Exploring the Roles of Phosphoinositides in the Biology of the Malaria Parasite Plasmodium Falciparum

Exploring the roles of phosphoinositides in the biology of the malaria parasite Plasmodium falciparum

Thèse

Zeinab Ebrahimzadeh

Doctorat en microbiologie-immunologie Philosophiæ doctor (Ph. D.)

Québec, Canada

Exploring the roles of phosphoinositides in the biology of the malaria parasite Plasmodium falciparum

Thèse

Zeinab Ebrahimzadeh M. Sc.

Dave Richard, directeur de recherche

Résumé Plasmodium falciparum est un parasite appartenant au phylum Apicomplexa et est à l’origine de la forme la plus sévère de la malaria. Dans les zones endémiques d'Afrique subsaharienne, la plupart des victimes sont des enfants de moins de cinq ans. L’entrée de P. falciparum dans sa cellule cible, le globule rouge, repose sur la sécrétion de protéines par des organites spécialisés : les micronèmes, les rhoptries et les granules denses. Les mécanismes de biogenèse de ces organites et la coordination de la libération de leur contenu lors de l'invasion sont cependant pour la plupart inconnus. Il a été toutefois été démontré que les protéines destinées à ces organites apicaux se concentrent dans des microdomaines de l’appareil de Golgi, dont la composition en lipides et en protéines détermine leur destination finale. À ce jour, les mécanismes de sélection et de transport des protéines apicales vers les organites d'invasion ainsi que leurs mécanismes de sécrétion durant l’invasion sont pour la plupart inconnus. Nous avons donc posé l’hypothèse que les phosphoinositides (PI) et leurs protéines effectrices sont impliqués dans ces processus chez P. falciparum.

Les PI sont sept lipides phosphorylés retrouvés de façon minoritaire dans les différentes membranes cellulaires. Chaque membrane subcellulaire contient une espèce caractéristique de PI qui peut être reconnue et liée spécifiquement par des protéines effectrices. Une large gamme de processus biologiques sont régulés par les PI, tels le trafic vésiculaire, les canaux ioniques, les pompes d’efflux et les transporteurs, ainsi que certains processus endocytiques et exocytaires. Des études antérieures ont été en mesure de détecter seulement cinq des sept espèces de PI chez P. falciparum. Dans le cadre d’un premier projet, nous avons étudié la distribution de six PI, à savoir PI3P, PI4P, PI5P, PI (4,5)P2, PI(3,4)P2 et PI(3,4,5)P3, chez P. falciparum. Pour ce faire, nous avons exprimé chez le parasite des rapporteurs spécifiques correspondant à des domaines humains de liaison aux PI, fusionnés à une protéine fluorescente. Cette méthode nous a permis de confirmer des rapports antérieurs sur la localisation du PI3P dans la membrane de la vacuole alimentaire, dans de petites vésicules près ou sur la membrane plasmique du parasite ainsi qu’à l’apicoplaste. De plus, nous avons révélé pour la première fois la

iii présence de PI5P chez P. falciparum et montré qu’il se localisait à la membrane plasmique, au noyau et potentiellement dans le réticulum endoplasmique de transition. Nous avons aussi montré que le PI4P est localisé dans la membrane plasmique ainsi que dans l’appareil de Golgi et que le PI(4,5)P2 est présent dans la membrane plasmique tout au long du cycle érythrocytaire. Cette carte de la distribution subcellulaire des PI constitue un excellent outil pour mieux déchiffrer les rôles de ces lipides chez le parasite P. falciparum.

Dans le cadre d’un second projet, nous avons caractérisé une protéine possédant un domaine conservé chez les Apicomplexa, le domain d’homologie de la Pleckstrine, la protéine PfPH2. En utilisant la stratégie de Knock-sideways pour inactiver conditionnellement la protéine d’intérêt, nous avons montré que PfPH2 est impliquée dans l’attachement initial du mérozoite à la surface du globule rouge. Cet effet est directement lié à un défaut de sécrétion d'une population spécifique de micronèmes en l’absence de la protéine PfPH2. Enfin, nous avons mis en évidence que le domaine PH de PfPH2, lorsque exprimé sous forme de protéine recombinante, se lie aux PI avec une grande spécificité. Pris ensemble, nos résultats démontrent le rôle essentiel des PI dans le processus d’invasion et proposent un modèle mécanistique pour l'exocytose des micronèmes.

Abstract

Plasmodium falciparum belongs to the phylum of Apicomplexa and causes the most severe form of malaria. In endemic areas of sub-Saharan Africa, most of the victims are among children under the age of five. P. falciparum relies on proteins released from sophisticated invasion organelles called micronemes, rhoptries and dense granules to enter human erythrocytes. The mechanism of biogenesis of invasion organelles and the coordinated release of their contents during invasion are mostly unknown. It has been shown that proteins targeted to the apical organelles accumulate in microdomains of the Golgi apparatus with specific lipid and protein composition that determine the final destination of their cargo. To date, the mechanisms of transport of the cargo molecules to the invasion organelles and their release mechanism are mostly unknown. We proposed that phosphoinositides (PIPs) and their effector proteins could be involved in these processes in P. falciparum.

PIPs are seven minor phosphorylated lipids in cellular membranes. Each subcellular membrane contains a characteristic species of PIPs that are specifically bound by PIP- interacting proteins. A wide range of biological processes regulated by PIPs such as vesicular trafficking, ion channels, pumps, and transporters and control both endocytic and exocytic processes. Based on previous reports five out of seven PIP species have been detected in P. falciparum. In my first project, we have studied the distribution of six PIPs namely PI3P, PI4P, PI5P, PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3 using expression of specific reporters made up of human PIP-binding domains fused to a fluorescent protein. Here, we have confirmed previous reports on PI3P localization to the food vacuole membrane, small vesicles close/on the parasite plasma membrane and the apicoplast. Also, we have reported for the first time the presence of PI5P in P. falciparum and showed that it localizes to the PM, nucleus and potentially transitional ER. PI4P shows localization to the PM and Golgi and PI(4,5)P2 localizes to the PM all over the erythrocytic cycle. The resulting map of the subcellular distribution of PIPs will now be a great tool to further decipher the roles of these lipids in P. falciparum,

In the second project, we have characterized a Pleckstrin Homology domain-containing protein (PfPH2) conserved in all apicomplexan parasites. Using the knock sideways

v strategy to conditionally inactivate the protein, we show that PfPH2 is involved in an early step of the invasion process, when the merozoites initially attach to red blood cells. We further demonstrate that this is due to the abrogated secretion of a specific population of micronemes. Finally, we reveal that recombinantly expressed PfPH2 binds PIPs with a broad specificity. Taken together, our results present evidence for the role of PI in invasion and propose a mechanistic model for the exocytosis of micronemes.

Table of contents

Résumé ...... iii Abstract ...... v Table of contents ...... vii List of Tables ...... x List of Figures ...... xi Abbreviation list ...... xii Avant-Propos ...... xvi Acknowledgments ...... xvi Contributions ...... xviii Introduction ...... 1 Malaria ...... 1 Etiology and Epidemiology ...... 1 Disease and Pathology of Plasmodium Infection ...... 4 Malaria vector ...... 4 Plasmodium falciparum life cycle ...... 5 Erythrocytic stage ...... 5 Atypical organelles ...... 8 Molecular bases of invasion ...... 10 Malaria Treatment ...... 14 Diagnosis, Treatments and Resistance ...... 14 Prevention and Vaccine Development ...... 15 Drug Resistance and Discovery ...... 18 Phosphoinositides ...... 21 PIP-binding proteins ...... 23 Phosphoinositide species ...... 25 Phosphoinositide metabolism ...... 31 PI kinases ...... 31 Phosphatases ...... 41 Chapter 1: Hypothesis and problem statement ...... 47 1.1 Hypothesis and objectives ...... 47 Chapter 2: A map of the subcellular distribution of phosphoinositides in the erythrocytic cycle of the malaria parasite Plasmodium falciparum ...... 50 Avant-propos ...... 50 Résumé ...... 51 Article ...... 52 Abstract ...... 53 Introduction ...... 54 Materials and methods ...... 57

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Results and Discussion ...... 59 Acknowledgments ...... 68 References ...... 68 Tables ...... 78 Figure legends ...... 79 Figures ...... 82 Supplementary Table ...... 86 Supplementary figure legends...... 88 Supplementary figures ...... 90 Chapter 3: A pan-apicomplexan phosphoinositide-binding protein acts in malarial invasion-microneme exocytosis...... 94 Avant-propos ...... 94 Résumé ...... 95 Article ...... 96 Abstract ...... 97 Introduction ...... 98 Results and Discussion ...... 100 Conclusion ...... 108 Materials and Methods ...... 108 References ...... 118 Competing interest ...... 125 Materials and Correspondence ...... 125 Acknowledgments ...... 125 Author contributions ...... 125 Data availability ...... 126 Figure legends ...... 127 Expanded View Figure legends ...... 130 Figures ...... 132 Appendix ...... 141 Appendix figure legends ...... 143 General Discussion, Conclusion and Perspectives ...... 153 A map of subcellular distribution of phosphoinositides in P. falciparum ...... 155 PI3P distribution ...... 155 PI4P distribution ...... 156 PI5P distribution ...... 157 PI(4,5)P2 distribution ...... 157 PI(3,4)P2 and PI(3,4,5)P3 distribution ...... 158 Conclusion on the subcellular PIP distribution and general pitfalls ...... 159 A pan-apicomplexan phosphoinositide-binding protein acts in malarial invasion- microneme exocytosis...... 159 PfPH2 is a PH-containing protein with a relaxed PIP-binding specificity...... 159 PfPH2 localizes to a structure close to the apical tip of the merozoite ...... 160

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PfPH2 is essential for the erythrocytic cycle and its absence affects merozoite invasion due to a default in microneme exocytosis...... 161 Conclusions on PfPH2 mechanism of action and future experiments ...... 164 References ...... 166

List of Tables

Table 1 Malaria vaccines in preclinical development or in clinical trial...... 17 Table 2 Phosphoinositide kinases and phosphatases in P. falciparum compared to yeast and T. gondii...... 37

List of Figures

Figure 1The malaria parasite life cycle...... 2 Figure 2Endemic area of malaria from 1900 to 2002. [6] ...... 3 Figure 3Erythrocytic cycle...... 6 Figure 4Smear of erythrocytic stages under light microscope...... 6 Figure 5Atypical organelles of merozoite...... 9 Figure 6Invasion ligands and their receptors involved in the invasion of erythrocyte by Plasmodium falciparum ...... 11 Figure 7An illustration of the seven known PIPs, and the enzymes involved in PI P metabolism...... 21 Figure 8A map of the subcellular localization of Pl in higher eukaryotic cells...... 23 Figure 9 PIP-recognizing effectors...... 24 Figure 10Kinases involved in phosphoinositide metabolism in yeast, mammalian cells and apicomplexan parasites...... 32 Figure 11The role of the PI-PLC pathway and calcium signaling at different stages of the Plasmodium life cycle and downstream stage-specific effectors...... 46

Abbreviation list

A Anopheles ACTs ART-based combination therapies AMA1 Apical membrane antigen 1 ANTH AP180 N-terminal homology AP Apicoplast AP-1 Adaptator protein-1 AP-2 Adaptator protein-2 ARF1 ADP-ribosylationfactor 1 ARM Armadillo ART Artemisinin ARTs Artemisinin and its semi-synthetic derivatives Atg Autophagy related protein Atg14 Autophagy related protein14 Atg Autophagy related protein BATS Barkor/Atg14(L) autophagosome targeting sequence BIP Binding immunoglobulin protein cKO conditional Knocking-Out C2 Conserved region-2 of protein kinase C Ca2+ Calcium CDPKs Calcium-dependent protein kinases CDPK1 Calcium-dependent protein kinase 1 CDPK5 Calcium-dependent protein kinase 5 CR1 Complement receptor 1 CyRPA GPI-anchored antigen D Dense granule DAG Diacylglycerol DBL Duffy binding-like domain DGK1 Diacylglycerol kinase-1 DHA Dihydroartemisinin DHR-1 Dock homology region-1 DOC2.1 Double C2 domain protein DOK5 Docking Protein 5 EBAs Erythrocyte binding antigens EBA-140 Erythrocyte binding antigens 140 kDa EBA-175 Erythrocyte binding antigens 175 kDa EBA181 Erythrocyte binding antigens 181 kDa EBL1 Erythrocyte-binding ligand 1 EE Early endosome ENTH Epsin N-terminal homology

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ENR Enoyl acyl carrier protein reductase ER Endoplasmic reticulum Fab1 Forms aploid and binucleate cells FAPP1 Four-phosphate-adapter proteins 1 FAPP1 Four-phosphate-adapter proteins 2 FERM 4.1, ezrin, radixin, moiesin FK506 a drug molecule (Immunosuppressor) FV Food vauole FYVE Conserved in Fab1, YOTB, Vac1 and EEA1 GAP45 Glideosome-associated protein 45 kDa GAP50 Glideosome-associated protein 50 kDa GlyA Glycophorin A GlyB Glycophorin B GlyC Glycophorin C GOLPH3 Golgi phosphoprotein 3 GPI Glycosylphosphatidylinositol GSK GlaxoSmithKline GTP Guanosine triphosphate heme Hemozoin HMW High molecular weight HPLC High-performance liquid chromatography IMC Inner membrane complex INPP4A Inositol polyphosphate 4-phosphatases A INPP4B Inositol polyphosphate 4-phosphatases B Ins(1,4,5)P3 or IP3 Inositol 1,4,5-trisphosphate IPP Isopentenyl pyrophosphate IPZ Imidazopyrazines iRBC infected Red blood cell iRBCM infected Red blood cell-membrane KD Knock-down KO Knock-out KS Knocksideways M Microneme MSP1 Merozoite surface protein 1 MTM Myotubularin MVBs Multivesicular bodies P. Plasmodium PA Phosphatidic acid PAS Pre-autophagosomal structure PDK-1 Phosphoinositide-dependent kinase 1 PDZ Postsynaptic density 95, disk large, zonula occludens

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PCR Polymerase chain reaction PEXEL Protein export element PfATG8 P. falciparum autophagy protein 8 PfATG8 P. falciparum autophagy protein 18 PfEMP-1 P. falciparum erythrocyte membrane protein-1 PfFCP P. falciparum FYVE-containing protein Pfs25 Post-fertilization antigen 25 Pfs230 Post-fertilization antigen 230 PfRH1 P. falciparum reticulocyte-binding like homolog 1 PfRH2a P. falciparum reticulocyte-binding like homolog 2a PfRH2b P. falciparum reticulocyte-binding like homolog 2b PfRH4 P. falciparum reticulocyte-binding like homolog 4 PfRH5 P. falciparum reticulocyte-binding like homolog 5 PfRIPR PfRH5 interacting protein PH Pleckstrin homology domain PI Phosphoinositides PiK1 Phosphatidylinositol kinase PIKs PI kinases PI3Ks PI3-kinases PI4Ks PI4-kinases PI(4)K Phosphatidylinositol-4-OH kinase PIKfyve Phosphoinositide kinase containing FYVE domain PI3P Phosphoinositide 3-phosphate PI4P Phosphoinositide 4-phosphate PI5P Phosphoinositide 4-phosphate PI(4,5)P2 Phosphoinositide 4,5-biphosphate PI(3,5)P2 Phosphoinositide 3,5-biphosphate PI(3,4)P2 Phosphoinositide 3,4-biphosphate PI(3,4)P2 Phosphoinositide 3,4,5-triphosphate PIPKs Phosphatidylinositol phosphate kinases or PIP kinases PIP5Ks PIP 5-kinases PIP4Ks PIP 4-kinases PI4P5K Phosphatidylinositol 4-phosphate 5-kinase PI-PLC PI-specific phospholipase C PKA cAMP-dependent protein kinase A PKC Protein kinase C PKG Protein kinase G PLCD Phospholipase C-delta PM Plasma membrane P4M PI(4)P binding of SidM/DrrA PM-V Plasmepsin V

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PM-X Plasmepsin X PM-IX Plasmepsin IX PROPPINs β-propellers that bind PIs PTB Phosphotyrosine binding Phosphatase and tensin homologue deleted on chromosome PTEN 10 PtIns Phosphatidylinositol PV Parasitophorous vacuole PVM Parasitophorous vacuole-membrane PX Phox homology R Rhoptry Rab Ras-related RAMA Rhoptry-associated RAP1 Rhoptry associated protein1 RBC Red blood cell RBCM Red blood cell-membrane RDTs Rapid diagnostic tests RHs Reticulocyte-binding like homologs RON2 Rhoptry neck protein2 RON complex Rhoptry neck protein complex SHIP SH2 domain-containing inositol 5-phosphatase SERA Serine repeat antigen SERA Serine repeat antigen5 SERA Serine repeat antigen6 SLI Selection-linked integration SNARE Soluble NSF Attachment Protein REceptor SP Signal peptide Stt4 Staurosporine and temperature sensitive SUB1 Subtilisin-like protease 1 SYLF SH3YL1, Ysc84p/Lsb4p, Lsb3p and plant FYVE proteins TAPP1 Tandem PH domain-containing protein 1 TBVs Transmission-blocking vaccines TGN Trans-Golgi network T. Gondii Toxoplasma gondii TgPH1 T. Gondii PH domain 1 TLC Thin-layer chromatography Vps15 Vacuolar protein sorting 15 WHO World Health Organization

Avant-Propos Acknowledgments

Ph.D. is a great opportunity from scientific and professional perspective but it can be very difficult since it affects both personal and social life. However, I find myself very lucky and glad for having nice companies that I encountered in the path. My supervisor, Dave, is one of those that I grant my luck and this work would not been possible without his friendly guidance, support and expert advice. His advice and support have been invaluable in advancing my project. I am also grateful for the time he spent in revision of my thesis and his kind consideration of my pregnancy and slow-moving! writing. Beside his professional and science expertise, he is always a good friend to his student with a great sense of humor that make the long hours of work, easy and cheerful in the lab. I would like to thank the rest of my thesis committee: Prof. Josée Lavoie, Dr. Denis Leclerc, and Dr. Christopher Fernández Prada, not only for their insightful comments and encouragement, but also for the questions which incented me to widen my research from various perspectives. I would especially like to thank Dr. Josée Lavoie as my teacher and mentor, she has shown me, by her example, how a great scientist and person should be. I take this opportunity to thank my fellow labmates for stimulating discussions, for the sleepless nights we were spending together before deadlines, and for all the fun we had in the last seven years. In particular, I am grateful that I had Dominic Gagnon as a collegue for both his friendly attitude and professional work during my thesis. I do appreciate your generous support and collaboration in all these years, something that indeed contributed to the success of my work. I thank my fellow labmate, Stephanie Hallee for her cunning and responsible personality. Also wish to express my deep gratitude to Dr. Angana Mukherjee for her involvement in the second project and her invaluable comments and suggestions which contributed greatly to the improvement of my thesis. I also want to thank all of those whom I had the pleasure to work with during my PhD and my projects, Catherine, David, Eamim, Mari-Eve and Audrey. Quebec has definitely offered me a unique experience which is useless to count but something unquestionable is the opportunity of meeting with amazing people from around the world. I want to thank Carole Dumas for her kindness and unconditional support, Dr.

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Ouafa Zghidi-Abouzid for her wise and kind advices and Dr. Prasad Padmanabhan for his generosity and unique jokes. Nobody would have been more important to me in pursuing my Ph.D studies than my family. I would like to thank my mother, whose love and guidance is always with me. Also my sister, Serva, which I am proud to have her always by my side. I would not forget the part that my two children played in my PhD too. Avin and Aso, who are the source of unending inspiration for me, came to this world in the hardest step of my life and filled my life with joy and love. Special thanks are due to my ultimate love, Hiva for his continuous support and understanding. I am lucky to have him by my side and I owe him my successes and happiness.

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Contributions

This doctoral thesis focuses on phosphoinositides’ localization and also characterization of a phosphoinositide effector in Plasmodium falciparum. The thesis contains two main projects, the first part provides a map of phosphoinositide distribution and the second part presents a new molecular player in the invasion process of erythrocytic cells by Plasmodium falciparum. The presented thesis is divided into seven chapters. First chapter is a review of literature on malaria parasite and phosphoinositide metabolism in the parasite and model organisms. The introduction starts with the characterization of malaria, geographical distribution and the general life cycle of the parasite. Continually, there are detailed description of parasite morphological changes during erythrocytic cycle, atypical organelles and their role in invasion erythrocytes. After, malaria treatment and prevention methods with their pitfalls have been described and new developments in these areas have also been reviewed. In the second part of the introduction, phosphoinositide structure, function and the enzymes responsible for their metabolism and regulation are reviewed. In the second chapter, the hypothesis and my objectives of my doctoral thesis have been presented. In answer to my two objectives, a manuscript has been already published and a manuscript is currently undergoing review. The paper presented in Chapter 3, entitled “A map of the subcellular distribution of phosphoinositides in the erythrocytic cycle of the malaria parasite Plasmodium falciparum” has been written as a ''scientific paper''. The paper has been published in the ''International Journal for Parasitology'' (IJP) in January 2018. In the manuscript presented in Chapter 4 entitled “A pan-apicomplexan phosphoinositide-binding protein acts in malarial invasion-microneme exocytosis”. It is a scientific paper and I am the first author. The article has been published in the EMBO Reports in May 2019. The final chapter is the discussion and general conclusions and perspectives on the two projects.

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Introduction Malaria Etiology and Epidemiology Malaria has plagued humanity for millennia after finding evidence from as early as 2700 BC in China to 400 BC in Greece, where it was described as periodic fevers and enlarged spleen [1]. From over a 100 Plasmodium species, only five are able to infect humans and cause malaria. These include Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi. The relevance of the malaria fevers to the presence of a parasite was first reported by Rastori in 1816. However, it was not clear until Laveran in 1880 observed Plasmodium gametocyte stage parasites in the blood of patients. Not long after, the method of transmission was revealed (1887-89) when Ross and Grassi together linked the female Anopheline mosquito vector to malaria transmission [1, 2]. Later, Shortt and Garnham in 1947 showed that a liver stage development comes before the erythrocytic stage. An important step which demonstrates where parasite resides during 8-30 days after infection [1]. Overall, the entire life cycle of Plasmodium was gradually unveiled over 100 years. Our current knowledge of the Plasmodium life cycle is presented in the Fig. 1.1, which is common among all species only with some differences in the details.

Malaria parasite belongs to the phylum Apicomplexa and shares two hosts: human and female Anopheles mosquito. An infected female Anopheles mosquito takes a blood meal from a human host and inoculates approximately 100 Plasmodium sporozoites into the dermis [3]. The sporozoites travel through the dermis until they reach a blood vessel. They then migrate to the liver where they invade, traverse and develop in hepatocytes via schizogony. Schizogony is an asexual reproductive process used by some apicomplexans. During the process, the parasites undergo nuclear division preceding cytokinesis. After a seven to ten day development, mature liver schizonts rupture and release merozoites into the bloodstream where they start the next phase of their life cycle called erythrocytic cycle. Merozoites invade erythrocytes and start a cycle of maturation from a ring to a metabolically active, hemoglobin degrading trophozoite. This is followed by another round of schizogony where they release new merozoites for reinvasion of new erythrocytes. Some early erythrocytic stages undergo a different route of development into the sexual gametocyte stages of the parasite. In P. falciparum, in particular, there are five

1 morphologically distinct stages of gametocyte development. Mature gametocytes are taken up in a blood meal by Anopheles vectors where male and female gametes mature and undergo fertilization in the midgut to form a zygote. These zygotes elongate and become motile, developing into ookinetes that invade the mosquito midgut wall and develop into oocysts. The oocysts then mature and produce sporozoites, which upon oocyst rupture, migrate to the salivary glands of the mosquito, where they are ready for inoculation into a new host.

Figure 1 The malaria parasite life cycle.

(malariasite.com)

Over 10,000 years the relationship between Plasmodium and humans has undergone evolutionary changes as each organism exerted selective pressure on the other. As humans scattered around the world, Plasmodium distributed geographically and expanded to other continents (Fig. 1. 2). During this time, human knowledge about the parasite grew and led to the use of malaria prevention methods. The latter along with climate changes, once again decreased the affected area to certain degrees [4, 5].

Figure 2 Endemic area of malaria from 1900 to 2002. [6]

In general the geographical area at risk for human malaria has been reduced, from around 53% of the total earth land area to 27% [6]. Additionally, malaria deaths have also experienced a drop over the past two decades. Fortunately, malaria-related child mortality has also decreased over 30% in sub-Saharan Africa from 2004 [7, 8]. Similarly, a continuous fall has been observed in global malaria deaths outside of Africa since 1990 [8]. A significant number of countries have also moved toward malaria elimination from 2000. Among 106 countries with ongoing malaria transmission in 2000, 15 countries reached malaria elimination and more than 50 lowered the number of new malaria cases to at least 75% by 2015 and eighteen countries reduced their malaria cases by 50- 75% [9].

Nevertheless, malaria still is a public health threat. Every year, 3.3 billion people are at risk of malaria infection. Only in 2016, 216 million cases have been estimated which resulted in approximately 445,000 deaths. Malaria transmission continues in South and South East Asia, Central and South America, and Africa. Among which, the most deadly malaria outbreaks occur in Sub-Saharan Africa due to P. falciparum [9]. Over the past decade, big steps have been made to reduce mortality and morbidity, however, malaria control alone will not be sufficient. While the available antimalarial interventions are effective and should be kept to control malaria (restrain disease, prevent death and interrupt transmission), treatment methods need improvement due to the new wave of resistance against current treatments. The ultimate goal in the control of malaria is to design a vaccine that is effective against a broad range of malaria species. This needs a big

3 investment in both control and new drug strategies on the one hand and continued pressure on the vector and parasite for future success and elimination efforts.

Disease and Pathology of Plasmodium Infection Malaria causes symptoms that typically include fever, shivering, tiredness, vomiting, and headaches [10]. Patients with uncomplicated malaria may present with fever, enlarged liver or spleen, and mild jaundice or anemia. The symptoms of malaria are related to the asexual erythrocytic stage. Along with the rupture of schizont and destruction of erythrocytes, numerous known and unknown waste substances such as hemozoin and other toxic factors are released into the blood stream which initiates an inflammatory response by the host immune system [10].

More severe disease occurs when malaria infection is complicated by organ failure or blood or metabolic abnormalities which include cerebral malaria, severe anemia, hemoglobinuria, renal failure and acute respiratory distress syndrome [11]. Severe symptoms are often the result of parasite sequestration, often seen with late asexual stages of P. falciparum, which bind to endothelial surfaces in capillaries and small blood vessels via the P. falciparum erythrocyte membrane protein-1 (PfEMP-1), a protein present on the erythrocyte membrane. The result of such a sequestration is the blocking of blood flow and oxygen deprivation in tissues [12]. Primary symptoms usually begin ten to fifteen days after being bitten [13]. In those who have recently survived an infection, reinfection usually causes milder symptoms. This partial resistance disappears over months to years if the person has no continuing exposure to malaria [13]. Dormant liver stage hypnozoite forms of the parasite are found in P. vivax and P. ovale infections and can reactivate, resulting in relapse after patients have recovered from the illness months or years after the original infection [14].

Malaria vector More than 400 different species of Anopheles mosquito are recognized of which around 30 commonly transmit parasites of the genus Plasmodium [9]. Among them, Anopheles gambiae is one of the best known, because of its major role in the transmission of P. falciparum to human [15]. An important behavioral factor for a mosquito vector is the degree to which an Anopheles

species prefers to feed on humans (anthropophily) or animals. Both A. gambiae and A. funestus, the primary malaria vectors in Africa, are strongly anthropophilic. Consequently, they are two of the most efficient malaria vectors in the world [15, 16].

Plasmodium falciparum life cycle As discussed above, the life cycle of Plasmodium species is overally similar (reviewed in early introduction) with some differences in the duration of incubation time and of different sub-cycles. Duration of the erythrocytic cycle in P. vivax, and P. falciparum is 48 hours. While in P. ovale and P. malariae, it takes longer between 50 and 72 hours, respectively [17]. It is worth mentioning, in P. vivax and P. ovale, hepatocytic stage can be dormant and persist in the liver cells for weeks, or even years. Among all three different stages of Plasmodium infection, the erythrocytic stage has driven the most attention due to its role in all malaria complications.

Erythrocytic stage Invasion of erythrocyte begins when a merozoite attaches to the host plasma membrane and penetrates into the erythrocyte using proteins released from the invasion organelles (Fig. 1. 3). Invasion organelles are a set of secretory organelles namely micronemes, rhoptries and dense granules which reside at the apical end of the merozoite [18]. The merozoite initial attachment is weak and it is followed by merozoite rotation to its apical end, which results in a stronger bond with host membrane [19]. This close contact is known as "tight-junction" which moves from the apical to the posterior end of the merozoite during invasion, an active process played by the parasite actin-myosin motor and proteases [20]. The invasion ligands mediating the tight-junction are removed proteolitically via rhomboid family of serine proteases, a process known as "shedding" event [21-23]. As the merozoite advances into the host cell; the parasite fabricates a parasitophorous vacuole (PV). The PV isolates the parasite from the cytosol of the red blood cell (RBC).

Figure 3 Erythrocytic cycle.

[24]. Under the light microscope, the newly invaded parasites have a ring-like structure in the giemsa- stained thin smears which gives the stage its name (Fig. 1. 4) [25]. Rings may possess one or two chromatin dots. The parasite begins to feed on the surrounding RBC through a small, dense ring on the surface of the parasite, the cytostome [26-29]. As the parasite grows during first 24 hours, the area of the PV surrounding the parasite also increases, and extends tubular-like projections into the surrounding RBC [30, 31]. The ring eventually changes shape to a more rounded trophozoite (Fig. 1. 4).

Figure 4 Smear of erythrocytic stages under light microscope.

Hrs= hours. (Ebrahimzadeh)

A distinctive point between the ring and trophozoite stages is the appearance of a pigmented vacuole within the parasite (Fig. 1. 3-arrow). The vacuole contains ingested hemoglobin with brown crystals called hemozoin (heme) that originally accumulates within small vacuoles but later fuse to form a single larger vacuole called food vacuole (FV) (Fig. 1. 4-arrowhead) [32]. The parasite exports various parasite proteins into RBC which gradually alter its appearance and results in some of known trophozoite stage manifestations, including Maurer's clefts and knob structures. In the infected-red blood cell (iRBC) cytoplasm, Maurer's clefts are believed to act as a sorting station to export parasite proteins toward RBC-membrane (RBCM) [33-35]. Later, the knob structures are dense and rigid membranous protrusions that appear during trophozoite and schizont stage on the iRBC-membrane. Owing to the hypervariability and the adhesive feature of parasite antigens in the knob structures, the parasite is able to evade the host immune system by its sequestration in capillaries and the formation of rosettes [36]. Sequestration allows the parasite to avoid splenic clearance however it also leads to blood flow blockage, which is known to be one of the cause of cerebral and placental malaria [37, 38]. Second, cytoadhesion to uninfected erythrocytes, also called rosetting [39], makes it easy for new released merozoites to quickly invade new host cells while hiding between uninfected erythrocytes. This is also a cause of microvascular blockage and severe anemia [40, 41]. Therefore, knobs are central to the virulence of P. falciparum. In general, during trophozoite stage the parasite increases protein synthesis and enlarges in size and prepares for nuclear and organellar multiplication. Another special feature of this stage is the branching of mitochondria and apicoplast in preparation for division [25]. The synthesis of some of the molecules needed for parasite multiplication starts from the trophozoite stage. In theory, a schizont is an intraerythrocytic parasite that is undergoing or has undergone repetitive nuclear division. As the parasite approaches the end of the cycle, it continues to consume hemoglobin. As a result, the parasite produces more hemozoin crystals and the FV grows bigger in size [42]. The nucleus divides about four times or more to produce about 16-32 nuclei. Nuclear division is endomitotic, division of chromosomes without nuclear division, a common feature in unicellular eukaryotes. Therefore, the segregating chromosomes and the spindle apparatus remain within the nuclear envelope throughout the process [26, 27, 43, 44].

Nuclear division is accompanied by multiplication of mitochondria, Golgi, and the apicoplast in the cytoplasm. The Golgi apparatus consists of a single disc-shape cisterna which is originated from nuclear envelope vesicles [43, 44]. It is believed apical organelles are the result of coated vesicles initiated from the Golgi [43, 44] that fuse to create the two rhoptries [44], or they stay individually to create micronemes or dense granules [25].

