DOCSLIB.ORG

Comparative Functional Analysis of Secreted Autotransporter Toxin (Sat) in Uropathogenic E

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Comparative Functional Analysis of Secreted Autotransporter Toxin (Sat) in Uropathogenic E Comparative Functional Analysis of Secreted Autotransporter Toxin (Sat) in Uropathogenic E. coli CFT073 and probiotic E.coli Nissle 1917 in mammalian cells A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Taylor Michelle Halsey 2018 SIGNATURE PAGE THESIS: COMPARATIVE FUNCTIONAL ANALYSIS OF SECRETED AUTOTRANSPORTER TOXIN (SAT) IN UROPATHOGENIC E. COLI CFT073 AND PROBIOTIC E. COLI NISSLE 1917 IN MAMMALIAN CELLS AUTHOR: Taylor Michelle Halsey DATE SUBMITTED: Fall 2018 Department of Biological Sciences Dr. Christos Stathopoulos __________________________________________ ___ Thesis Committee Chair Biological Sciences Dr. Nancy Buckley __________________________________________ ___ Biological Sciences Dr. Junjun Liu ___________________________________________ __ Biological Sciences ii Acknowledgements I would like to sincerely thank my entire support system for walking with me through this journey. To my support system at Cal Poly Pomona, a place that I have called a home for the past eight years, I say thank you for your collective belief in my academic process and for giving me an education that will allow for so many opportunities in the future; the faculty and staff at Cal Poly Pomona have provided me with a tremendous gift and I promise to use this gift to continue to help others. To the faculty that have helped me the most, including members of my thesis committee, Drs. Buckley, Liu, and Stathopoulos (thesis committee chair), as well as the coordinators for the NIH-RISE program Drs. Adler and Valdes, thank you all for investing your precious time in me as a student and a researcher; your guidance has shaped my path and I hope to be able to inspire a new generation of scientists just as you all have inspired me. Finally, to my family and friends, you each hold a different piece of my heart and I know I could not have gotten through this program without all of your love and support. Through all of the long days and nights in the lab and all of the times I thought I could not complete this task, it was you that made it easier and allowed me to keep my goals in sight; this great project could have never been completed without all of you. iii Abstract Autotransporter proteins, typically associated with virulence in pathogenic bacteria, are thought to serve a different function in probiotic bacteria. One such protein, secreted autotransporter toxin (Sat), has been shown in numerous pathogenic bacteria to cause vacuolization and cytotoxic effects on cells. Sat is also secreted in high amounts by the non-pathogenic, probiotic Escherichia coli Nissle 1917 (EcN). The use of EcN is under review as a candidate probiotic supplement in the United States and approval has been delayed in part because of genomic similarities to uropathogenic E. coli (UPEC) CFT073, which is responsible for approximately 70% of non-hospital acquired urinary tract infections nationwide. Previous generalized studies have indicated that when Sat functions collaboratively with other native proteins in EcN, its cytotoxic effects are negated, but the mechanisms behind these actions are unknown. To independently study the effects of Sat, we used a recombinant sat overexpression plasmid derived from UPEC CFT073 (pSat) transformed into the non-pathogenic E. coli strain HB101 (HB101 pSat). Concentrated cell-free supernatant was obtained from wildtype EcN and wildtype UPEC CFT073, as well as EcNDSat for assays and an SDS-PAGE and Bradford protein concentration assay were performed to confirm the expression, concentration, and secretion profiles. In vitro infection challenges using the HeLa and Vero cell lines were performed and analyzed using the MTT cytotoxicity assay. Our preliminary results showed that Sat of UPEC CFT073 decreased viability of undifferentiated epithelial cells when subjected to challenges with concentrated cell-free supernatant. When the same challenges were given to Vero cells, a greater level of toxicity was conferred on the differentiated epithelial cell line. Cytotoxic effects in Vero cells were amplified following crude purification of cell- iv free supernatant samples using ammonium sulfate precipitation. These results suggest that Sat of UPEC CFT073 confers damage to host epithelial cells and may have greater effects on relevant differentiated cell lines. It is also suggested that purified Sat protein from both EcN and UPEC CFT073 confer similar levels of cytotoxicity to Vero cells and reduce viability compared to the concentrated supernatant samples alone. Further experiments are needed to determine the mechanisms of action behind these results and target the specific factors that prevent Sat of EcN from displaying cytotoxic features in host cells. v TABLE OF CONTENTS SIGNATURE PAGE .......................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii ABSTRACT ....................................................................................................................... iv INTRODUCTION ............................................................................................................ 1 1. Escherichia coli ............................................................................................................ 1 2. E. coli Nissle 1917 ........................................................................................................ 4 3. Probiotic Bacteria ....................................................................................................... 10 4. Uropathogenic E. coli strain CFT073 ......................................................................... 