of Plasmodium falciparum resistance to the first-line drug artemisinin has created an imperative to develop therapeutics with novel modes of action. Recent work has identified the Plasmodium proteasome as a promising drug target, as inhibitors of this proteolytic complex inhibit growth at all stages of the parasite life cycle. Moreover, the recent publication of a high resolution cryo-electron microscopy-based structure of the P. falciparum proteasome has enabled the development of inhibitors that are clearly selective for the parasite enzyme over its human counterpart. Here, we report that WLL- vs and WLW-vs, two highly selective peptide vinyl-sulfone parasite proteasome inhibitors, potently inhibit genetically diverse P. falciparum asexual blood stage parasites, including K13-mutant, artemisinin-resistant strains. WLL-vs and WLW-vs are particularly active against the early ring-stage parasites that are traditionally difficult to treat. Unlike many advanced antimalarial candidates, the potency of WLL-vs and WLW-vs is not compromised by existing mechanisms of antimalarial drug resistance, and these compounds do not readily select for parasite resistance in vitro. Selection studies reveal that mutations in the ß2, ß5 or ß6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield at most five-fold decreases in parasite susceptibility to the selection compound. These results compare favorably against previously published non-covalent inhibitors of the P. falciparum proteasome that can select for resistant parasites with hundred-fold decreases in susceptibility. We observe no cross-resistance between WLL-vs and WLW- vs; in fact, partial resistance to one compound often creates hypersensitivity to the other. Lastly, these inhibitors potently synergize multiple chemically diverse classes of antimalarial agents, underscoring the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as part of next-generation antimalarial combination therapies to treat multidrug-resistant parasites. 343

260 - Human immune response differences between protected and not-protected participants vaccinated with radiation- attenuated Plasmodium falciparum sporozoites.

Stuart, Ken (Seattle Children's Research Institute); Du, Ying (Seattle Children's Research Institute); Hertoghs, Nina; DeRosa, Stephen (Fred Hutchinson Cancer Research Center); Schwedhelm, Katharine (Fred Hutchison Cancer Research Center); Carnes, Jason; Duffy, Fergal; Neal, Maxwell (Seattle Children's Research Institute); McDermott, Suzanne (Seattle Children's Reearch Institute); Daubenberger, Claudia (Swiss TPH); Epstein, Judith (Navy Medical Research Center); Richie, Thomas; Hoffman, Stephen (Sanaria); Aitchison, John (Seattle Children's Research Institute); Gottardo, Raphael; McElrath, Julie (Fred Hutchinson Cancer Research Center)

A vaccine that prevents infection with malaria parasites would be a valuable tool to combat the numerous annual cases and deaths due to malaria gloabally. We analyzed samples from trials in which participants were immunized with radiation-attenuated Plasmodium falciparum sporozoites and subsequently challenged by controlled human malaria infection (CHMI). The vaccine dose and schedule in the IMRAS trial, our major focus, resulted in 55% of one cohort being protected from challenge, providing an opportunity to identify immune responses that differ between protected (P) and not-protected (NP) participants. Whole blood RNA-seq, flow cytometry with a total of 97 markers, and serological analyses were performed on samples obtained before the first vaccination, at multiple times after each immunization, and after CHMI. Thousands of gene- specific, vaccine-induced transcriptome responses were identified by RNA- seq after each immunization and CHMI. Hundreds of these differed between P and NP participants. Clustering and gene module analyses of the transcripts that differed between P and NP participants revealed associations with various immunological processes and cell types. Our analyses identified a progression from innate to adaptive processes and a complex pattern of changes over the vaccination series and after CHMI. Since RNAseq was carried out using blood, changes in cell numbers, expression levels, or trafficking could not be distinguished. Extensive immune phenotyping with our flow cytometry panels revealed changes in immune cell populations which will be integrated with the RNA-seq and serological data in preparation for specific immune cell analyses. This work is intended to identify immune responses and processes that will advance our understanding of radiation-attenuated sporozoite-induced protective immunity and aid malaria vaccine development.

344

261 - Dissecting Pathogenesis of Central Nervous System (CNS)-parasites by Electron Cryo-tomography (Cryo-ET)

Sun, Stella Y. (Stanford University); Chen, Muyuan (baylor college of medicine); Kaelber, Jason T. (Rutgers University); Dong, Xiaoduo (National University of Singapore); Shi, Jian; Nematbakhsh, Yasaman; Lim, Chwee Teck (National University of Singapore); Schmid, Michael F.; Zarko, Li-av S.; Boothroyd, John C. (Stanford University); He, Cynthia Y. (National University of Singapore); Chiu, Wah (Stanford University)

Electron Cryo-tomography (Cryo-ET) is a bioimaging technique that combines electron microscopy with cryopreservation, without chemical fixation or dehydration. Cryo-ET reveals the 3D ultrastructure and function of interest objects of a biological system in their native environment. The size and complexity of the systems can range from nanometers to micrometers and can be combined with other specimen preparation and imaging modalities. Protozoan parasites have evolved diverse mechanism to cross the host barriers and reach deeper tissues where they proliferate and lead to severe infectious diseases. Trypanosoma brucei (T. brucei), a flagellated protozoan parasite as the causative agent of human African sleeping sickness, is a highly invasive pathogen. They are able to penetrate deeply into the host central nervous system (CNS). To understand how flagellar motility facilitates cell penetration, we used cryo-ET to visualize two genetically anucleate mutants with different flagellar motility behaviors. We found that the T. brucei cell body is highly deformable in response to flagellar beating and environmental conditions. Based on the cryo- ET models, we proposed a mechanism of how flagellum motility is coupled to cell shape changes, which may facilitate penetration through size-limiting barriers. To date and give the size and complexity, studying parasite invasion by Cryo-ET has been extremely challenging, especially for the intracellular parasite Toxoplasma gondii (a congenital pathogen) involving entering a host cell. With the development of Cryo- electron microscopy, we are able to visualize a 3D structure of the remarkable invasion machinery in Toxoplasma tachyzoites. The resulting 3D structures provide a testable model for its role in the invasion process.

345

262 - Vesicular mechanisms provide phenotypic assays of artemisinin resistance in Plasmodium falciparum malaria

Suresh, Niraja (University of Notre Dame); Khair nima, Maisha (University of Notre Dame); Coppens, Isabelle (Johns Hopkins University); Bhattacharjee, Souvik (Jawaharlal Nehru University); Ghorbal, Mehdi; Haldar, Kasturi (University of Notre Dame)

Artemisinin resistance threatens world-wide malaria control and elimination. Our studies show that P. falciparumKelch13 (K13), a primary marker of artemisinin resistance and mutations in which cause resistance, localizes to vesicles in the parasite’s endoplasmic reticulum (ER), as detected by cryo-immunoelectron microscopy. Emerging data from multiple labs suggest additional mechanisms that regulate ER vesiculation and export, independent of K13 mutation, also induce artemisinin resistance. Together these data suggest that ER vesicular dynamics may provide phenotypic markers of resistance in both K13- dependent and independent resistance. But phenotypic assays that may be used to detect K13-dependent and independent pathways of resistance have remained elusive. We integrated findings of proteomic studies with transcriptomic and metabolomics analyses to reveal just two parasite pathways that are selectively refractory to artemisinins in drug-resistant but not sensitive ring-stage parasites, the stage of clinical artemisinin resistance. Validation of the first, an ER proteostatic pathway suggests multiple substrates, whose dynamics in presence of both mutation as well as drug provide novel phenotypic readouts of resistance that may be deployed without establishing (long or short term) cultures from clinical samples.