Subsequently, a cleavage furrow forms around each nascent merozoite containing a nucleus, mitochondrion, Golgi and plastid. A constriction ring then separates each merozoite from the residual body of the schizont containing the food vacuole. The separated merozoites group within the parasitophorous vacuole. Finally, the PV-membrane (PVM) and RBCM are disrupted following an increase in cGMP levels, which results in microneme secretion into the PV and onto the merozoite surface [45]. A key protease, subtilisin-like protease 1 (SUB1) processes several substrates that are important for downstream events [46]. Among these are members of the serine repeat antigen (SERA) family. SERA5 and SERA6 are the most abundant SERAs in blood stages. Recent work has revealed that both SUB1 and SERA6 are essential for successful egress. SUB1 is required for PVM breakdown while SERA6 is needed to disrupt the RBCM [47]. Recent observations also strongly suggest SUB1 is involved in proteolytic activation of several other proteins, including SERA6 and the merozoite surface protein 1 (MSP1) [48, 49]. Breakdown of the PVM and RBCM allows the merozoites to egress from iRBCs. The new liberated merozoites now invade new RBCs. The synchronous release of merozoites and toxic material from the iRBCs are responsible for the cyclical symptoms of the disease, including fever, chills, nausea, body aches and headaches, which can lead to serious complications mentioned earlier.

Atypical organelles Micronemes Micronemes are about 120 nm long and vary in shape and numbers between Plasmodium species (Fig. 1. 5). They are enclosed in a bilayer membrane, and have a fine granular interior [18]. During merozoite egress, micronemes release partially their content and their complete discharge happens later during the invasion process. Microneme discharge happens presumably by membrane fusion into the rhoptry duct/the plasma membrane (PM) and consequently to the exterior [50, 51].

Structural evidence suggests micronemes are originated from budding vesicles from the Golgi apparatus [52].

Rhoptries These organelles are pear-shaped membrane-bound and found at the apical end of the merozoite (Fig. 1. 5). Based on electron microscopy results, each rhoptry consists of two distinct parts: an electron-dense rounded basal bulb and a less dense narrow duct [44]. Like for micronemes [44, 53], rhoptries appear to be formed initially as small vesicles originating from the Golgi, which eventually fuse together and grow in size [43, 44]. This has also been demonstrated in Toxoplasma sp. [54]. The membrane surrounding the rhoptry is a bilayer membrane [44]. During invasion, rhoptry ducts fuse with each other at the tips as well as with the merozoite plasma membrane [55], and they crumble as they completely empty their content.

Figure 5 Atypical organelles of merozoite.

M= microneme, R= rhoptry, D= dense granule, AP= apicoplast. (Ebrahimzadeh)

Dense granules These organelles are spheroidal membranous vesicles, which in P. knowlesi are about 80 nm in diameter [50, 56, 57], and of similar appearance in P. falciparum (Fig. 1. 5) [58-60]. They are

9 situated between the rhoptries and the merozoite nucleus, freely within the cytoplasm. After invasion of a red blood cell, the dense granules move to the merozoite surface where they fuse with the membrane and liberate their contents into the nascent parasitophorous vacuole, which originate finger-like projections that extends into the red blood cytoplasm. [50, 56-60].

Apicoplast The discovery of the vestigial plastid (apicoplast) in apicomplexan parasites such as malaria and Toxoplasma gondii gave us new insight into the origin of the phylum Apicomplexa. The first hints on the existence of the apicoplast were images of circular, extra chromosomal DNA molecules in P. lophurae, a malarial parasite of ducks, published by Kilejian [61]. Then Iain Wilson in collaboration with an expert mitochondriologist, Donald Williamson, and researchers Malcolm Gardner and Jean Feagin commenced to study malarial extra chromosomal DNAs. In 1991, the group published a paper in Parasitology Today entitled "Have malaria parasites three genomes?" [62]. This title was intentionally provocative. Two plant scientist, Geoffrey McFadden and Ross Waller, which they did not typically read journals on parasitology, quite accidentally saw the paper. As plant scientists, they were well aware of the dogma that only algae and plants have three genomes. So, when they read the paper's title about malaria parasites having three genomes, this was equivalent to saying that malarial parasites were plants [63]. We now know that the Apicomplexan family originated from photosynthetic ancestors, probably similar to modern dinoflagellate zooxanthellae [64, 65]. The apicoplast has four bounding membranes [66], a characteristic of secondary endosymbiosis, in which the plastid is derived by eukaryote-eukaryote endosymbiosis (Fig. 1. 5) [67]. We have also learned that the apicoplast contains an ensemble of bacteria-like pathways to replicate and express its genome plus an anabolic capacity generating fatty acids, heme and isoprenoid precursors. Apicoplasts are essential, and perturbing them, usually results in parasite death, thus making apicoplast metabolism an attractive target for drugs (reviewed in ref. [68]).

Molecular bases of invasion Merozoites are probably the smallest form of all the Plasmodium spp, with dimensions close to a large bacterium ( ≈1.6 μm long and 1.0 μm wide) [25]. This stage of the parasite is important

immunologically because it is exposed to the human immune system even if for as short as one minute or so. Merozoites also share special features with other invasive forms of Plasmodium, the sporozoite and ookinete form. The common characteristics of all are having a polarized morphology and apical organelles, where invasion proteins are located [18]. This gives a good opportunity for drug and vaccine designing to possibly target all three stages.

Invasion is a complex, multistep process and highly regulated. It is categorized into three stages: pre-invasion, internalization, and post-invasion (Fig. 1. 6) [52]. In vitro the entire process, from egress to the end of the invasion, is usually accomplished in less than a minute. Early interactions of merozoite-erythrocyte appear to be mediated by MSP1 complex and is a random binding on merozoite's sides (Fig. 1. 6). In this step, the interaction between invasion ligands and their receptors is cumulative which cause the erythrocyte membrane to indent [69].

After the primary attachment, the merozoite reorients to its apical end where its invasive organelles will be in close contact with the erythrocyte surface. Two family of proteins, the erythrocyte binding antigens (EBAs) and the reticulocyte-binding like homologs (RHs), mediate reorientation of merozoite [70, 71]. The EBAs and PfRhs are held in reserve until needed in

Figure 6 Invasion ligands and their receptors involved in the invasion of erythrocyte by Plasmodium falciparum [70].

11 micronemes and rhoptries, respectively. The EBA protein family includes EBA-175, EBL1, EBA- 140, and EBA-181 and they bind to erythrocyte receptors glycophorin (Gly) A, GlyB, GlyC, and putative erythrocyte membrane protein band 4.1 respectively [72]. The binding of most EBA ligands to their erythrocyte glycophorin receptors is dependent on sialic acid residues on these glycoproteins.

The PfRHs family has five members: PfRH1, PfRH2a, and PfRH2b and PfRH4. The first three members have no known receptors and PfRH4 binds to complement receptor 1 (CR1) [73]. The fifth family member, PfRh5, has a different function downstream of the other family members (see below). The EBAs and PfRHs are referred to as alternative pathway ligands because they are functionally redundant and replaceable in part if not totally [73-75]. It has been shown the deletion of EBA-175 in W2mef parasite strain results in the upregulation of PfRH4 which indicates to functional substitution of EBA-175 [74]. Recent data suggest that alternative pathway ligands work together with a combination of overlapping function and cooperation [71]. The parasite can epigenetically silence or upregulate particular invasion-related genes, such as PfRH4 [74, 76, 77], resulting in divergent ligand expression between isolates. Due to this plasticity, the parasite is able to rapidly adjust to erythrocyte receptor polymorphisms in human populations [76, 78, 79].

Microneme and rhoptry content release occurs in multiple steps and it is speculated that EBAs and PfRHs interactions with their respective receptors stimulate downstream invasion events [80]. Rhoptry release starts with early discharge during initial merozoite contact and ends when the parasite has completed internalization into the host cell [81]. In the case of micronemes, there is accumulating evidence suggesting they are composed of heterogeneous populations with specific functions in egress and/or invasion[82]. The trigger for protein release from micronemes and rhoptries is not well-known. There is some evidence hinting at changes in ion concentration like potassium and Ca2+ during egress which trigger PfRH1 and PfEBA-175 release [83-85]. Interestingly, live cell imaging has revealed two calcium fluxes during the invasion. The first flux in the merozoite is observed as a faint and weak signal upon egress. While the second flux is often intense and punctate at the point of contact between the merozoite apex and its erythrocyte. The timing of the second flux is immediately after merozoite contact with erythrocyte and before proceeding to the invasion [69].

The most irregular member of PfRH family is PfRH5. Unlike other PfRHs, it is smaller in size, has no transmembrane domain and is expressed in all parasite strains. The gene is refractory to disruption attempts, a sign of its essentiality [86, 87]. It has two partner proteins, termed PfRH5 interacting protein (PfRIPR), and a GPI-anchored antigen (CyRPA). Through interaction with PfRIPR and CyRPA, it is anchored to the merozoite surface [86, 88-90]. In this step, live cell imaging on merozoites blocked by antibodies to PfRH5 or its erythrocyte receptor, basigin, shows the complete reorientation of the merozoites [69, 88]. This is the distinction between the attachment and entry of the parasite. Recently, a calcium-regulated phosphatase, calcineurin, has been shown to play a role in host cell attachment too. Although it is not precisely clear how calcineurin affects the attachment step, the findings show calcineurin is required for the extracellular parasite to strongly attach to the host before intracellular entry [91, 92].

For entry, another step of rhoptry release is needed during which the rhoptry neck protein complex (RON complex) is embedded in the erythrocyte membrane. The RON complex serves as a docking site for apical membrane antigen 1 (AMA1) to hold on the erythrocyte membrane and forms the tight-junction [93-96]. Once the tight-junction is established, the merozoite advances into the erythrocyte membrane and the PV, mediated by rhoptry proteins, forms around the merozoite [55, 97]. Video microscopy of merozoites treated with antibodies against RON2 reveals there is an important difference compared to the PfRH5 block [69, 98-100]. In addition to erythrocyte deformation and merozoite reorientation, the RON2-antibody-treated merozoites cause echinocytosis of their target erythrocyte, indicating RH5 complex triggers the echinocytosis of the erythrocyte [69]. An interesting work of Volz et al. confirmed PfRH5/PfRIPR/CyRPA complex binding to host receptor basigin is required for Ca2+ release and establishing the tight-junction [88]. This finding plus the earlier calcium flux observations indicate a stage of rhoptry release immediately upstream of the AMA1–RON2 interaction that is triggered by Ca2+ release results in the tight-junction formation [69, 88].

In brief, the following order of events are characterized during recent advances in dissecting P. falciparum invasion:(a) A weak, reversible interaction mediated by MSP1 complex results in slight deformation in the binding area on the targeted erythrocyte. (b) Then release of the alternative-

13 pathway EBA/PfRH ligands which interact strongly and irreversibly with their receptors leading to the merozoite reorientation. (c) Later, PfRH5 binds to the erythrocyte receptor basigin which causes a further stage of rhoptry release indicated by a calcium flux at the parasite-host interface. (d) Upon RON complex embedding in the erythrocyte membrane, AMA1-RON2 form a tight- junction in the entry site [81] (e) Finally, as the merozoite invades the erythrocyte with the force of actin-myosin motor, rhoptry proteins and lipids form the PV around the parasite, and protease proteins degrade the used ligand-receptor bands in the tight-junction. In the end, the tight-junction will be sealed and the parasite will be isolated in the PV from its surrounding erythrocyte [69].

Malaria Treatment Diagnosis, Treatments and Resistance Clinical diagnosis is based on the patient’s symptoms and on physical findings at examination. The classic symptom of malaria is a cyclical occurrence of sudden coldness followed by shivering and then fever and sweating [10]. The time of periodic cycle is different in Plasmodium species [17]. It occurs every two days (tertian fever) in P. vivax and P. ovale infections, and every three days (quatrain fever) for P. malariae. In case of P. falciparum infection, the recurrent fever is the shortest and every 36-48 hours. Sometimes it is almost continuous fever and hard to diagnose from other infections [17]. Malaria parasites can be identified by examining under the microscope [10, 101]. A drop of the patient’s blood is spread out as a “blood smear” on a microscope slide. Both, thick and thin smears, must be provided and examined by a laboratory technician. Prior to examination, the specimen is stained by the Giemsa stain (or other available staining methods) to visualize the intracellular parasites. The microscopy technique is still the gold standard for laboratory confirmation of malaria. However the efficiency of the test varies, depends on the quality of the reagents, of the microscope, and on the experience of the laboratorian [101]. The results of microscopy can be deceiving especially in case of falciparum malaria. The degree of parasitemia or the parasite stage can be underestimated due to partial antimalarial treatment or by sequestration of parasitized cells deep into vascular walls. Therefore, double-checking of the infection by other methods, if available, is recommended before proceeding to diagnostic.

Various test kits are available to detect antigens derived from malaria parasites and provide results in 2-15 minutes. These “Rapid Diagnostic Tests” (RDTs) offer a useful alternative to microscopy in situations where a reliable microscopic diagnosis is not available [10, 101, 102]. However, their accuracy needs to be improved. Importantly, their cost in most of the malaria-affected areas are not affordable. Parasite nucleic acids can be detected using polymerase chain reaction (PCR) [10, 101, 102]. Although this technique is more sensitive than smear microscopy, it needs a standard healthcare center in malaria endemic area. Even so, the PCR results are often not available quickly enough to be of value to diagnose the type of infection. However, it is the best to detect the species of malarial parasite after the primary diagnosis by either smear microscopy or RDT [101, 102].

Treatment of malaria depends on many factors, including the species of parasite(s) causing malaria, the area of the world where the disease was contracted which could indicate which drugs the parasites would be resistant to, disease severity, age, weight and if the patient is pregnant or not. Malaria can be a severe, potentially fatal disease, especially when caused by P. falciparum, and treatment should be initiated as soon as possible [103]. The current recommendation for treatment is with artemisinin and its semi-synthetic derivatives (ARTs). For more efficiency, they are prescribed with partner drugs in ART-based combination therapies (ACTs) [104]. Most of the prescribed drugs are active only against the blood stage parasite and cannot block parasite transmission. Moreover, the biggest challenge for malariologists is resistance to anti-malaria drugs. Therefore, drug resistance testing, in case of availability, is recommended before doctors proceed to any treatments.

Prevention and Vaccine Development The first line of malaria elimination is prevention methods. Individual protection for mosquito control like bednets, insecticides, and repellents are being recommended in endemic areas. Also, protective clothing can help at times of the day when vectors are active [10]. Draining still waters and using insecticides have an important impact on the vector control. Even so, disease elimination is out of reach in many areas without a vaccine. The path to develop an effective malaria vaccine is a very demanding process both due to parasite related-complexity and economically [105]. Malaria parasites have a complex life cycle and the polymorphic nature of its antigens make it even more

15 difficult to find effective and universal candidates with required immunogenic property. Economically, malaria affects mainly people in low-income countries leading to a lower interest in the vaccine development investments.

An effective and durable vaccine for malaria must be able to target all three life stages of the parasite: pre-erythrocytic stage, blood stage, and mosquito stage [106]. Though, such a vaccine seems to be far from being available and existing; vaccine strategies are mostly targeting just one stage of the parasite life cycle. Pre-erythrocytic vaccines target the clinically silent stages of Plasmodium during sporozoite and liver stages intending to eliminate parasite at the first step of parasite entrance. The advantage of such vaccines, in case of success, is prevention of disease and most importantly interrupting parasite transmission. As of today, four candidates are in different stages of clinical trials with the most promising being the RTS,S vaccine (Table. 1. 1) [106]. In the last malaria vaccine symposium 2017, the World Health Organization (WHO) announced that the RTS,S malaria vaccine completed its Phase III clinical trials and the pilot implementation programs would begin in Ghana, Kenya, and Malawi in 2018 (Phase IV). The RTS,S vaccine is composed of the repeat region of the circumsporozoite protein fused to the Hepatitis B virus surface antigen, and is adjuvanted with AS01 adjuvant. Findings on RTS,S demonstrate it is safe and efficient in adults, children, and young infants in sub-Saharan Africa [106]. More than 15,000 children were vaccinated with RTS,S in 11 centers across seven African countries during the phase III trials which is been estimated 829 clinical malaria episodes per 1000 children were prevented over 18 months of follow-up [106]. Though, RTS,S vaccine still shows moderate effects and its efficacy declines over time [107].

Blood stage based vaccine development faces some restricting points such as antigenic polymorphism of infected erythrocyte surface proteins, redundancy in the merozoite invasion pathways (alternative pathways), and expressing conformationally correct parasite antigens [108]. Even so, exciting findings have recently identified two new protein candidates promising a high level of specificity: PfRH5 and the AMA1-RON2 complex. RH5 is the first highly conserved target from the merozoite which is susceptible to neutralizing antibody induced by vaccine [109]. It is an invasion ligand which is common between alternative pathways [88]. In Aotus monkeys, PfRH5- based vaccines induced antibody response and rose protection against a heterologous P. falciparum

challenge [110]. A first generation PfRH5-based vaccine is being also tested in clinical trials, but more studies are required to improve vaccine efficacy, including identification of new RH5 epitopes [111] and a proper cell line for the large scale production of PfRH5 in a conformationally correct form [112]. The AMA1-RON2 complex is another erythrocyte stage vaccine candidate that targets merozoite invasion. The complex mediates merozoite invasion through tight-junction formation that is a stage subsequent to PfPRH5. Initial reports demonstrate antibody response raised against AMA1-RON2 complex peptide can protect against virulent P. yoelii and P. falciparum infection in mice and Aotus monkeys, respectively [113]. The vaccine development is still in preclinical trials.

Table 1 Malaria vaccines in preclinical development or in clinical trial.

[106]

Parasite stage Vaccine classification Current status PfSPZ vaccine Whole organism Phase II (radiation attenuation)

GAP vaccines Whole organism Phase I (genetic attenuation)

RTS,S Subunit Phase IV

Pre-erythrocytic stage Pre-erythrocytic CVac Whole organism Phase I (chemical attenuation)

Chemically attenuated Whole organism Preclinical parasites

AMA1-RON2 Subunit Preclinical Blood stage Blood Subunit Subunit

Pfs25 Subunit Phase I

Pfs230 Subunit Phase I (TBVs)

Mosquito stage stage Mosquito Pfs47 Subunit Phase I

The third category of vaccine strategies, transmission-blocking vaccines (TBVS), strive for parasite transmission blocking via targeting parasites at the mosquito stage. Currently, Pfs25 (post- fertilization antigen), Pfs230 (pre-fertilization antigen) and Pfs47 (an immune suppressor of A. gambiae) showed good properties in preclinical studies as vaccine candidates for mosquito stage

[106]. Ongoing trials combine Pfs25 and Pfs230 vaccine antigens to test their efficiency and find the best formulation for vaccine activity [114, 115]. In one case, both Pfs25 and Pfs230 conjugate vaccines administered with AS01 adjuvant from the GlaxoSmithKline (GSK). Based on preclinical studies this formulation might notably increase antibody titer and therefore serum functional activity after vaccination [106].

Maintaining durable protection after immunization is one of the key limitations for the success of malaria vaccines [106]. This could be due to the suppressing mechanisms of the parasite towards host’s immune system and also poor immunogenic property of antigens. Combination of different antigens from different stages, new methods of administration as well as the dosage and vaccine schedules may potentially lead to a more durable and effective vaccine.

Drug Resistance and Discovery The emergence of resistance to ARTs was reported several years ago and is now spread out in six countries in South East Asia [116]. Malaria drug resistance is best described for P. falciparum as it is the most deadly infection and a quick treatment is crucial for survival. The historical and genetic origins of P. falciparum resistance to artemisinin, chloroquine, and sulfadoxine-pyrimethamine have consistently been found within Western Cambodia [117]. Chloroquine-resistant P. falciparum is the most widespread form in the world. Hence, combination therapies like antifolate, mefloquine, and atovaquone were introduced, but later resistance against chloroquine combination therapies also emerged [118]. This was coincident with the discovery of artemisinin in 1972 by a Chinese scientist Tu Youyou who was awarded later half of the 2015 Nobel Prize in Medicine [119]. Artemisinin is extracted from Artemisia annua a herb employed in Chinese traditional medicine [120]. Artemisinin and its derivatives are exceptionally fast acting against intra-erythrocytic asexual blood-stage malaria parasites [120]. But with a big disadvantage; their very short half-life in vivo (about one hour in human). As a result, they are co-administered with longer half-life partner drugs, such as lumefantrine, amodiaquine, piperaquine, mefloquine, sulphadoxine- pyrimethamine or pyronaridine as ACTs [104, 119].

The molecular basis for chloroquine, antifolate and atovaquone resistance has been well established. Mutations in the target transporter or enzymes of these drugs allow for the parasite to 18

become resistant by a number of mechanisms, including increased efflux ability or inability to inhibit the enzyme [119]. In the case of ARTs, the exact resistance mechanism is still elusive but recent findings showed resistant parasites have mutations in Kelch13 gene affecting encoded propeller, BTB/POZ domains [121, 122]. It has been shown that Kelch13 gene mutations are associated with a slow parasite clearance rate after treatment with artemisinin derivatives [116, 121]. Following, scientists noticed a correlation between Kelch13 mutations and mutations in at least four other genes (fd, arps10, mdr2, and crt). Whenever the kelch13 mutation was present in the genome of a resistant parasite, the other four mutations almost invariably seemed to be there too [121].

With the appearance of ART resistance, the need for new drug strategies is now more urgent than ever. Many parasite pathways have been targeted due to their uniqueness including hemoglobin digestion and the folate pathway [123, 124]. Mitochondrial function and respiration are also interesting targets since the electron transport chain in Apicomplexa species differs from that of the mammalian host mitochondria [125-127]. Inhibitors of the apicoplast, a plastid-like organelle found in most Apicomplexa, which target the synthesis of crucial precursors like isopentenyl pyrophosphate (IPP) are another route for anti-malarials [128]. Apicoplast pathways obviously do not exist in the human host and there has been considerable excitement about targeting the apicoplast pathways as a parasite Achilles' Heel for drug inventory with no or fewer side effects for its infected host [68, 129, 130]. Plasmodium proteases also represent potential high-value targets [49]. Partly, due to our knowledge of their enzymatic mechanisms and active site structures. More importantly, they have been shown to be involved in a variety of pathways that are essential for parasite survival [49]. For example, aspartic proteases called plasmepsins which are involved in diverse cellular processes include interesting hits like Plasmepsin V, IX and X. In the ER lumen, Plasmepsin V (PM-V) is involved in a specific cleavage downstream of an export signal. Most Plasmodium exported proteins contain a protein export element (PEXEL) motif and its cleavage by PM-V exposes a specific signal [131, 132] that is detected by a translocon at the parasitophorous vacuole membrane [133]. PM-V is therefore a very promising target and its inhibition will likely affect most extracellular functions of the parasite which are related to the parasite virulence. Recently Nasamu A.S. et al. showed both PM-IX and PM-X are important for erythrocyte invasion [134]. PM-X, in addition to invasion, controls egress and merozoite maturation via processing

SUB-I [134]. Of importance, the same group has identified compounds with potent antimalarial activity targeting PM-X [134]. Plasmepsin X has also been suggested to play a role in midgut transversal by ookinetes [135]. Therefore, a PM-X inhibitor has the potential of targeting two or three stages. However, there are other parasite pathways that have yet to be explored for drug discovery.

Currently, an area of focus for P. falciparum drug development is kinase inhibitors [136-139]. With the success of kinase inhibitors as a treatment for other disease models such as cancers, much research is going on to develop kinase inhibitors specifically targeting P. falciparum kinase activity. Employing bioinformatic analysis, putative kinases and signaling pathways in P. falciparum have been identified [140, 141]. Many parasite kinases have been found to be different from mammalian kinases, which may indicate their usefulness as a drug target, specially kinases that affect phosphoinositide metabolism [142-144]. Despite disappointing news of malaria resistance to ACTs, phosphatidylinositol-4-OH kinase (PI(4)K) inhibitors have brought back hope to the battle camp of malaria [145-149]. Fortunately, recent advances in malaria research in the fields such as forward genetics and structure-activity relationship chemistry are powerful assistant for drug designers to potentially target unique enzymes of P. falciparum. Along with that, basic knowledge on Plasmodium spp. biology has considerably improved owing to recent progresses in the fields of genetic modification methods. In particular, the development of conditional genetic approaches in P. falciparum is allowing us for the first time to validate essential genes and study their molecular functions. DiCre recombinase [150, 151] and selection-linked integration (SLI) [152] are currently efficient available methods for P. falciparum. The DiCre recombinase system allows for conditionally Knocking-Out (cKO) genes [153] while SLI system increases our ability to select integrants in a shorter time compared to the traditionally used drug cycling method. Also SLI system in combination with knocksideways permits mislocalization of native proteins [152]. Hopefully the new approaches will help to develop a better understanding of the basic biology of the parasites which is vital to develop novel medical therapeutics.

Phosphoinositides In eukaryotic cells, cellular membranes are a station for cell-signal transduction, and they undergo frequent changes like budding, fission, and fusion [154]. These require highly regulated dynamic interactions between cytosolic protein complexes and membrane components. A key mechanism in such a regulation is the recruitment of protein complexes to certain membranes at specific time points. To a large extent, this recruitment is arranged by phosphorylated versions of the membrane lipid phosphatidylinositol (PtIns), known as phosphoinositides (PIPs) (Fig. 1. 7) [155, 156].

Figure 7 An illustration of the seven known PIPs, and the enzymes involved in PI P metabolism. kinases (blue), phosphatases (violet) and phospholipases (green). DAG, diacylglycerol; MTMR, myotubularin-related phosphoinositide phosphatase; SHIP, SH2 domain-containing inositol 5- phosphatase; OCRL, oculocerebrorenal syndrome of Lowe; PIKfyve, phosphoinositide kinase with specificity for the five position containing a FYVE finger. Pathways that have been proposed but not characterized are indicated with dashed arrows [157].

Traditionally phosphorylated lipids were known as precursors of the two messengers inositol 1,4,5- trisphosphate (Ins(1,4,5)P3 or IP3) and diacylglycerol (DAG) in calcium signaling events [156].

However, deeper investigations revealed the tiny lipids and their effector enzymes are the regulators of vast cellular functions in eukaryotic cells such as signal transduction, cell motility, cytoskeletal reorganization, DNA synthesis, cell cycle, adhesion, membrane transport, permeability, and trafficking [155, 156]. They are especially the focus of many studies in drug discovery field from cancers to infection [156].

Upon specific stimulation, PI-kinases and -phosphatases enrich or deplete certain subcellular membranes from specific PIP (Fig. 1. 7) [158]. Most unicellular organisms like yeast produce only five phosphoinositides PI3P, PI4P, PI5P, PI(3,5)P2, and PI(4,5)P2. Among these, only PI4P and PI(4,5)P2 are essential for viability [156, 158]. Higher eukaryotes produce two more PIP isoforms namely PI(3,4)P2 and PI(3,4,5)P3. The distribution of phosphoinositides establish organelle identity known as the "PIP" code (Fig. 1. 8) [155, 158]. The PIP code is then “read” by specific effector proteins, which contain domains with the ability to detect individual PIPs. These interactions eventually lead to the recruitment of host proteins to specific intracellular compartments [156].

Due to the importance of these tiny lipids, some studies started focusing on the role of PIPs in P. falciparum. However, the molecular identity and the combination of the PIP profile in infected RBC were not clear until recently. Uninfected RBCs only produce PI4P and PI(4,5)P2 when labeled with 32P-phosphate but after infection with P. falciparum, the production of PIPs considerably changes in both PIPs level and diversity [159, 160]. In 2010, using a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), Tawk et al. gave a detailed characterization of the PIPs profile in infected erythrocytes by P. falciparum [160]. Their results showed that, in addition to PI4P and PI(4,5)P2, the infected erythrocytes produce high levels of PI3P and low levels of PI(3,4)P2 and PI(3,4,5)P3 [160]. PI(3,5)P2 was not detected and PI5P could not have been detected with the method used. The presence of PI(3,4,5)P3 was unexpected as unicellular organisms generally do not produce this lipid. Recently, PI(3,4,5)P3 production has been re-confirmed in both P. falciparum schizonts and in P. berghei ookinetes via lipidome analysis [161]. Surprisingly, in T. gondii, only PI3P and PI4P have been identified in high quantities [162]. However, unpublished observations of Wengelnik et al. have suggested the higher phosphorylated species are present but only in small amounts [163].

Figure 8 A map of the subcellular localization of Pl in higher eukaryotic cells. PIPs are concentrated in distinct pools of cytosolic membranes and serve as markers of various cell compartments and regions. EE= early endosome; MVB= multivesicular bodies [155]. PIP-binding proteins The identification of protein effectors of PIPs was a revolutionary step in the understanding of the importance of the PIPs code in the regulation of different biological processes inside cells. In particular, those with PIP-binding domains that can recognize individual PIPs specifically [155]. Since the recognition of the first protein module in 1994, the list of PIP-binding domains has grown larger (Fig. 1. 9) [164].

Figure 9 PIP-recognizing effectors. [154]

Presently, there are 16 modules that exhibit a wide range of affinities and selectivities toward lipid derivatives in the membranes [154, 165]. These include PH (Pleckstrin homology domain), ANTH (AP180 N-terminal homology), BATS (Barkor/Atg14(L) autophagosome targeting sequence), C2 (conserved region-2 of protein kinase C), DHR-1 (Dock homology region-1), ENTH (Epsin N- terminal homology), FERM (4.1, ezrin, radixin, moiesin), FYVE (conserved in Fab1, YOTB, Vac1 and EEA1), GOLPH3 (Golgi phosphoprotein 3), P4M (PI(4)P binding of SidM/DrrA), PDZ (postsynaptic density 95, disk large, zonula occludens), PROPPINs (β-propellers that bind PIPs), PTB (phosphotyrosine binding), PX (Phox homology), SYLF (SH3YL1, Ysc84p/Lsb4p, Lsb3p and plant FYVE proteins), and Tubby modules (reviewed in [154]). Among them, proteins containing PH domains are one of the largest families of signaling proteins. PH domain-containing family are important players of many biological processes such as membrane dynamics, protein trafficking, intracellular signaling, cytoskeletal alteration and lipid metabolism [155, 158, 166-169]. Most of the PH domains are promiscuous and can bind to a variety of PIPs but with different affinities. Nevertheless, 10-20% of the PH domains show specificity for individual PIPs such as PI(3,4,5)P3, PI(4,5)P2, and PI(3,4)P2 [169, 170]. Domains such as the FYVE domain and PX domain often preferably bind to PI(3)P [171, 172]. Proteins with FYVE domains are implicated in the regulation of endocytosis mostly [154, 173]. PX domains are found in many signaling and regulatory proteins mediating essential biological processes such as endocytosis, protein sorting, membrane trafficking, transcription, and cell polarity [154, 174]. Fluorescent molecular made up of PIP- binding domains fused to a reporter protein are very useful tools to study PIP dynamics and allow imaging of the PIP location in live cells [175].

Phosphoinositide species The structure of PIPs are based on glycerol, a three-carbon alcohol with the formula CH 2 OH– CHOH–CH 2 OH and an inositol ring. PtdIns synthase is responsible for the synthesis of PtdIns which is located on the cytoplasmic face of the endoplasmic reticulum (ER), the Golgi, mitochondria, and microsomes in both human [176] and yeast [177]. Partly, lipid phosphatases are also involved in recycling PtdIns via dephosphorylation of mono-phosphoinositides [178, 179].

PI3P PI3P accounts for about 30% of the total PIPs in yeast and is as abundant as PI4P [180]. Contrarily, in human cells, it represents less than 15% of monophosphorylated PIPs and is much less abundant than PI4P [181]. PI3P is produced by the phosphorylation of PtdIns at position D3 of inositol or by the dephosphorylation of PI(3,4)P2 or PI(3,5)P2 via 4- or 5-phosphatases [182]. In yeast and mammalian cells, PI3P is enriched at endosomal membranes and membranes of the multivesicular body (MVB) [173]. At the early endosomes, it plays a central role in recruiting endosomal effector proteins which are involved in the endosomal sorting of proteins and the formation of the MVB [183]. One of the physiological roles of PI3P is to serve as a precursor for PI(3,5)P2 and PI(3,4)P2 synthesis [181]. Furthermore, it is involved in autophagy through the formation of PI3P-enriched regions at the ER via a PI3-kinase, a process termed pre-autophagosome formation [184, 185]. The latter then serves as platforms to recruit autophagosomal proteins [186-188]. In fact, mutations affecting the binding of human pre-autophagosome proteins to PI3P has been observed in cancers and neurodegenerative diseases, indicating an important biological role of PI3P in the regulation of autophagy [187]. Additional functions of PI3P include endocytic trafficking, macroautophagy, phagocytosis, cytokinesis, and nutrient sensing [189].