13 5. Similarities between EcN and UPEC CFT073 ........................................................... 15 6. Bacterial Toxins ......................................................................................................... 16 7. Gram-negative Bacteria Secretion Mechanisms ........................................................ 19 8. Type V (Autotransporter) Protein Secretion .............................................................. 21 9. Adhesin Involved in Diffuse Adherence (AIDA-I) and Trimeric Autotransporter Adhesins (TAA) ......................................................................................................... 28 10. Serine Protease Autotransporters of Enterobactericiaea (SPATE)...……......…........29 11. Secreted Autotransporter Toxin (Sat) ......................................................................... 31 vi 12. In vitro Cell Models .................................................................................................... 35 13. Previous Stathopoulos Lab Findings .......................................................................... 36 14. Thesis Scope and Focus .............................................................................................. 40 MATERIALS AND METHODS ................................................................................... 41 1. Bacterial Strains and Plasmids ................................................................................. 41 a) Bacteria Media Preparation ................................................................................ 41 b) Preparations of Bacterial Overnight Cultures .................................................... 42 c) Preparations of Bacterial Day Cultures .............................................................. 42 2. Bacterial Protein Work ............................................................................................. 42 a) Supernatant Isolation and Concentration ........................................................... 42 b) Isolation and Partial Purification of Secreted Autotransporter Toxin ................ 43 c) Acetone Protein Precipitation ............................................................................. 43 d) SDS-PAGE/Silver Stain Analysis ...................................................................... 44 3. Cell Lines Used in Study .......................................................................................... 47 a) Cell Culture Media Preparation .......................................................................... 47 4. Oligopeptide Cleavage Assay .................................................................................. 48 5. MTT Cytotoxicity Assay .......................................................................................... 48 6. Data Analysis and Statistics ..................................................................................... 49 RESULTS ........................................................................................................................ 50 1. Concentration of Sat protein from select strains using Filtered Centrifugation ....... 50 2. Effects of concentrated Sat of UPEC CFT073 on cell viability of HeLa cell line ... 54 vii 3. Concentrated Sat of UPEC CFT073 decreases cell viability in Vero cell line ........ 60 4. Supernatants of Wildtype EcN and Wildtype UPEC CFT073 and their effects Vero cell line viability in vitro .......................................................................................... 63 5. Partially purified Sat of UPEC CFT073 effects on Vero cell line from recombinant HB101/pSat .............................................................................................................
Load more

Recommended publications
  • Autodisplay: One-Component System for Efficient Surface Display And
    JOURNAL OF BACTERIOLOGY, Feb. 1997, p. 794–804 Vol. 179, No. 3 0021-9193/97/$04.0010 Copyright q 1997, American Society for Microbiology Autodisplay: One-Component System for Efficient Surface Display and Release of Soluble Recombinant Proteins from Escherichia coli 1 2 1,2 JOCHEN MAURER, JOACHIM JOSE, AND THOMAS F. MEYER * Downloaded from Abteilung Infektionsbiologie, Max-Planck-Institut fu¨r Biologie, 72076 Tu¨bingen,1 and Abteilung Molekulare Biologie, Max-Planck-Institut fu¨r Infektionsbiologie, 10117 Berlin,2 Germany Received 26 August 1996/Accepted 24 September 1996 The immunoglobulin A protease family of secreted proteins are derived from self-translocating polypro- tein precursors which contain C-terminal domains promoting the translocation of the N-terminally attached passenger domains across gram-negative bacterial outer membranes. Computer predictions identified the C-terminal domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) as a member of the autotransporter family. A model of the b-barrel structure, proposed to be responsible for http://jb.asm.org/ outer membrane translocation, served as a basis for the construction of fusion proteins containing heterologous passengers. Autotransporter-mediated surface display (autodisplay) was investigated for the cholera toxin B subunit and the peptide antigen tag PEYFK. Up to 5% of total cellular protein was detectable in the outer membrane as passenger autotransporter fusion protein synthesized under control of the constitutive PTK promoter. Efficient presentation of the passenger domains was demonstrated in the outer membrane protease T-deficient (ompT) strain E. coli UT5600 and the ompT dsbA double mutant JK321. Surface exposure was ascertained by enzyme-linked immunosorbent assay, immunofluorescence microscopy, and immunogold electron microscopy using antisera specific for the passenger domains.