346

263 - The disulfide bonds as functional motifs of the Myb3 transcription factor in Trichomonas vaginalis

Tai, Junghsiang (Institute of Biomedical Sciences); Chen, Shu-Hui (National Chen-Kung University); Tai, Junhg-Hsiang (National Taiwan University); Huang, Jessica (National Chen-Kung University)

In Trichomonas vaginalis, the biochemical properties of the Myb3 transcription factor have been explored to study cell biology of the primordial eukaryote with regards to transcription regulation, signal transduction and protein trafficking. In the present study, Myb3 was found to form monomers and multimers via the inter- and intra- molecular disulfide bonds linking Cys46-Cys46, Cys91-Cys91, Cys46- Cys91, Cys46-Cys243 and Cys91-Cys243. Mutation of individual Cys or disruption of all disulfide linkages by double-mutation rendered differential effects on cytoplasmic retention, protein-protein interactions and phosphorylation of Myb3. Interestingly, Myb3(C91S), but not Myb3(C46S) or Myb3(C243S), was restrained in hydrogenosomes, indicating that the inter-disulfide linkage, C91-C91, is essential for the release of Myb3 from hydrogenosmes. In addition, phosphorylation of Myb3 at two potential casein kinase I sites, Thr57 and Thr109, was identified, and Myb3(T109A) was restrained in some tubular network previously defined as Myb3IPhmw compartment in the cytoplasm. Together, these observations suggest that Myb3 is matured in endoplasmic reticulum for subsequent trafficking towards the nucleus for its proper functions. Yet, in this cellular pathway, Myb3 is subjected to the regulation of a few functional motifs and posttranslational modifications.

347

264 - Pathogen Box Chemical Library as an antimalarial toolbox for phenotypic and chemogenomic studies on Plasmodium falciparum.

Tanala Patra, Alok (Singapore University of Technology and Design)

The Pathogen Box consists of a diverse library of compounds assembled by Medicines for Malaria Venture (MMV), as an open resource to facilitate antimicrobial drug discovery. This small molecule collection contains approximately 400 drug-like molecules, many of them with established in-vitro activity against neglected diseases like malaria, leishmaniasis, and pathogenic bacteria, e.g., Mycobacterium tuberculosis. Our study focuses on screening the pathogen box library in an effort to identify chemical scaffolds that induce unique phenotypic consequences during intraerythrocytic development of Plasmodium falciparum. This approach has led to the discovery of promising lead compounds with nanomolar efficacy. Furthermore, we identified several new inhibitors of parasite proliferation through hindering merozoite release from the infected host cells. To understand the intricacies of the drug-induced blockage of parasite stage transition and drug-target engagement, we have used chemogenomic and proteomics approaches Collectively, this approach has derived new insights into small molecule mediated egress inhibition as a way to facilitate target-based drug design against human malaria infection.

348

265 - Lifecycle progression and sexual development of the apicomplexan parasite Cryptosporidium parvum

Tandel, Jayesh (University of Pennsylvania); English, Elizabeth; Sateriale, Adam; Gullicksrud, Jodi; Beiting, Daniel (University of Pennslyvania); Pinkston, Brittain (University of Georgia); Striepen, Boris (University of Pennslyvania)

The apicomplexan parasite Cryptosporidium is one of the leading causes of diarrhea-induced mortality in children and immunocompromised individuals. Currently there are no drugs or vaccines against the parasite due to limited understanding of the biology and lack of a continuous culture system. Infection is initiated by ingestion of meiotic spores called oocysts. Sporozoite emerging from oocysts infect enterocytes followed by unfolding of asexual and sexual development in the same host. Sex results in the production of infectious, mature oocysts that can be transmitted or infect the same host. We have engineered numerous reporter strains of the parasite that allow us to discern different stages and study the kinetics of sexual development both in culture and in mice. Our in vitro analyses have revealed that Cryptosporidium undergoes robust sexual differentiation (~80%) in culture but fail to have sex. However, parasites have sex in mice and produce autoinfectious oocysts that maintain continuous infection. We have validated our findings independently by using a cre-lox based reporter assay for sex. Our findings strongly suggest that Cryptosporidium is programmed to undergo limited asexual replication & obligatory sexual differentiation. And oocyst biogenesis seems to be an obligatory requirement to maintain continuous infection, which also explains lack of continuous amplification in the culture model. This implies that targeting the transition from sexual stages to oocyst by therapeutic interventions might be an attractive approach to prevent subsequent infection and transmission. Hence, we engineered male- & female-specific fluorescent lines to flow sort sexual stages for RNA seq to study the biology of sexual stages. Our transcriptional analyses of sexual stages have revealed conserved and novel biological processes, and some of which are potential drug targets. Our transcriptional analysis has allowed us to identify a female-specific ApiAP2 protein that we have validated by gene-tagging. We have further identified this female specific AP2 gene to be essential and are currently investigating the effect of conditional ablation of this gene on infection and transmission in a mouse model.

349

266 - The use of oxidative stressors and inducible targets for assessing the viability of foodborne protozoa

Temesgen, Tamirat Tefera (Norwegian University of Life sciences); Tysnes, Kristoffer; Robertson, Lucy (Norwegian University of Life sciences)

Assessing the infectivity of foodborne protozoa is of both public health and trade relevance. Animal bioassay is the gold standard for evaluating infectivity, but is expensive, ethically challenging, time consuming, and not possible for protozoa for which relevant lab hosts are not available (e.g., ). Therefore, viability assessment has been proposed as an alternative to in vivo infectivity assays. Reverse transcription quantitative PCR (RT-qPCR) targeting the sporozoite surface antigen (SporoSAG) for Toxoplasma gondii, HSP70 for Cryptosporidium parvum, and beta giardin for Giardia duodenalis has been proposed. Using inducible target genes could be a relevant approach for viability assessment, as the low numbers of parasites expected as contaminants of fresh produce require sensitive methods. Our aim has been to develop RT-qPCR viability protocols for foodborne protozoa, such as T. gondii and Cryptosporidium spp., based on gene targets that can be induced by oxidizers. In the present study, gene expression was induced by exposing parasite transmission stages to oxidative stressors that produce reactive oxygen species. Menadione sodium bisulfite (MSB) was investigated for its inducing effect on the target mRNA of T. gondii. Dynabeads mRNA Direct kit was used for mRNA extraction, followed by quantification with RT- qPCR. The target genes evaluated included superoxide dismutase (SOD3), heat-shock proteins (HSP70, HSP90), lactate dehydrogenase (LDH1), and eukaryotic translation initiation factor kinase (eifk2B). Preliminary studies have shown promising results with MSB, with at least 10-fold difference in gene expression of the tested targets compared with control samples. This indicates the potential for development of an RT-qPCR protocol for viability assessment of T. gondii oocysts, with the aim to investigate the application to other foodborne protozoa.

350

267 - Ribozyme-mediated, multiplex CRISPR gene editing and CRISPR interference (CRISPRi) in rodent-infectious Plasmodium yoelii

Walker, Michael P. (Penn State University); Lindner, Scott E. (Pennsylvania State University)

Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causative agent, the Plasmodium parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite’s growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene-of-interest to understand its importance and function. However, these approaches can be limited by the availability of sufficient selectable markers, by the time required to generate transgenic parasites, and by exogenous sequences left behind. Moreover, there also is a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) single-guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious Plasmodium yoelii, we demonstrate that gene disruptions and coding sequence insertions are efficiently generated, producing marker-free and scar-free parasites with homology arms as short as 80-100bp. Additionally, we find that the common practice of using one sgRNA for genome editing can produce both unintended plasmid integration and the desired locus replacement editing events, while the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding catalytically-dead SpCas9 (dCas9) to targets in the gene control region of a gene-of-interest, resulting in a position-dependent, but strand-independent reduction in gene expression. This robust and flexible system facilitates in-depth and efficient genetic characterizations in rodent-infectious Plasmodium species. 351

268 - Regulation of PIP39 expression by mitochondrial protein translocase, Tim50, and its role in Trypanosoma brucei infectivity

TRIPATHI, ANUJ (MEHARRY MEDICAL COLLEGE); SINGHA, UJJAL K.; Chaudhuri, Minu (Meharry Medical College)

Trypanosoma brucei, the infectious agent for African trypanosomiasis, undergoes various developmental processes during its digenetic life cycle that includes a major alteration in mitochondrial activities. It is known that changes in mitochondrial functions is critical for the parasite to adapt in different environmental conditions, however, the communication mechanism between this single organelle with the rest of the cell is not clear in T. brucei. Here we showed that the mitochondrial inner membrane protein translocase, TbTim50, plays a critical role in this signaling pathway. Depletion of TbTim50 in the procyclic or the insect form of T. brucei reduced mitochondrial membrane potential and ATP production by oxidative phosphorylation. As a consequence, cellular AMP/ATP ratio increased, and AMPK was activated by phosphorylation. Interestingly, we also observed ~5-fold increase in the levels of PIP39, which is a developmentally regulated protein phosphatase similar to TbTim50 and is primarily localized in glycosomes. PIP39 upregulation was correlated with the increased tolerance of T. brucei to oxidative stress and double knockdown of TbTim50 and PIP39 diminished this tolerance. In addition, we found that TbTim50 down-regulation in the mammalian bloodstream form of T. brucei though minimally affected its growth in vitro, but greatly hampered the parasite infectivity in a mouse-model. Together our results showed for the first time that TbTim50 and PIP39, two similar protein phosphatases in mitochondria and glycosomes, respectively, crosstalk via AMPK pathway to maintain cellular homeostasis and Tim50 is critical for T. brucei infectivity.Supported by RO1AI125662.