The lipid has been studied extensively in apicomplexan parasites. Radioactive labeling experiments revealed high levels of PI3P synthesis in P. falciparum-infected erythrocytes, approximately 30% of the total PIP-monophosphates [160], or even higher, according to unpublished data from Wengelnik et al. which is a condition more similar to yeast than mammalian cells [163]. Similarly, in T. gondii, PI3P levels are relatively high [162]. In the malaria parasite, PI3P localizes to the food vacuole, at the apicoplast and in the membranes of small vesicles clustered near the food vacuole and/or the apicoplast [160]. A fluorescent PI3P reporter also localizes to the apicoplast in T. gondii [162]. However, constitutive expression of the PI3P reporter was not tolerated in T. gondii and led

25 to the disturbance of apicoplast biogenesis. Consequently, the parasites lost their apicoplast and died in a delayed death mode [190]. This finding brought up a probable role for PI3P in vesicular trafficking toward the apicoplast and also an evolutionary link between phagosome formation, ER- enriched PI3P membranes, and the apicoplast membrane. As mentioned before, the apicoplast is originally derived from the phagocytic compartment during the secondary endosymbiosis event. And the analysis of the outermost membrane of the apicoplast and the ER-enriched membranes indicated the presence of autophagy proteins [191, 192]. An unusual function has also proposed for PI3P in P. falciparum parasites as its involvement in protein export into parasite-host [193]. According to this finding, PI3P is present in the ER lumen and interacts with certain sequence motifs that are important for the export of parasite proteins to the host erythrocyte [193]. Since PIPs generally reside in the cytoplasmic leaflet of cellular membranes, the ER lumen is not a common location for PIPs. This finding has been later challenged with a work that our lab has contributed where it was showed that the PEXEL (an export motif) sequence does not bind PI3P and that a PI3P-reporter containing PEXEL sequence was targeted into the PV, the default secretory pathway, without accumulating in the ER lumen [194]. In a recent study, high PI3P levels were found to be related to a specific mutation in Kelch13 in artemisinin resistance parasites. Kelch13 mutation is one of the known molecular markers for artemisinin resistance in the malaria field. It is hypothesized that Kelch13 regulates PfPI3Kinase (PfVps34) via ubiquitination and control its abundance in the parasite [195]. Thus, Kelch13 modulates PI3P levels indirectly. Some effector proteins containing PI3P-binding domain have been also studied in P. falciparum. A PI3P- binding FYVE-containing protein (PfFCP) has been identified in the FV [196]. Recent findings on an autophagy protein, PfATG18, also showed its importance for the apicoplast inheritance through interaction with PI3P [197]. Its ortholog, TgATG18 binds to PI3P as well but it is not localized to the apicoplast unexpectedly [197]. Another autophagy protein, PfAtg8, has been shown to localize to the apicoplast [191]. The presence of autophagy proteins plus Vps34 suggests a role for PI3P in apicoplast biology.

PI4P PI4P accounts for around 30% of the total PIPs in yeast and ≈ 45% in Human [181]. It is mostly produced by the phosphorylation of PtdIns by PI4-kinases [198] and some parts are the result of the dephosphorylation of diphosphorylated PIPs by 3- and 5-phosphatases [182]. In yeast, PI4P is

enriched mostly in the PM and the Golgi [199]. Some pools of PI4P are also found in endosomal compartments and in the trans-Golgi [200]. The Golgi is a central station in the secretory pathway where PI4P is involved in Golgi trafficking toward the PM [201], and the retrograde transport from the Golgi to the endoplasmic reticulum [199]. Many proteins interacting with PI4P have been localized to the Golgi [202] and most of them were shown to transport lipids in association with PI4P [203]. At the ER, PI4P is hydrolyzed to PtdIns by the SAC1 phosphatase and the released energy is, consequently, used for sterol transfer [204]. PI4P is also required for receptor sorting in the early endosomes [205]. In addition, it is a key intermediate in the biosynthesis of PI(4,5)P2 at the PM. The PH domain of FAPP1 and FAPP2 (four-adaptor-phosphate proteins 1 and 2) proteins, Golgi resident proteins, are broadly used as PI4P-reporters in different organisms due to their specificity for PI4P-binding [202, 206].

The intracellular distribution of PI4P is traced to foci and the parasite plasma membrane in P. falciparum [145, 207]. In a study in P. berghei, parasites expressing a PI4P-reporter treated with PI4K inhibitors showed redistribution of the PI4P-reporter signal from intracellular foci to the parasite PM [145]. These parasites also showed a failure in membrane ingression during merozoite formation, an evidence for the role of PI4P in cytokinesis in Plasmodium.

PI5P PI5P is the last identified monophosphorylated PIPs due to its low basal concentration in quiescent mammalian cells, and due to technical difficulties in separating it from PI4P [208]. In basal mammalian cell conditions, PI5P represents less than 10% of monophosphorylated PIPs [181] but its level increases following specific stimuli. In mammalian cells, PI5P is mostly synthesized from PtdIns by PIKfyve and some results from the dephosphorylation of P(3,5)P2 by 3-phosphatases [209-211]. A fraction of PI5P is found in the nucleus, where it could be involved in stress responses [212]. The PH domain of DOK (downstream of tyrosine kinase) proteins shows a strong binding affinity to PI5P, and this binding activates the phosphorylation of DOK proteins in T cell signaling [213]. Moreover, PI5P was also shown to localize in the PM and involved in PM and endosomal functions [214, 215]. It has been shown the latter is employed by Shigella bacteria to regulate membrane dynamics during host cell invasion. Shigella bacteria express a bacterial phosphatase, IpgD, which dephosphorylates PI(4,5)P2 into PI5P [216, 217]. In addition, an

27 increase in the number of autophagosomes has been observed after PI5P addition to cells treated with the Vps34 inhibitor wortmannin or knocked down for Vps34, indicating a link between PI5P and autophagy [218].

To date, there is no indication for the presence of PI5P in P. falciparum, and the only detected putative PIKfyve-like orthologue does not contain a FYVE domain.

PI(4,5)P2 PI(4,5)P2 is the most abundant PIPs in both yeast and human and it represents about 45% of total PIPs [158]. PI(4,5)P2 is involved in a vast variety of biological processes including endocytosis, phagocytosis, cell adhesion, and cell motility [219, 220]. PI(4,5)P2 is most abundantly present in the cytosolic face of the PM, where it acts as an anchoring point and a major regulator of membrane fusion events such as vesicle endocytosis and exocytosis [158, 182, 221]. It also interacts with cytoskeletal proteins and bridges between the cytoskeleton and the PM [221]. In yeast, PI(4,5)P2 is the result of the action of the PI5-kinase (Mss4) and the PI4-kinase (Stt4) at the PM from PtdIns [182, 222]. Different effector proteins required for the internalization step of endocytosis have ENTH, ANTH or PH domains that interact specifically with PI(4,5)P2 [158, 223]. PI(4,5)P2 has also been detected in the nucleus, where it is involved in the control of the expression of targeted genes [224, 225]. Further functions for PI(4,5)P2 is coming from the fact that the lipid is the precursor of intracellular signaling molecules IP3 and DAG through the action of PIP-specific phospholipase C (PI-PLC).

In Plasmodium, PI(4,5)P2 is the most abundant PIPs [160]. It might be produced via phosphorylation of PI4P by the single type I phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) [163, 226]. Most of our knowledge about the role of PI(4,5)P2 in Plasmodium comes from calcium/PI(4,5)P2/PI-PLC signaling. Through this signaling cascade, PI(4,5)P2 is implicated in many cellular functions such as ookinete motility, merozoite egress, and sporozoite gliding motility [161, 227-229].

PI(3,4)P2 In human cells, PI(3,4)P2 counts for less than 10% of the total PIPs in quiescent cells while it is not detectable in yeast. Upon cell stimulation, PI(3,4)P2 levels transiently increase via dephosphorylation of PI(3,4,5)P3 by 5-phosphatases [181]. Alternatively, PI(3,4)P2 is synthesized by the phosphorylation of PI4P via class II PI3K lipid kinases at the PM [182]. PI(3,4)P2 acts as a secondary messenger by recruiting protein kinases such as Akt (protein kinase B) and PDK1 (phosphoinositide-dependent kinase 1) through their PH domain [230]. A transforming mutation in the PH domain of Akt1 is associated with several cancers [231]. The link between PI(3,4)P2 and the PI3K/Akt signaling pathway suggests that this PIP could be involved in numerous biological processes, such as controlling the cell cycle, cell survival, and clathrin-dependent endocytosis [232]. The balance between PI(3,4)P2 and PI(3,4,5)P3 is essential in modulating signaling pathways downstream of Akt [233]. Therefore, the interaction between lipid kinases and phosphatases keeps this balance and deregulations of molecular players could lead to numerous problems [234]. Among the various protein domains binding PI(3,4)P2, only the PH domains of TAPP1 (tandem PH domain-containing protein 1) interact specifically with PI(3,4)P2 [235, 236].

In Plasmodium, PI(3,4)P2 has been detected at low levels by metabolic labeling. However, there is no evidence of its function or localization yet.

PI(3,4,5)P3 In yeast, there is no detectable PI(3,4,5)P3 and in human, it represents less than 5% of total PIPs in quiescent cells. However, following various stimuli, its intracellular levels rapidly and transiently increase up to 100-fold [237]. PI(3,4,5)P3 is mainly localized in the PM and is synthesized by class I PI3-kinases from PI(4,5)P2. Small pools of PI(3,4,5)P3 can be synthesized in other subcellular membranes in response to agonists [181]. It is a signaling molecule in many signaling pathways and its regulation is critical. PTEN (phosphatase and tensin homologue deleted on chromosome 10), a PI-phosphatase, is among its regulators. It has been categorized as a tumor suppressor since mutations in the pten gene are linked to many cancers [238]. PI(3,4,5)P3 regulates important cellular functions such as cell proliferation and cell survival, cytoskeleton dynamics, cell motility, membrane trafficking and apoptosis [202, 239].

In Plasmodium, PI(3,4,5)P3 has been detected in low levels [160, 161]. Apicomplexan parasites only possess a single PI3-kinase (class III) which is known to synthesize only PI3P in other organisms. Phosphorylation assays using the immunoprecipitated PfPI3-kinase revealed that the enzyme was able to phosphorylate PtdIns, PI4P, and PI(4,5)P2 [240]. However, a recent work has challenged this result and suggested that trace levels of PI(3,4)P2 and PI(3,4,5)P3 in P. falciparum are produced by other related kinases found in the P. falciparum genome [241].

PI(3,5)P2 PI(3,5)P2 is a rare PIP and represents less than 5% of the total PIPs in both yeast and human. In response to osmotic stress, PI(3,5)P2 synthesis is stimulated, and its intracellular level increases 20-fold compared to non-stressed cells [242]. In yeast, PI(3,5)P2 is synthesized from PI3P to generate PI(3,5)P2 via PI3P 5-kinase Fab1 [182]. It is mostly enriched in vesicular and tubular domains in late endosomes [158, 243]. At the endosomes, yeast epsins Ent3 and Ent5 interact with PI(3,5)P2 through their ENTH domain and are required for endosomal sorting of ubiquitylated cargos and endosomal recycling of SNARES [244-246]. To date, the yeast Atg18/Svp1 and Hsv2 proteins which are involved in autophagy show the highest affinity and specificity for PI(3,5)P2 in vitro [247, 248]. In human, PI(3,5)P2 synthesis is similar to yeast, and it is catalyzed by PIKfyve, the sole PI3P 5-kinase [249]. PI(3,5)P2 plays an essential role in protein sorting at the late endosomes/MVB, lysosomal homeostasis or signaling pathway regulations [181, 250].

In P. falciparum, PI(3,5)P2 is not detected. Although a PIKfyve-like protein is present in Plasmodium genomes, it does not contain a FYVE domain in contrast to PIKfyve of all other organisms, and there is no evidence that it is capable of PI(3,5)P2 production [160]. In T. gondii, the single PIKfyve has been studied and revealed enlargement of the apicoplast upon its conditional disruption [190]. Comparable results were obtained after depletion of an ATG18 homologue, an autophagy protein in T. gondii [197]. TgATG18 was also described to bind both PI(3,5)P2 and PI3P in lipid blots and liposome binding assays. Similar observations have been seen with PI3P depletion following PI3K inhibition [251]. Altogether, these data suggest a common role of TgPIKfyve, ATG18 and PI3K in apicoplast homeostasis and inheritance along with PI3P and PI(3,5)P2 lipids. In another study, using a pull-down approach in T. gondii, an interactor protein of PI(3,5)P2 was detected and denoted TgPH1 [251]. However, it binds PI3P in vivo and, similarly,

co-localized with common PI3P reporters like the PX domain of p40phox and FYVE domain of EEA1. Thus far, there is no detectable function of TgPH1 in apicoplast homeostasis or parasite survival. Yet, PI(3,5)P2 is left to be detected in T. gondii [190] and P. falciparum.

Phosphoinositide metabolism Three types of enzymes are involved in phosphoinositide metabolism: PI kinases, PI phosphatases and PI lipases [156]. The PI kinases (PIKs) convert PtdIns to PtdIns monophosphate and PtdIns monophosphate to PtdIns pyrophosphate in separated activities called “PI-kinases” and “PIP kinases” [156]. Based on the place of action on the inositol ring PIKs can be categorized into 3 types: PI3-kinases (PI3Ks), PI4-kinases (PI4Ks), and PI-phosphate (PIP) kinases (PIP5Ks and PIP4Ks).

PI kinases Phosphatidylinositol-3 kinases (PI3Ks) There are three PI3K classes (I, II and III) which can phosphorylate the D3-position hydroxyl of the D-myo-inositol head group and generate specific phosphoinositide forms [252]. In vivo, class I PI3K syntheses PI3P, PI(3,4)P2, and PI(3,4,5)P3. Class II and III PI3K are both able to synthesize PI3P from PtdIns whilst class II PI3K also synthesize PI(3,4)P2 [232, 252-254]. The PI3K family appears restricted to eukaryotes, and only the class III PI3K is conserved from yeast to human [252]. The homologue of class III PI3K in yeast is vesicle protein sorting 34 (Vps34) and exists in a cytosolic and a vesicular form (Fig. 1. 10) [186]. A phylogenetic study suggests the co-evolution of Vps34/PI3KIII and its regulatory subunit Vps15 in most eukaryotes [255]. Vps34 is the key molecular player in trafficking in the endosomal/lysosomal system, the membrane invaginations in multivesicular bodies (MVB) and the various forms of autophagy [156, 184, 247, 256, 257]. The essential role of Vps34 in vesicular trafficking is to ensure the proper sorting of proteins to a prevacuolar compartment and, subsequently, to the vacuole by the production of PI3P [183, 258]. In each of these functions, different proteins are associated with the Vps34p-Vps15 complex through interaction with its product PI3P [257]. In human, the lipid kinase activity of Vps34 leads to the formation of PI3P-enriched regions at the ER [185, 259]. This results in the recruitment of endosomal effector proteins with FYVE domains, PX domains, and other less characterized PIP- binding domains via PI3P [156, 183, 260]. Consequently, a protein complex comprising Vps34,

Vps15, Beclin1 and ATG14 forms at the ER membranes, which is the core for autophagosome formation. Chemical inhibitors such as Wortmannin and LY294002 have been useful tools to evaluate PI3K functions. They can block the activities of all PI3K enzymes and induce cell cycle arrest and cell death [259]. Some new specific inhibitors targeting PI3K function in cancer cells have been also developed [259].

Based on genome analysis prediction, the P. falciparum genome encodes only one PI3K (PF3D7_0515300) (Table- 2). Since PfPI3K is related to class III PI3K it is denoted by PfVps34 [160, 162, 190, 240]. PfVps34 is much bigger compared to its yeast homologue.

Figure 10 Kinases involved in phosphoinositide metabolism in yeast, mammalian cells and apicomplexan parasites. At left, it is the chemical structure of phosphatidylinositol with the hydroxyl-groups that can be phosphorylated highlighted in red. The kinases responsible for phosphoinositides synthesis are indicated on the arrows. The color code on the top right indicating their organisms of origin. In the bottom right phosphoinositide-specific phospholipase C (PI-PLC) and its products (in yellow box) are mentioned. Solid redlines are represented pathways that have been confirmed in apicomplexan parasites and black dotted lines and dotted boxes representing to reactions and products described in other organisms which have not currently any proof in apicomplexan parasites. Finally, red dotted lines used for hypothetical reaction pathways in Plasmodium. DAG, diacylglycerol; IP3, inositol 3-phosphate; Fab1, forms aploid and binucleate cells; Lsb6, Las Seventeen binding; Mss4, multicopy suppressor of Stt4 mutation; Pik1, phosphatidylinositol kinase; PIKfyve, FYVE domain-

containing phosphoinositide kinase; Stt4, staurosporine and temperature sensitive; Vps34, vacuolar protein sorting [163].

In addition to the main PI3K domains, PfVps34 also contains several different repetitive peptide motifs of six to eight amino acids [160, 240]. A comparative analysis of PI3K sequences in Plasmodium showed a high conservation between the main domains, but not the repetitive sequences [160, 240]. Previously, It has been shown PfVps34 can produce all three forms of D3 phosphorylated products (PI3P, PI(3,4)P2, PI(3,4,5)P3) in vitro [240] but recently a new study showed it is not true. Thus far, PfVps34 and its product PI3P have been shown to be involved in hemoglobin endocytosis [240], apicoplast biogenesis [162, 190], autophagy and artemisinin resistance [195].

In a study, immunoprecipitation-purified PfVps34 showed a significant decrease in its activity in the presence of PIK inhibitors [240]. Employment of the latter on P. falciparum parasites resulted in parasite growth defect by the disturbance of the trafficking of hemoglobin-containing vesicles revealing a role for PfVps34 in the endocytosis of hemoglobin from the host cytoplasm to the FV [240]. Localization studies demonstrated endogenous PfVps34 localized to the FV, the FV membrane, vesicular structures concentrated near the PVM/PM and erythrocyte membrane during the blood stage. The localization profile provides additional support for the function of PfVps34 in hemoglobin metabolism [160, 240, 261]. However, Wengelnik et al. have challenged the FV and erythrocyte localization. They reasoned both localizations are very surprising for a generally cytosolic enzyme, and they also considered the possibility of cross-reactivity of used antisera with other parasite proteins in this experiment [163].

PfVps34 is also reported to be found at the apicoplast and its product (PI3P), in addition to the apicoplast, is enriched at the FV membrane and nearby single-membrane vesicles [160]. Over- expression of a PI3P reporter, GFP-2xFYVE, in T. gondii parasites led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast. This eventually resulted in the loss of the apicoplast most likely due to the deleterious effect of the abundance of the PI3P reporter which might compete with natural ligands of PI3P; therefore, interfering with vesicular transport of lipids or proteins to the apicoplast [162]. Consistently, conditional depletion

33 of TgVps34 disturbed apicoplast biogenesis and led to apicoplast loss [162, 190]. The parasites lacking their apicoplast also lost their ability to divide their DNA, which eventually resulted in parasite death [162, 190]. Despite that, over-expression of a tagged version of TgVps34 under the control of the tubulin promoter led to a scatter punctate staining in the cytosol (possibly at the ER) but not at the apicoplast. Moreover, the gene was refractory to gene disruption attempts in T. gondii [190], P. berghei and P. falciparum indicating its essentiality ([163]- unpublished data), [160, 190, 226].

Another role for PfVps34 and its product PI3P has been proposed in the autophagy process. Their localization status at the FV membrane, the ER [262] and apicoplast is consistent with the role of Vps34 in autophagy in yeast and mammals. Interestingly, PfAtg8, an autophagy protein has been frequently observed to localize to the apicoplast which coincides with PfVps34 and PI3P localization [191]. Consequently, the apicoplast membrane is proposed as the site of phagophore formation [263]. On the other hand, due to the lack of some autophagy effector proteins in Plasmodium genomes, post-translational modifications of ATG proteins are suspected to play a role in regulating autophagy [264]. Accordingly, cAMP-dependent protein kinase A (PKA) is proposed to play a regulatory role. As the phosphorylation status of Vps34 and some other autophagy proteins collected from PlasmoDB are at the typical PKA sites in infected red blood cells [264]. Another evidence that points to the Vps34 role in the autophagy process arose from the characterization of TgPROP1 and TgPROP2, homologues to ATG18/WIPI [265]. The two proteins contain WD-repeat that can bind PI3P, the Vps34 product, for their recruitment to vesicular structures upon stress and are known to be important for the autophagic process.

The versatile functions of PfVps34 remain to be fully elucidated in P. falciparum as recently a new function proposed from Mbengue A. et al. work on artemisinin-resistant parasite strains. In a cell- based screen study to find new PfVps34 inhibitors, they identified three more inhibitors in addition to Wortmannin and LY29400. One of them was dihydroartemisinin (DHA), the active form of the artemisinin family of potent anti-malarial drugs [195]. Unexpectedly, they observed a correlation between PI3P level and point mutations in Kelch13 in the artemisinin-resistant strains of both laboratory produced and clinical parasites [195]. A specific mutation in Kelch13 (C580Y) found to be associated with an increased level of PfVps34 (about ~2.5 fold) compared with the wild-type

strains [195]. Kelch13 is hypothesized to be a part of E3 ligase and modulates PI3P levels through modification of PfVps34 (ubiquitination) and, thus, enzyme abundance in the parasite [195]. The same team reported recently PI3P localization to the ER lumen and adjacent vesicles, both by cryo- IEM/PI3P antibody and PI3P-fluorescent fused reporters [262]. Interestingly, PI3P enriched regions at the ER also include Kelch13 protein [262]. Altogether, these findings indicate a new mechanism of resistance by PI3P-vesicle amplification and PfVps34 level augmentation in the artemisinin resistance parasites with Kelch 13 mutants [262].

Phosphatidylinositol-4 kinases (PI4Ks) The phosphatidylinositol 4-kinases (PI4Ks) phosphorylate the D4 position of the inositol ring of PtdIns and produce PI4P. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β) that have different domain organization (Fig. 10). PI4Ks are conserved in all the eukaryotes from yeast to human [23]. Type II PI4K, the most abundant PI4K in mammalian cells and mainly localized in the trans-Golgi network (TGN) and endosomes, produces more than half of the Golgi PI4P [266-268] and is involved in many cellular pathways, including PI(4,5)P2 synthesis, membrane trafficking, signal transduction, phagocytosis, and the exo-endocytic cycle of synaptic vesicles [156, 266, 269, 270]. The most well-known function of PI4KII is the regulation of vesicle trafficking toward the PM from the Golgi/TGN by the production of PI4P. The latter includes the recruitment of the clathrin adaptor AP-complex to the membrane [266]. Its yeast homologue has been implicated in the regulation of actin polymerization and in endosome mobility, but is not an essential gene for yeast survival [271]. Homologues of type III PI4 kinases are Stt4p and Pik1p and both are essential genes in yeast [272]. Stt4p and Pik1 correspond to the human PI4K IIIα and PI4K IIIβ enzymes, respectively. Stt4p functions are mainly in the cytoplasm and provide the PI4P substrate for the Mss4p, a PI4P-5 kinase that synthesizes the cytoplasmic pool of PI(4,5)P2 [273]. PI4K IIIα like Stt4p localizes to the PM. While Pik1p was identified to function mainly at the Golgi and nucleus [274]. In the Golgi, PI4K IIIβ/Pik1 regulates trafficking in the secretory pathway along with the four-phosphatase-adaptor proteins 1 and 2 (FAPP1 and FAPP2) by synthesizing PI4P [275, 276]. The PH domain of FAPP protein is commonly used to detect PI4P lipid in live cells [277]. Disturbance with FAPP expression leads to transport inhibition between TGN and the PM. In downstream, PI4K simultaneously recruits Rab11 to the membrane [278]. Inhibition of this interaction abolishes Rab11 localization to the Golgi and blocks transport from the Golgi to the PM

[279]. Golgi to the PM [279]. New functions have also been discovered in the regulation of autophagy, where Pik1p was shown to regulate Atg9p trafficking from the trans-Golgi to the pre- autophagosomal structure (PAS) [274]. PI4Ks type III are sensitive to PI3K inhibitors such as Wortmannin [280]. They regulate PI(4,5)P2 pools which are sensitive to hormone signaling. Thus, they control the intracellular concentrations of second messengers like IP3, DAG, and Ca2+ indirectly [281].

Table 2 Phosphoinositide kinases and phosphatases in P. falciparum compared to yeast and T. gondii. [163]

Name Annotaion Yeast P. falciparum T.gondii References PI3K/Vps34 PI3 kinase Vps34 PF3D7_0515300 TGME49_215700 Tawk et al., 2010, 2011; 875aa 2133aa 2935aa Vaid et al., 2010; Daher et al., 2015

PIKfyve PI3-phosphate 5-kinase PF3D_1412400 TGME49_258960 Daher et al., 2015 3216aa 5839aa TGME49_258920 5839aa

PI4K II PI4 kinase II Lsb6p PF3D_0311300 TGME49_276170 607aa 953aa 1228

PI4K IIIα PI4 kinase IIIα Sttp4 PF3D_0419900 TGME49_226690 1900aa 5035aa 6780aa

PI4K IIIβ PI4 kinase IIIβ Pik1 PF3D_0509800 TGME49_296010 Kruger et al., 2010; 1066aa 1559aa 1583aa McNamara et al., 2013; Paquet et al., 2017

PI4P5K (A) PI4-phosphate 5-kinase PF3D_0110600 TGME49_230490 Leber et al., 2009; 1710aa 1293 Brochet et al., 2014 TGME49_245730 2144aa

PI-PLC PI-specific phospholipase PF3D_1013500 TGME49_248830 Fang et al., 2008; 1385aa 1097aa Raabe et al., 2011a, 2011b; Bullen et al., 2016

SAC1 (A) Inositol-phosphate 5-phosphatase PF3D_1354200 TGME49_316230 Thiearult and Richard, 2017 803aa 1006aa

SAC-like (B) Inositol-phosphate phosphatase PF3D_0705500 TGME49_238400 2814aa 2708aa

SAC-like (C) Inositol 5-phosphatase PF3D_0802500 TGME49_256630 1419aa 2264aa *The color code corresponds to observed phenotypes: red, essential; blue, fitness; green, dispensable; black, not analysed

In P. falciparum, there are three genes that code for phosphatidylinositol 4-kinases: PI4KII (PF3D7_0311300), PI4KIIIɑ (PF3D7_0311300), and PI4KIIIβ (PF3D7_0509800) (Table. 2). A knock-out screen performed in P. berghei demonstrated that only PI4KIIIβ is likely essential

37 during the asexual stage and the knock-out of two other genes can be tolerated, although they showed a considerably slower growth compared to their control parasite lines [226]. Most of the information on the PI4K role in Plasmodium comes from PfPI4KIIIβ. With 1559 amino acids, it contains armadillo (ARM) repeats in addition to several other structural features characteristic of PtdIns kinases [282]. ARM repeats are involved in protein-protein interactions in other systems [282]. Orthologs of PI4KIIIβ are found in all Plasmodium species and are extensively conserved at the amino acid level, including 97% identity in the catalytic domain between the P. falciparum and P. vivax orthologs [145]. It has been shown the Pik domain of PfPIKIIIβ is capable of complementing a yeast temperature-sensitive pik1 mutant and localizes to the Golgi and the nucleus as its endogenous homologue [282]. In P. berghei, PI4KIIIβ is described to be involved in ookinete gliding motility [161]. Specific point mutations in PI4KIIIβ have been shown to be related to a significant decrease in gliding. Later on, the same group found out these sites are regulatory phosphorylation sites by protein kinase G (PKG) upon gametocyte activation [161].

A study on new antimalarials namely imidazopyrazines (IPZ) has also led to the discovery of several mutations in PI4KIIIβ in laboratory-generated resistant strains. The mechanism of resistance to IPZ was consequently demonstrated to act through their association with cytokinesis in the late stage of Plasmodium parasite [145]. McNamara et al. observed a disruption of plasma membrane ingression around the developing daughter merozoites due to PI4KIIIβ inhibition in parasites treated by an IPZ (specifically KDU407). Using a parasite line expressing a PI4P reporter, they showed that this was the result of an alteration in PI4P pools from an unknown compartment to the PM. The discovery of PI4K inhibitors in Plasmodium has been very fruitful also in the Plasmodium treatment field. For example, IPZ not only show a strong potency against blood-stage field isolates of P. falciparum and P. vivax, but also IPZ molecules like KDU691, GNF179, and BQR695 inhibit parasite-derived PfPI4KIIIβ at very low nanomolar concentrations [145]. In addition, some IPZ derivatives such as GNF179 also eliminate quiescent rings which are resistant to dihydroartemisinin (DHA) [283]. Its analog, KAF156, has demonstrated efficacy in early clinical trials against a broad range of stages of the Plasmodium life cycle, including liver and asexual/sexual blood stages as well as parasites bearing Kelch13 (K13) propeller mutations (reviewed in [283]). These two compounds opened up an opportunity of using PI4K inhibitors as a combination drug in malaria treatment against dormant rings resulting from DHA treatment and

more importantly in blocking malaria transmission. Two other notable PI4K inhibitors are MMV390048 [145] and BRD73842 [284]. They have been shown to have inhibitory activity against strains resistant to other antimalarials in all stages of the parasite life cycle excluding MMV390048 which is not active against liver-dormant stage [147].

Phosphatidylinositol phosphate kinases or PIP-Kinases (PIPKs) PIP kinases are soluble peripherally membrane-bound proteins that have a highly conserved catalytic core but vary only in their C-terminal [156]. They also do not have considerable homology with other protein kinases [259]. Phosphatidylinositol phosphate kinases (PIPKs) are classified into three different categories based on their substrate specificity and sequence similarity (Fig. 10) [156, 259]. Type I and II PIPKs both produce PI(4,5)P2 though using two different routes. It is important to mention that the majority of the PI(4,5)P2 pool is produced by type I PIPKs [259]. Type III PIPK, on the other hand, is responsible for PI(3,5)P2 production [156]. Type I PIPKs (PIP 5-kinases (PIP5K) synthesize PI(4,5)P2 via phosphorylation of D5 position of PI4P at the inositol ring [285]. Whereas type II (PIP 4-kinases (PIP4K)) use PI5P to produce PI(4,5)P2 [156, 286]. In mammalian cells, three isoforms exist for type I and II PIPKs [156, 259]. The relative tissue distribution and the cellular localization of the various isoforms are unique. Type I PIP5Ks isoforms are found mostly in the PM [287, 288] which is found to be uniquely responsible for the generation of the PI(4,5)P2 pools linked to Ins(1,4,5)P3 synthesis and Ca2+ signaling in HeLa cells [289]. PIPKI’s main function has been associated with endocytosis via binding to the AP-2 adaptor and clathrin [290] and by promoting actin polymerization at the PM [291]. The only homologue of type I PIPKs in yeast is Mss4 which is also important for actin organization and membrane morphogenesis [222, 292]. Mss4 also has a role in the late stages of secretion. PIPKII is found in the cytosol, the nucleus and mainly the cis-Golgi [293, 294]. Recent findings on type II PIPKs revealed its main function is to control PI5P levels via converting PI5P into PI(4,5)P2 [156, 259]. In this capacity, PIPKIIs are implicated in the regulation of various processes like proliferation, secretion, ion channel function, stress responses and insulin sensitivity [295, 296].

In Plasmodium, there is a single PIP5K which is likely an essential gene in P. berghei (Table. 2) [161, 226]. Interestingly, in addition to a PPI 5-kinase domain in the C-terminal, it has also an N- terminal domain of the neuronal calcium sensor family [297]. The 5-kinase activity of the enzyme

39 was validated using recombinant expression of the Plasmodium PI-kinase domain. Expectedly, the recombinant protein is as well triggered by the small G protein ADP-ribosylation factor 1 (ARF1) both from P. falciparum and from mammals [297]. It has been speculated the N-terminal calcium sensor domain in this enzyme could be a sign of regulation of PI(4,5)P2 production by calcium [163].

The type III PIPK (PI3P5K) family member is PIKfyve, a single-copy gene that is conserved from yeast to mammals during evolution [259]. PIKfyve phosphorylates PI3P to produce PI(3,5)P2 [209]. In mammalian cells, the disruption of PI3P5K function leads to a major disturbance in the dynamics of cellular membranes which results in vacuolation and enlargement of early and late endosomes [259]. Similarly, its yeast homologue Fab1 is necessary for normal vacuolar morphology and functions [298, 299]. As to its function, it has been reported PI3P5K localizes to early endosomes and vacuolar membranes [300-302]. Fab1 acts in a larger signaling complex that contains Vac14, Vac7, Atg18 [303] and Fig4, a phosphatase that dephosphorylates PI(3,5)P2 into PI3P [304, 305]. Findings conclude Vac14, Vac7, and Fig4 are regulators of Fab1 and their coordinated activity control PI(3,5)P2 levels in the multivesicular body (MVB) sorting pathway and autophagy [248, 298, 300, 306, 307].

In P. falciparum, there is a putative homologue for PIKfyve and its regulator ArPIKfyve (Vac14) but it is not analyzed yet. PfPIKfyve does not have FYVE domain as its yeast and mammals homologue. In T. gondii, conversely, the PIKfyve gene encodes a 5639 amino acid protein with an N-terminal FYVE and a C-terminal 5-kinase domain [190]. Epitope tagging of endogenous TgPIKfyve did not permit detection or localization of the protein. Over-expression of TgPIKfyve under control of the tubulin promoter demonstrated a dot-like staining in the cytoplasm and some co-localization with the apicoplast [190]. The gene is essential as the gene knock-out attempt failed and conditional disruption of TgPIKfyve resulted in enlarged apicoplast showing its critical role in apicoplast homeostasis. Interestingly, knock-down of TgArPIKfyve led to the same defect of apicoplast homeostasis [190].

Phosphatases Phosphoinositide 3-phosphatases All phosphoinositide 3-phosphatases contain a phosphatase domain containing a catalytic motif called CX5R in their active sites. The PIP 3-phosphatases are classified into two families: PTEN (phosphatase and tensin homologue deleted on chromosome 10) and multiple members of the myotubularin family. PTEN protein is localized in both the cytosol and the nucleus [308]. The most important function of PTEN is the regulation of Akt-kinase via PI(3,4,5) dephosphorylation specifically at the D3 position on the inositol ring. Therefore, PTEN controls cell survival signaling through negative control of Akt activation and its downstream signaling pathway [259, 309, 310]. Loss of PTEN protein function is been observed in some human cancers [311, 312]. Hence, it is also known as a tumor suppressor [259].

The myotubularin (MTM) family consists of 15 members containing a characteristic phosphatase domain. Myotubularin phosphatases favor PI3P and/or PI(3,5)P2 as their substrate [259]. They are involved in many processes including cell proliferation and differentiation, autophagy, cytokinesis, and cytoskeletal and cell junction dynamics [313].

Phosphoinositide 4-phosphatases Thus far, only four proteins have been identified with 4-phosphatase activity in mammalian cells: two inositol polyphosphate 4-phosphatases A and B (INPP4A and INPP4B) and two transmembrane proteins denoted as TMEM55A and TMEM55B (Table. 4) [156, 259]. INPP4s dephosphorylate the D4 position of PI(3,4)P2, whereas the TMEM55 proteins dephosphorylate the D4 position of PI(4,5)P2 inositol ring [156].

INPP4A is localized to the endosomes in quiescent cells and in the PM in stimulated cells [364]. Upon activation, INPP4A promotes receptor-mediated endocytosis [314]. However, INPP4B shows a diffused distribution in the cytoplasm. Both INPP4A and INPP4B are shown to be a negative regulator of Akt signaling [315]. INPP4B is been also suggested being a tumor suppressor in human cancers [316].

TMEM55s show cytosolic and late endosomal membrane localization [317]. Nonetheless, DNA damage induces TMEM55B translocation from the cytosol to the nucleus [318]. As a result, an increase in PI5P level is observed, suggesting that TMEM55B plays a role in the control of nuclear levels of PI5P [319]. This is believed to be related to the p53-dependent apoptosis through PI5P- ING2 interaction.

Phosphoinositide 5-phosphatases This enzyme family consists of 10 mammalian and five yeast members [320]. In yeast, INPP5 types I-IV family proteins contain a central catalytic ‘5- phosphatase’ domain, except for 5- phosphatase I [238, 321]. Therefore, only types II-IV hydrolyze phosphoinositide substrates as well as soluble inositol phosphates. The type II enzymes include the synaptojanins, OCRL1, INPP5B, INPP5J, and SKIP. The type III INPP5 enzymes are SHIP1, SHIP2, and the sole type IV enzyme is INPP5E. INPP4s are involved in a variety of cellular events, such as protein trafficking, phagocytosis, and synaptic vesicle recycling [238, 322, 323]. Several other enzymes can act as phosphoinositide 5-phosphatases.

Sac family phosphatases There are five Sac1 domain-containing proteins in both human and yeast [320]. In mammalian cells, the Sac domain is found in the synaptojanins and in the Sac family phosphatases Sac1, Sac2 and Sac3 [324, 325]. Sac domain containing proteins sometimes show PI-phosphatase activity to different PIP species [259]. For instance, mammalian Sac1 acts on PI3P, PI4P and PI(3,5)P2 but not on PI(4,5)P2 as yeast Sac1p [259]. In mammals, the Sac1 protein is found associated with both the ER and the TGN [326]. Through control of PI4P levels, Sac1 directly modulates growth- dependent secretion at the Golgi [327]. In yeast, loss of function of Sac1 causes a variety of cellular defects [320] such as actin cytoskeleton disorganization, drug sensitivities, vacuolar function, cell wall maintenance and ATP uptake into the ER [328-332]. Sac3 and its yeast homologue Fig4p are the PI-5-phosphatase that specifically convert PI(3,5)P2 back to PI(3)P [305, 333]. As mentioned above, Sac3/Fig4p is part of a complex with the PI(3)P-5-kinase Fab1p and the scaffold protein Vac14p. This complex is involved in regulating the subcellular levels of PI(3,5)P2 [303, 306].

Bacterial PI-phosphatases Several studies on host-bacterial infections have clearly shown a common mechanism of infection via the exploitation of the host PIP metabolism by bacterial virulence factors [148, 149]. One way by which several bacterial pathogens do this is by injecting PIP metabolizing enzymes like PI phosphatases into the host cell. The enterobacteria Salmonella enterica injects the PI phosphatase SigD/SopB into the host and this is essential for enteropathogenicity [334]. Similarly, Shigella flexneri [335] and Mycobacterium tuberculosis [336], by secreting PI phosphatases, exploit their host cells. The growing body of data demonstrates the importance of this strategy employed by bacterial pathogens [337, 338].

PI phosphatases in apicomplexan parasites In P. falciparum, there are three homologue sequences to the Sac family including Sac1 (A), Sac- like (B) and Sac-like (C) (Table. 2). Only Sac1 has been studied recently [339] and was found to be localized to the ER and transitional ER. It is likely essential according to the unsuccessful knock- out effort. In systematic knock-out analysis, PbSac-like B was found to be essential, and the PbSac- like knock-out resulted in a slower growth [226]. And finally, there are four putative Sac family phosphatases in T. gondii [340]. Among them, the homologue of Sac-like B and C are essential, Sac1 A shows slower growth, and Sac-like D is dispensable for parasite survival.

PLC and PI signaling Phospholipase C, PLC is the enzyme that produces two signaling molecules IP3 and DAG via the hydrolysis of the phosphodiester bond at the SN3 position of PI(4,5)P2 (Fig. 10) [156]. IP3 is responsible for the liberation of calcium ions from the internal stores such as the ER while DAG activates protein kinase C (PKC) [341, 342]. PLC is an important player in receptor-regulated signal transduction [343]. PLCs are detected both in membranes and soluble fractions, and they mostly work in the PM [156]. Each PI-PLC contains a common basic core such as a PH domain, four EF-hands, and a C2 domain [344]. PLC recruitment, as well as its regulation, are mediated through interactions of effector molecules with the PH domain and the C2 domain [156]. A PLC homologue, Plc1, has also been identified in yeast [345, 346]. Yeast deficient in the plc1 gene demonstrate growth defects, impaired cell wall integrity and decreased osmotic resistance [346]. The PH domain of Plc1 has been structurally characterized and exhibits binding to P(4,5)P2, which

43 has been employed by many studies to investigate the roles of PI(4,5)P2 and its localization [347- 349].

Many critical events, such as gametocyte activation [350-353], ookinete motility [161], egress [161, 354] and sporozoite gliding motility [227, 228] are controlled via PLC/PI(4,5)P2/Ca2+ signaling in the Plasmodium life cycle (Fig. 11). A single plc gene has been studied in both Plasmodium and T. gondii [51, 352, 353, 355] which is essential for parasite survival and localizes to the PM. In T. gondii, PLC is also detected in the parasite cytosol and apical accumulations [51]. It has been shown that gametocyte activation is the result of PI(4,5)P2 hydrolysis by the calcium- dependent PI-PLC activity [350, 351]. Moreover, recent investigations in P. berghei gametocytes revealed redistribution of a PI(4,5)P2-reporter from the PM to the cytosol upon gametogenesis stimulation suggesting a link between PI(4,5)P2 cleavage and calcium-dependent PI-PLC signaling in preparation for gametogenesis [353].

In T. gondii, PLC/PI(4,5)P2/Ca2+ is implicated in the regulated exocytosis of the invasion organelles [51]. During invasion, following external stimuli, PLC produces IP3 and DAG. IP3 causes Ca2+ release from internal stores and CDPKs activation. In parallel, DAG is converted to phosphatidic acid (PA) by Diacylglycerol kinase-1 [51]. This PA is then bound by a specific binding protein (TgAPH) on the microneme surface which leads to microneme exocytosis [51, 356]. Consequently, PLC localizes to the invasion organelles in extracellular parasites [51]. In P. falciparum a rise in cytosolic Ca2+, potentially through the phospholipase C (PLC) pathway, triggers microneme release [84]. Since P. falciparum APH (PfAPH) also binds selectively to PA both on PIPstrips and in liposome assays [51], exocytosis of the invasion organelles might follow the same principles. In other words, the PLC/PI(4,5)P2/Ca2+ pathway and following PA sensing events might be a conserved mechanism in apicomplexan. Therefore, it is proposed that PfAPH interacting with PA, bring the microneme and PM to a closed position to fuse together [51, 356] via the involvement of SNARE-like proteins such as DOC2.1 [357, 358]. It is important to note that there is no predicted PKC orthologue in apicomplexan parasites [163].

PKG/Ca2+/PIP signaling in Plasmodium The key regulatory molecule in the PI-PLC/PI(4,5)P2/Ca2+ signaling is protein kinase G (PKG). PKG modulates PIP metabolism events that lead to the synthesis of IP3 and eventually Ca2+ release (Fig. 11) [161]. To synthesize PI(4,5)P2 from phosphatidylinositol, enzymes like PI4KIIIβ and PI4P5K are required to be activated. Consequently, the resulting PI(4,5)P2 is hydrolyzed by PI-specific phospholipase C (PI-PLC) [156]. Eventually, the release of Ca2+ from intracellular stores activates specific Ca2+-dependent protein kinases (CDPKs) depending on the parasite stage. There is good evidence for the regulation of PI kinases, PI-PLC and CDPKs by phosphorylation through a cascade involving protein kinase G (PKG) [161, 228]. Pieces of evidence for PKG in controlling this signaling cascade are also arising in many other processes. Redistribution of an epitope-tagged version of P. berghei PI4P5K from the cell periphery of ookinetes to the cytosol by PKG inhibitor suggests its regulatory role through the subcellular localization of PI4P5K [161]. In gliding ookinetes, PKG inhibition also causes a specific decrease in the relative abundance of PIP- mono- and -bisphosphates. Similarly, in P. falciparum schizonts, a decrease in PIP-mono-, bi-, and trisphosphates, and an increase in the PIPs were observed upon PKG stimulation which lead to the premature egress of the merozoites [161]. Moreover, the presence of an N-terminal domain of the neuronal calcium sensor family in PfPI4P5K could be an indication that PI(4,5)P2 production is also controlled by calcium [297].

Figure 11 The role of the PI-PLC pathway and calcium signaling at different stages of the Plasmodium life cycle and downstream stage-specific effectors. PKG= cGMP-dependent protein kinase G , cGMP= , PIP2= PI(4,5)P2 , SUB1= Subtilisin-like 1 serine protease, PKA= Protein kinase A, CDPK1 or 3 or 4= Ca-dependent protein kinase1or 3 or 4, Calcinurin= a Ca21-dependent phosphatase , DOC2= double C2 domain protein [228].

Chapter 1: Hypothesis and problem statement Malaria affects more than 200 million people each year and is the most widespread parasitic disease confronted. The new wave of resistance to current medicines has already arisen from South-East-Asia, while the malaria vaccine, RTS,S, is still far from being fully effective. Accordingly, there is an urgent need for new therapeutic methods and vaccine strategy. Plasmodium is an intracellular parasite and evades the human immune system by hiding and exploiting erythrocytic cells. The parasite is only exposed to the host immune system for a very short period of time, once before migrating from the dermis to the hepatocytic cells and once when newly formed merozoites are released into the blood stream to find a new host cell. Therefore, many protein candidates for vaccine-based strategies are proteins found in the apical organelle which are molecular players involved in erythrocyte invasion. Similarly, understanding of the pathways involved in the biogenesis of these organelles could potentially lead to new drug targets since without them, the parasite cannot invade. Despite this, the mechanism governing the secretory pathway toward apical organelles and their biogenesis are still poorly known. Work from our lab has proposed a Golgi-to-apical organelle protein-sorting model in asexual blood stage parasites. In this model the presence of specific microdomains inside the Golgi apparatus has been suggested that determine the final destination of secretory proteins. Based on this theory, the microdomains have a specific composition of lipid and protein, which license the selection of proteins destined for the different cell compartments. A specific signal in the microdomains recruits cytosolic effector proteins, which lead to vesicle budding and the departure of cargo protein to its next destination. However, the lipid and protein composition of such microdomains are mostly unknown.

1.1 Hypothesis and objectives In 2009, Richard et al. have demonstrated that the rhoptry-associated membrane antigen, (RAMA) through an interaction with the N-terminus of rhoptry-associated protein 1 (RAP1), recruited RAP1, 2 and 3 into rhoptry-destined protein complexes [359]. Disruption of this interaction resulted in the mistargeting of RAP1 to the PV, the default secretory pathway. The role of RAMA in recruiting these proteins is reminiscent of escorter proteins in higher eukaryotic cells that target cargo proteins to different membrane destinations through their interaction with special adaptor

47 proteins. These data suggested that one way that proteins could be differentially targeted to the apical complex would be by clustering of protein complexes in membrane microdomains. However, how these microdomains would be differentiated from one another and the mechanisms behind the actual trafficking from the Golgi to the apical organelles is currently unknown. In eukaryotes, there are seven different phosphoinositides, which define the identity of subcellular membranes and act as a code that can be read by PIP effector proteins. Uninfected erythrocytic cells only produce two PIP species, PI4P and PI(4,5)P2. This changes dramatically after infection by P. falciparum since five PIP species are detected in iRBCs which imply the importance of these tiny phospholipids for parasite survival. Recently, an interesting study from Bullen et al. proposed that in Toxoplasma, PIP signaling plays a role in invasion through induction of microneme exocytosis [51]. Based on their model, following external stimuli, TgPI-PLC activity increased and led to IP3 and DAG production. Consequently, IP3 production induces calcium release, which in turn stimulates calcium-dependent kinases that starts a cycle of phosphorylation of their downstream substrates. Concurrently, the generated DAG is phosphorylated to PA, which is detected by a PH-containing protein called TgAPH. TgAPH is on the microneme membrane and has been shown to be essential for microneme exocytosis. P. falciparum possesses a homologue of TgAPH but whether it plays the same role is currently unknown. Consistently with this, TgPI-PLC is observed in merozoite apex [51] which suggests that PIPs might also play a role in RBC invasion by P. falciparum. Our working hypotheses were thus:

1. Phosphoinositides are involved in defining the identity of subcellular membranes in the malaria parasite P. falciparum and therefore play a central role in the differential trafficking of proteins to the apical complex.

2. Effectors of phosphoinositides are among the molecular players involved in protein trafficking toward the apical organelles and they might play essential roles in their biogenesis and their exocytosis.

To test the hypotheses, I pursued the following specific objectives: 1. To develop fluorescent probes to monitor the intracellular distribution of phosphoinositides in P. falciparum.

2. To characterize a putative PIP-binding protein and investigate its potential role in trafficking of proteins to the apical complex and the invasion process.

Chapter 2: A map of the subcellular distribution of phosphoinositides in the erythrocytic cycle of the malaria parasite Plasmodium falciparum Avant-propos The paper presented in Chapter 3, entitled “A map of the subcellular distribution of phosphoinositides in the erythrocytic cycle of the malaria parasite Plasmodium falciparum” has been written as a ''scientific paper''. I performed and analyzed all the experiments. I also designed some of the experiments and wrote the manuscript. Angana Mukherjee supervised the project and wrote the manuscript. Dave Richard designed and supervised the project, and wrote the manuscripts. The paper has been published in the ''International Journal for Parasitology'' (IJP) in January 2018.

Résumé Bien qu’ils représentent un faible pourcentage des lipides cellulaires des cellules eucaryotes, les phosphoinositides (PIPs) sont impliqués dans divers processus essentiels tels le trafic intracellulaire et la transduction de signal. Au centre de leurs diverses fonctions se retrouve la distribution différentielle des différentes espèces de PIP dans des compartiments membranaires spécifiques, grâce l’actions de différentes kinases, phosphatases et lipases. Malgré leur importance dans le cycle de vie du parasite de la malaria, la distribution subcellulaire de la plupart des espèces de PIP est encore inconnue. Les travaux présentés ici montrent la localisation subcellulaire de plusieurs espèces de PIP tout au long du cycle érythrocytaire de Plasmodium falciparum. En effet, nous avons montré que le PI3P se trouve principalement à l’apicoplaste et à la membrane de la vacuole alimentaire, et que le PI4P est associé à l'appareil de Golgi et à la membrane plasmique. Quant au PI (4,5) P2, il est plutôt détecté au niveau de la membrane plasmique et à l’intérieur de structures sphériques en forme de cavité. Finalement, nous avons montré que le PI5P se localise à la membrane plasmique, au noyau et potentiellement dans le reticulum endoplasmique de transition. Notre carte de la distribution subcellulaire des différentes espèces de PIP chez P. falciparum est un outil important qui permettra de mieux comprendre la dynamique de ces lipides chez ce parasite morte.

Article

A map of the subcellular distribution of phosphoinositides in the erythrocytic cycle of the malaria parasite Plasmodium falciparum

Zeinab Ebrahimzadeh, Angana Mukherjee, Dave Richard*

Centre de recherche en infectiologie, CRCHU-Université Laval, 2705 Boul. Laurier Québec (QC), Canada, G1V 4G2

*Corresponding author. Dave Richard, Tel.: 1-418-525-4444 ext 47975; fax: 1-418-654-2715.

E-mail address: [email protected] (DR)

Note: Supplementary data associated with this article.

Abstract Despite representing a small percentage of the cellular lipids of eukaryotic cells, phosphoinositides (PIPs) are critical in various processes such as intracellular trafficking and signal transduction. Central to their various functions is the differential distribution of PIP species to specific membrane compartments through the actions of kinases, phosphatases and lipases. Despite their importance in the malaria parasite lifecycle, the subcellular distribution of most PIP species in this organism is still unknown. We here localize several species of PIPs throughout the erythrocytic cycle of Plasmodium falciparum. We show that PI3P is mostly found at the apicoplast and the membrane of the food vacuole, that PI4P associates with the Golgi apparatus and the plasma membrane and that PI(4,5)P2, in addition to being detected at the plasma membrane, labels some cavity-like spherical structures. Finally, we show that the elusive PI5P localizes to the plasma membrane, the nucleus and potentially to the transitional endoplasmic reticulum (ER). Our map of the subcellular distribution of PIP species in P. falciparum will be a useful tool to shed light on the dynamics of these lipids in this deadly parasite.

Keywords: Malaria, Phosphoinositide, Subcellular distribution

Introduction Despite recent progress in reducing mortality and morbidity, malaria still takes a tremendous toll on human health. The disease is caused by five species of the genus Plasmodium with Plasmodium falciparum as the causative agent of the most virulent form of malaria. In 2015, there were 438,000 deaths from malaria, mostly children in sub-Saharan Africa [360]. The symptoms of human malaria are caused by the asexual red blood cell (RBC) stages of the parasite. Within the RBC, the parasite develops in its own compartment surrounded by the parasitophorous vacuolar membrane (PVM). During this symptomatic phase, the intraerythrocytic parasite modifies the host RBC to make it suitable for its survival and growth. The ~48 h asexual life cycle of a parasite is complex, with three successive distinct morphological stages (ring, trophozoite and schizont). Ultrastructural studies have indicated that ring stage parasites are cup shaped [26, 43]. At this stage, host cell modification is at its peak with the parasite exporting remodeling and virulence proteins into the RBC [361]. The trophozoite stage, from approximately 24 to 36 h after invasion, is characterised by rapid parasite growth, repeated DNA replication and the appearance of hemozoin (inert remnant of digested hemoglobin) in the food vacuole. Finally, the parasite enters the schizont stage (~36-48 h) during which individual nuclei and associated organelles are partitioned to produce daughter parasites during a specialised type of cytokinesis called schizogony, and generates up to 32 daughter merozoites [362]. The host cell then ruptures and releases the merozoites which may then go on to invade new RBCs. The parasite, apart from possessing the classically observed organelles of eukaryotic cells, contains some more atypical compartments, probably driven by its need to survive in an unusual biological niche. These include the apicoplast, a four-membrane bounded plastid-like organelle, the food vacuole, a lysosome-like compartment that contains proteolytic enzymes that degrade host cell hemoglobin internalised by the parasite, and finally specialised secretory organelles forming the apical complex (micronemes, rhoptries and dense granules), located at the apical pole of the merozoite and playing critical roles during host cell invasion. Uncovering the principles governing the biogenesis of these various cellular compartments is of great interest because they likely represent sources of new potential targets for the development of antimalarial therapeutic drugs.

Phosphoinositides are phospholipids found on the cytosolic surface of a variety of intracellular membranes in eukaryotic cells. They contain an inositol head group that can be reversibly

phosphorylated at three positions. Although they account for less than 1% of total cellular lipids, they are of paramount significance in a variety of processes such as signal transduction, cell motility, cytoskeletal reorganisation, DNA synthesis, cell cycle, adhesion, membrane transport, permeability and trafficking [158, 363, 364]. Phosphorylation and dephosphorylation of inositol head groups by different kinases and phosphatases result in seven different phosphatidyl inositol phosphate (PIP) species including three phosphatidylinositol monophosphates (PI3P, PI4P and PI5P), three phosphatidylinositol biphosphates (PI(3,4)P2, PI(4,5)P2 and PI(3,5)P2) and one phosphatidylinositol triphosphate (PI(3,4,5)P3 [365]. Each of these seven PIPs has a unique subcellular membrane distribution (see below). Furthermore, within a given membrane, the localization of specific PIPs can be heterogeneous. Altogether, these enzymatic reactions lead to a specific PIP code, where certain subcellular membranes are enriched or depleted of specific PIPs, creating a membrane identity (reviewed in [155]).

To shed light on the respective roles and localisation of the different species of PIPs, live-cell imaging studies have provided relatively clear maps of the intracellular PIP distribution in several types of eukaryotic cells (reviewed in [155]) [366]. PI3P largely resides in early endosomes [173] and contributes to endosomal maturation, cargo protein degradation and cell signaling [367-369], and autophagy [370]. Some pools of specific PIPs can be found at more than one location in a cell. For example, PI4P is highly enriched at the Golgi membrane where it is involved in trafficking events and a deficiency in PI4P has been shown to affect Golgi structure and function (reviewed in [371]) while another pool is found at the plasma membrane where it acts as a precursor for the synthesis of PI(4,5)P2 [372-374]. PI(4,5)P2 exists predominantly on the plasma membrane although it can also be delivered from the Golgi complex by membrane carriers [375, 376]. PI(4,5)P2 is implicated in a number of cell surface related events such as exocytosis, endocytosis, phagocytosis, cell motility, cell adhesion, microtubule capture, regulation of integral membrane proteins and has a central role in cell signalling through its degradation into inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG) by the action of Phospholipase C (reviewed in [158]). Unlike constitutive PIPs, others such as PI5P, PI(3,4)P2 and PI(3,4,5)P3 are rapidly and transiently produced in response to the activation of cell surface receptors and other stimuli. PI(4,5)P2 is the precursor for the synthesis of PI(3,4,5)P3, critical in signalling pathways involved in cell proliferation, migration, chemotaxis, phagocytosis and micropinocytosis (reviewed in [377, 378]),

55 which, together with PI(3,4)P2, accumulate in the plasma membrane but only after specific signalling activation [156, 379]. PI(3,4)P2 is also found at the early endocytic pathway and although poorly characterised, specific roles of PI(3,4)P2 have been described in both clathrin- dependent and independent endocytosis [232, 380]. There are only small amounts of PI5P in resting cells and so its function remains poorly characterised but after certain types of stimuli, PI5P accumulates at the plasma membrane and in the nucleus and nuclear PI5P has been proposed to act as a stress response element [319, 381]. As for PI(3,5)P2, it is found to be enriched in the late compartments of the endosomes where it may regulate endosomal operations (fission and fusion) that maintain endomembrane homeostasis and proper performance of the trafficking pathways emanating from or traversing endosomes in yeast, mammalian and plant cells [298, 301, 382-384]. Despite their central role in the cellular biology of several types of eukaryotic cells, comparatively little is known about the role of PIPs in the malaria parasite P. falciparum. Normal mature mammalian RBCs have only small amounts of detectable PIPs, however upon infection with P. falciparum, the phosphoinositide profile undergoes profound changes with important increases in PI3P, PI4P and PI(4,5)P2, and the detection of some small amounts of PI(3,4)P2 and PI(3,4,5)P3 [160, 385]. The latter finding is interesting since these two PIP species are usually not detected in other unicellular organisms where only class III PI3-kinases are present [160, 386]. What functions the individual PIPs might play has recently become the focus of a number of studies. PI3P is thought to play a role in processes such as hemoglobin uptake to the food vacuole [159, 160, 196, 240, 387], biogenesis of the apicoplast [160, 162], resistance to artemisinin [195] and export of proteins to the erythrocyte [388] although the latter data have recently been challenged [194]. Inhibition of a P. falciparum PI4kinase with imidazopyrazines and quinoxalines has revealed that PI4P was likely critical for proper plasma membrane ingression during schizogony [145]. Much of what is known with regards to PI(4,5)P2 in malaria seems to be related to its involvement in calcium signalling cascades as a substrate for Phospholipase C in processes such as male gametocyte exflagellation [350, 351], gametocyte activation [353], synchronization of the erythrocytic cycle by the hormone melatonin [389, 390], sporozoite gliding motility [227], merozoite egress [45, 229] and potentially subsequent erythrocyte invasion [391]. Finally, PI(3,5)P2 has not been detected in P. falciparum-infected RBCs (iRBCs) [160] and PI5P has also not yet been reported for any Plasmodium spp.

In an effort to gain a deeper understanding of the multiple roles that PIPs are likely to play in the P. falciparum erythrocytic cycle, we undertook a comprehensive analysis of the subcellular localisation of each individual PIP species throughout the asexual blood stages. Our results show that the distribution of most PIPs is quite dynamic between the different steps of the cycle. We confirm that PI3P is found at the digestive vacuole membrane and the apicoplast and reveal that PI4P is found at the Golgi apparatus and, similar to PI(4,5)P2, at the plasma membrane. Furthermore, we demonstrate the presence of PI5P in P. falciparum and show that it localises to the plasma membrane and potentially the transitional endoplasmic reticulum. Our map will be a useful tool in the further unravelling of the numerous roles played by PIPs in the malaria parasite P. falciparum.

Materials and methods This study was approved by the Canadian Blood Services (CBS) research ethics board, project number 2015.001 and by the Centre Hospitalier Universtaire (CHU) de Québec institutional research board, Canada, project number 2015–2230, B14-12-2230, SIRUL 104595. Written consent was obtained by the CBS for all study participants.

2.1. Parasite culture Plasmodium falciparum 3D7 parasites were originally obtained from David Walliker at Edinburgh University, Scotland. Plasmodium falciparum asexual stage parasites were cultured under standard conditions in RPMI-HEPES medium at 4% hematocrit (human erythrocytes of O+ group) and 0.5% (w/v) AlbumaxTM (Invitrogen, Canada) [392]. Parasites were kept at 37°C in a gas mixture of 5% oxygen, 5% carbon dioxide and 90% nitrogen. Synchronisation was performed using sorbitol as previously described [393].

2.2. Parasite transfection Tightly synchronous ring stage parasites were transfected with 100 µg of purified plasmid DNA (Promega) as described previously[394]. The transfectants were selected using blasticidin (BSD) at 2.5 µg/ml. Co-transfections were performed with 100 µg of each plasmid and parasites were selected with BSD together with 10 nM WR99210 (Jacobus Pharmaceuticals, USA) or 5 nM DSM1 (MR-4), depending on the selectable marker. 57

2.3. Construct generation To create the PIP sensors, PBDs were PCR amplified with flanking restriction sites from human cDNA (PXP40, PHFAPP, PHBTK) or from a series of PIP sensor plasmids previously designed for use in Arabidopsis thaliana (2xPH PLCδ and PHTAPP1) [366]. In the case of the DOK5 PH domain, PCR was done on a plasmid containing the domain fused to GST [395]. Most PIP sensors were cloned in the pGFP-glmS vector from Prommana et al. [396]. This plasmid allows the expression of genes of interest with GFP fused at their C-terminus. For the triple hemagglutinin (HA) tagged PHFAPP and PHDOK5, the domains were cloned using 5’ AvrII and 3’Xho1 in a pLN-GFP vector where the GFP had been replaced by a 3HA tag [397]. Primer sequences and restriction sites used are listed in Supplementary Table S1.

2..4 Fluorescence imaging The PBD expressing cell lines were imaged using a GE Healthcare Applied Precision Deltavision Elite microscope with a sCMOS camera and the obtained images analyzed with the SoftWorx software. For live cell imaging, infected RBCs were incubated with 0.1 μg/ml of DAPI (Invitrogen) for 10 min at 37˚C. For IFAs, parasites were fixed either in solution using 4% paraformaldehyde- 0.0075% glutaraldehyde (ProSciTech, Australia) [398] or on slides using 100% methanol. After blocking in 3% BSA (Sigma Aldrich, Germany) the cells were incubated for 1 h with rabbit polyclonal anti-ERD2 1:2000 [399]; mouse monoclonal anti-HA 1:2000; mouse monoclonal anti- RAP1 1:2000 [400]; rabbit polyclonal anti-RON4 1:1000 [359, 401]; rabbit anti-AMA1 1:1000 [402]. Bound antibodies were then visualised with Alexa Fluor-594 anti-rabbit IgG and Alexa Fluor-488 anti-mouse IgG diluted 1:1000. Parasites were mounted in Vectashield (Vecta Laboratories, USA) containing with 0.1 μg/ml of DAPI. Images represent single optical slices except where indicated.

2.5. Western blot analysis Mixed-stage P. falciparum parasite cultures were saponin-lysed and resuspended in SDS protein sample buffer. Proteins were separated on 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, USA). Monoclonal mouse anti-GFP (Roche Canada, clones 7.1 and 13.1), rabbit polyclonal anti-GFP (Abcam, USA, AB6556) or rabbit

polyclonal anti-mCherry (Abcam; AB167453) were used at a 1/500 dilution in 1% skimmed milk (w/v). Appropriate horseradish peroxidase- coupled secondary antibodies were used and immunoblots were revealed by chemiluminescence (ECL, Amersham Biosciences, Canada).