352

269 - In vitro and in vivo evidence that GDV1 regulates sexual differentiation upstream of ap2-g

Usui, Miho (Uniformed Services University of the Health Sciences); Prajapati, Surendra K. (Uniformed Services University of the Health Sciences); Ayanful-Torgby, Ruth; Acquah, Festus K.; Cudjoe, Elizabeth; Kakaney, Courage; Amponsah, Jones A. (Noguchi Memorial Institute for Medical Research); Obboh, Evans (School of Medical Sciences, University of Cape Coast); Reddy, Deepti K.; Barbeau, Michelle C. (Uniformed Services University of the Health Sciences); Simons, Lacy M. (Loyola University Chicago); Czesny, Beata (Loyola University Chicago); Raiciulescu, Sorana; Olsen, Cara (Uniformed Services University of the Health Sciences); Abuaku, Benjamin K.; Amoah, Linda E. (Noguchi Memorial Institute for Medical Research); Williamson, Kim C. (Uniformed Services University of the Health Sciences)

The development of sexual stage parasites is critical for malaria transmission, yet much remains unknown about how this process is regulated. Recently, epigenetic repression and expression of the transcription factor AP2-G have been shown to play important roles in sexual commitment. However, the mechanisms that release repression and allow gametocyte production are still a mystery. Our previous work demonstrated that Pfgdv1 is required for gametocyte production. Here, we assess gametocytogenesis in vitro and demonstrate that GDV1 protein expression during schizogony is critical for gametocyte commitment and modulates transcript levels of ap2-g, as well as msrp1. In the next generation of ring stage parasites, ap2-g and msrp1 RNA levels remain elevated and there is a GDV1-dependent increase in transcript levels of gexp5, an ap2-g independent gene. Ring stage RNA levels of all three GDV1-regulated genes, ap2-g, msrp1 and gexp5 correlated positively with gametocyte differentiation in vitro (R2 = 0.45 to 0.92). Transcript levels for these genes were also significantly higher in field samples collected in Ghana during two malaria seasons, 2016 and 2017, that had high ex-vivo gametocyte conversion rates (GCR) (Day4 GCR > 4.9 %; n=20) than those with low to undetectable ex-vivo GCR (n=20). Interestingly, the frequency of a GDV1 mutant allele (Histidine217) which predominated in an area of limited seasonal malaria in the Gambia, is significantly higher in high GCR samples (Fisher exact test, p=0.022) while msp2 alleles are randomly distributed in both groups. Together the data suggests GDV1 acts as a rheostat for sexual differentiation upstream of ap2-g in vitro and plays a regulatory role in vivo.

353

270 - Functional characterization of a DNA sequence motif associated with Plasmodium falciparum gametocyte- associated transcripts

Van Biljon, Riëtte (Department of Biochemistry & Molecular Biology and the Huck Center for Malaria Research); Russell, Timothy J.; Llinás, Manuel (Pennsylvania State University, Department of Chemistry)

The mature gametocyte stages of Plasmodium parasites are critical to malaria transmission. In Plasmodium falciparum, gametocytogenesis is a considerably longer process (~14 days) than almost all other Plasmodium species (~48 h). Previous high-resolution transcriptome data from P. falciparum sexual stage commitment and gametocytogenesis shows distinct shifts in transcript abundance associated with morphological stage transitions in developing gametocytes. As expected, downstream of PfAP2-G, the well- characterized regulator of gametocyte commitment, additional mechanisms controlling gametocyte development are at play. Specifically, we have identified an enriched DNA sequence motif (AGACA) upstream of genes whose mRNA abundance peaks during the intermediate to late stage gametocytes. This motif has previously been implicated in P. falciparum sexual development by several other transcriptome studies and has been linked to transcription of a gametocyte-specific gene in P. gallinaceum. Our preliminary studies have determined that this motif is bound by nuclear lysate extracted from P. falciparum gametocytes. Currently DNA pull-down assays using biotinylated oligomers containing the AGACA motif are underway to identify the DNA binding proteins/ complexes that interact with this gametocyte-specific DNA motif. Follow up studies include using CRISPR-Cas 9 to tag these protein/s to determine their functional relevance via ChIP-seq as in vivo validation of the DNA pull-down experiments and phenotypic evaluation following genetic modification/ ablation of these interactions in Plasmodium falciparum parasites.

354

271 - Development of an ookinete and zygote inhibition assay for prescreening of transmission-blocking candidates

Varadharajan Suresh, Ragavan (National Institute of Health); Miura, Kazutoyo; Deng, Bingbing; Zhou, Luwen (National Institute of Health); Tachibana, Mayumi (Ehime University); Long, Carole A. (National Institute of Health)

Transmission-Blocking Vaccines (TBV) are a promising means of malaria eradication, however, the screening of candidates via Standard Membrane Feeding Assay (SMFA) carries with it logistical and technical limitations in the form of housing live infected mosquitoes. In our assay, we induce ex vivo gametogenesis from lab cultured Plasmodium falciparum (P. falciparum) NF54 parasites in the presence of candidate inhibitors. Twenty-four hours after gametogenesis, we quantify the numbers of zygotes and ookinetes. We have opted to perform this quantification via direct observation through fluorescent microscopy. Rabbit polyclonal antibody against P. falciparum alpha tubulin II serves as the marker for gametocytes while mouse monoclonal antibody (Mab) against Pfs25 serves as the marker for post fertilization stage parasites. Using fluorescently labeled secondary antibodies, we can quantify these stages and determine the ratio of fertilized forms to unfertilized parasite. When testing our anti-Pfs230 Mab with known transmission-blocking activity, our assay shows a dose dependent reduction of fertilized forms from 11.98% at 1.5µg/mL Mab to as low as 2.89% at 50µg/mL Mab (r2=0.95 by a linear regression); serving as a proof of principle. We aim to miniaturize this assay to reduce the volume of test reagent required and allow the use of standard multi-well plates. We also aim to automate the quantification process via image processing software to minimize manual counting time. Together, this will enable high-throughput screening of TBV candidates without any need to house infected mosquitoes. We hope that our assay will accelerate TBV candidate discoveries and may even be adapted to identify drugs that reduce malaria transmission.