Results and Discussion 3.1. Generation of a set of PIP biosensors expressed throughout the P. falciparum asexual blood stages Proteins involved in various aspects of PIP metabolism often have very well-defined lipid binding domains through which they interact with specific species of PIPs. Sensors made up of isolated lipid-binding domains with the ability to recognize a single type of PIP paired with fluorescent reporters have been used to determine the subcellular distribution of PIPs in a variety of living cells [156, 366, 373]. Here, in order to localise the various PIP species in P. falciparum asexual blood stages, we selected six different PIP binding domains (PBDs) with extensive literature supporting their PIP specificity (Supplementary Table S2). We did not choose any PBD binding to PI(3,5)P2 as this species was not detected in P. falciparum iRBCs and a P. falciparum homologue of PIKfyve/Fab kinase, the enzyme responsible for the synthesis of PI(3,5)P2 has not yet been identified [159, 160]. Our initial strategy was to generate a set of PIP sensors where each PBD would be fused separately to GFP and to mCherry, cloned in plasmid vectors with different selectable markers (at least two different selectable markers, for a total of four plasmids for each PBD) to allow co-transfection and subsequent colocalization analysis of multiple PIP species in the same cell. However, this turned out to be much more complicated than we anticipated. In the end, most of the data obtained were with a plasmid containing the blasticidin deaminase selectable marker. In addition, for some of the PBDs, we never managed to obtain good expression levels with either GFP, mCherry or mCitrine fusion. All our sensors were placed under the control of the well characterised constitutive P. falciparum Heat shock protein 86 promoter [403, 404]. A caveat of using PIP sensors is that these will compete with native PIP effectors, which might result in dominant negative effects. One method that has been used to decrease the likelihood of this happening is through the use of mild promoters [175, 373]. In our experience with P. falciparum transgenics, this has never been critical since the parasite seems very capable of decreasing the expression of any transgene to non-detrimental levels, with no measurable decrease in parasite

59 growth. This translates into a smaller number of parasites with detectable levels of fluorescence or even no fluorescence at all if the transgene is not tolerated (see Section 3.2).

3.2. Subcellular localisation of PI3P To determine the localisation of PI3P, we chose the Phox (PX) domain of the mammalian P40phox protein that has been successfully used in various organisms to visualize PI3P [366, 405] (Supplementary Table S2). In ring stage parasites, the GFP signal was thin, rod shaped and somewhat away from the nucleus (Fig. 1A). As the parasite matured to the trophozoite and schizont stages, the sensor was detected at the membrane surrounding the food vacuole, recognisable by the dark crystals of hemozoin in the bright field image (Fig. 1B, C). As previously reported by Tawk and colleagues (2010) using a PI3P sensor made of 2xFYVE domains, some regions of the food vacuole membrane had more intense GFP labelling that sometimes appeared as small circles or rod shaped structures (Fig. 1B, Supplementary Fig. S1A, arrows). Some parasites also displayed cytosolic vesicle-like structures at or near the parasite membrane (Supplementary Fig. S1A, arrowheads). Western blot was performed on parasite extracts to control for the integrity of the sensor and this revealed a major band around the expected size of 46.4 kDa for PXP40-GFP and a very faint band around 26 kDa, potentially some degradation product (Fig. 1D). Several lines of evidence strongly support a critical role for the sole PfPI3K in hemoglobin transport from the host cell cytoplasm to the parasite food vacuole: localisation studies using a specific antibody revealed that PfPI3K is found inside the food vacuole and in vesicular structures near the parasite membrane/PVM [240]; incubation of P. falciparum parasites with the PI3K inhibitors wortmannin and LY294002 results in a significant reduction of parasite growth potentially through a defect in the transport of hemoglobin from the host cell to the food vacuole [240, 406, 407]. Whether the PI3P found as these locations results directly from the activity of PfPI3K is not known, however.

From the trophozoite stage, a bright spot of fluorescence not associated with the food vacuole was also seen and it increased in number as the parasite went through schizogony (Fig. 1B, C, arrowheads). In a few late trophozoites and schizonts, we sometimes observed the PXP40 –GFP signal in a more complex, branched form, reminiscent of the apicoplast (Supplementary Fig. S1B) [408]. The PI3P focus had previously been shown to represent the apicoplast [160] and to further confirm this, we co-transfected a PXP40-mCherry sensor in a parasite line expressing GFP fused to

Enoyl acyl carrier protein reductase (ENR), an apicoplast resident protein [66, 409]. Observation of doubly-labelled cells was a very rare event but when it occurred, the signal from ENR-GFP was always colocalizing with the foci of PXP40, confirming an enrichment of PI3P at the apicoplast membrane (Supplementary Fig. S2). Further support for the critical role of PI3P in apicoplast homeostasis comes from an elegant study in the related apicomplexan Toxoplasma gondii where conditional ablation of TgPI3K expression resulted in altered morphology of the organelle and delayed parasite death, proving the essential contribution of this lipid kinase in preserving apicoplast integrity [190]. Whether the same is true in Plasmodium remains to be demonstrated. Control parasites transfected with GFP alone showed a cytosolic distribution throughout the blood stages (Supplementary Fig. S3).

In conclusion, our results confirm that PI3P is found at numerous intracellular locations throughout the P. falciparum asexual blood stages where it likely plays critical roles in a variety of processes such as apicoplast biogenesis and hemoglobin endocytosis. Of interest, PI3P levels do not fluctuate greatly in most eukaryotic cells so the fact that P. falciparum increases PI3P levels up to four-fold in proportion compared with total phosphoinositides is very unusual [160]. Targeting the enzymes responsible for the generation of PI3P might therefore represent an attractive antimalarial strategy and recent results suggesting that PfPI3K might be a target of artemisinins supports this [195].

3.3. Subcellular localisation of PI4P To investigate the subcellular distribution of PI4P we used the well-characterised Pleckstrin homology (PH) domain of the human Four-phosphate-Adaptor Protein 1 (FAPP1) [235]. We initially transfected parasites with a sensor containing a single FAPP1 PH domain but capturing images turned out to be difficult as the signal faded out quickly (results not shown). To increase the avidity of our PI4P sensor, we used two FAPP1 PH domains in tandem as this was shown to provide a better and more stable signal in other systems [275, 366]. In ring stage parasites, the sensor labelled the plasma membrane together with an intense focus of GFP staining (Fig. 2A). As the ring matured to the trophozoite stage, distinct intracellular foci arose, in addition to the plasma membrane signal. This distribution was also observed by McNamara and colleagues using the PI4P probe GFP-PHOsh2 [145]. Some cytosolic signal could also be observed in some parasites which is sometimes indicative of the degradation of a portion of the sensor resulting in the presence of a sole

GFP protein (Fig. 2B). However, western blot with an anti-GFP antibody on parasite extracts revealed only a single band at the expected size of approximately 80 kDa and no degradation product (Fig. 2E). In schizonts, each individual merozoite contained one focus together with some plasma membrane labelling (Fig. 2C). Ruptured merozoites seemed to have lost the plasma membrane signal and intriguingly, in a few of them, the fluorescence seemed to cap one end of the parasite (Fig. 2D, arrows). A few studies have shown that in vitro, PHFAPP also has the ability to bind to PI(4,5)P2 although with lower affinity than PI4P [203, 410]. It is therefore possible that some of the plasma membrane labelling is due to PI(4,5)P2 (see below). The recently characterised PI4P-specific binding domain of the Legionella pneumophila SidM protein could potentially help in quantifying the true amount of PI4P present at the membrane [411, 412]. A role for PI4P generated by Golgi apparatus-localised PI4Ks during regulation of membrane trafficking events and cytokinesis has been shown in a variety of systems such as mammalian cells [276, 413, 414], Drosophila [415] and yeast [416]. To determine whether the focus of fluorescence could represent the Golgi, we performed IFAs with an antibody to Endoplasmic reticulum Retention Defective protein 2 (ERD2), a marker of this structure [399, 417-419]. When observing parasites that had been fixed with paraformaldehyde-glutaraldehyde, we noticed that the fluorescence signal was no longer found as dots and at the plasma membrane but instead was redistributed throughout the cytosol, suggesting that the centrifugation steps required in this fixation protocol had an impact on PI4P location (results not shown). To circumvent this, we reduced the time between harvesting and fixation by using methanol fixation on parasite smears. For this analysis, we used parasites transfected with a FAPP PH domain fused to a triple HA tag since our anti-GFP antibody does not work on methanol fixed slides. Analysis of the transfectants showed that the distribution of the foci mirrored what was seen in live parasites, however the plasma membrane labelling was somewhat less obvious although some overlap with the plasma membrane marker Merozoite surface protein 1 (MSP1) could still be observed (Supplementary Fig. S4 versus Fig. 2). This might potentially be explained by the fact that methanol fixation is known to deplete phospholipids [420]. Colocalisation experiments revealed that PHFAPP-3HA overlapped extensively with Golgi ERD2 throughout the erythrocytic cycle (Fig. 3A, B: Pearson’s correlation: Rings: 0.63±0.03, Trophozoites: 0.63±0.03, Schizonts: 0.60± 0.03). As a control for proteins residing in separate organelles, we used ERD2 and Rhoptry associated protein 1 (RAP1) and obtained a coefficient of 0.12±0.01. To assess if the PHFAPP-3HA signal localised to the apical complex, we next performed

IFAs with antibodies labelling different organelles forming this structure. The micronemes contain effectors involved in diverse processes such as merozoite egress, formation of the tight junction and as adhesions and evidence suggest that they form a heterogeneous population [19, 421]. IFAs performed with antibodies to Apical membrane antigen 1 (AMA1) and Erythrocyte binding antigen 175 (EBA175), markers of two different micronemal populations, failed to show any strong overlap with PHFAPP-3HA (Fig. 3Ca,b). The rhoptry is the most prominent secretory organelle in the parasite and it is subdivided into two regions, the bulb, which is the larger lipid-rich region of the organelle and the apical duct known as the rhoptry neck [422]. IFA using an anti-RON4 antibody, a marker of the rhoptry neck [401], and co-transfection of the 2xPHFAPP-GFP line with mCherry-tagged Rhoptry associated membrane antigen (RAMA), a marker of the bulb, showed an absence of colocalization in both cases (Fig. 3Cc, d). Quantification revealed that the R coefficients between PHFAPP and any of the apical markers was not statistically different from the negative control ERD versus RAP1 (Fig. 3D: PHFAPP-3HA vs AMA1, 0.15±0.03; PHFAPP-3HA versus EBA175, 0.13±0.02; PHFAPP-3HA versus RON4, 0.10±0.02; PHFAPP-GFP versus mCherry-RAMA, 0.17±0.03; ERD2 versus RAP1, 0.12±0.02). Plasmodium falciparum possesses three putative PI4kinases and incubation of parasites with the antimalarial compounds imidazopyrazines and quinoxalines, targeting the PfPI4KIIIβ homologue, led to a major defect in cytokinesis due to perturbed membrane biogenesis and ingression around developing merozoites [145]. This is in line with other systems where Golgi-generated PI4P plays a critical role in regulating vesicular trafficking in the late secretory pathway and in cytokinesis (Audhya et al., 2000; Polevoy et al., 2009). PfPI4KIIIβ is found spread throughout the cytosol in trophozoites but foci of concentrated fluorescence, hypothesised to be the Golgi apparatus, are seen in schizont stages [145]. It is tempting to speculate that PfPI4KIIIβ might be the enzyme responsible for the generation of the Golgi-associated PI4P we detect in schizont stages and the fact that the PIP4 foci disappear in parasites treated with imidazopyrazines supports our observation. However, the broad cytosolic distribution of PfPI4KIIIβ in trophozoites makes it unlikely to be involved in the synthesis of the Golgi PI4P we detect in earlier stages. As to the origin of the plasma membrane associated PI4P pool seen throughout the erythrocytic cycle, it could be generated in situ by other PI4Ks or perhaps by dephosphorylation of PI(4,5)P2 [423], or coming from Golgi-derived PI4P containing vesicles, or both possibilities, as seen in other systems [424]. In addition to serving as a precursor for PI(4,5)P2 synthesis, plasma membrane PI4P contributes directly to a number of signalling

63 pathways in mammalian cells (reviewed in [425]). Through its contribution to the total negative charge unique to the cytoplasmic leaflet of the plasma membrane, PI4P might also be involved in the recruitment of polybasic motifs-containing effectors requiring an enrichment in polyanionic lipids [423, 426, 427]. Recently, several studies have uncovered a critical role for PI4P in the bidirectional transfer of certain lipid types such as sterols and phosphatidylserine between the endoplasmic reticulum (ER) and the plasma membrane at what are called membrane contact sites. This action is performed by lipid transfer proteins of the oxysterol-binding protein-related protein family that take advantage of the energy created by the PI4P gradient between the ER and the plasma membrane [428-430], reviewed in [431]. We have identified at least one homologue of oxysterol-binding proteins in P. falciparum (PF3D7_1131800, www.plasmodb.org) and proteomics analyses have detected that it is expressed in schizont stage parasites [432, 433]. Whether it plays a similar role remains to be investigated. It is interesting to note that although not much is known with regards to membrane contact sites in P. falciparum, there is evidence that they occur between the ER and the apicoplast, and between the mitochondria and the apicoplast [434-436]. Furthermore, the extensive branching of the parasite ER in trophozoites and developing schizonts make contact sites between the organelle and the plasma membrane a possibility [434].

In conclusion, our results show that PI4P is found at the Golgi apparatus and at the plasma membrane in all stages of the erythrocytic cycle.

3.4. Subcellular localisation of PI5P Being the last identified species, PI5P is the most enigmatic of the PIPs, even though it is not the least abundant in cells (reviewed in [437]). The pathways regulated by PI5P in mammalian cells and its effectors/binding proteins are still poorly understood, and monitoring the intracellular localisation of PI5P has been a challenge to date. The PI5P pool was long thought to be confined to the nucleus, where it is involved in various processes such as protection against UV stress, apoptosis, transcriptional control and the cell cycle [381] [318, 438](reviewed in [156, 437]). Only recently have extranuclear pools of PI5P been identified in the plasma membrane, ER, Golgi [208] and endosomes [439]. Since nothing is known about PI5P in Plasmodium parasites, we sought to determine whether it could be detected in P. falciparum by using a sensor containing the PH domain of mammalian Downstream of tyrosine kinase 5 (DOK5), a well described domain with

high affinity and specificity for PI5P [213, 395]. Examination of the PHDOK5-GFP transgenics revealed that PI5P was detected throughout the asexual lifecycle of the parasite. In ring stages, the GFP signal seemed to be more pronounced in certain regions of the plasma membrane, together with some diffused cytoplasmic signal (Fig. 4Aa, arrow and Supplementary Fig. S5A, star). An anti-GFP western blot revealed a single band around the expected size of 41.3 kDa and no degradation product (Fig. 4Da). As the parasite matured to the trophozoite stage, the plasma membrane associated signal seemed to become concentrated in some regions whilst a significant portion remained cytosolic (Fig. 4Ab, arrow). In some instances, some PI5P signal was found very close to, and sometimes overlapping with, DAPI in the nucleus (Supplementary Fig. S5B). At >40 h in late schizonts, we no longer saw colocalization with the nucleus (Fig. 4Ac). In the developing daughter merozoites, the signal seemed to be enriched as a focus close to the nucleus of the daughter cells with some fluorescence sometimes wrapping around the DAPI (Fig. 4Ac and Supplementary Fig. S5C, arrowheads) and sometimes almost capping the apical tip (Fig. 4Ac, star). To try to identify the structure corresponding to the foci of fluorescence in schizonts, we performed IFAs on a parasite line expressing PHDOK5-3HA. In most individual developing merozoites, the signal partially overlapped with the Golgi marker ERD2 (Fig. 4B). Quantification of the colocalization revealed an R coefficient of 0.50±0.02 which is significantly less than the 0.60± 0.03 obtained for PHFAPP-3HA versus ERD2 (Fig. 4C). The PHDOK5 pattern of fluorescence is reminiscent what is observed with PfSEC13p, a marker of the transitional ER [440]. To determine whether PI5P colocalized with PfSEC13p, parasites were transfected with plasmids expressing PHDOK5-GFP and PfSEC13p-mCherry but unfortunately we never recovered doubly-labelled parasites. Perhaps this is due to some sort of steric hindrance between the fusion proteins which prevented binding of both proteins at the same time. Of note, as we saw with the PI4P probe, the plasma membrane associated fluorescence detected in live cells was much less prominent by IFA although it could still be observed in some cells. The detection of PI5P leads to the intriguing question as to how it is generated in malaria parasites. Recent reports have provided evidence that its direct synthesis from PI by the enzyme PIKfyve is the major route in mammalian cells [211, 441, 442]. However, P. falciparum does not have a direct PIKfyve homologue. PfPI3K, with its demonstrated relaxed specificity [240] or perhaps some of the other PfPIkinases might play a role. Another way through which PI5P was proposed to be generated is by the dephosphorylation of PI(4,5)P2 by myotubularin/myotubularin-related phosphatases but again, this class of enzymes is

65 not found in P. falciparum. Four putative PI-phosphatases are found in the P. falciparum genome but so far, nothing is known about either their subcellular localisation or their substrate specificity [443].

In conclusion, our data suggest that PI5P is indeed found in the malaria parasite asexual blood stages and that it localises to the plasma membrane, to a structure potentially corresponding to the transitional ER in schizonts, and sometimes in the nucleus. This varied subcellular distribution of PI5P suggests that it might potentially play pleitrophic roles in the malarial erythrocytic cycle.

3.5. Subcellular localisation of PI(4,5)P2 PI(4,5)P2 is by far the most abundant PIP, corresponding to 70% of total PIPs in P. falciparum asexual stages [160]. To determine the location of PI(4,5)P2, parasites were transfected with a fusion of the phospholipase C δ PH domain and a fluorescent reporter [348]. When using a single PH domain, the signal was too faint to be imaged, whether it was fused to GFP, mCitrine or mCherry (results not shown) as was previously described in other cell types [203, 372]. To increase the avidity of our probe, we then used a sensor containing two PLCδ PH domains in tandem. Analysis of the mCherry-2xPHPLCδ parasite line revealed a very strong labelling of the plasma membrane at all stages of the cycle (Fig. 5A), confirmed by the extensive colocalization with MSP1 (Fig. 5B), an abundant merozoite surface protein expressed from the late trophozoite stage onwards [444]. It is worth noting that unlike the other sensors we used, mCherry-2xPHPLCδ proved to be stable enough to perform IFAs with paraformaldehyde/glutaraldehyde fixation. The integrity of the sensor was next confirmed by western blot on parasite extracts and a single band of the expected size of approximately 100 kDa was observed (Fig. 5C). We often observed large vesicle- like structures at the plasma membrane in ring stages that increased in numbers as the parasites transitioned to trophozoites and schizonts (Fig. 5Aa, b; Supplementary Fig. S6, arrows). These could potentially represent cytostomes, endocytic structures involved in hemoglobin transport from the host cytoplasm to the parasite digestive vacuole [28, 435, 445]. Furthermore, a cavity was previously shown to originate from an invagination of the PVM and the parasite plasma membrane and to contain host cell cytosol, however the structure disappeared as the parasites entered schizogony [446]. Moreover, a similar kind of spherical structure had also been shown to remain connected to the RBC cytoplasm and was hypothesised to be involved in hemoglobin endocytosis

[447]. Whether the mCherry-2xPHPLCδ labelled features we saw in early stage parasites are the same as those in schizonts is unknown but it is assumed that hemoglobin uptake is much less important once cell division has started [448]. Whatever cellular processes occur at these structures, our results suggest that there is no massive decrease in PI(4,5)P2 levels present on their membranes, at least as detectable using our sensor. Whether this is because the lipid plays no role, that its function does not require its modification or that it is resynthesized as it is consumed remains to be seen. Finally, in very late schizonts, once cytokinesis is completed, each daughter merozoite displays a very uniformly labelled plasma membrane (Fig. 5Ac, d). Control parasites transfected with mCherry alone showed a cytosolic distribution throughout the blood stages (Supplementary Fig. S7).

As described earlier, in other systems, plasma membrane-localised PI(4,5)P2 is implicated in several cell surface related actions (reviewed in [158]). Plasmodium falciparum possesses a PI- PLC [159] and inhibitor studies have implicated it in processes such as gametocyte activation and differentiation [351, 353], sporozoite motility [227], synchronization of the erythrocytic cycle [449], schizont egress [229] and invasion of merozoites (Vaid et al., 2008), although the latter finding was subsequently questioned (Jones et al., 2009). Recently, a PH domain-containing ccalcium-dependent protein kinase (PfCDPK7) was shown to specifically bind to PI(4,5)P2 and to localise to vesicles in close proximity to the parasite ER [450]. Close inspection of some of our 2xPHPLCδ parasites sometimes showed some minor fluorescence close to the DAPI-stained nucleus but it was certainly not as defined as the PfCDPK7 pattern published by Kumar and colleagues (2014) (Fig. 5Aa, Max proj panel, arrowhead). Perhaps the tandem nature of our sensor precludes its efficient binding to these intracellular vesicles. In addition, it has been reported that PH domains with different affinities to the same PIP species sometimes show slightly different intracellular localisation [366]. Of note, in other eukaryotes, although the major pool of PI(4,5)P2 is locally produced at the plasma membrane by the action of a PIP5K on PI4P [451], small subcellular pools have also been found at the Golgi, the ER and endosomes [424, 452].

Our data show that PI(4,5)P2 is found all over the parasite plasma membrane and at some vesicular structures potentially corresponding to cytostomes or the previously described cavity [446].

In summary, our data, summarised in Fig. 6, reveals that the distribution of most PIP species is quite stable during the erythrocytic cycle of P. falciparum except for PI5P which is found potentially associated to the transitional ER (tER) only in schizonts but otherwise at the plasma membrane. Remarkably, the repartition of most PIPs in the parasite is quite similar to what was previously observed in other eukaryotic cell types (Table 1). Of interest, despite some minor amounts of PI(3,4)P2 and PI(3,4,5)P3 previously detected in schizonts [160], only a broad cytosolic signal identical to the fluorescent protein alone was detected when we used sensors specific to each of these PIP species (results not shown). It will now be of great interest to identify the mechanisms behind the specific localisation of each PIP, whether through the action of kinases, phosphatases and lipases or through the activity of lipid transfer proteins. This will in turn shed some light on the various roles likely played by these critical but tiny lipids.

Acknowledgments We would like to thank Philip Shaw (National Center for Genetic Engineering and Biotechnology, Thailand) for the gift of the original pGFP-glmS plasmid, Yvon Jaillais (Université Claude Bernard Lyon 1, France) the PIPline plasmids and Jacques Nunes (Centre de Recherche en Cancérologie e Marseille, France) for plasmid GST-PH DOK5. We also thank Jacobus Pharmaceuticals for WR99210 and Dominic Gagnon for technical assistance. The following reagents were obtained through MR4 as part of the BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA: Polyclonal Anti-Plasmodium falciparum PfERD2 (antiserum, Rabbit), MRA-1; plasmid pLN-ENR-GFP, MRA-846, contributed by David A. Fidock; DMS1, MRA-1161. We would also like to acknowledge the Canadian Blood Services for providing human erythrocytes. The authors declare no competing financial interests. This study was funded through a Canadian Institutes for Health Research (CIHR) operating grant MOP 130359. DR was a Fonds de la Recherche du Québec-Santé Junior 1 fellow.

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Tables Table 1. Subcellular distribution of phosphoinositides (PIPs) in various types of eukaryotic cells.

Localisation Saccharomy PIP species Mammalian Plasmodium References ces Plant cells cells falciparum cerevisiae Endosome/v Late endosome, Apicoplast, FV, single Gillooly et al., 2000, Simon et al., 2013, PI3P Early endosome acuole tonoplast membrane vesicles Tawk et al. (2010), this study Levine and Munro, 1998, Balla et al., PM, Golgi, post- PI4P Golgi Golgi, PM Golgi and PM 2005, Simon et al., 2013, McNamara et Golgi/endosome al., 2013, this study PM,ER,Golgi, Jones et al., 2006, Sarkes and Rameh, PI5P No data No data PM, nucleus and tER? nucleus 2010, this study Not PM upon Marshall et al., 2002, Simon et al., PI(3,4)P2 Cytosol Cytosol detected stimulation 2013, this study Endosome/ Late endosome, Late endosome, pre- Gary et al., 1998, Rudge et al., 2004, PI(3,5)P2 multivesicul multivesicular vacuole, cortical Not detected van Gisbergen et al., 2012, Tawk et al., ar bodies bodies patches of the PM 2010

PM, Golgi, PM and large vesicular Stefan et al., 2002, Hammond et al., PI(4,5)P2 PM endosome, ER, PM structures 2014, Rameh et al., 1997, Simon et al., nucleus (Cytostomes?, cavity?) 2013, this study

Not PM upon Várnai et al., 1999, Cantley, 2002; this PI(3,4,5)P3 Not detected Cytosol detected stimulation study PM, plasma membrane; ER, endoplasmic reticulum; tER, transitional ER.

Figure legends Fig. 1. Subcellular localization of phosphatidylinositol 3-monophosphate (PI3P) in Plasmodium falciparum. (A) In ring stage parasites, the PXP40-GFP sensor is found as a single structure close to the nucleus. (B) In trophozoites (Troph), the signal now surrounds the food vacuole and an intense dot close to the plasma membrane (arrowhead) is also observed. In addition, some large vesicular structures were often seen in close proximity to the food vacuole membrane (arrow). (C) In schizonts (Schiz), the number of fluorescent dots increased along with the number of nuclei. The food vacuole membrane and the large vesicles seen in trophozoites were still present (arrowheads and arrow, respectively). (D) Western blot of mixed stages parasite extracts confirms expression of the full-length PXP40-GFP sensor. Blue, DAPI stained nuclei; BF, bright field. Scale bar: 5 m.

Fig. 2. Subcellular localization of phosphatidylinositol 4-monophosphate (PI4P) in Plasmodium falciparum. (A) In ring stage parasites, the 2xPHFAPP-GFP sensor is found around the plasma membrane and as a focus on fluorescence. (B) In trophozoites (Troph), in addition to the plasma membrane associated signal, two foci of intense fluorescence are now seen on each side of the DAPI stained nucleus. (C) As the parasite proceeds through schizogony (Schiz), each individual merozoite potentially possesses a single spot of GFP, together with some plasma membrane labeling. (D) In free merozoites (Mero), the plasma membrane GFP is no longer seen whilst the focus is still there. In certain cases, the signal seemed to cap one side of the merozoite (arrows). (E) Western blot of mixed stages parasite extracts confirms expression of the full-length 2xPHFAPP-GFP sensor. Blue, DAPI stained nuclei; BF, bright field. Scale bar: 5 m.

Fig. 3. Phosphatidylinositol 4-monophosphate is found at the Golgi apparatus throughout the Plasmodium falciparum erythrocytic cycle. (A) The PHFAPP-3HA sensor colocalizes extensively with the Golgi marker Endoplasmic reticulum Retention Defective protein 2 (ERD2) in rings (a), trophozoites (Troph) (b), schizonts (Schiz) (c) and free merozoites (Mero) (d). (B) Quantification of the colocalization between PHFAPP-3HA and ERD2 at each stage of the erythrocytic cycle. ERD2-Rhoptry associated protein 1 (RAP1) is used as a control for proteins residing in different organelles. The coefficients were calculated by the intensity correlation of Alexa fluor 488 and 594. Each dot on the graph represents an individual cell. Horizontal line represents the mean. The

79 mean ± S.E.M. is represented for each condition. ****P <0.0001. Number of cells: PHFAPP versus ERD2: Ring, n=15; Troph, n=14; Schiz, n= 15; ERD2 versus RAP1, n=20. An unpaired t-test was used to calculate the P value. (C) PHFAPPsensors do not colocalize with micronemal markers Apical membrane antigen 1 (AMA1) (a) and Erythrocyte binding antigen 175 (EBA175) (b), the rhoptry neck marker RON4 (c) and the rhoptry bulb marker RAMA (d). (D) Quantification of the colocalization between PHFAPP-3HA and the apical markers. Number of cells: PHFAPP-3HA versus AMA1, n=7; PHFAPP-3HA versus EBA175, n=7; PHFAPP-3HA versus RON4, n=7; 2xPHFAPP-GFP versus mCherry-RAMA, n=10; ERD versus RAP1, n=20. NS, non-significant in unpaired t-test. (E) Western blot of mixed stage parasite extracts confirms expression of the full-length PHFAPP- 3HA sensor. Blue, DAPI stained nuclei; BF, bright field; mC-RAMA, mCherry-RAMA. Scale bar: 5 m.

Fig. 4. Subcellular localization of phosphatidylinositol 5-monophosphate (PI5P) in Plasmodium falciparum. (A) In ring (a) and trophozoite (Troph) (b) stage parasites, the PHDOK5- GFP sensor is found in the cytosol and at the plasma membrane with an enriched region (arrow). In schizonts (Schiz) (c), the GFP signal was often seen as capping the apical region of individual merozoites (star) and sometimes as a single dot per merozoite (arrowhead). (B) IFA with an anti- ERD2 antibody shows that the PHDOK5-3HA sensor overlaps partially with the Golgi in schizonts. Blue, DAPI stained nuclei; BF, bright field. Scale bar: 5 m. (C) Quantification of colocalization shows that PHDOK5-3HA overlaps significantly less with the Golgi marker ERD2 than PHFAPP- 3HA. Horizontal line represents the mean. The mean ± S.E.M. is represented for each condition. *P <0.05. Number of cells: PHDOK5-3HA versus ERD2, n=15; PHFAPP-3HA versus ERD2, n=15. An unpaired t-test was used to calculate the P value. (D) Western blot of mixed stages parasite extracts confirms expression of the full-length (a) PHDOK5-GFP and (b) PHDOK5-3HA sensors.

Fig. 5. Subcellular localization of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in Plasmodium falciparum. (A) The mCherry-2xPHPLCδ sensor labels the parasite plasma membrane throughout the erythrocytic cycle. In rings (a) and trophozoites (Troph) (b), the mCherry signal was additionally labeling a cavity and some membrane protrusions (arrows). Maximum projection shows more than one structure in the ring. (B) IFA with an anti-merozoite surface protein 1 (MSP1) antibody shows that the PHPLCδ sensor overlaps extensively with the plasma membrane in

early (a) and late (b) schizonts (Schiz). (C) Western blot of mixed stages parasite extracts confirms expression of the full-length mCherry-2xPHPLCδ sensor. Blue, DAPI stained nuclei; mC, mCherry; BF, bright field; Max proj, Maximum projection. Scale bar: 5 m.

Fig. 6. Subcellular distribution of phosphoinositide (PIP) species during the erythrocytic cycle of Plasmodium falciparum. PI3P is found at the apicoplast (AP), food vacuole (FV) membrane and vesicles (V). PI4P is found at the plasma membrane (PM) and the Golgi apparatus (G). PI5P is found at the PM in all stages but also potentially at the transitional endoplasmic reticulum (tER) in schizonts. PI(4,5)P2 is found at the PM and large vesicular structures potentially corresponding to the cytostome/cavity (C). RBCm, red blood cell membrane; N, nucleus. Note that the ring and the schizont have been magnified for clarity, and that the distinct regions of PM labeling for PI4P and PI(4,5)P2 do not represent distinct domains and are just for clarity.

Figures Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

Supplementary Table Table S1: List of primers and plasmids used in this study

Selectable PIP sensor Primers Sequence (5'->3') Expression Systema Enzymes marker

5- AGATCTTAT ATATAATGG CTGTGG CCC AGC AGCTG -3 PHPX40-GFP pglmS-Rep20-Hsp86 BglII-KpnI BSD 5- GGTACCTGA GTC ATA GGG CGA CTG G -3 5- AGATCTTAT ATATAATGG CTGTGG CCC AGC AGCTG -3 PHPX40-Cherry pglmS-Rep20-Hsp86 BglII-KpnI yDHODH 5- GGTACCTGA GTC ATA GGG CGA CTG G -3

5- AGATCT TAT ATATAATGG AGG GGGTGT TGT ACA AGT GGA CCA AC -3 2xPHFAPP-2xGFP pglmS-Rep20-Hsp86 BglII-KpnI BSD 5- GGTACC CCT TGT ATC AGT CAA ACATGC TTT -3 5-TAT ATATAATGGCTTCCAATTTTAATGACATAG-3 PHDOK5-GFP pglmS-Rep20-Hsp86 BglII-KpnI BSD 5-GGTACCGATCCGTGTTCCTACACACTCCATCTG-3 5-CCTAGGTATATATAATGGTGAGCAAGGGCGAGGAGC-3 mCITRINE-PHTAPP1 pglmS-Rep20-Hsp86 BglII-KpnI BSD 5-CTGCAGCTAACCGGGGGGATGCTCAG-3 5-AGATCTTATATATAGCAGGCTTAACCATGCTAGACCCTTTG-3 2xCherry-2xPHPLC pglmS-Rep20-Hsp86 AvrII-PstI BSD 5-CAACTATGTATAATAAAGTTGCCTCGAGCTACTGGATGTTGA-3 5- AGATCT TAT ATATAATGG CCG CAGTGA TTCTGG AGA G -3 PHBTK-GFP pglmS-Rep20-Hsp86 BglII-XhoI BSD 5- GGT ACC AGGTTT TAA GCT TCC ATT CCT GT -3

5-CCTAGGTATATATAATGAGTAAAGGAGAAGAACTTTTCACTG-3 GFP pglmS-Rep20-Hsp86 BglII-KpnI BSD 5-CTCGAGCTACTGGATGTTGAGCTCCTTCAGG-3 5-ACGCGTATATATAATGGTGAGCAAGGGCGAGGAG-3 mCITRINE pglmS-Rep20-Hsp86 AvrII-XhoI BSD 5-CTGCAGTTACCTAGGCTTGTACAGCTCGTC

5-ACGCGTATATATAATGGTGAGCAAGGGCGAGGA-3 Cherry pglmS-Rep20-Hsp86 MluI-AvrII-stop-PstI BSD 5-CTGCAGTTACCTAGGCTTGTACAGCTCGTC-3 5-CCTAGGTATATATAATGG AGG GGGTGT TGT ACA AGT GGA CCA AC -3 PHFAPP-3HA pLN-Cam AvrII-XhoI BSD 5-CTCGAGCCTTGTATCAGTCAAACATGCTTT -3 5-CCTAGGTATATATAATGGCTTCCAATTTTAATGACATAG-3 PHDOK5-3HA pLN-Cam AvrII-XhoI BSD 5-CTCGAGGATCCGTGTTCCTACACACTCCATCTG-3 a: pglmS-Rep20-Hsp86 allows the expression of a fusion protein using the Heat shok protein 86 promoter. pLN-Cam allows the expression of a fusion protein through the Calmodulin promoter.

Table S2: Phosphoinositide binding domains used in this study.

Domain Gene Protein PIP detected Amino acids Reference Kanai, F., Liu, H., Field, S.J., Akbary, H., Matsuo, T., Brown, G.E., PX p40phox PX40 (h) PtdIns(3,4)P2 1-140 Cantley, L.C., Yaffe, M.B., 2001. Nat Cell Biol 3, 675-678. phosphatidylinositol- Dowler, S., Currie, R.A., Campbell, D.G., Deak, M., Kular, G., four-phosphate- FAPP1(h) PtdIns(4)P 1-99. Downes, C.P., Alessi, D.R., 2000. Biochem J., 351, 19-31. adaptor-protein-1 Guittard, G., Gérard, A., Dupuis-Coronas, S., Tronchère, H., docking protein 5 DOK5(h) PtdIns(5)P 1-116 Mortier, E., Favre, C., Olive, D., Zimmermann, P., Payrastre, B., Nunès, J.A., 2009. J Immunol 182, 3974-3978. Kimbar, W.A., Trinkle-Mulcahy, L., Cheung, P.C.F., Deak, M., tandem PH-domain- Marsden, L.J., Kieloch, A., Watt, S., Javier, R.T., Gray, A., TAPP1(h) PtdIns(3,4)P2 182-305 PH containing protein-1 Downes, C.P., Lucocq, J.M., Alessi, D.R., 2002. Biochem J, 361 (Pt3) 525-536

Lemmon, M.A., Ferguson, K.M., O'Brien, R., Sigler, P.B., phospholipase Cδ1 PLCδ1(r) PtdIns(4,5)P2 1-175 Schlessinger, J., 1995. Proc Natl Acad Sci USA 92, 10472-10476.

Bruton Salim, K., Bottomley, M.J., Querfurth, E., Zvelebil, M.J., Gout, I., agammaglobulinemia Btk1(h) PtdIns(3,4,5)P3 1-177 Scaife, R., Margolis, R.L., Gigg ,R., Smith, C.I., Driscoll ,P.C., tyrosine kinase Waterfield, M.D., Panayotou, G., 1996. EMBO J. 15(22):6241-6250 h, human; r, rat

Supplementary figure legends Supplementary Fig. S1. More examples of the subcellular localization of phosphatidylinositol 3-monophosphate (PI3P) in Plasmodium falciparum. (A) An early schizont stage parasite shows numerous large vesicular structures close to the food vacuole (arrows) together with some smaller vesicles close to the plasma membrane (arrowheads). (B) In a later schizont, the PXP40-GFP sensor labels a reticulated structure reminiscent of the apicoplast. Blue, DAPI stained nuclei; BF, bright field. Scale bar: 5 m.

Supplementary Fig. S2. The foci of phosphatidylinositol 3-monophosphate (PI3P) colocalize with the apicoplast in Plasmodium falciparum. Imaging of parasites expressing PXP40-mCherry and a fusion of the apicoplast protein Enoyl acyl carrier protein reductase (ENR) with GFP shows strong overlap between the PI3P foci and the apicoplast in trophozoites (A), and early schizonts (B, C). Note that very few doubly-transfected parasites were observed and none in ring or in late schizont stages. Blue, DAPI stained nuclei; mC, mCherry; BF, bright field. Scale bar: 5 m.

Supplementary Fig. S3. Subcellular localization of GFP in Plasmodium falciparum. (A) The GFP is found throughout the cytosol with some enrichment close to the nucleus (arrows) in rings (A), trophozoites (Troph) (B) and schizonts (Schiz) (C). Blue, DAPI stained nuclei; BF, bright field. Scale bar: 5 m.

Supplementary Fig. S4. Additional images of phosphatidylinositol 4-monophosphate (PI4P) in Plasmodium falciparum. (A) IFA showing PHFAPP-3HA sensor shows a similar pattern to PHFAPP-GFP in Fig. 2 (B) IFA showing some overlap between PHFAPP-3HA and the parasite plasma membrane marker merozoite surface protein 1 (MSP1) in early (a) and in late schizonts (Schiz) (b). Blue: DAPI stained nuclei; BF: bright field; Troph, trophozoite. Scale bar: 5 m.

Supplementary Fig. S5. Additional images of phosphatidylinositol 5-monophosphate (PI5P) in Plasmodium falciparum. (A) Another example of a parasite with a zone of the plasma membrane enriched with the PHDOK5-GFP sensor (star). (B) A trophozoite (Troph) with an accumulation of the PHDOK5-GFP sensor in the nucleus and in two spots very close to it (arrow).

(C) An example of a schizont (Schiz) where the foci of fluorescence are closely associated with the DAPI stained nucleus in individual merozoites. In some merozoites, the signal is not concentrated in a spot but instead looks more like a slightly curved line around the nucleus (arrowhead). Blue, DAPI stained nuclei;BF, bright field. Scale bar: 5 m.

Supplementary Fig. S6. An example of the phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 large vesicular structures in schizonts in Plasmodium falciparum. Early schizont stage parasites with large vesicular structures associated with the parasite plasma membrane (arrows). Blue, DAPI stained nuclei; mC, mCherry; BF, bright field; Max proj, Maximum projection. Scale bar: 5 m.

Supplementary Fig. S7. Subcellular localization of mCherry in Plasmodium falciparum. (A) The mCherry protein is found throughout the cytosol with some enrichment close to the nucleus (arrows) in rings (A), trophozoites (Troph) (B) and schizonts (Schiz) (C). Blue, DAPI stained nuclei; BF, Bright field. Scale bar: 5 m.

Supplementary figures Supplementary Fig.1

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Chapter 3: A pan-apicomplexan phosphoinositide- binding protein acts in malarial invasion-microneme exocytosis. Avant-propos In the article presented in Chapter 4 entitled “A pan-apicomplexan phosphoinositide-binding protein acts in malarial invasion-microneme exocytosis”. It is a scientific paper and I am the first author. I did most of the parasite manipulation, phenotypic analysis of the results related to the knocksideways (Fig. 1A, 3, 4A, B&C, EV1, 2, 3 S4, S5, S6, S7A) and designed some experiments. I also contributed to the writing of the manuscript. Angana Mukherjee performed the invasion assays with enzyme-treated erythrocytes (Fig.4F, S8) and contributed to the writing of the manuscript. Marie-Ève Crochetière and Audrey Sergerie analyzed the secretion of PfAMA1 by IFA and performed the secretion assay with free merozoites. (Fig. 2, 4A, EV2B, 3B, S3). Souad Amiar performed the protein-lipid interaction analyses and contributed to the writing of the manuscript (Fig. 1, S1, 2). L. Alexa Thompson and Joel B. Dacks performed the bioinformatics analysis (Fig.5, EV4, S8) and contributed to the writing the manuscript, David Gaumond and Dominic Gagnon performed parasite manipulations. R.V.S. interpreted results and contributed to the writing of the manuscript (Fig.3B). Dave Richard conceptualized the study, designed experiments, supervised the project, interpreted results, contributed to some of experiments (Fig.4D, S7B) and wrote the manuscript. The article has been published in the EMBO Reports in May 2019.

Résumé L'invasion des globules rouges par le parasite de la malaria Plasmodium falciparum, est une étape essentielle dans le développement de la maladie. Par conséquent, les acteurs moléculaires impliqués dans l'invasion des cellules hôtes représentent des cibles importantes pour le développement d'inhibiteurs et le de vaccins anti-malariaux. Le processus d’invasion du globule rouge par les mérozoïtes est médié par la sécrétion de protéines contenues dans les organites du complexe apical. Peu de choses sont connues quant aux mécanismes derrières l’exocytose de leur contenu. Ici, nous avons identifié une protéine contenant un domaine conservé de liaison aux phosphoinositides et avons montré que celle-ci est importante pour l’attachment et les étapes subséquentes d’invasion de l'érythrocyte par le mérozoïte. De plus, nous avont montré que retirer la protéine de son site d’action, en utilisant la technique de knock sideways empêche la sécrétion d’une population spécifique de protéines de micronèmes liées à l’invasion. Nos résultats fournissent donc des preuves du rôle important des phosphoinositides dans le processus d’invasion par le parasite de la malaria et apporte un éclairage nouveau quant aux mécanismes de sécrétion différentielle des populations de protéine de micronèmes, un concept qui pourrait être applicable aux autres parasites de la famille Apicomplexa.

Article A pan-apicomplexan phosphoinositide-binding protein acts in malarial invasion- microneme exocytosis.

Zeinab Ebrahimzadeh1, Angana Mukherjee1,4, Marie-Ève Crochetière1,4, Audrey Sergerie1,4, Souad Amiar2, L. Alexa Thompson3, Dominic Gagnon1, David Gaumond1, Robert V. Stahelin2, Joel B. Dacks3 and Dave Richard1*

1 : Centre de recherche en infectiologie, CRCHU de Québec-Université Laval, 2705 Boul. Laurier Québec (QC), Canada, G1V 4G2

2: Department of Medicinal Chemistry and Molecular Pharmacology and the Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907 USA

3: Division of Infectious Disease, Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta.

4: These authors contributed equally to this work.

*Corresponding author. Dave Richard, Tel.: 1-418-525-4444 ext 47975; fax: 1-418-654-2715.

E-mail address: [email protected] (DR)

Running title: Malaria microneme exocytosis

Abstract

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is an essential step in the development of the disease. Consequently, the molecular players involved in host cell invasion represent important targets for inhibitor design and vaccine development. The process of merozoite invasion is a succession of steps underlined by the sequential secretion of the organelles of the apical complex. However, little is known with regards to how their contents is exocytosed. Here, we identify a phosphoinositide-binding protein conserved in apicomplexan parasites and show that it is important for the attachment and subsequent invasion of the erythrocyte by the merozoite. Critically, removing the protein from its site of action by knock sideways preferentially prevents the secretion of certain types of micronemes. Our results therefore provide evidence for a role of phosphoinositide lipids in the malaria invasion process and provide further insight into the secretion of microneme organelle populations, which is potentially applicable to diverse apicomplexan parasites.

Introduction With more than 216 million cases and 445,000 deaths in 2016, malaria still represents a devastating disease[1]. The widespread occurrence of antimalarial drug resistance and the lack of a commercialized vaccine highlight the need for novel therapeutics. Plasmodium spp., the etiological agents of the disease, are obligate intracellular parasites and their invasion of human red blood cells (RBCs) is an essential part of their lifecycle. The invasion process debuts with the initial recognition of the erythrocyte membrane by merozoite surface proteins[2-4] after which the merozoite reorientates so that its apical tip becomes juxtaposed to the RBC membrane. Tight attachment of the parasite then occurs through the binding of parasite invasion ligands to RBC surface receptors. These ligands are the erythrocyte-binding-like (EBAs) and reticulocyte binding- like (RHs) proteins to their cognate receptors[5-8]. Subsequently, a tight junction is formed through an interaction between the apical membrane antigen 1 (AMA1) and the rhoptry neck (RON) complex[9-11]. Through an acto-myosin molecular motor, the parasite pulls itself into a parasitophorous vacuole in which it will reside [12-14].

Several of the effector proteins involved in the process of merozoite invasion are stored in the apical complex organelles and released in a controlled fashion[15]. How apical organelles are secreted by the merozoite is poorly known but evidence suggests that calcium and cGMP signaling are implicated[16,17]. Indeed, studies have shown that microneme discharge through the activation of calcium-dependent protein kinases (CDPKs)[18,19] and the cyclic GMP- dependent protein kinase (PKG)[20,21] is required for egress of merozoites from the schizont. Of interest, it was proposed that the disparate roles of PKG throughout the malaria parasite lifecycle could potentially be explained by its regulation of phosphoinositide metabolism and its effect on calcium signalling and potentially vesicular trafficking[17]. Exposure to low potassium levels as found in human plasma leads to a rise in intracellular calcium that then triggers the secretion of the micronemal proteins PfAMA1 and PfEBA175 and the subsequent interaction of the latter with glycophorin A on the RBC surface then results in the exocytosis of the rhoptries [22,23]. The interaction of PfRH1 with its as yet unknown receptor also results in an increase in intracellular calcium and secretion of PfEBA175[24]. The ability of the PLC inhibitor U73122 to abrogate microneme secretion suggests that the P. falciparum PLC homologue is implicated in the process[22]. 98

Recent work in the related apicomplexan parasite Toxoplasma gondii further suggested that recognition of phosphatidic acid produced through the action of TgPI-PLC by an acylated Pleckstrin-homology protein (TgAPH) present on the surface of the micronemes led to their exocytosis and parasite egress. The authors further showed that recombinantly expressed P. falciparum APH also bound to PA but whether it plays an equivalent role in microneme exocytosis is unknown [25,26]. Finally, the snare-like C2 domain-containing protein PfDOC2.1 was shown to be required for the secretion of the micronemal protein PfEBA175 and the rhoptry neck protein PfRH2a[27,28].

Until recently and despite early evidence suggesting that the P. falciparum micronemes were composed of heterogenous populations with specific functions in egress and/or invasion[29], most studies extrapolated results obtained while studying one micronemal protein (most often PfAMA1) to apply to all the others. Of interest, this heterogeneity of micronemes is also conserved in T. gondii[30,31]. A few studies have now started to take this into consideration. For example, recent results have shown that PfCDPK1 knockdown parasites have no defect in egress but cannot invade erythrocytes due to a specific defect in the secretion of PfEBA175-containing micronemes but not of PfAMA1 micronemes[32]. Absalon and colleagues revealed that the signaling cascade containing PfPKG and PfCDPK5 led to the secretion of PfAMA1 on the surface of merozoite before egress but not of PfEBA175 which remains in the micronemes and led them to suggest the inclusion of additional subsets of egress-specific micronemes [18]. The mechanisms underlying this differential exocytosis are currently unknown.

Intriguingly, despite representing only a minor fraction of the total lipids of eukaryotic membranes, phosphoinositides (PIPs) are critical components involved in a variety of cellular processes and recent work has shown that it was also the case for apicomplexan parasites (reviewed in [33]). More specifically for P. falciparum, roles for PIPs have been shown in cytokinesis and merozoite formation[34], apicoplast biogenesis and inheritance[35-37], hemoglobin endocytosis[38], merozoite egress[17], gametocyte activation [39-41] and ookinete motility[17], the latter two occurring in the mosquito vector, and finally in resistance to artemisinin[42,43]. In line with these varied functions, the determination of the subcellular distribution of several species of PIPs in the malaria parasite asexual erythrocytic stages showed

99 localizations to structures such as the Golgi apparatus, the plasma membrane, the food vacuole membrane, the endoplasmic reticulum and the apicoplast[34,35,44,45].

While hydrolysis of PI(4,5)P2 by the Toxoplasma gondii PI-phospholipase C is critical for the invasion of this parasite (see below,[25]), a direct role for PIPs in the malaria merozoite invasion process has not yet been described.

We here identify a P. falciparum PH domain-containing protein that is important for the secretion of micronemes containing PfEBAs. This protein has a relaxed phosphoinositide binding ability and we show, using knock sideways (KS), that it is required for merozoite attachment and invasion. Furthermore, we show bioinformatically that this protein is present in diverse apicomplexans suggesting that it plays a role in pathogenesis beyond Plasmodium. Our results therefore provide further insight into the secretion of micronemal populations.

Results and Discussion

The Pleckstrin homology domain of PF3D7_1337700 has a relaxed phosphoinositide binding specificity As part of our efforts to investigate potential roles for PIPs in the invasion process, we identified PF3D7_1337700 (www.plasmodb.org), a putative PIP-binding protein containing a PH domain. (Figure 1A). Less than 10% of the characterized PH domains possess the ability to bind PIPs but a common feature of PIP binding PH domains is the presence of a basic sequence motif KXn (K/R)XR involved in the binding to the head group of the inositol moiety[46]. Inspection of the PF3D7_1337700 PH domain sequence revealed that such a motif was present

(84KANIFYIYKLR94, Figure 1A). To determine whether the PH domain had the capacity to bind to PIPs, we recombinantly produced the WT PH domain and a version where residues K84, K92 and R94 were mutated to alanines, fused to an N-terminal glutathione-S-transferase tag. Coomassie staining of the purified proteins revealed a band at the expected size of 38 kDa along with a 26 kDa degradation product likely corresponding to GST alone (Appendix Figure S1). Incubation of the GST-WT PH domain with PIP-Strips showed that the protein interacts significantly with PI(3)P, whereas the well-established PLC PH domain bound PI(4,5)P2

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[47](Figure 1B). The WT PH domain also showed some residual interaction with PI(4)P, PI(5) (Figure 1B).The PH domain triple mutant was used to confirm the specificity of this domain to phosphoinositides (PIPs). Lipid overlay showed the ability of the PH domain mutant to interact the same PIPs than the wildtype with higher affinity to PI(3)P (Figure 1B).

PIP strips contain lipids spotted on nitrocellulose, which cannot recapitulate a membrane bilayer. To determine if the PH domain and triple mutant could interact with PIPs in a membrane bilayer, we employed a liposome-binding assay using a 5% molar ratio of PIPs or DPPA (phosphatidic acid) containing liposomes. The liposome-binding assay indicated the selective binding of WT PH domain to the phosphonoisitol monophosphate species. Indeed, as shown in Figure 1C, about 20% of the protein was found in the pellet fraction of PI(3)P, PI(4)P, or PI(5)P. The WT PH domain also exhibited significant binding to PI(4,5)P2 and a small amount of detectable binding to

PI(3,4,5)P3 and PA containing vesicles. However, no binding of WT PH domain was observed for two other phosphonoisitol bisphosphate containing membranes (PI(3,4)P2 and PI(3,5)P2) (Figure 1C, top panel). In contrast, the triple mutation of the PH domain showed a clear loss of the ability to bind liposomes containing different PIPs (Figure 1C, bottom panel). Taken together, these data indicate that the WT PH domain can interact robustly with several PIP species in lipid bilayers, whereas the triple mutant had a greatly diminished ability to interact with PIP containing membranes. Since the liposome-binding assay showed that the triple mutant lost the ability of a stable binding to specific PIP species, this suggests that the basic sequence motif identified (K84, K92 and R94) may be involved in the binding to the PIP head group in the lipid bilayer. To confirm this hypothesis, we performed surface plasmon resonance (SPR) analysis to determine the binding affinity of both WT and the PH domain triple mutation to PI(3)P-containing lipid vesicles. We employed lipid vesicles on the surface of a L1 sensor chip. The SPR assay confirmed the ability of the WT PH domain to bind lipid vesicles containing PI(3)P with an apparent disassociation constant (Kd) of 630 nM (Figure 1D and Appendix Figure S2). In contrast, the PH domain triple mutant had weak binding to PI(3)P containing vesicles and we were not able to determine an apparent Kd for the triple mutant (Figure 1D and Appendix Figure S2). The apparent PI(3)P membrane binding affinity of the WT PH domain is on par with other well characterized PIP binding domains[48]. Promiscuous PIP binding has been described for 67% of yeast PH domains and their specific subcellular distribution requires coincidence detection of

101 additional factors such as another protein or membrane curvature for example [49]. These results demonstrate that PF3D7_1337700 is a true PIP-binding protein and we renamed it PfPH2 following the uncharacterized PfPH1 named after a T. gondii PI(3,5)P2-binding protein [50].

PfPH2 is likely essential for the asexual erythrocytic cycle To further characterize PfPH2, we endogenously tagged its C-terminus with GFP by single cross-over recombination using the recently developed selection-linked integration (SLI) strategy[51]. To allow the functional analysis of PfPH2 by KS (see below), a double FK506 binding protein domain (2xFKBP) tag was also appended[51] (Figure EV1A). Proper integration of the vector and the absence of a WT allele were verified by polymerase chain reaction (PCR) demonstrating that we had successfully tagged the pfph2 gene (Figure EV1B). Time course analysis of PfPH2-2xFKBP-GFP expression by Western blot using an anti-GFP antibody on parasite protein extracts taken throughout the asexual erythrocytic cycle (from the ring through to the schizont stage) revealed a single band at the expected size of around 133 kDa for the PfPH2-2xFKBP-GFP fusion protein in schizont stage parasites. An antibody against the constitutive protein PfHSP70 was used as staging control (Figure EV1C). Immunofluorescence assays (IFA) of PfPH2-2xFKBP-GFP parasites showed a faint punctate signal in late schizont stages that did not colocalize with any of the markers investigated (micronemes: PfAMA1, PfEBA175 and PfEBA140 (Figure EV2A), rhoptry bulb: PfRAP1 or neck: PfRON4, RH1, RH4 AND RH5 (Figure EV2B), dense granules: PfRESA (Figure EV2C) or the Golgi apparatus: PfERD2 (Figure EV2D). To try to get a less crowded view than with schizonts, IFAs were also performed on free merozoites recently egressed. Again, no strong colocalization could be seen between PfPH2 and any of the markers (Figure EV3A and B). However, the PfPH2 signal often seemed to be more apical (Figure EV3Aii and iii and B). To look at this in a more quantitative manner, we calculated the distance between the farthest edge of the DAPI and PfPH2, PfRON4 and PfEBA175 and used this to infer how apical a protein potentially is i.e. the bigger the distance between the marker and the DAPI, the more apical a protein likely is. These data show that PfPH2 is farther from the DAPI than PfEBA175 (91.341.78 vs 75.601.72 pixels, Figure 2 Ai and B) and potentially more than PfRON4 (90.832.83 vs 83.192.49 pixels) though the latter difference was not statistically significant (Figure 2Aii and B). PfRON4 is a well described

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marker of the rhoptry neck so our results suggest that PfPH2 localizes to a structure close to the apical tip of the merozoite.

To investigate the essentiality of PfPH2 for the asexual erythrocytic cycle, we first tried to knock out (KO) its gene by single cross-over recombination using the SLI for targeted gene disruption strategy (SLI-TGD)[51]. The fact that we could never detect proper integration of the vector by PCR and the persistence of the WT allele in three KO attempts suggest that PfPH2 might be required for the asexual stage growth (unpublished observations). This is further supported by a recent whole genome functional screen using saturation mutagenesis by the piggyBac transposon[52]. To gain insight into the role of PfPH2 in the asexual blood stages, we performed KS, a strategy that allows the conditional removal of a protein of interest from its site of action[53]. To do so, we transfected the PfPH2-2xFKPB-GFP parasite line with an episome expressing a nuclear mislocalizer consisting of a triple nuclear localization signal fused to a double FKBP12-rapamycin binding domain and the cherry fluorescent protein (3xNLS-2xFRB- cherry)[51]. We next tested the ability of the mislocalizer to translocate PfPH2-2xFKPB-GFP to the nucleus in the presence of rapamycin (Rapa), thus removing it from its normal site of action. As seen in figure 3Ai, in absence of Rapa, the mislocalizer colocalizes with the DAPI stained nucleus whilst PfPH2-2xFKPB-GFP shows its normal punctate pattern. When adding Rapa at the ring stage, before the expression of PfPH2 is turned on, and letting the parasites mature to late schizonts, a substantial proportion of the GFP signal was now observed in the nucleus instead of the apical foci (Figure 3Aii). We quantified the levels of KS by calculating Pearson’s correlation coefficient for PfPH2-2xFKPB-GFP vs the mislocalizer and also vs the DAPI staining, in the presence and absence of Rapa. Our results show that the addition of Rapa does lead to a significant increase in the colocalization between GFP and both the mislocalizer (0.240.02 vs 0.580.02) and the DAPI (0.210.01 vs 0.500.01) at the population level though there is some variability when looking at individual cells (Appendix Figure S3). This shows that we succeeded in performing KS on PfPH2.

We next determined the effect of the PfPH2 KS on the ability of the parasite to proliferate. To do this, tightly synchronous PfPH2-2xFKPB-GFP+3xNLS-2xFRB-cherry (PfPH2- GFP+mislocalizer) parasites were incubated with or without Rapa and growth was monitored over 2 cycles. We first checked the effect of adding Rapa at different times and found that growth inhibition was 103 maximal when the compound was added at the ring stage and that adding it shortly before egress decreased the effect by around 50% (unpublished observations). We hypothesize that this is due to the fact that PfPH2 is potentially strongly attached to an apical structure so that it cannot easily be “extracted” by the mislocalizer. For efficient mislocalization to occur, PfPH2 would need to be captured as it is synthesized. Based on this, all subsequent experiments were performed with Rapa added at the ring stage. As a control, we used the PfPH2-2xFKPB-GFP without the mislocalizer (PfPH2-GFP). As shown in Figure 3Bi and ii, the KS resulted in an around 65% decrease in parasitemia over one reinvasion cycle and up to 86% after the second reinvasion cycle whilst Rapa had no effect on the control line without mislocalizer. This suggests that PfPH2 is required for optimal proliferation of asexual stages.

Having confirmed that the KS was dependent on the presence of the mislocalizer and that the concentration of Rapa used in our assays was not toxic in itself, the remaining experiments were only performed with the PfPH2+mislocalizer line incubated with or without Rapa. We first looked at the integrity of various subcellular structures by IFA and could see no obvious difference in the PfPH2-GFP+mislocalizer incubated with Rapa (referred to as the KS line onwards) (Appendix Figure S4). Next, to check whether the reduced parasitemia was due to a failure of the parasites to egress from the RBC, the number of schizonts and rings was followed over a 16-hour period, at every 4 hours, by Giemsa staining of parasite smears. As seen in Figure 3C and Appendix Figure S5, there was no difference in the evolution of schizont rupture between the KS and the control line however, there was an important decrease in the number of rings formed. These data show that egress proceeds normally in the KS line. We next looked at whether the KS line produced fewer merozoites per schizont by counting DAPI-stained cells by fluorescence microscopy but again, no difference was seen (Figure 3D) which suggests that a defect in merozoite invasion was potentially the cause of the reduced parasitemia. To directly address this, invasion assays were performed with +Rapa or -Rapa PfPH2+mislocalizer parasites and this revealed a 672% decrease in the formation of new rings (Figure 3E). To determine what step of the invasion process was affected, we monitored the ability of the merozoites to bind to RBCs in the presence of the actin polymerization inhibitor cytochalasin D, which blocks entry but not attachment (Appendix Figure S6 for schematic of the experiment)[54,55]. This

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showed that the KS had a 3510% reduction in its ability to attach (Figure 3E). Globally, this suggests that a major consequence of the KS of PfPH2 is a default in the ability of the merozoites to strongly bind to erythrocytes and to proceed to invasion.

KS of PfPH2 potentially more strongly affects the secretion of a subset of microneme proteins. The sequential secretion of apical complex organelles is central to invasion with the effectors contained in each set playing specific roles at distinct steps[15,55,56]. The lack of a measurable egress defect in the PfPH2 KS line indicates that secretion of the egress protease Subtilisin 1 from the exonemes[57] occurred normally. To investigate whether PfPH2 had a role in microneme secretion, we first looked at the translocation of PfAMA1 to the merozoite surface in late stage schizonts with fully mature daughter merozoites and saw no difference (Figure 4A). This shows that the population of micronemes containing PfAMA1, recently referred to as egress-related[18], is properly secreted in the PfPH2 KS line. Several of the invasion effectors are cleaved off from the merozoite surface and released into the culture supernatant as invasion proceeds[58]. In order to investigate whether PfPH2 had a role in microneme secretion, equal numbers of tightly synchronous PfPH2 KS and control schizonts were let to rupture and reinvade over a 12- hour period and the resulting culture supernatant was collected and probed with antibodies against a variety of micronemal proteins. To control for schizont number and parasite egress we normalized the secreted apical protein levels to PfSERA5, a parasitophorous vacuole protein that is released upon schizont rupture[57]. As seen in Figure 4B, the shedding of PfEBA175 and PfEBA140 was much reduced in the presence of Rapa. Quantification of three independent biological replicates revealed decreases of around 90% for PfEBA175 and 79% for PfEBA140 (Figure 4C and Appendix Figure S7). To verify that the decrease of PfEBA175 in the supernatant correlated with an increase in the unprocessed form in free merozoites, we next quantified the amount of unprocessed PfEBA175 using purified merozoites stimulated with the calcium ionophore A23187 to induce microneme secretion[22]. The results show that indeed, a higher amount of

105 unprocessed PfEBA175 is seen in Rapa+ merozoites (1.390.10 for Rapa+ vs 0.450.23 for Rapa-, normalized ratio to PfHSP70 levels) (Figure 4D and E). Though we could not detect a defect in PfAMA1 translocation to the merozoite surface in schizonts, the protein was decreased by around 24% in the supernatant (Figure 4B and C and Appendix Figure S6). This is likely due to the reduced invasion of the KS merozoites i.e less PfAMA1 is used up and released in the supernatant since fewer merozoites successfully attach and invade. Taken together these results suggest that PfPH2 perhaps plays a more critical role in the secretion of micronemes containing EBA ligands than of PfAMA1-containing micronemes. Of interest, it was recently shown that PfCDPK1 was specifically implicated in the secretion of PfEBA175[32] so it is tempting to speculate that PfPH2 could potentially be regulated by phosphorylation through this kinase.

The interaction between parasite ligands and their cognate receptors on the RBC surface defines the host cell tropism of Plasmodium parasites[59]. In P. falciparum, an interesting dichotomy is based on the dependency, or lack thereof, of parasite ligands to the presence of sialic acid on RBC receptors. Since PfEBA175 and PfEBA140 are involved in the sialic acid-dependent pathway and 3D7 PfEBA175 KO parasites were previously shown to be severely impaired in their capacity to invade erythrocytes treated with chymotrypsin[60], we expected that their severely decreased levels in the PfPH2 KS line would impact its host cell tropism. However, invasion assays performed in enzyme- treated erythrocytes showed that there was no difference in the sensitivity to either trypsin, chymotrypsin or neuraminidase treatment between the control and KS line suggestive of a more general invasion defect (Appendix Figure S8). Perhaps the depletion of PfEBA175 in the PfPH2 KS line is not severe enough to completely phenocopy the 3D7 PfEBA175 KO parasites. Interestingly, the decrease in invasion obtained with the PfPH2 KS line was slightly higher than any of the enzymatic treatments though the difference was not statistically significant compared to chymotrypsin (Figure 4F). These data could mean that, at least for the 3D7 parasite line that we have used in our study, decreasing the levels of EBA ligands has a more profound impact on invasion than removing sialic acid on erythrocyte receptors. This might be potentially explained by the fact that the invasion ligands functionally interact with one another as was previously shown[61]. What

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precise role PfPH2 plays in microneme exocytosis is unknown but it might interact with the calcium-activated snare-like PfDOC2.1 previously suggested to be involved in the membrane fusion event between PfEBA-175 containing micronemes and the parasite plasma membrane[28].Since PfRH1 has been shown to be required for PfEBA175 exocytosis[24], another possibility could be that PfPH2 is implicated in the secretion of PfRH1 instead of PfEBAs directly.