355

272 - High Molecular Weight Chitinase Invasion Complex Secreted by Plasmodium Ookinetes Enables Mosquito Midgut Invasion

Vinetz, Joseph M. (Yale School of Medicine); Patra, Kailash P. (UCSD); Prieto, Helena (Western Connecticut State University); Yates, John R. (Scripps Research Institute); Kaur, Hargobinder (Yale School of Medicine)

The Plasmodiumookinete depends on chitinase to penetrate the peritrophic matrix thereby invading the mosquito midgut. The P. falciparumgenome encodes one chitinase gene, PfCHT1, comprising of solely of a TIM barrel- structured chitinase catalytic domain (short form), while the genome of the avian malaria parasite, —a model system for studying the ookinete—encodes two chitinase genes (a long form—PgCHT1and a short form—PgCHT2). The long form of chitinase (PgCHT1) has prodomain and chitin-binding domains at the N-terminal and C-terminal end of the protein respectively, and is the sole form of chitinase found in all non-P. falciparum chitinases, including that of the rodent-infecting parasite, P. berghei. The short form chitinases (PgCHT2and its ortholog PfCHT1) lack both pro and chitin- binding domains. We used a novel chitin bead method to pull-down native chitinase(s), along with associated interacting proteins, from P. gallinaceumand P. falciparumookinete culture supernatants. Immunoblot analysis and high resolution chromatographic fractionation of concentrated ookinete supernatants demonstrated that native chitinases form a high molecular weight protein complex (above 1,300 kDa) in both Plasmodiumspp. Mass spectrometry analysis of this complex showed other micronemal proteins including von Willebrand factor A domain-related protein (WARP), enolase and secreted ookinete adhesive protein (SOAP) that associated with chitinase to form the high molecular chitinase-containing protein complex. Site-directed mutagenesis of three conserved cysteines in the predicted rPgCHT1 chitin- binding domain significantly reduced binding to chitin. Soluble benzamidine hydrochloride prevented prodomain cleavage of the native PgCHT1 which confirms that endogenous trypsin-like protease activates the long form of chitinase. Allelic exchange of the short-form PfCHT1 for the long-form PbCHT1 will enable mechanistic understanding of the composition, assembly and micronemal targeting of Plasmodium chitinases. Targeting the ookinete- secreted chitinase invasion complex, its relation to organellar biogenesis and mechanisms of secretion may lead to next-generation strategies to block malaria transmission

356

273 - Molecular mechansims of host translational control during Toxoplasma gondii infection

Louis-Philippe Leroux1, Julie Lorent2, Tyson E. Graber3, Visnu Chaparro1, Laia Masvidal2, Tommy Alain3, Ola Larsson2, Maritza Jaramillo1 1Institut National de la Recherche Scientifique, Quebec, Canada 2Karolinska Institutet, Stockholm, Sweden 3University of Ottawa, Ontario, Canada

Toxoplasma gondii (T. gondii), the etiological agent of toxoplasmosis, is an obligate intracellular protozoan parasite that alters core host cell functions. We recently demonstrated that infection of primary murine macrophages with type I or II T. gondii strains caused a profound perturbation of the host cell translatome (i.e. efficiently translated mRNAs). Translation of transcripts encoding proteins involved in metabolic activity and components of the translation machinery were activated upon infection in an mTOR-dependent fashion. As such, mTOR signaling was increased and sustained upon infection, while inhibition of mTOR activity suppressed T. gondii replication. C. To further characterize the role of eIF4E-dependent translation in T. gondii-infected cells, we assessed the activity of the cap-binding protein eIF4E and its upstream kinases mitogen-activated protein kinases (MNK)1/2. Unlike the phosphorylation of the mTOR downstream targets, we observed an inhibition of the phosphorylation of MNK1/2 and eIF4E in Toxoplasma-infected macrophages. Chemical inhibition of host phosphatases with okadaic acid restored the phosphorylation of MNK1/2 and eIF4E despite infection. Mice harboring a non-phosphorylable form of eIF4E (S209A knock-in) were more susceptible to acute toxoplasmosis compared to their wild-type counterparts and displayed elevated levels of the inflammatory cytokine interferon-gamma (IFN). Interestingly, genetic deletion of the translational repressors eIF4E-binding proteins (4E-BP) 1 and 2 had a substantially negative impact whereby mice became highly susceptible to the infection. These data hint a prominent role on eIF4E-dependent translational control in the outcome of T. gondii infection. Thus, host mRNA translation represents an important parasite subversion strategy. Ultimately, identifying the molecular underpinnings will help pave the way to new therapeutic treatments against intracellular pathogens. 357

The host cell secretory pathway mediates the export of Leishmania virulence factors out of the parasitophorous vacuole

Guillermo ARANGO DUQUE1, Armando JARDIM2, Étienne GAGNON3, Mitsunori FUKUDA4 and Albert DESCOTEAUX1* 1 INRS-Centre Armand-Frappier Santé Biotechnologie, Université du Québec, Laval, Québec, Canada. 2 Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Québec, Canada. 3 Département de Microbiologie et Immunologie, Institut de Recherche en Immunologie et Cancer, Université de Montréal, Montréal, Québec, Canada. 4 Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.

The protozoan parasite Leishmania causes the leishmaniases, a spectrum of human diseases affecting the skin and viscera. Leishmania parasites replicate within the phagolysosomal compartment of host phagocytes. To colonize those cells, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan (LPG) and the GP63 metalloprotease. Whereas the impact of these virulence glycoconjugates on host cell function has been investigated, the mechanism by which they are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. This is an important issue since Leishmania does not possess a specialized secretion system, analogous to those used by bacteria and apicomplexan parasites, to deliver effectors into host cells. Here, we show that LPG and GP63 are released from the parasite surface post-phagocytosis and redistribute to endoplasmic reticulum (ER)-derived vesicles of macrophages. Pharmacological disruption of ER-Golgi transport hindered the exit of these molecules from the PV, and dampened the cleavage of host proteins by GP63. RNA interference-mediated silencing of the soluble N- ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and Stx- 5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediate the spread of Leishmania effectors within host cells. Our findings unveiled a mechanism whereby a vacuolar pathogen co-opts the host cell’s secretory pathway to promote virulence molecule egress beyond the PV. Improved understanding of this virulence strategy may help identify host factors to be potentially targeted for chemotherapy. Supported by CIHR and by the Canada Research Chair on the Biology of Intracellular Parasitism.

358

ABDELKRIM ÉP. GUEDICHE, Basson, Travis Yosser zina Swiss TPH, University of Basel [email protected] [email protected]

Absalon, Sabrina Batoff, William Indiana University School of Medicine [email protected] [email protected] Beck, Josh Acevedo Ospina, Hamlet Iowa State University INRS-Armand-Frappier Santé [email protected] Biotechnologie [email protected] Belfetmi, Anissa Harvard Medical School Agrawal, Palak [email protected] [email protected] Bello, Fabiola Ahiya, Avantika Centro de Investigación y Estudios Drexel university, College of Medicine Avanzados, IPN [email protected] [email protected]

Asady, Beejan Ben Chaabene, Rouaa University of Georgia UNIGE [email protected] [email protected]

Assaraf, Shany Bennink, Sandra hebrew university of jerusalem Division of Cellular and Applied [email protected] Infection Biology, RWTH Aachen University Balestra, Aurélia [email protected]

Université de Genève [email protected] Beri, Divya [email protected] Balouz, Virginia Institute for Research in Biotechnology Beverley, Stephen [email protected] Washington University School of Medicine Bandini, Giulia [email protected]

Boston University [email protected] Bhabha, Gira Skirball Institute, NYU School of Medicine [email protected]

Baptista, Carlos Bhanot, Purnima Department of Microbiology and Rutgers - New Jersey Medical School Immunology, University at Buffalo [email protected] [email protected]

Bhatnagar, Suyash Bullen, Hayley Drexel University College of Medicine Burnet Institute [email protected] [email protected]

Billker, Oliver Burleigh, Barbara Umeå University Harvard School of Public Health [email protected] [email protected]

BISHNOI, RITIKA Calixto, Abigail [email protected] [email protected]

Bohmer, Monica camara, maria de los University of Central Florida milagros [email protected] [email protected]

Bonnell, Victoria Carneiro Alves Ferreira, Eliza Department of Biochemistry and Molecular Biology, Huck Center for Vanessa Malaria Research, Pennsylvania State [email protected] University, University Park, PA 16802 [email protected] , Mark University of Cambridge, Department Bopp, Selina of Biochemistry Harvard T.H.Chan School of Public [email protected] Health [email protected] Carter, McKenzie The George Washington University Botté, Cyrille [email protected] Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, Chaddha, Kritika INSERM U1209, Université Grenoble [email protected] Alpes [email protected] Chahine, Zeinab UC Riverside Brown, Audrey [email protected] University of Virginia [email protected] Chakrabarti, Debopam University of Central Florida Brühlmann, Francis [email protected] [email protected]

Brunk, Brian Chandra, Monica University of Penn German Cancer Research Center [email protected] (DKFZ) [email protected] Buhrow, Leann University of Texas Southwestern Chasen, Nathan Medical Center Center for Tropical and Emerging [email protected] Global Diseases, University of Georgia [email protected]

[email protected] Clements, Rebecca Das, Anish Harvard University Rutgers - New Jersey Medical School [email protected] [email protected]

Cobb, David Dass, Sheena University of Georgia Apicolipid Team, IAB, CNRS UMR5309 [email protected] [email protected]

Cobbold, Simon Dass, Swati University of Melbourne Drexel University College of Medicine [email protected] [email protected]

Collins, James de Graffenried, Christopher UT Southwestern Medical Center Brown University [email protected] [email protected]

Collins, Jennifer Deitsch, Kirk University of Central Florida: Burnett Weill Cornell Medical College School [email protected] [email protected] Deng, Yue Wen Connacher, Jessica [email protected] University of Pretoria [email protected] Desai, Sanjay NIAID, NIH Connelly, Sean [email protected] [email protected] Descoteaux, Albert , Roland INRS- Institut Armand-frappier Dominican University of California [email protected] [email protected] Dobson, Deborah Costa, Elizabeth Washington University Whitehead Institute [email protected] [email protected] Drozda, Allison Crater, Anna [email protected] NIAID, National Institutes of Health [email protected] Dumaine, Jennifer University of Pennslyvania [email protected] Cruz, Angela Dumoulin, Peter University of Sao Paulo Harvard T.H. Chan School of Public [email protected] Health [email protected] Cruz Talavera, Irene [email protected] Duraisingh, Manoj Harvard T.H. Chan School of Public Dacheux, Denis Health [email protected]

Dvorin, Jeff Fagbami, Lola Harvard Medical School / Boston Harvard University Children's Hospital [email protected] [email protected] Falekun, Seyi Dzikowski, Ron Department of Biochemistry, University Hebrew University of Utah [email protected] [email protected]

Egan, Elizabeth Ferreira, Tiago Stanford University School of Medicine National Institute of Allergy and [email protected] Infectious Diseases, National Institutes of Health Ekiert, Damian [email protected] [email protected] Ferrel, Abel Ekloh, William [email protected] West African Centre for Cell Biology of Infectious Pathogens, University of Fierro, Manuel Ghana The University of Georgia [email protected] [email protected]

Ellis, Angela Figueiredo, Luisa NIAID Instituto de Medicina Molecular, [email protected] University of Lisbon [email protected] Ellis, Cameron University of Texas at El Paso Filarsky, Michael [email protected] [email protected] Elsworth, Brendan Harvard School of Public Health Florentin, Anat [email protected] University of Georgia [email protected] Engstler, Markus University of Wuerzburg Florini, Francesca [email protected] IZB, University of Bern, Switzerland wuerzburg.de [email protected]

Esherick, Lisl Fraser, James Massachusetts Institute of Technology [email protected] [email protected] Fu, Yong Etheridge, Ronald Washington University School of University of Georgia Medicine [email protected] [email protected]

Evers, Felix Ganter, Markus Radboud Institute for Molecular Life Center for Infectious Disease, Sciences Parasitology, Heidelberg University [email protected] [email protected]

[email protected] Garcia Lopez, Laura Lorena Universidad del Quindio Guizani, Ikram [email protected] Institut Pasteur de Tunis [email protected] Garten, Matthias SIB/NICHD/NIH, Bethesda, MD Guler, Jennifer [email protected] University of Virginia [email protected] Gezelle, Jeanine [email protected] Gull, Keith University of Oxford Ghosh, Swagata [email protected] [email protected] gupta, devendra kumar Gibson, Alexis Novartis Institute for Tropical Diseases University of Pennsylvania [email protected] [email protected] Gurung, Pratima Gkeka, Anastasia LPHI, University of Montpellier [email protected] [email protected]

Glennon, Elizabeth Gwira, Theresa Manful Seattle Children's Research Institute [email protected] [email protected] Hagenah, Laura Glushakova, Svetlana [email protected] SIB/NIH/NICHD, Bethesda, MD [email protected] Hakimi, Hassan Department of Protozoology, Institute Goldberg, Daniel of Tropical Medicine (NEKKEN), Washington University Nagasaki University [email protected] [email protected]

Gomes, Ana Rita Haldar, Kasturi DIMNP, Université de Montpellier University of Notre Dame [email protected] [email protected]

Gonzalez Baradat, Bernardo Harb, Omar Hunter College [email protected] [email protected] Goswami, Debashree Hart, Melissa Seattle Children's Research Institute LSHTM [email protected] [email protected]

Goyal, Manish Hasan, Md Mahmudul [email protected] University of Vermont [email protected] Guerin, Amandine UPENN Heaslip, Aoife

University of Connecticut Jaramillo, Maritza [email protected] [email protected]

Heinberg, Adina Jeffers, Vicki [email protected] [email protected]

Ho, Chi-Min Jeneby, Maamun UCLA Dept. of Microbiology, [email protected] Immunology & Molecular Genetics [email protected] Jennison, Charlie

[email protected] Hollin, Thomas University of California, Riverside [email protected] Jimenez, Veronica Center for Applied Biotechnology Studies and Department of Biological Hovel-Miner, Galadriel Science, College of Natural Sciences The George Washington University and Mathematics, California State [email protected] Universi [email protected] Hsu, Hong Ming The Division of Tropical Medicine and Joy, Deirdre Parasitology, College of Medicine, National Institutes of Health National Taiwan University [email protected] [email protected]

Kafsack, Björn HUANG, ZHOU Weill Cornell Medical College Washington University in St. Louis [email protected] [email protected]

Kashif, Mohammad Huet, Diego National Institute of Immunology University of Georgia [email protected] [email protected] Hussain, Tahir Katris, Nicholas Iowa State University Apicolipid Team, IAB, CNRS UMR5309 [email protected] [email protected]

Huston, Christopher Kaur, Hargobinder University of Vermont [email protected] [email protected]

Imlay, Leah Kaushansky, Alexis UT Southwestern Medical Center Center for Infectious Disease Research [email protected] [email protected]

Ishizaki, Takahiro Ke, Hangjun Graduated school of Biomedical Drexel University College of Medicine Science, Nagasaki University [email protected] [email protected] Kegawa, Yuto

National Institute of Halth International Centre For Genetic [email protected] Engineering and Biotechnology New Delhi Kellermeier, Jacob [email protected] [email protected] Lacombe, Alice Keroack, Caroline Wellcome Centre for Integrative [email protected] Parasitology [email protected]

Kieft, Rudo Department of Biochemistry and Lahree, Aparajita Molecular Biology, University of Instituto de Medicina Molecular- Joao Georgia Lobo Antunes [email protected] [email protected]

Kim, Chi Yong Lamb, ian [email protected] Center for Molecular Parasitology, Department of Microbiology and Kirkman, Laura Immunology, Drexel University College Weill Cornell Medical College of Medicine. Philadelphia, PA, United [email protected] States o [email protected] Kissinger, Jessica University of Georgia Lander, Noelia [email protected] University of Georgia [email protected] Klaus, Severina [email protected] Lane, Kristin NIAID/NIH Klemba, Michael [email protected] Virginia Tech [email protected] Lavazec, Catherine Inserm U1016/CNRS UMR8104 Klingbeil, Michele [email protected] University of Massachusetts, Amherst [email protected] Lawong, Aloysus UT Southwestern Medical Center Knuepfer, Ellen [email protected] The Francis Crick Institute [email protected] Leger-Abraham, Melissa Harvard Medical School melissa_leger- Kongsomboonvech, Angel [email protected] UC Merced Lim, Caeul [email protected] [email protected]