PfPH2 is Apicomplexan Specific and the PIP-Binding Sequence Motif is Partially Conserved. Finally, to assess the extent to which PfPH2 is a broader feature of apicomplexan invasion/attachment, we took an informatics approach. BLASTp and HMMer homology searching was undertaken to search for orthologues in a wide variety of taxa, with at least two organisms from each of the six supergroups and with emphasis on apicomplexan organisms and their outgroups (Dataset EV1). Initial BLASTp searches using the full length PfPH2 protein as a query generated multiple positive results in organisms both within and outside the apicomplexa phylum. Notably most of these protein hits were from a variety of different protein families that also contained PH domains. In order to mitigate the possibility of false positives due to the conserved PH domain, we performed the homology searching using the query with the PH domain removed. This only retrieved positive hits from proteomes of apicomplexan organisms (Dataset EV1). To mitigate the possibility of false negatives due to sequence divergence, we further searched the predicted proteomes using HMMer. We identified orthologues of PfPH2 in all apicomplexan genomes examined, notably including C. parvum (Figure 5A and B). All orthologues also shared the domain structure of an N-terminal PH domain followed by a putative SMC-N domain and were within ~25% of the size of the P. falciparum protein. By contrast, we were unable to identify orthologues in any of the outgroup taxa that we searched. Searching by tBLASTn into the nuclear scaffolds of these outgroup taxa, in order to mitigate false negatives due to mis-prediction of the protein similarly failed to identify and PfPH2 orthologues (Table EV1). These outgroups include the chromerids which is the closest sister lineage to apicomplexans, indicating the protein was

107 gained within the apicomplexan phylum, potentially as an adaptation to a parasitic specific lifestyle.

We next wanted to determine if the PIP-binding KXn(K/R)XR sequence motif identified within the PH domain of PfPH2 is conserved between all orthologues to give some sense of functional homology (Figure EV4). The initial lysine residue was largely conserved, as was a terminal positively charged residue R/H/K in all orthologues with the exception of those from T. annulata and B. microti. This raises the possibility that the method of microneme secretion by means of PfPH2 binding PIP residues may be conserved across apicomplexans. Interestingly, results from a genome-wide knock out screen suggests that, contrarily to PfPH2, the T. gondii orthologue is not essential for in vitro growth[62].

Conclusion In conclusion, our work has uncovered a potential role for phosphoinositide lipids in the process in merozoite invasion of red blood cells. The presence of PfPH2 proteins in diverse relatives may indicate that this mechanism could have broader relevance to understanding apicomplexan invasion mechanisms. Determining whether PfPH2 is a substrate of PfCDPK1 and identifying proteins interacting with PfPH2 are now logical steps to further our understanding of the cascade of effectors involved in the secretion of micronemes.

Materials and Methods The study was approved by the Canadian Blood Services (CBS) research ethics board, project number 2015.001 and by the CHU de Québec IRB, project number 2015–2230, B14-12-2230, SIRUL 104595. Written consent was obtained by the CBS for all study participants. All experiments were performed in accordance with relevant guidelines and regulations.

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Production and purification of the recombinant PfPH2 PH domains The PH domain of PfPH2 (amino acids 70 to 175) was amplified on 3D7 WT mixed stages cDNA using primers 5’BamH1-210-PfPH2 and 3’Xho1-525-PfPH2 (WT version) and 5’BamH1-210-PfPH2mut and 3’Xho1-525-PfPH2 (mutated version) and subsequently cloned in pGEX-6P3 (GE Healthcare) to be expressed as a recombinant GST fusion following standard procedures. pGST-PfPH2-PHWT, pGST-PfPH2-PHK84A/K92A/R94A and empty vector were transformed into Escherichia coli BL21 (DE3) competent cells and

◦ bacterial cultures were grown at 37 C and further induced at an OD600 of 0.6-0.65 with 0.5 mM isopropyl -D-1- thiogalactopyranoside overnight at 16 ◦C. Bacterial pellets were resuspended in Gibco PBS buffer (pH 7.4) containing 1 mM EDTA, 50 mM DTT, 100 µg/ml lysozyme and and 1x Thermo Scientific Halt protease inhibitor cocktail, and then sonicated four times for 30 s at 4°C. Further, protein solubilization was performed with 0.1% Triton-100X for 15 min at 4°C then centrifuged at 30,000 g for 25 min at 4°C. Proteins were purified with Sigma glutathione- agarose according to the manufacturer’s instructions, eluted with 50 mM Tris (pH 9.0), and 10 mM reduced glutathione, and analyzed by SDS/PAGE and Coomassie staining for purity check. For lipid overlay assay, the proteins were washed three times with cold 50 mM Tris (pH 8.0) with Amicon Ultra 30K MWCO to remove free glutathione. For SPR studies, the proteins were buffer exchanged in 20 mM HEPES containing 160 mM NaCl, pH 7.4. For Liposome-Binding assays, extensive washes were performed to remove any residual detergent post-agarose beads that may interfere with the lipid binding of liposome stability. However, the extensive washes resulted in reduction of protein yield and stability during the binding experiments. To overcome this issue, an additional solubilization step of the protein from inclusion bodies was performed prior to sonication as described previously[63]. The protein was then washed with cold 50 mM Tris (pH 8.0) with an Amicon Ultra 30K MWCO to remove free glutathione.

Protein-lipid overlay assay Lipid overlay assay was carried out using PIP StripsTM (Echelon Biosciences) and the protein- lipid binding experiment were performed according to the manufacturer’s

109 instructions[64]. Briefly, the PIP strips were blocked overnight with PBS buffer containing 1% milk and 0.1% Tween 20 at 4°C. The membranes were then incubated with

2 µg/ml of recombinant protein (GST-PfPH2-PHWT, GST-PfPH2-PHK84A/K92A/R94A, GST- alone) or 0.5 µg/ml of GST-PLC-PH (Echelon Biosciences) in the same buffer for 1 h at room temperature. After three washes with the same buffer, the membranes were incubated for 1 h at room temperature with anti-GST antibody (Bethyl Laboratories 1:10,000). After three more washes with the same buffer, the membranes were incubated for 1 h at room temperature with HRP-conjugated Anti-rabbit antibody (Abcam 1:10,000) followed by three washes. The bound proteins were detected with Clarity™ Western ECL kit from Bio-Rad Laboratories.

Liposome-Binding assay The preparation of liposomes was performed based on the previously described method[65]. Briefly, the chloroform mixture of lipid POPC:POPE:TopFluor-PC (8.9:1:0.1 mol:mol:mol ratio), POPC:POPE:POPS:TopFluor-PC (7.9:1:2:0.1) or POPC:POPE:POPS:X:TopFluor-PC (7.4:1:2:0.5:0.1), where X is one of tested PIPs or DPPA, were mixed and dried under a stream of lipid nitrogen. The dried lipid films were resuspended in liposome buffer (250 mM raffinose pentahydrate, 20 mM HEPES containing 160 mM NaCl, pH 7.3) to hydrate the lipid films for 15 min at 37°C. The multilamellar vesicles formed were extruded through 100 nm polycarbonate membrane to form large unilamellar vesicles. The liposomes were then resuspended in 3 times volume of liposome-binding buffer (20 mM HEPES, pH 7.3, containing 160 mM NaCl) and centrifuged at 50,000 g for 15 min at 22°C using a Beckman TLA-55 rotor. The pelleted liposomes were resuspended in corresponding volume of liposome-binding buffer. The protein was centrifuged for 5 min at 16,000 g to pellet any possible aggregation and the protein in the soluble fraction was diluted in liposome-binding buffer. For the liposome- binding assay, 50 µM of lipid vesicles were mixed with 500 ng of protein and incubated for 30 min at room temperature with continuous and gentle shaking. The mixture was then centrifuged at 75,000 g for 30 min at 22°C using a Beckman TLA-100 rotor. Supernatants (SN) corresponding to the soluble fraction were separated from the

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pellet (P) (lipid-bound fraction) and analyzed with Western blot using an anti-GST antibody (Bethyl Laboratories 1:10,000) and HRP-conjugated Anti-rabbit antibody (Abcam 1:10,000). The protein fractions were detected with a Clarity™ Western ECL kit from Bio- Rad Laboratories

Surface plasmon resonance analysis SPR-based lipid binding experiments were performed at 25 °C on a Biacore™ X100 (GE Healthcare) as described previously[66]. Briefly, the L1 sensor chip (GE Healthcare) was equilibrated in running buffer (20 mM HEPES containing 160 mM NaCl, pH 7.4). Large unilamellar vesicles (LUVs) with ~50 nm diameter were prepared from POPC/POPE (9:1 mol:mol-control LUVs) or POPC/POPE/PIP (8.5:1:0.5 mol:mol-active LUVs) by the extrusion method in SPR running buffer at a final concentration of 0.5 mM. The L1 sensor chip was then coated by injecting lipid vesicles at 5 µl/min for a response of 6000-7000 resonance unites (RU) either with control LUVs or active LUV preparations. Lipid coating was then stabilized by injecting 4 µl of 50 mM NaOH three times at 30 µl/min following lipid coating followed by a blocking with 0.1 mg/ml fatty acid free BSA at 10 µl/min for three times 100 min. SPR measurements were done at the flow rate of 5 µl/min with increasing protein concentrations (from 10 nM to 2000 nM) in freshly prepared SPR running buffer with a contact time of 600 sec to give the protein association time required time to reach saturation. Data were analyzed with BiaEval Software (GE Healthcare) and plotted with Kaleidagraph. The apparent Kd of vesicle binding was determined using a non- linear least squares analysis using the equation Req= Rmax/(1+ Kd/[C]). Where Req was measured in response units (RU) and was plotted versus protein concentration, [C], for protein concentration injected in each experiment. Rmax is the theoretical maximum RU response and Kd is the apparent membrane affinity[67].

Parasite Culture P. falciparum 3D7 asexual stage parasites (from David Walliker, Edinburgh University) were cultured under standard conditions in RPMI-HEPES medium at 4% hematocrit

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(human erythrocytes of O+ group) and 0.5% (w/v) AlbumaxTM (Invitrogen) and kept at 37°C in a gas mixture of 5.0% oxygen, 5.0% carbon dioxide and 90% nitrogen[68].

Vector Construction and Transfection To tag the endogenous PfPH2 with 2xFKBP-GFP, around 500 bp of the C-terminus of PfPH2 was amplified with primers 5'Not1-1700-PfPH2 and 3'AvrII-stopless-PfPH2 and cloned in frame with 2xFKBP-GFP in pSLI-2xFKBP-GFP-hDHFR digested NotI- AvrII[51]. Parasites were transfected and integrants were selected as described previously with some modifications[51]. Briefly, P. falciparum 3D7 parasites were transfected with 100 µg of purified pSLI-PfPH2-2xFKBP-GFP plasmid. Positive selection for transfectants was achieved using 5nM WR99210 (WR). Then drug resistant parasites were split into 3 separate wells with 2-4% parasitemia and went under another round of selection using 400µg/ml neomycin (NEO) to select for integrants. After parasite re- emergence (after around 10 days) WR was put back in the culture medium. Genomic DNA was prepared from NEO and WR resistant parasites. Integration was monitored by PCR using the forward 5'upstream-1250-PfPH2-F (primer 1) and the reverse 3'90-GFP-R primer (primer 2) for 5' integration and 5'pARL-F (primer 3) with the reverse 3’-3UTR- PfPH2-R primers (primer 4) for the 3' integration. Primer 1 was used primer 4 to detect the WT version of the gene. (see Appendix Table S1 for primer sequences).

To generate the parasite line for the knock sideways, the PfPH2-2xFKBP-GFP line was transfected with 100 µg of purified p3xNLS-FRB-mCherry-BSD plasmid and selected 2 µg/ ml blasticidin (Sigma-Aldrich) to obtain the PfPH2-GFP+mislocalizer line.

Western Blotting For the time-course of expression analysis, parasites were synchronized twice at an 18- hour interval (between 18 and 22 hours ring stages), with a 0.3 M alanine-10mM HEPES solution (as described in [69]). Synchronous parasites were then harvested by saponin lysis at 8, 30, 36 and

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42h post-reinvasion, the pellets solubilized in SDS protein sample buffer and separated on a 7.5% SDS-PAGE gel under reducing conditions and transferred to PVDF membranes (Millipore). The antibodies rabbit polyclonal anti-PfHSP70 (SPC-186C; StressMarq Bioscience Inc., 1:20000) [70] and mouse monoclonal anti-GFP, (Roche, 1:1000, JL8), were diluted in 0.1% (v/v) Tween 20-phosphate-buffered saline with 1% (w/v) skim milk. Appropriate HRP-coupled secondary antibodies were used and immunoblots were revealed by ECL (Amersham Biosciences). For all expression analyses, proteins extracted from an equal number of cells were used for each time point.

Fluorescence Imaging Fluorescence images of parasites were captured using a GE Applied Precision Deltavision Elite microscope with 100x 1.4NA objective and with a sCMOS camera and deconvolved with the SoftWorx software. Chromatic calibration of the microscope was performed prior to imaging experiments. For immunofluorescence assays, parasites were fixed on slides using 4% paraformaldehyde (ProSciTech)[71]. After blocking in 3% bovine serum albumin (Sigma Aldrich) the cells were incubated for 1 hour with rabbit polyclonal anti-PfERD2 (1:2000)[72], mouse monoclonal anti-PfRON4 (1:2000)[73], mouse monoclonal anti-PfRAP1 (1:2000)[74], mouse monoclonal anti-PfRESA (1:2000)[75], rabbit anti-PfEBA175 (1:1000)[76], rabbit anti- PfEBA140 (1:1000)[61], rabbit anti-PfAMA1 (1:2000)[29], rabbit anti-PfGAP45 (1:2000)[70] and rabbit anti- PfMSP1 (1:1000)[77]. Bound antibodies were then visualized with Alexa Fluor- 594 anti-rabbit IgG and Alexa Fluor-488 anti-mouse IgG diluted 1:1000. Parasites were mounted in Vectashield (Vecta Laboratories) containing 0.1 μg/ml 4', 6–diamidino-2- phenylindole (Dapi, Invitrogen). Images shown represent a single optical slice from a deconvolved z-stack.

To measure the distance between the Dapi and the different markers, a line was first drawn across the center of the merozoite, always starting at the extreme end of the DAPI. In the resulting line intensity profile (Plot Profile, Fiji), the relative distance in pixel was defined as the maximum-intensity point of the other channel (green = PfPH2, red = EBA175 or

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RON4), considering the 0 is always the extreme end of the Dapi. The distance was then put in GraphPad Prism 7 and a One-way ANOVA was performed as a statistical test.

Growth assays Tightly synchronous ring stage PfPH2-2xFKBP-GFP+mislocalizer parasites were seeded at 0.2% parasitemia and grown +/- 250 nM rapamycin. After 28, 60 and 120 hours in culture, the cells were harvested and analyzed by fluorescence-activated cell sorting (FACS) on a BD FACSCanto A to calculate the parasitemia as previously described[78]. Briefly, the cells were stained with SYBRGold (Invitrogen-Molecular Probe) and then fixed with 1% paraformaldehyde for 1 hour. 100 000 events were acquired on the FACSCanto A using the FACSDiva software. The data were analyzed with the FlowJo software. The percentage of survival was obtained by normalizing to untreated parasites in the same experiment, which was taken as 100% survival. Uninfected red blood cells were used to determine the threshold for FITC signal. Experiments were performed with a minimum of 3 biological replicates.

Merozoite number assay Tightly synchronous ring stage PfPH2-2xFKBP-GFP+mislocalizer parasites were grown +/- 250 nM rapamycin until they reached the schizont stage after which 10 µM of the protease inhibitor E64 was added for 4-6 hours to prevent schizont rupture. When the majority of parasites had developed into well-segmented merozoites, the number of merozoite nuclei per schizont was counted. Only single parasite-infected red blood cells with one food vacuole were counted in this experiment. Nuclei of a total of 20 parasites were counted per condition and the experiment was repeated in 4 biological replicates.

Monitoring of schizont rupture Tightly synchronized ring stage PfPH2-2xFKBP-GFP+mislocalizer parasites which were grown +/- 250 nM rapamycin until the schizont stage, Percoll-purified and then plated at a 2% parasitemia and incubated at 37°C with or without rapamycin. Parasites

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were smeared every 4hours for 16 hours and the number of schizonts and newly formed rings was counted. The experiment was performed in biological triplicates.

Attachment/Invasion assay Tightly synchronized PfPH2-2xFKBP-GFP + mislocalizer parasites which were grown +/- 250 nM rapamycin until the schizont stage, Percoll-purified and then plated at a 2% parasitemia in 48 well plates containing 100 µl of a pre-made mix of medium plus drugs and 2% hematocrit. For the attachment assay, 1 µM cytochalasin D was used to prevent invasion following merozoite attachment. Plates were shaken for 3 min and then incubated for 12 hours. For the merozoite attachment assays, the number of merozoites bound to red blood cells was counted while for the invasion assay, the number of rings was counted. All counts were performed on Giemsa-stained parasite smears. All experiments were performed in biological triplicates.

Microneme secretion assay Tightly synchronous ring stage PfPH2-2xFKBP-GFP+mislocalizer parasites were incubated +/- 250 nM rapamycin, until they reached the schizont stage at which point they were let to rupture and reinvade overnight and the invasion supernatant collected. The resulting supernatants were solubilized in SDS sample-buffer to extract the proteins and analyzed by Western blot. The antibodies used were: rabbit anti-PfSERA5 (1:5000)[79], rabbit anti-PfEBA175 (1:2500)[76], rabbit anti-PfEBA140 (1:1000)[61] and rabbit anti- PfAMA1[29], were diluted in 0.1% (v/v) Tween 20-phosphate-buffered saline with 4% (w/v) skim milk. Appropriate HRP-coupled secondary antibodies were used and immunoblots were revealed by ECL (Amersham Biosciences). The signal for each antigen was normalized to PfSERA5 to control for schizont number and egress efficiency. To determine the amount of full length PfEBA175 remaining in merozoites after induction of microneme exocytosis, free merozoites of PfPH2-2xFKBP- GFP+mislocalizer parasites incubated +/- 250 nM rapamycin were prepared as described previously[80]. Purified merozoites were incubated for 30 min at room temperature with 10uM of the calcium ionophore A23187 to induce microneme secretion[22] after which

115 they were pelleted and resuspended in SDS sample buffer and analyzed by Western blot using rabbit anti- PfEBA175 (1:2500)[76]. A rabbit polyclonal anti-PfHSP70 (SPC-186C; StressMarq Bioscience Inc., 1:20000) [70] was used as a loading control. To analyze the amount of unprocessed PfEBA175, free merozoites were purified according to [55,80] and incubated for 15 minutes at 37 οC with 10 μM of the calcium ionophore A23187 to stimulate microneme exocytosis. The merozoites were then pelleted, resuspended in SDS sampled buffer and analyzed by Western blot.

Invasion assays in enzyme-treated erythrocytes Invasion assays in enzyme-treated RBCs were performed as described previously with modifications[81,82]. Uninfected RBCs were washed with incomplete RPMI (iRPMI) and 100 μl was treated with 10 volumes of 66 mU/ml neuraminidase (Sigma), 1mg/ml chymotrypsin (Sigma), 1mg/ml trypsin (Sigma) or iRPMI (vehicle) for 1 hour at 37°C. All samples were washed with iRPMI and then trypsin and chymotrypsin samples were treated with 0.5mg/ml soybean trypsin inhibitor (Sigma) at 37°C for 10 minutes. All samples were then extensively washed (3-4x) in iRPMI. Ring infected RBCs were synchronized with sorbitol once at 0-4hpi and then 16 hours later. 34-38hpi infected RBCs (iRBC) from the same cycle were then purified on a 65%-35% Percoll gradient, the purified iRBC pellet was washed 3-5 times in iRPMI and mixed with enzyme-treated RBCs at 2% hematocrit and 0.2-0.5% parasitemia and plated in 96 well plate in media containing +/- rapamycin (250nM). Following incubation in standard parasite culture conditions for 30-36 hours, parasitemia was measured by flow cytometry in the FITC channel after cells were stained with SYBRGold and fixed with 1% paraformaldehyde for 1 hour. The experiments were performed in at least 3 biological replicates.

Statistical analysis Prism 7(GraphPad) was used for all statistical analyses. Depending on the assay, one-way ANOVA or two-tailed unpaired t-tests were performed. A minimum of 3 biological replicates was done for each experiment. A P value of < 0.05 was considered statistically significant.

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Homology Searching PfPH2 (PF3D7_1337700) was used a query for searches using BLAST[83] into the protein databases of Acanthamoeba castellani, Arabidopsis thaliana, Babesia microti, Bigelowiella natans, Cyclospora cayetanensis, Cryptosporidum parvum, Cystiospora suis, Chromera velia, Dictyostelium discoideum, Eimeria necatrix, Guillardia theta, Hammondia hammondi, Homo sapiens, Neospora caninum, Plasmodium falciparum, Plasmodium vivax, Perkinsus marinus, Phytophthora sojae, Saccharomyces cerevisiae, Symbiodinium minutum, Theileria annulata, Trypanosoma brucei, Toxoplasma gondii, Tetrahymena thermophila, Trichomonas vaginalis and Vitrella brassicaformis using inhouse scripts. Potential orthologues were then used as queries for a homology search using BLAST into the P. falciparum protein database. Candidates were considered positive orthologues when both forward and reverse BLAST hits generated an E- value below a 0.05 threshold, if the candidate protein in question retrieved the relevant query with e-values two orders of magnitude better compared to the next non-redundant hit, and if the top reverse BLAST hit was PF3D7_1337700. HMMer (v3.2.1)[84] searches were also conducted using a HMMer profile made of PF3D7_1337700 known orthologues into the protein databases of the organisms listed above. The HMMer profile was generated using MUSCLE alignment software (v3.8.31) [85]and Fetch software (v5.7.7 downloaded from https://fetchsoftworks.com) was used to search into the protein databases. Potential orthologues were validated using the above procedure. BLAST and HMMer searches were performed for both the full length PF3D7_1337700 query as well as PF3D7_1337700 without the PH domain (amino acids 71-172). In the case where searches were performed without the PH domain, CDD on NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) identified the location of the PH domain and Emboss:extractseq (http://www.bioinformatics.nl/cgi-bin/emboss/extractseq) was used to trim the PH domain from the query. Conserved domain structures of the orthologues were identified using Phyre v2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). tBLASTn searches were also performed using PF3D7_1337700 without the PH domain (amino acids 71-172) as the

117 query into the nuclear scaffolds of the non-apicomplexan taxa, with homology assessed using the above criteria.

References

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Competing interest The authors declare no competing interests.

Materials and Correspondence Requests for materials should be addressed to Dave Richard.

Acknowledgments We would like to thank Tobias Spielman for the pSLI plasmids and Robin Anders, James Beeson, Michael Blackman, Alan Cowman and Julian Rayner for antibodies. We also thank Jacobus Pharmaceuticals for WR99210. The following reagent was obtained through MR4 as part of the BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA: Polyclonal Anti-Plasmodium falciparum PfERD2 (antiserum, Rabbit). We would also like to acknowledge the Canadian Blood Services for providing human erythrocytes. The authors declare no competing financial interests. This study was funded through a Canadian Institutes for Health Research (CIHR) operating grant MOP 130359 to DR. DR is a Fonds de la Recherche du uébec- Santé unior 2 fellow. Work in the Dacks Lab is supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (RES0021028). JBD is the Canada Research Chair (Tier II) in Evolutionary Cell Biology.

Author contributions

Z.E. performed most of the knock sideways parasite manipulation and phenotypic analysis and contributed to the writing of the manuscript. A.M. performed the invasion assays with enzyme- treated erythrocytes and contributed to the writing of the manuscript. M.E.C and A.S. analyzed the secretion of PfAMA1 by IFA and performed the secretion assay with free merozoites. SA performed the protein-lipid interaction analyses and contributed to the writing of the manuscript. L.A.T. and J.B.D. performed the bioinformatics analysis and contributed to the writing the manuscript, DGau and DGag performed parasite

125 manipulations. R.V.S. interpreted results and contributed to the writing of the manuscript. D.R. conceived the study, designed experiments, interpreted results and wrote the manuscript.

Data availability

The data supporting the findings of this study are available within the paper and its Appendix and are also available from the corresponding author upon request.

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Figure legends

Figure 1: PF3D7_1337700 is a phosphoinositide-binding protein with a relaxed specificity. A) Schematic of PF3D7_1337700 showing the PH domain with the conserved PIP-binding motif. B) Lipid blots showing that the WT PH domain and triple mutant PH domain bind several species of PIPs. A negative (GST) and positive control (PLC-PH) are also shown for respective lipid binding. C) Liposome-binding assays of GST-tagged WT and triple mutant PH domains. GST- tagged PfPH2-PH proteins (500 ng) were incubated with 50 µM liposomes composed of POPC:POPE, POPC:POPE:POPS, or liposomes containing 5% molar ratio of one of seven PIP species or DPPA (POPC:POPE:POPS:DPPA or POPC:POPE:POPS:PIPs). kDa indicates molecular weight, P: pellet fraction, SN: supernatant fraction. D) SPR analysis of the WT PfPH2-PH domain and PH domain triple mutant demonstrates binding to lipid vesicles (POPC:POPE) containing 5 mol% PI(3)P. The response values shown (determined by subtracting the binding signal from control lipid vesicles) were plotted versus PH domain concentration to determine the apparent affinity

(Kd) of vesicle binding.

Figure 2: PfPH2 localizes close to the apical tip.

A) IFA showing the distance between the farthest edge of the DAPI and PfPH2 and (Ai) PfEBA175 and (Aii) PfRON4. Two images are shown for EBA175 to demonstrate how much variety can be observed between different cells. Green: PfPH2. Red: EBA175 (Ai) or RON4 (Aii). Blue: DAPI stained nucleus. Scale bar: 0.5 µm. B) Quantification of the distances reveals that the distance between PfPH2 and the DAPI is significantly bigger than PfEBA175 and the latter but not with PfRON4. ****: p- value<0.0001. ns: non significant. One-way ANOVA. Horizontal lines: Median. Box limits: 25tth to 75th percentile. Whiskers: Min to max values. Error bars: stander error of the mean.

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Figure 3: PfPH2 is important for attachment to and subsequent invasion of the red blood cell. A) Live cell microscopy showing that in the presence of 250 nM Rapa, PfPH2- 2xFKBP-GFP (GFP) is translocated from the apical pole to the nucleus in the presence of the nuclear mislocalizer. Scale bar represents 5m. Blue: DAPI stained nucleus. BF: bright field. B (Bi) Growth curve analysis showing that the KS of PfPH2 severely decreases the asexual

replication of the parasite. The PfPH2-GFP parasite line (black square) not transfected with the nuclear mislocalizer is used as a control to show that the reduced growth is dependent on the presence of both Rapa and the mislocalizer. Full line: -Rapa. Dashed line: +Rapa. Mean  SEM of 3 biological replicates are shown. (Bii) Data from (Bi) represented as the percentage of growth of parasites incubated with Rapa compared to their control without Rapa. Mean  SEM of 3 biological replicates are shown. C) Time course of schizont rupture (red) and subsequent new ring formation (blue) showing that the PfPH2 KS line has no egress defect but an important decrease in the formation of new rings. Results from one experiment representative of 3 biological replicates are shown. Full line: -Rapa. Dashed line: +Rapa. D) Quantification of the number of merozoites formed per schizont. n= 4 biological replicates with 20 schizonts counted per condition. n.s: non significant. Unpaired t-test. p- value= 0.1524. Error bars: Standard error of the mean. E) Merozoite attachment and invasion is decreased in the PfPH2 KS line. Attachment was measured by incubating parasites with 1 µM cytochalasin D. CytD: cytochalasin D. n=3 biological replicates for both attachment and invasion assays. One-way ANOVA followed by Fisher’s LSD test. Error bars: Standard error of the mean.

Figure 4: PfPH2 is required for the secretion of PfEBA-micronemes. A) IFA showing that the PfPH2 KS line has no defect in the exocytosis of PfAMA1 containing micronemes as measured by the translocation of PfAMA1 from the apical tip to

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the merozoite surface. n= 3 biological replicates with 150 cells counted for each condition. n.s.: non significant in a 2-tailed t-test. p-value=0.9061. Error bars: Standard error of the mean. Scale bar: 5 µm B) Western blot showing the proteolytic shedding of micronemal proteins in the supernatant following merozoite invasion. The parasitophorous vacuole protein PfSERA5 is used as a control for schizont rupture. Sn: supernatant. C) Densitometric analysis of reveals that the PfPH2 KS line has a severe defect in the shedding of PfEBA175 and PfEBA140 but not PfAMA1. Data normalized to PfSERA5 and then expressed as a percentage of the control incubated without Rapa. Data from 3 independent biological replicates. s: significant. An unpaired t-test was performed and showed a significant difference of invasion between the PfAMA1 and both PfEBA175 (p-value=0.0047) and PfEBA140 (p-value=0.0019). Error bars: Standard error of the mean. D) Western blot showing that the amount of unprocessed PfEBA175 associated with free merozoites after stimulation of microneme exocytosis. Biorep: Bioreplicate E) Densitometric analysis reveals that free merozoites of the PfPH2 KS line have increased levels of unprocessed PfEBA175. Data normalized to PfHSP70. Data from 3 biological replicates is shown. p-value=0.0201. Unpaired t-test. Error bars: Standard error of the mean. F) Comparison of the effect of the PfPH2 KS and enzymatic treatments on invasion. Values represent % of invasion relative to -Rapa PfPH2 line in normal untreated erythrocytes. Rapa=rapamycin. s: significant. ns: non significant. Data from 3 biological replicates is shown. An unpaired t-test was performed and showed a significant difference of invasion between the PfPH2 KS and trypsin (p-value=0.0041) and neuraminidase (p- value=0.0069). Error bars: Standard error of the mean. Untr: Untreated. Ezn: Enzyme. T: trypsin. C: Chymotrypsin. NM: Neuraminidase. Rapa: Rapamycin. Biorep: Bioreplicate

Figure 5: PfPH2 is conserved throughout apicomplexan parasites.

A) Schematic of PfPH2 showing the PH domain with the conserved PIP-binding motif.

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B) PfPH2 proteins are conserved but restricted to apicomplexans. This cartoon illustrates the presence of PfPH2 orthologues mapped onto the tree of major apicomplexan representatives with genome sequences available. Relationships between the taxa are based on [86]. The phylogenetic distribution of PfPH2 orthologues demonstrates apicomplexan specificity. Positive orthologues were identified in all apicomplexans sampled (red) and could not be identified denoted by an asterisk in Ciliates (green), Dinoflagellates (purple) or the Chromerids outgroup to the Apicomplexa (yellow), as demonstrated by the E-values for the candidate orthologue into the P. falciparum proteome (Column Reverse BLAST E-values). Relationships based on[86]. Conserved domain structure in all orthologues is shown based on Phyre v2.0. Black arrow represents the gain of PfPH2 at the base of Apicomplexa coincident with the shift to parasitism. Orange box; PH domain. Blue box; SMC_N superfamily domain.

Expanded View Figure legends

Figure EV1: Generation of the PfPH2-2xFKBP-GFP parasite line.

A) Schematic showing the tagging strategy by single cross-over recombination using SLI.

B) PCR on parasite genomic DNA showing the proper integration of the tagging vector at (5’ junction: primers P1 and P3, 3’junction: primers P2 and P4) and the disappearance of the WT allele (primers P1 and P4) in all 3 SLI attempts. C) Time course of expression of parasite protein extracts taken throughout the erythrocytic cycle shows that PfPH2 is expressed in schizonts. PfHSP70 is used as a control for a constitutive protein. Cartoon shows the time points in hours post invasion (hpi). 1: 8 hpi, 2: 30 hpi, 3: 36 hpi, 4: 42 hpi.