Kuchipudi, Arunakar Lindner, Scott [email protected] Pennsylvania State University [email protected] Kumar, Pradeep Ling, Liqin

1, Department of Microbiology and Immunology, Drexel University College McConville, Malcolm of Medicine; 2, West China Hospital, University of Melbourne Sichuan University [email protected] [email protected]

McDermott, Suzanne Llinas, Manuel Seattle Children's Research Institute Penn State University suzanne.mcdermott@seattlechildrens. [email protected] org

Lourido, Sebastian McGovern, Victoria Whitehead Institute for Biomedical [email protected] Research [email protected] McGugan, Glen National Institutes of Health Lourido, Sebastian [email protected] Whitehead Institute for Biomedical Research [email protected] Meister, Thomas Stanford University Mach, Jan [email protected]

Charles University [email protected] Merrick, Catherine Cambridge University Mageswaran, Shrawan [email protected]

Kumar Miao, Jun Department of Entomology, Mandt, Rebecca Pennsylvania State University, Harvard T.H. Chan School of Public [email protected] Health [email protected] Millar, Scott

University of Glasgow Marcolino, Monica [email protected] Stanford University [email protected] Miskinyte, Migla

[email protected] Marquez-Nogueras, Karla University of Georgia [email protected] Modrzynska, Katarzyna University of Glasgow [email protected] Martynowicz, Jennifer Indiana University School of Medicine [email protected] Mather, Michael Moon, Robert Drexel University College of Medicine London School of hygiene and Tropical [email protected] Medicine [email protected]

M'Bana, Viriato Instituto de Medicina Molecular, João Moonah, Shannon Loo Antunes, Lisboa University of Virginia [email protected] [email protected]

Neveu, Gaëlle Mowat, Kaitlyn INSERM, Cochin Institute [email protected] [email protected] mukherjee, sumit Okada, Megan [email protected] University of Utah [email protected] Mulaka, Maruthi Drexel University College of Medicine Olias, Philipp [email protected] [email protected]

Munro, Justin Pagura, Lucas Chemistry Department, Center for [email protected] Malaria Research (CMaR), The Pennsylvania State University Painter, Heather [email protected] Biochemistry and Molecular Biology Department, Huck Center for Malaria Muralidharan, Vasant Research, Pennsylvania State University of Georgia [email protected] [email protected] Panas, Michael Murillo_Solano, Claribel Stanford University [email protected] [email protected] Murithi, James CUIMC Pangburn, Sarah [email protected] CUNY Graduate Center [email protected] Murphy, Robert University of Kentucky Papavasiliou, Nina [email protected] DKFZ-Heidelberg and Rockefeller Univ [email protected] Nair, Sethu Johns Hopkins University Paredez, Alexander [email protected] University of Washington [email protected] Naor, Adit Stanford Parker, Shelbi [email protected] [email protected]

N'DRI, Marie-Esther Parveen, Nikhat Inserm U1016, Institut Cochin, CNRS, Rutgers-New Jersey Medical School UMR8104, Université Paris Descartes, [email protected] [email protected] Pasaje, Charisse Flerida Negreiros, Raquel Massachusetts Institute of Technology State University of Campinas [email protected] [email protected] Pellisson, Mélanie Nessel, Timothy [email protected] Iowa State University [email protected] Phillips, Meg

UT Southwestern Med Center Ramanathan, Aarti [email protected] Drexel university [email protected] Pollastri, Michael Northeastern University Ramirez-Montiel, Fatima [email protected] Universidad de Guanajuato [email protected] Pontikos, Michael [email protected] Raper, Jayne City University of New York Portugal, Silvia [email protected] Heidelberg University [email protected] Rathod, Pradipsinh University of Washington Power, B. Joanne [email protected] Pennsylvania State University [email protected] Rawat, Mukul [email protected] Prajapati, Surendra Uniformed Services University of the Ray, Sneha Health Sciences [email protected] [email protected] Reese, Michael Preiser, Peter UT Southwestern Medical Center Nanyang Technological University [email protected] [email protected] Remcho, T. Parks Prigge, Sean [email protected] Johns Hopkins SPH [email protected] Richard, Dave

Centre de Recherche en Infectiologie, Primo, Vincent Université Laval Boston College [email protected] [email protected] Rijo-Ferreira, Filipa Quinn, Jennifer [email protected] Institute of Microbiolgy - University Clinic Erlangen [email protected] Rios, Kelly Pennsylvania State University [email protected]

Rivera-Cuevas, Yolanda Radke, Joshua University of Michigan in Ann Arbor Washington University School of [email protected] Medicine [email protected] Roos, David Univ Pennsylvania Ramanantsalama, Miharisoa [email protected] Rijatiana [email protected] Rosenberg, Alex

Washington University in St. Louis [email protected] [email protected] Seizova, Simona Rudlaff, Rachel [email protected] Harvard Medical School [email protected] Serre, David University of Maryland School of Ruivo, Margarida Medicine [email protected] [email protected]

Russell, Timothy Shakya, Bikash Department of Biochemistry and Stanford University Molecular Biology and Center for [email protected] Malaria Research (CMaR), Pennsylvania State University Sharma, Aabha [email protected] Harvard T. H. Chan School of Public Health Sa, Juliana [email protected] NIAID [email protected] Sharma, Manu Stanford University Saggu, Gagandeep [email protected] LMVR, NIAID, National Institutes of Sheiner, Lilach Health University of Glasgow [email protected] [email protected]

Sambamurthy, Shukla, Anurag Chandrasekaran [email protected] [email protected] Sidik, Saima Sardinha-Silva, Aline Whitehead Institute Molecular Parasitology Section, [email protected] Laboratory of Parasitic Diseases, National Institute of Allergy and Sievert, Mackenzie Infectious Diseases, NIH Eck Institute for Global Health, [email protected] Department of Biological Sciences, University of Notre Dame Sateriale, Adam [email protected] [email protected] Sigala, Paul Schiano, Irio University of Utah School of Medicine [email protected] [email protected] Schneider, Victoria Silva, Thiago [email protected] National institute of health [email protected] Schureck, Marc NIAID Silva Pereira, Sara [email protected] Instituto de Medicina Molecular - João Lobo Antunes Segev Zarko, Li-av [email protected] Stanford University

simantov, karina [email protected] Stephens, Dylon The University of Georgia Sinai, Anthony [email protected] University of Kentucky College of Medicine Stokes, Barbara [email protected] Columbia University Irving Medical Center Department of Microbiology Sladewski, Thomas [email protected] [email protected] Striepen, Boris Small-Saunders, Jennifer University of Pennsylvania [email protected] [email protected]

Smith, Tyler Striepen, Josefine [email protected] Columbia University [email protected] Soldati-Favre, Dominique University of Geneva Stuart, Ken [email protected] Seattle Children's Research Institute [email protected] Sollelis, Lauriane Welcome Center for Integrative Su, Xin-zhuan Parasitology National Institutes of Health, USA [email protected] [email protected]

SOMEPALLI, MASTANBABU Summers, Robert University of Pennsylvania Harvard T.H. Chan School of Public [email protected] Health [email protected] SRINIVASAN, PRAKASH Johns Hopkins Malaria Research Sun, Stella Institute, Dept. Molecular Microbiology Stanford University and Immunology [email protected] [email protected] Sur, Aakash Srivastava, Anubhav University of Washington Monash Institute of Pharmaceutical [email protected] Sciences, Monash University, Melbourne, VIC 3052, Australia Suresh, Niraja [email protected] University of Notre Dame [email protected] Stadler, Rachel Swearingen, Kristian Department of Microbiology and Institute for Systems Biology Molecular Genetics, University of [email protected] Vermont Larner College of Medicine [email protected] Tagoe, Daniel Boston College Stasic, Andrew [email protected] US Food and Drug Administration [email protected] Tanala Patra, Alok

Singapore University of Technology and Design Vinetz, Joseph [email protected] Yale School of Medicine [email protected] Tandel, Jayesh University of Pennsylvania von Grüning, Hilde [email protected] University of Pretoria [email protected] Thomas, Elizabeth University of Washington Voss, Till [email protected] SwissTPH [email protected] Tripathi, Jaishree Nanyang Technological University, Walczak, Marta Singapore Stanford University [email protected] TRIPATHI, ANUJ MEHARRY MEDICAL COLLEGE Waldman, Benjamin [email protected] [email protected]

Tschudi, Christian Walker, Michael Yale School of Public Health Penn State University [email protected] [email protected]

Ullah, Imran Walrad, Pegine B. Department of Pediatrics, University of York Biomedical Research Institute, Texas Southwestern Medical Center, Univ. of York 6001 Forest Park Road, Dallas, 75235 [email protected] Texas, USA [email protected] Wang, Fengrong [email protected] Usui, Miho Henry M Jackson Foundation/USUHS [email protected] WANG, KAI-HSUAN [email protected]

Vaidya, Akhil Drexel University College of Medicine Ward, Honorine [email protected] [email protected]

Van Biljon, Riëtte Ward, Gary Pennsylvania University of Vermont [email protected] [email protected] Varadharajan Suresh, Warrenfeltz, Susanne University of Georgia Ragavan [email protected] National Institute of Health [email protected] Warrenfeltz, Susanne University of Georgia Verdi, Joey [email protected] Hunter College, City University of New York [email protected] Waters, Andy

Wellcome Trust Centre for Molecular [email protected] Parsitology [email protected] Xu, Rui [email protected] Watzlowik, Maria [email protected] Yeh, Ellen Stanford Wetzel, Dawn [email protected] UT Southwestern Medical Center [email protected] Yun, EJun [email protected] Widmer, Giovanni [email protected] Ženíšková, Katerina Wilkinson, Mark Department of Parasitology, Faculty of [email protected] Science, Charles University in Prague [email protected] Williamson, Kim Uniformed Services University of the Zhang, Xu Health Sciences Weill Cornell Medical College [email protected] [email protected]

Winiger, Rahel R. Zhang, Yang Laboratory of Molecular Parasitology [email protected] [email protected]

Zimmann, Nadine BIOCEV [email protected] Wirth, Dyann Harvard School of Public Health Zimmerberg, Joshua [email protected] NIH [email protected] Xia, Jing

Author Index

A - B 17, 18, 33, 38, 39, 92, ABDELKRIM Botté, Cyrille 105, 179, 190, 208 ÉP. Brown, GUEDICHE, Audrey 36, 192 Yosser zina 34, 181 Brühlmann, Absalon, Francis 24, 47, 146, 237 Sabrina 37, 199 Brunk, Brian 49, 247 Acevedo Bullen, Hayley 36, 193 Ospina, Burleigh, Hamlet 17, 93 Barbara 28, 55, 172, 286 Ahiya, Avantika 35, 183 C Asady, Beejan 21, 46, 122, 236 Calixto, Assaraf, Shany 64, 323 Abigail 36, 194 Balestra, camara, maria Aurélia 16, 84 de los Balouz, milagros 25, 150 Virginia 25, 65, 150, 325 Carter, Bandini, Giulia 26, 60, 161, 298 McKenzie 14, 75 Baptista, Chahine, Carlos 27, 167 Zeinab 37, 195 Basson, Travis 65, 326 Chakrabarti, Batoff, Debopam 37, 54, 201, 280, 282 William 65, 327 Chandra, 14, 22, 28, 35, 39, 46, Monica 37, 196 48, 76, 130, 175, 183, Chasen, Beck, Josh 209, 236, 243 Nathan 37, 197 Bello, Fabiola 26, 60, 162, 298 Clements, Ben Rebecca 37, 198 Chaabene, 21, 37, 46, 122, 200, Rouaa 36, 191 Cobb, David 236 Bennink, Cobbold, Sandra 61, 300 Simon 28, 52, 173, 270 Beri, Divya 25, 60, 155, 298 Collins, Jennifer 37, 54, 201, 280 Beverley, Stephen 24, 143 Connacher, Jessica 38, 202 Bhabha, Gira 21, 120 Connelly, Bhanot, Sean 38, 203 Purnima 51, 263 Costa, Bhatnagar, Elizabeth 38, 204 Suyash 27, 35, 169, 183 Crater, Anna 34, 20, 114, 180 BISHNOI, RITIKA 52, 264 25, 40, 60, 154, 218, Cruz, Angela 298 Bohmer, Monica 54, 282 Cruz Talavera, Irene 38, 205 Bonnell, Victoria 36, 189

Bopp, Selina 62, 65, 311, 331

D Fierro, Dacheux, Manuel 21, 46, 122, 236 Denis 61, 302 Figueiredo, 17, 33, 41, 96, 17, Luisa 229 Dass, Sheena 17, 36, 92, 190 Florentin, Dass, Swati 40, 51, 219, 261 Anat 55, 67, 287, 341 de Florini, Graffenried, Francesca 25, 151 Christopher 16, 40, 87, 220 Fraser, James 23, 137 25, 49, 60, 153, 246, Fu, Yong 55, 288 Deitsch, Kirk 298 20, 34, 65, 114, 180, Desai, Sanjay 330 G Descoteaux, Ganter, Albert 17, 93 Markus 16, 85 Dobson, Garten, Deborah 24, 143 Matthias 28, 175 Drozda, Ghosh, Allison 21, 118 Swagata 54, 55, 277, 289 Dumaine, Gibson, Alexis 41, 56, 222, 290 Jennifer 41, 222 Gkeka, Dumoulin, Anastasia 15, 32, 80, 178 Peter 28, 55, 172, 286 Glennon, Duraisingh, Elizabeth 56, 291 Manoj 18, 41, 103, 225 Glushakova, 15, 20, 32, 34, 37, 40, Svetlana 39, 209 79, 111, 178, 180, Goldberg, 14, 22, 28, 46, 76, Dvorin, Jeff 198, 199, 219 Daniel 130, 175, 236 Dzikowski, 49, 56, 64, 250, 293, Gomes, Ana Ron 323 Rita 16, 32, 88, 178 Goswami, E - F Debashree 27, 166 Egan, Goyal, Manish 56, 293 Elizabeth 21, 39, 121, 211 Guerin, Ekiert, Amandine 21, 119 Damian 21, 120 Guizani, Ikram 23, 34, 47, 138, 181 Ellis, Angela 41, 223 Guler, Ellis, Cameron 41, 54, 224, 286 Jennifer 36, 192 Elsworth, gupta, Brendan 18, 41, 103, 225 devendra Engstler, kumar 63, 314 Markus 56, 294 Gurung, Pratima 63, 315 Esherick, Lisl 20, 34, 111, 180

Evers, Felix 56, 295 Fagbami, Lola 41, 226 Falekun, Seyi 17, 33, 95, 179 14, 25, 41, 60, 77, Ferreira, Tiago 154, 227, 298 Ferrel, Abel 41, 63, 228, 316