Figure EV2: Localization of PfPH2 in schizonts. A–D IFA showing that PfPH2 does not colocalize with markers of the micronemes: PfAMA1, PfEBA175 and PfEBA140) (A), of the rhoptries: PfRAP1, PfRON4, PfRH1,

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PfRH4 and PfRH5 (B), of the dense granules: PfRESA (C) or of the Golgi apparatus: PfERD2 (D). Scale bar represents 5m. Blue: DAPI stained nucleus. BF: bright field.

Figure EV3: Localization of PfPH2 in free merozoites. A, B IFA showing that PfPH2 does not colocalize with markers of the micronemes: PfAMA1 (secreted on the parasite surface in free merozoites), PfEBA175 and PfEBA140 (A) or of the rhoptry markers PfRON4, PfRAP1, PfRH1, and PfRH5 (B). Scale bar represents 5m. Blue: DAPI stained nucleus.

Figure EV4. Alignments of the KXn(K/R)XR binding motif in Apicomplexan orthologues of PfPH2. Alignments of a portion of the PH domains from PF3D7_1337700 identified orthologues in Apicomplexa are shown. The three arrows outline the alignments of the sequence motif from PF3D7_1337700 with similar amino acid positions of the other proteins.

Notably the first and terminal positions of the KXn(K/R)XR motif show conservation.

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Fig. EV4

P.falciparum 1 WERYYCFIKANIFYYKLRGDYKPHVIFLL P.vivax 1 WVKCFCFVKSNFFYYKERGDYRPSIIFLL T.annulata 1 WKELNLEIRGNCLLCDASDECGPVMGWIL B.microti 1 WEENNVEIRGNALFYPQYDQCVIGRGYLL E.necatrix 1 WLKRYCEIRGNLLLYTSHAEAAFEGAYML C.cayetanensis 1 WLKRYCELKGNLLLYAPHSDAAFEGAFML N.caninum 1 WVKRFCTVKANLLGFAPHSDAPFEGAYLL T.gondii 1 WVKRFCTVKANLFGFAPHSDAPFEGAYLL H.hammondi 1 WVKRFCTVKANLFGFAPHSDAPFEGAYLL C.suis 1 WIKRFCVVKANMLGFAPHSDAPFEGAYLL C.parvum 1 WEQYYFNLKGGMMFTSKKDGSTLEVVYVL

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Appendix -Appendix Table S1. Page 141 -Appendix figure titles. Page 142 to 143 -Appendix figures. Page 144 to 151

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Appendix Table S1. List of primers used in this study Primer name Primer sequence (5' to 3')

5'NotI-1700-PfPH2-F CGCGGCCGCCGTTTAACTTATTATACTAC 3'AvrII-stopless- GCCCTAGGATACATCTCATGTACTTGTAAATC PfPH2-R 5’XhoI-1250-PfPH2-F CGAGATCTCTACAGAAGAGTGCTACAAG (P1) 3'-3UTRPfPH2-R (P4) GCGACTAGTCTTAGCAGCTTGTTTAGATCATAG pARL-F (P3) GGAATTGTGAGCGGATAACAATTTCACACAGG 3'180-GFP-R (P2) AGGTAGTTTTCCAGTAGTGC 5'NotI-NtermPfPH2-F CGCGGCCGCTAATTCAATGTAAGTTCATTAAT 3'MluI-NtermPfPH2-R GCACGCGTGGGAGAAAACAAATTTGTGCAC

PfPH2-290-up-F GTGTATGTTCATTTATGGACCATCC PfPH2-1361-R CGTAAGTCTTTCTGGATCGC

5'BamHI-210-PfPH2-F CGCGGATCCATGTTCAATGTAAGTTC GCGCTCGAGTTAATACATCTCATGTACTTGTAAATCA 3'XhoI-525-PfPH2-R TCC

-restriction sites underlined

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Appendix figure legends Fig. S1: Purification of the recombinant GST fusion of the PH domain of PF3D7_1337700. Coomassie stained gel showing soluble GFP-PH fusion protein at the expected size of around 40 kDa. The lower 26 kDa band likely represents a degradation product of GST alone.

Fig. S2: Generation of the PfPH2-2xFKBP-GFP parasite line. A) Schematic showing the tagging strategy by single cross-over recombination using SLI. B) PCR on parasite genomic DNA showing the proper integration of the tagging vector at (5’ junction: primers P1 and P3, 3’junction: primers P2 and P4) and the disappearance of the WT allele (primers P1 and P4) in all 3 SLI attempts. C) Time course of expression of parasite protein extracts taken throughout the erythrocytic cycle shows that PfPH2 is expressed in schizonts. PfHSP70 is used as a control for a constitutive protein. Cartoon shows the time points in hours post invasion (hpi). 1: 8 hpi, 2: 30 hpi, 3: 36 hpi, 4: 42 hpi.

Fig. S3: IFA showing that the PfPH2 knock sideways does not noticeably impact the appearance of various subcellular structures by IFA. A) PfRAP1: rhoptry bulb. B) PfRON4: rhoptry neck. C) PfRESA: dense granules. D) PfAMA1: egress-related micronemes E) PfEBA175: invasion-related micronemes. F) PfMSP1: Merozoite surface. G) PfGAP45: Inner membrane complex. Scale bar represents 5m. Blue: DAPI stained nucleus.

Fig. S4: Bioreplicates for the egress assay, related to figure 3C.

Fig. S5: Schematic explaining the attachment-invasion assay related to figure 3E. In normal conditions (-Rapa, -CytD), the molecular motor is functional, the merozoites invade the erythrocytes and become rings. Counting the number of rings in -Rapa vs +Rapa shows the decrease in invasion (i.e. less rings are formed) in the PfPH2 KS. When adding the actin polymerization inhibitor cytochalasin D, the merozoites attach to the surface of the red blood cell (red arrows) but since the molecular motor is not functional, they cannot proceed

143 to invasion. This allows the uncoupling of the attachment and invasion steps. Counting the number of merozoites bound to the surface of red blood cells in -Rapa vs +Rapa, both in the presence of CytD, shows the decrease in merozoite attachment (i.e. less merozoites are found associated with the red blood cell surface) in the PfPH2 KS. Rapa: Rapamycin. CytD: Cytochalasin D.

Fig. S6: Bioreplicates for the microneme secretion assay, related to figure 4B.

Fig. S7: Invasion assays using enzyme-treated erythrocytes show that host cell tropism of the PfPH2 KS line is not affected. Data expressed as the % of invasion compared to the control without enzyme treatment. T= trypsin. C= chymotrypsin. NM= neuraminidase. n= 3 biological replicates with at least 3 technical replicates for each condition. n.s; non- significant in a multiple t-test.

Figure S8. Alignments of the KXn(K/R)XR binding motif in Apicomplexan orthologues of PfPH2. Alignments of a portion of the PH domains from PF3D7_1337700 identified orthologues in Apicomplexa are shown. The box outlines the alignments of the sequence motif from PF3D7_1337700 with similar amino acid positions of the other proteins. Notably the first and terminal positions of the KXn(K/R)XR motif show conservation.

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Appendix Fig. S7, related to Fig. 4C

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General Discussion, Conclusion and Perspectives The apical organelles are a set of secretory organelles that contain proteins whose function is necessary for invasion of the red blood cell by the parasite [445]. Therefore, a disturbance in their secretion results in egress or invasion defects. Due to the uniqueness of these organelles and their essential role in parasite survival, a better understanding of the mechanisms behind the biogenesis and transport of proteins to these organelles are necessary since identification of the molecular players has the potential to be used for therapeutic purposes and vaccine design strategies. The Plasmodium genome contains homologues of many coat, adaptor and regulatory proteins that are involved in vesicle traffic and protein transport. There are also Plasmodium specific proteins which might play a role in the protein transport machinery that is specific to the parasite. However, the protein transport machinery and biogenesis of these organelles are poorly understood. PIPs have been shown to play important roles in the Plasmodium life cycle. Recent findings have revealed the role of PIPs in gametocyte activation [353], merozoite egress [45, 229], resistance to artemisinin [262], cytokinesis [145], and male gametocyte exflagellation [350, 351], most of which were related to the hydrolysis of PI(4,5)P2. As mentioned earlier, in P. falciparum, a model of shuttling cargo molecules has been proposed by Richard et al. that posits the existence of specific microdomains in the Golgi membrane [359]. Based on the model, proteins destined to the PM or apical organelles accumulate into distinct microdomains with specific lipid compositions. Then, specific escorters that are exposed to the cytosolic face of microdomains recruit coat and adaptor proteins to form a shuttle vesicle. Our lab has also shown a rhoptry protein, RAMA, interacts with another rhoptry protein RAP1 to escort specifically to the rhoptry destined vesicles. However, RAMA does not have a cytosolic domain that recruits vesicle components to the sub-domain. Subsequently, recent work from our lab has shown that the more general sorting escorter is a homologue of Sortiline. Inside the Golgi lumen, PfSortiline interacts either directly or indirectly with the receptor of the protein cargo inside the Golgi lumen to induce vesicle budding [453, 454]. Interestingly, the conditional knockdown of PfSortiline leads to disturbed protein transport that disrupts the biogenesis of apical organelles and the IMC. PfSortiline has been shown to bind to RAMA directly in the case of rhoptry proteins and

153 also to be involved in sorting of micronemes and dense granule and potentially IMC proteins [455]. In the cytosolic face of eukaryotic membranes apart from the cytosolic domain of Sortiline, PIPs also act as a recruiting signal for the vesicle components. Many PIP effectors either have a PIP-binding or kinase domain, most of which are not characterized in P. falciparum. In addition, several of the parasite’s putative PIP effectors don't show any homology to known proteins in the vesicle traffic machinery of the other organisms other than the predicted PIP-binding domain. Therefore, some functions of PIPs and their effector molecules might be specific to Plasmodium given the existence of unique cellular compartments, specifically in the case of apical organelles. The goal of my work was to generate a map of the distribution of PIPs inside the parasite and to identify PIP interacting partners implicated in the biogenesis of the invasion organelles or their secretion during the process of invasion.

We hypothesized that phosphoinositides are part of the protein transport machinery in P. falciparum and create a distinct membrane identity in different subcellular membranes. The results obtained from the PIP mapping in P. falciparum will be discussed in the section 5. 1. For my second project, I have characterized the protein of PF3D7_1337700 which is an apicomplexan specific gene with unknown function. Apart from a PH domain there is no other known domains, therefore, based on this and the fact that the protein is the second studied Plasmodium protein with a PH domain we renamed it to PfPH2[251]. As PfPH2 has a PIP-binding domain we proposed that it might be involved in the membrane transport or PIP-signaling. At the end of this work, we will show that PfPH2 is an essential gene that is important for the exocytosis of a specific population of micronemes. This will be discussed in the section 5. 2.

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A map of subcellular distribution of phosphoinositides in P. falciparum PI3P distribution In eukaryotic cells, PI3P localizes on early endosome membranes and is involved in endocytosis and vesicular trafficking toward lysosomes [156]. In P. falciparum, our results confirmed what has been previously reported by Tawk et al. [160] and showed PI3P localizes to the food vacuole membrane, vesicles at or the vicinity of the food vacuole and the PM, and the apicoplast. However, we did not detect any trace of PI3P at the ER vicinity despite the recent evidence of PI3P-enriched regions at the ER [262]. This could be explained by this fact that the PX domain that we used here as PI3P binding reporter for some reason cannot detect the PI3P pools at the ER or it is possible that the level of PI3P is so low that is out of the range of our detection tool. In yeast, PI3P production is mostly the result of the kinase Vsp34 and the lipid phosphatases Fig4 (also known as Sac3), Sjl2/Inp52, Sjl3/Inp53 and Sac1[238]. Lipid phosphatases all possess a SAC catalytic domain able to dephosphorylate PtdIns(3,5)P2 in PtdIns3P. So far the only homologue identified is a PfSAC1 phosphoinositide-phosphatase recently characterized in our lab [456]. Although, its phosphatase activity is unknown, it localizes to the ER and Golgi where it might be involved in the synthesis of PI3P pool at the ER. The lipid counts for 30% of total monophosphorylated PIPs in the parasite. The unusually high level of the lipid suggests that it might have critical functions for the erythrocytic cycle especially during late stages of parasite development [160]. A recent example of a such role has been revealed recently in the resistance mechanism to ARTs [262]. It has been shown that parasite strains resistant to ARTs have increased levels of PI3P. This might be related to the role of PI3P/Vsp34 in response to the drug stress as in yeast, the deletion of the VPS34 gene results in reduced resistance to numerous stress factors (temperature, pH and some drugs) [182]. So far, evidence of a role for PI3P has been found in important parasite pathways such as hemoglobin transport [240, 406, 407], autophagy [197, 264], apicoplast homeostasis [160, 162], and apicoplast inheritance [190, 197]. Moreover, PI3P/Vsp34 might use the apicoplast as the pre-phagosome station [190, 197] as many homologues of ATG proteins (autophagy proteins) are present in apicomplexans [264]. One of such homologues is PfATG18 which has been identified in both P. falciparum and T. gondii.

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PfATG binds to PI3P (and PI(3,5)P2) in lipid dot blot and liposome binding assays [197]. Although there is no evidence for the existence of PI(3,5)P2 so far in P. falciparum. However, conditional ablation of TgVsp34 expression in T. gondii [162] and the depletion of the only TgPIKfyve both result in apicoplast loss and delay death, suggesting PI3P might be used as a substrate to produce PI(3,5)P2. Because of the critical importance of PI3P in the parasite life cycle, targeting the enzyme responsible for PI3P production could be an interesting antimalarial strategy.

PI4P distribution The IFA results obtained from the parasite expressing a PI4P-tracker in our laboratory showed PI4P localizes to the Golgi apparatus and the PM during all life stages of the parasite. Upon addition of imidazopyrazines to the parasites expressing a different PI4P- tracker, McNamara et al. observed a redistribution of the PI4P signal from an internal foci to the parasite PM [145]. The affected parasites eventually showed a cytokinesis defect and perturbed membrane ingression around the developing merozoite which resulted in drastically decrease in the parasitemia in the next cycle. Similar to other systems [156], disturbance in PI4P leads to a major defect in cytokinesis. Plasmodium has three putative PI4kinases and McNamara et al. demonstrated that the target enzyme of imidazopyrazines and quinoxalines is the PI4KIIIβ homologue. Their microscopy results also revealed that PfPI4KIIIβ localizes through the cytosol and apical ends of nascent merozoites during trophozoite and late schizont stage, respectively. Therefore, we speculate that PI4P-residing Golgi is the result of PfPI4KIIIβ activity and that the lipid likely then delivered to the PM via vesicular traffic. Furthermore, IPZ molecules like KDU691, GNF179, and BQR695 inhibit parasite-derived PfPI4KIIIβ in a very low nanomolar activity compared to any human lipid kinase which is an advantage to use as a drug against the parasite [145]. These results also reveal that the PI4K target of KDU691 is different from KAF156 (or its analog GNF179) [283]. Since KDU691 is potent against ring stage and can only kill dormant rings while KAF156 inhibits ring stage parasite in addition to schizont and trophozoite stage. Having different PI4K inhibitors is an advantage to study parasite PI4K function. As for the role of PI3K/kelch13 in the ART resistance mechanism, PI4K target of GNF179, might

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play a regulatory function in the control or reactivation of the quiescent rings through regulation of PIP metabolism.

PI5P distribution Similar to PI3P, the lipid is present in many compartments throughout the cell, including nucleus, PM, Golgi, ER, and endosomes [437]. The available data on its role is mostly obtained from the nuclear pool which is involved in various processes such as protection against UV stress, apoptosis, transcriptional control and the cell cycle [437]. PI5P lipid has not been reported so far in Plasmodium, however, this could be simply due to limitations in current technologies being used to detect PIPs [163]. As to our findings with a PI5P-tracker we demonstrated, for the first time, pools of PI5P in the PM throughout the erythrocytic cycle, potentially transitional ER during schizont stage and sometimes in the nucleus. PI5P synthesis in mammalian cells is the result of the interplay of PIKfyve activity on PtdIns and 3-phosphatase activity on PI(3,5)P2 for which the first is the main route [211]. Nevertheless, the only detected PIKfyve-like orthologue does not contain a FYVE domain and there is no evidence of its 5-kinase activity, and as mentioned earlier PI(3,5)P2 is also not detected in Plasmodium. Moreover, there is no evidence of the substrate activity of four annotated phosphatases. Assuming the presence of PI5P in Plasmodium, it is therefore tempting to consider that PIKfyve-like orthologue can produce PI5P directly via phosphorylation of PtdIns. PI5P in P. falciparum might act as a second messenger in different signaling pathways such as regulation of cell cycle in nucleus and actin remodeling in the PM in response to parasite stimuli during different stages.

PI(4,5)P2 distribution PI(4,5)P2 is the most abundant PIP in mammals and yeast [156]. Although, PI(4,5)P2 is mostly in the PM there are small pools in the Golgi, ER and endosomes. Most of its functions are related to cell surface related actions. Our observation with the PI(4,5)P2 tracker showed the lipid localizes to the parasite PM during all the erythrocytic cycle showing no variation in its localization. This might be the result of its structural nature that is needed continuously and synthesized and consumed in the same time. We have also

157 observed lipid localization to large vesicular-structures on the parasite PM. Since PI(4,5)P2 is mediating endocytosis events in other systems, these structures might be involved in endocytosis and possibly the hemoglobin ingestion [458]. A small pool of PI(4,5)P2 was also detected close to the nucleus in early stages of the parasites. Whether there is a relation between this PI(4,5)P2 pool and a PH domain-containing calcium-dependent protein kinase (PfCDPK7) that localizes to vesicles in close proximity to the parasite ER is left to be investigated [450]. PfCDPK7 was shown to specifically bind to PI(4,5)P2. In P. falciparum, there are several PH domain/ENTH-containing protein and two PX/PDZ- containing proteins but there is no evidence so far of their ability for PIP-binding. Most of the evidence for the role of PI(4,5)P2 in P. falciparum comes primarily from studies using inhibitors of the single PfPI-PLC enzyme [159] which is implicated in many essential processes such as gametocyte activation [351, 353], ookinete motility [227], synchronization of the erythrocytic cycle [449], schizont egress [229] and invasion of merozoites [391]. However, a role for PfPI-PLC in merozoite invasion was later argued [459].

PI(3,4)P2 and PI(3,4,5)P3 distribution In eukaryotes, PI(3,4)P2 and PI(3,4,5)P3 are synthesized in response to cell stimuli and localize to the PM [156]. Very low amounts of these two lipids have been detected in the infected RBCs [160] though our attempts to localize PI(3,4)P2 and PI(3,4,5)P3 in P. falciparum were not successful. In our hands, only a diffused cytosolic signal was visualized like what we observed with GFP- or mCherry-alone all over the erythrocytic cycle. In the case of PI(3,4,5)P3, it was even rare to find parasites that expressed the reporter protein. It is likely that these lipids need special stimuli which might not have been present in our in vitro experiments in contrast to the stable signal that we observe from the structural lipids such as PI3P, PI4P and PI(4,5)P2. Such signals could be required for gametocytogenesis for example. It is also possible that the signals were so quick that we were not able to capture the time of their actions. Looking at parasite development using live-cell imaging could potentially reveal transient PI(3,4)P2 and PI(3,4,5)P3 containing structures. Another possibility would be to use different PI(3,4)P2- and PI(3,4,5)P3-binding domains that might help to visualize these lipids.

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Conclusion on the subcellular PIP distribution and general pitfalls Although PIP tracker expressing parasite lines were generally stable and showed no difficulty in their expression, the PIP tracker signal easily disappeared after processing the samples such as centrifugation steps which made microscopy studies difficult. To avoid this problem we used methanol-fixation for IFAs, which does not require centrifugation step. Also, double-transfectants with Plasmodium organellar-markers were in general a rare occasion and in the few successful ones, the parasite did not look healthy. Surprisingly, PIP distribution in P. falciparum is a reflection of what has been observed with other systems. As P. falciparum has unusual organelles like apicoplast and invasion organelles we expected to record some differences compared to model organisms like yeast and human (Fig. 3. 6). We were interested in determining whether invasion organelles were enriched in specific PIPs however none of the probes we have used labelled these structures. A different strategy such as BioID might help to better clarify the PIP composition of the parasite’s various subcellular features. BioID is based on the fusion of the promiscuous biotin ligase BirA to a protein of interest (PIP-binding domains in our case). Following its expression in cells, addition of biotin in the culture medium results in the biotinylation of proteins proximal to the BirA fusion. These can then be identified by proteomics. Identification of proteins from a particular organelle when using specific PIP- binding domain-BirA probe could mean that a particular PIP species is enriched. This strategy could also potentially allow the identification of new interactors of PIPs in P. falciparum.

A pan-apicomplexan phosphoinositide-binding protein acts in malarial invasion-microneme exocytosis. PfPH2 is a PH-containing protein with a relaxed PIP-binding specificity. The aim of my second project was to characterize a PH-containing protein, namely PfPH2 (PF3D7_1337700). It is a hypothetical protein conserved throughout apicomplexans. It does not have a transmembrane domain or a known secretion signal, but it does contain a basic sequence motif (KXn (K/R)XR), responsible for binding to the inositol head group

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[460]. Here, we showed that the recombinant PH domain of PfPH2 alone is able to interact with different PIP species both by lipid blot assays and liposome binding assays. The results of liposome binding assay seems reliable, as PIP strip cannot mimic membrane bilayer and showed stronger interaction with PI(4,5)P2 and monophosphate species and some residual interaction with PI(3,4,5)P3 and PA containing vesicles. We observed a clear loss of binding ability of a triple mutant version of the basic motif where residues K84, K92 and R94 were mutated to alanines, suggesting the role of the basic sequence motif (KXn (K/R)XR) in binding to the inositol head group[460]. Moreover, comparison of binding affinity of the wild type version and its mutant, incubated with PI3P-containing lipid vesicles reaffirmed our hypothesis. All together, our results demonstrate that PF3D7\1337700 is a genuine PIP-binding protein. A relaxed PIP-binding ability is known for a majority of PH domains [461]. For a specific PIP-interaction, the PH domain of PfPH2 might need auxiliary domains that are hidden in 3D-conformation of native protein or the presence of partner proteins which is referred as coincidence detection to lead to a specific subcellular distribution. To elucidate more its PIP-binding ability, it would be necessary to study the full-length protein with and without the PH domain as well. In addition, it would be interesting to perform pull-down assays using the full-length protein/the PH domain alone. Analysing these protein interactions might lead us to the PfPH2 potential partners and elucidating its molecular mechanism.

PfPH2 localizes to a structure close to the apical tip of the merozoite To analyze the localization of PfPH2, we tagged the protein at its C-ter by 2xFKBP-GFP and detected a band of ~133 kDa consistent with the tagged-protein size at the late schizont parasites. Fluorescence microscopy of 2xFKBP-GFP-tagged PfPH2 parasite line showed a punctate pattern of GFP signal in the late schizont, reminiscent of apical organelles pattern. Interestingly, IFA attempts revealed no colocalization of PfPH2-2xFKBP-GFP signal with apical organelles or any organellar markers tested. However, the PfPH2 signal is always farther from the DAPI than PfEBA175 and potentially more than PfRON4, though the difference with PfRON4 was not statistically significant.. To clarify the exact localization of PfPH2 we need to produce a specific PfPH2 antibody. A PfPH2-specific antibody will

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be very useful to reassert the IFAs with untagged parasite line and also perform immuno- electron microscopy (IEM) experiments.

PfPH2 is essential for the erythrocytic cycle and its absence affects merozoite invasion due to a default in microneme exocytosis. To investigate the function of PfPH2 we performed knock-sideways (KS) analysis on PfPH2-2xFKBPGFP line that allows dislocating the protein from its site of action and mistargeting it to the nucleus in presence of mTOR inhibitor, rapamycin. The proliferation of rapamycin-treated parasites, the KS line, showed around 65% decrease in the first cycle and up to 86% in the second cycle compared to the control line. Therefore, we observed few rings formed in the next cycle compared to the control line. At the same time, all our three knockout attempts failed, suggesting an essential role for the protein. Consequently, we analyzed different cellular processes including organelle formation, nuclear division, egress, and invasion to find out what stage of life cycle has been impaired. The analysis of the IFAs and egress assay proved that the absence of PfPH2 does not affect the events before egress such as nuclear division, merozoite formation, organellar integrity and egress itself but potentially affects the merozoite invasion. The invasion process is a rapid and timely controlled process during which invasion organelles secrete their contents in orderly steps. Invasion can be divided in the attachment of the merozoite and entry into the erythrocyte. Using an actin inhibitor we were able to stop merozoites at the entry and study their attachment which revealed a significant decrease in the attachment of the KS line. This observation suggests lack of PfPH2 from its site of action affects merozoite attachment to the erythrocyte which is necessary for entry. A micronemal protein (AMA1) and the RON complex are responsible for a tight junction that along with a motor-myosin mediate the parasite entry. In absence of PfPH2, the normal translocation of AMA1 to the merozoite surface confirmed that the entry process was not affected. Merozoites engage in a stable attachment with the erythrocyte membrane using two different routes regarded as the alternative pathways. The EBAs and RHs family proteins, which mediate the attachment would be held in reserve in micronemes and rhoptries respectively, until they are needed. However, during the invasion, their secretion is triggered to the merozoite surface leading to the interactions with their receptors on the

161 erythrocyte membrane after which they are cleaved and shed into culture supernatant via parasite proteases during invasion. It has been shown that some of the invasion-receptor interactions like Rh1 are related to Ca2+-dependent release of microneme proteins specifically EBA175 [85].The order of secretion of each set of ligands is critical and well- documented during invasion in P. falciparum. AMA1 is among the first set of invasion ligands, secreting into merozoite surface and its secretion appeared normal in both control and KS line, based on the IFAs results. To further investigate their secretion, we performed western blot on the collected supernatant of the KS and the control line cultures and detect their presence via antibodies specific to each ligand. The results showed significant decrease in shedding of EBA ligands into the supernatant of KS line (EBA-175~90% and EBA-140 ~79%) indicating a defect in secretion of microneme proteins. Micronemes release their content upon receiving a signal. It has been shown that the trigger is a change in the level of intracellular Ca2+, which activates a cascade of events that lead to microneme discharge via fusion of microneme membrane with the parasite PM. PfPH2 function appear to be necessary for microneme discharge but the exact mechanism of action of this protein in this process still need to be elucidated. AMA1, also showed decrease but with a lesser degree (around 24%) suggesting that, secretion of microneme proteins is generally affected but at different degrees. Moreover, we observed the decrease of EBA175 in the supernatant correlated with an increase in the unprocessed form in free merozoites of KS line. Notably, there was no increase in the unprocessed AMA1 pointing to a defect in EBA175 secretion alone. The discrepancy in exocytosis of the contents of same organelle can't be explained unless we assume there are different populations of micronemes. Recently, some evidence arose about the presence of at least two different populations of micronemes and AMA1 has been suggested to be related to the egress-related micronemes [82]. Here, our results also suggest the possibility of two populations of micronemes since we observed a significant decrease in EBAs ligands but not in AMA1. The 24% decrease in AMA1 shedding can be also explained by the fact that in the KS line, a low amount of successful invasion events happens compared to the control line leading to less shedding of AMA1. Therefor we suggest the possibility of a new group of micronemes that are invasion-related and contains EBA family proteins which display a defect in their exocytosis due to the absence of PfPH2. Lately, it has been shown inhibition of a PM

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resident protein, PfCDPK1, in the young and mature merozoites results in microneme discharge arrest [462]. PfCDPK1 is a calcium-dependent protein kinase and specifically involved in the EBA-175 secretion [84, 463]. After a rise in merozoite intracellular Ca2+, the kinase starts a cascade of phosphorylation that leads to invasion. Hence, we speculate PfPH2 might be a substrate of PfCDPK1 which is regulated through phosphorylation. This could be determined through use of phospho-specific antibody or by immunoprecitation techniques and looking into the phosphorylated peptides

EBA family proteins bind to a specific group of erythrocyte receptors, which has sialic acid (SA) branches in their structure and are sensitive to neuraminidase treatments. They differ from RH-receptors that are resistant to such treatments. Plasmodium species have different preferences in choosing the erythrocyte receptors during the merozoite invasion of RBCs [73-75]. Therefore, EBAs and RHs are referred as alternative pathway ligands or SA- dependent and SA-independent pathway ligands, respectively. For example, the deletion of EBA-175 in W2mef parasite strain results in the upregulation of PfRh4 which indicates the functional substitution of EBA-175 [74]. The parasite can epigenetically silence or upregulate particular invasion-related genes, such as PfRh4 [74, 76, 77], resulting in divergent ligand expression between isolates. Due to this plasticity, the parasite is able to rapidly adjust to erythrocyte receptor polymorphisms in human populations [76, 78, 79]. Considering the severe decrease in EBA-175 and EBA-140 level, we anticipated that if we treat erythrocytes with neuramidase enzymes to remove sialic-acid groups, the drastic diminution of EBAs due to absence of PfPH2 in KS line should affect the host cell tropism. However, the invasion assays performed on different enzyme treated erythrocytes showed no difference in the sensitivity to either trypsin, chymotrypsin or neuraminidase treatment between the control and KS line. This suggests that absence of PfPH2 leads to a more general invasion defect. Even, the invasion rate decrease in KS line was generally higher than control line and in some cases this was significant. This indicates that the effect of PfPH2 mislocalization is more severe than removing sialic acid groups from erythrocyte receptors. This could be potentially explained by considering overlap of alternative pathway ligands in function and cooperating together during invasion as it has been shown, previously [71].

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Conclusions on PfPH2 mechanism of action and future experiments The exact function of PfPH2 on microneme exocytosis is not clear yet. A recent work has shown a PH-containing domain in the related apicomplexan parasite T. gondii, TgAPH, is involved in phosphatidic acid (PA) sensing during microneme exocytosis events. Since PfPH2 contain PH domain and showed PIP-binding ability, it is tempting to speculate that PfPH2, like TgAPH, is involved in the fusion of invasion-related microneme membranes with the PM. An interesting candidate that might interact with PfPH2 is a calcium-activated snare-like protein named PfDOC2.1 which has a role in the fusion events. It has been shown PfDOC2.1 knockdown impairs merozoite invasion and blocks microneme secretion. Importantly it is specifically required for Ca2+-dependent release of EBA175 from the micronemes as well as PfCDPK1 and Rh1 [357]. Consequently, PfCDPK1 might be implicated in regulation of PfPH2 likely via its kinase activity. Also, Rh1 interaction could be a common point in a complex signalling network that might connect the alternative pathways and PfCDPK1 by triggering Ca+ signal after its interaction. Whether PfPH2 is a substrate to PfCDPK1 and would be phosphorylated during the activation process needs to be further investigated. Based on our results and what is discussed on the potential roles of other factors, we propose a model for differential exocytosis of the micronemes. During late schizont stage, PA sensing of P. falciparum homologue of TgAPH in the merozoite apical tips potentially leads to the liberation of AMA1 from egress-related micronemes. Later, upon egress and exposure of the merozoites to the extracellular environment, a signaling cascade starts through PfCDPK1 by phosphorylation of its substrates that results in the secretion of EBA- containing invasion micronemes. One of the PfCDPK1 substrates is potentially the PfPH2 protein, which might be involved in the exocytosis of invasion-related micronemes through interaction with an unspecified PIP species. In this model, absence of PfPH2 leads to a deficient attachment of merozoites to the erythrocyte membrane that leads to a drastic decrease in the invasion rate. To elucidate the exact function of PfPH2, it is necessary to identify its interacting partners. Also, whether PfPH2 is one of the substrates of PfCDPK1 may clarify the cascade of

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events that control the microneme exocytosis. Moreover, solubility assays can provide some information on the protein position in vivo. If the protein is membrane bound it could suggest it interacts with the membranes through the PIPs or other phosphorylated forms most likely via its PH domain. As the growth defect is a more intense defect compared to what we see with SA-dependent ligands, it would be recommended to analyze secretion of other invasion ligands. Finally, in a broader view, the function of PfPH2 in P. falciparum might be a common role in the exocytosis of micronemes and might help to better understand the invasion mechanism in all apicomplexans.

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