H 40, 51, 54, 219, 261, Hagenah, Ke, Hangjun 278 Laura 66, 337 Kegawa, Yuto 39, 209 Hakimi, Kellermeier, Hassan 24, 47, 145, 237 Jacob 39, 210 Haldar, Keroack, Kasturi 68, 346 Caroline 18, 41, 103, 225 Harb, Omar 49, 247 Kieft, Rudo 26, 158 Hart, Melissa 49, 53, 248, 276 Kim, Chi Yong 39, 211 Hasan, Md Kissinger, Mahmudul 49, 249 Jessica 39, 49, 212, 247 21, 39, 46, 65, 123, Klaus, Heaslip, Aoife 210, 236, 329 Severina 16, 85 Heinberg, Klemba, Adina 49, 250 Michael 39, 213 Ho, Chi-Min 14, 76 Knuepfer, Hollin, Ellen 22, 128 Thomas 49, 64, 251, 319 Kongsomboon Hovel-Miner, vech, Angel 40, 214 Galadriel 14, 75 Kumar, Hsu, Hong Pradeep 18, 33, 105, 179 Ming 48, 240 HUANG, L ZHOU 48, 241 Lahree, Huet, Diego 48, 242 Aparajita 40, 52, 217, 269 Hussain, Tahir 48, 243 Lamb, ian 50, 256 Huston, Lander, Noelia 17, 33, 97, 179 Christopher 48, 49, 244, 249 Lane, Kristin 50, 62, 257, 306 Lavazec, 24, 51, 55, 144, 258, I - J - K Catherine 284 Imlay, Leah 48, 245 Lawong, Ishizaki, Aloysus 51, 259 Takahiro 63, 313 Leger- Jaramillo, Abraham, Maritza 70, 357 Melissa 51, 260 23, 61, 62, 69, 137, Jeffers, Vicki 50, 252 Lindner, Scott 300, 307, 351 Jennison, 40, 51, 54, 219, 261, Charlie 50, 254 Ling, Liqin 278 Jimenez, 17, 23, 27, 38, 94, Veronica 50, 255 Llinas, Manuel 137, 169, 202 Kafsack, Björn 25, 60, 153, 298 20, 26, 34, 38, 48, Katris, 17, 36, 39, 92, 190, Lourido, 113, 159, 180, 204, Nicholas 208 Sebastian 239, 242 Kaur, Hargobinder 70, 356 Kaushansky, Alexis 56, 291

M N - O Mach, Jan 47, 52, 238, 266 Nair, Sethu 17, 54, 94, 279 Mandt, Naor, Adit 54, 63, 283, 316 Rebecca 52, 267 N'DRI, Marie- Marcolino, Esther 55, 284 Monica 18, 100 Negreiros, Martynowicz, Raquel 10, 28, 174 Jennifer 52, 268 Nessel, Mather, 40, 50, 51, 61, 219, Timothy 22, 46, 130, 236 Michael 256, 261, 303 Neveu, Gaëlle 24, 51, 144, 258 M'Bana, Okada, Megan 55, 285 Viriato 52, 269 McConville, Olias, Philipp 24, 47, 146, 237 Malcolm 28, 52, 173, 270 McDermott, P Suzanne 53, 68, 271, 344 Pagura, Lucas 28, 55, 172, 286 Meister, Painter, 27, 36, 67, 169, 189, Thomas 53, 272 Heather 340 Merrick, Panas, Catherine 53, 273 Michael 63, 316 Millar, Scott 53, 274 Pangburn, Miskinyte, Sarah 63, 317 Migla 19, 108 Papavasiliou, 15, 32, 37, 80, 178, Modrzynska, Nina 196 Katarzyna 53, 275 Paredez, 22, 49, 53, 128, 248, Alexander 63, 318 Moon, Robert 276 Parveen, Moonah, Nikhat 35, 184 Shannon 54, 55, 277, 289 Pasaje, 19, 20, 34, 49, 64, Mowat, Charisse 110, 111, 180, 251, Kaitlyn 18, 33, 104, 179 Flerida 319 mukherjee, Pellisson, sumit 36, 191 Mélanie 24, 142 Mulaka, 40, 51, 54, 219, 261, Pollastri, Maruthi 278 Michael 54, 280 17, 19, 54, 94, 110, Pontikos, Munro, Justin 279 Michael 64, 320 21, 37, 46, 55, 67, Portugal, Muralidharan, 122, 200, 236, 287, Silvia 23, 134 Vasant 341 Power, B. 24, 47, 53, 147, 237, Murillo_Solan Joanne 274 o, Claribel 54, 280 Prajapati, 22, 52, 70, 128, 265, Murithi, 19, 54, 64, 68, 110, Surendra 353 James 280, 281, 319, 343 Preiser, Peter 35, 185 Murphy, 17, 54, 55, 94, 279, Robert 35, 62, 188, 308 Prigge, Sean 285 Primo, Vincent 35, 186

Q - R Schureck, Quinn, Marc 65, 330 Jennifer 35, 187 Segev Zarko, Li-av 14, 74 Radke, Joshua 61, 301 Seizova, Ramanantsala Simona 22, 46, 129, 236 ma, Miharisoa Rijatiana 61, 302 Serre, David 16, 33, 89, 178 Ramanathan, Shakya, Bikash 21, 121 Aarti 61, 303 Sharma, Ramirez- Aabha 65, 331 Montiel, Sharma, Fatima 61, 304 Manu 66, 332 Raper, Jayne 22, 56, 126, 292 Sheiner, Lilach 15, 32, 81, 178 Rathod, Shukla, Pradipsinh 48, 245 Anurag 66, 333 Rawat, Mukul 62, 305 Sidik, Saima 20, 34, 113, 180 Remcho, T. Sievert, Parks 50, 62, 257, 306 Mackenzie 66, 334 Rijo-Ferreira, 17, 33, 55, 95, 179, Filipa 23, 135 Sigala, Paul 285 Rios, Kelly 62, 307 Silva, Thiago 66, 335 Rivera- Silva Pereira, Cuevas, Sara 17, 33, 96, 179 Yolanda 27, 168 simantov, Roos, David 49, 247 karina 66, 336 Rosenberg, Sinai, Anthony 35, 188 Alex 20, 34, 112, 180 Sladewski, Rudlaff, Thomas 16, 40, 87, 220 Rachel 15, 32, 79, 178 Small- Ruivo, Saunders, Margarida 22, 127 Jennifer 66, 337 Russell, Smith, Tyler 48, 239 Timothy 18, 70, 101, 354 Soldati-Favre, 32, 36, 67, 78, 178, Dominique 191, 338 S Sollelis, Lauriane 25, 152 16, 33, 41, 63, 89, Sa, Juliana 178, 223, 312 SOMEPALLI, MASTANBABU 64, 324 Sambamurthy , Srivastava, Chandrasekar Anubhav 18, 102 an 23, 136 Stadler, Sardinha- Rachel 67, 339 Silva, Aline 19, 109 Stasic, Sateriale, 41, 56, 69, 222, 290, Andrew 67, 340 Adam 349 Stephens, 21, 46, 65, 123, 236, Dylon 55, 67, 287, 341 Schiano, Irio 329 Stokes, Schneider, Barbara 68, 343 Victoria 25, 60, 153, 298

21, 41, 56, 64, 69, W 119, 222, 290, 324, Walczak, Striepen, Boris 349 Marta 35, 53, 182, 272 Striepen, Waldman, Josefine 19, 110 Benjamin 26, 159 Stuart, Ken 53, 68, 271, 344 Walker, Summers, 19, 62, 64, 110, 311, Michael 69, 351 Robert 319 Wang, Sun, Stella 14, 68, 74, 345 Fengrong 40, 216 Suresh, Niraja 68, 346 WANG, KAI- HSUAN 48, 240 T - U - V Ward, Gary 67, 339 Warrenfeltz, Tagoe, Daniel 21, 118 Susanne 49, 247 Tanala Patra, 24, 47, 53, 147, 237, Alok 68, 348 Waters, Andy 274, 275 Tandel, Jayesh 69, 349 Watzlowik, Thomas, Maria 41, 230 Elizabeth 26, 160 Williamson, TRIPATHI, Kim 52, 70, 265, 353 ANUJ 69, 352 Winiger, Rahel Usui, Miho 70, 353 R. 15, 32, 78, 178 27, 35, 40, 50, 61, 66, 19, 52, 62, 64, 65, 169, 183, 219, 256, 110, 267, 311, 319, Vaidya, Akhil 303, 333 Wirth, Dyann 331 Van Biljon, Riëtte 70, 354 X - Y - Z Varadharajan Suresh, Xia, Jing 42, 231 Ragavan 70, 355 Xu, Rui 42, 232 Verdi, Joey 22, 56, 126, 292 Yeh, Ellen 35, 53, 182, 272 Vinetz, Joseph 70, 356 Ženíšková, von Grüning, Katerina 47, 52, 238, 266 Hilde 61, 299 Zhang, Xu 49, 246 Voss, Till 65, 326 Zhang, Yang 26, 62, 158, 309 Zimmann, Nadine 62, 310 Zimmerberg, Joshua 28, 39, 175, 209