    [Show full text]
  • Protein Secretion Systems in Fusobacterium Nucleatum
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1713 (2005) 92 – 112 http://www.elsevier.com/locate/bba Protein secretion systems in Fusobacterium nucleatum: Genomic identification of Type 4 piliation and complete Type V pathways brings new insight into mechanisms of pathogenesis Mickae¨l Desvauxa,b, Arshad Khana, Scott A. Beatsona, Anthony Scott-Tuckera, Ian R. Hendersona,* aThe Institute for Biomedical Research (IBR), The University of Birmingham-The Medical School, Division of Immunity and Infection, Bacterial Pathogenesis and Genomics Unit, Edgbaston, Birmingham B15 2TT, UK bInstitut National de la Recherche Agronomique (INRA), Centre de Recherches de Clermont-Ferrand-Theix, Unite´ de Recherche 370, Equipe Microbiologie, F-63122 Saint-Gene`s Champanelle, France Received 23 February 2005; received in revised form 11 April 2005; accepted 2 May 2005 Available online 8 June 2005 Abstract Recent genomic analyses of the two sequenced strains F. nucleatum subsp. nucleatum ATCC 25586 and F. nucleatum subsp. vincentii ATCC 49256 suggested that the major protein secretion systems were absent. However, such a paucity of protein secretion systems is incongruous with F. nucleatum pathogenesis. Moreover, the presence of one or more such systems has been described for every other Gram- negative organism sequenced to date. In this investigation, the question of protein secretion in F. nucleatum was revisited. In the current study, the absence in F. nucleatum of a twin-arginine translocation system (TC #2.A.64.), a Type III secretion system (TC #3.A.6.), a Type IV secretion system (TC #3.A.7.) and a chaperone/usher pathway (TC #1.B.11.) was confirmed.
    [Show full text]
  • Supplementary Information
    Supplementary information (a) (b) Figure S1. Resistant (a) and sensitive (b) gene scores plotted against subsystems involved in cell regulation. The small circles represent the individual hits and the large circles represent the mean of each subsystem. Each individual score signifies the mean of 12 trials – three biological and four technical. The p-value was calculated as a two-tailed t-test and significance was determined using the Benjamini-Hochberg procedure; false discovery rate was selected to be 0.1. Plots constructed using Pathway Tools, Omics Dashboard. Figure S2. Connectivity map displaying the predicted functional associations between the silver-resistant gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S3. Connectivity map displaying the predicted functional associations between the silver-sensitive gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S4. Metabolic overview of the pathways in Escherichia coli. The pathways involved in silver-resistance are coloured according to respective normalized score. Each individual score represents the mean of 12 trials – three biological and four technical. Amino acid – upward pointing triangle, carbohydrate – square, proteins – diamond, purines – vertical ellipse, cofactor – downward pointing triangle, tRNA – tee, and other – circle.
    [Show full text]
  • Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family
    GBE Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family Eva Heinz1,2,y, Christopher J. Stubenrauch1,y, Rhys Grinter1,3, Nathan P. Croft4, Anthony W. Purcell4, Richard A. Strugnell5, Gordon Dougan2, and Trevor Lithgow1,* 1Department of Microbiology, Infection & Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, Australia 2 Wellcome Trust Sanger Institute, Hinxton, United Kingdom Downloaded from https://academic.oup.com/gbe/article-abstract/8/6/1690/2574022 by guest on 13 December 2018 3Institute of Microbiology and Infection, School of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom 4Department of Biochemistry and Molecular Biology, Infection & Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, Australia 5Department of Microbiology & Immunology, University of Melbourne, Parkville, Australia *Corresponding author: E-mail: [email protected]. yThese authors contributed equally to this work. Accepted: May 3, 2016 Abstract The bacterial cell surface proteins intimin and invasin are virulence factors that share a common domain structure and bind selectively to host cell receptors in the course of bacterial pathogenesis. The b-barrel domains of intimin and invasin show significant sequence and structural similarities. Conversely, a variety of proteins with sometimes limited sequence similarity have also been annotated as “intimin-like” and “invasin” in genome datasets, while other recent work on apparently unrelated virulence-associated proteins ultimately revealed similarities to intimin and invasin. Here we characterize the sequence and structural relationships across this complex protein family. Surprisingly, intimins and invasins represent a very small minority of the sequence diversity in what has been previously the “intimin/invasin protein family”.
    [Show full text]
  • Autodisplay of Enzymes—Molecular Basis and Perspectives
    Journal of Biotechnology 161 (2012) 92–103 Contents lists available at SciVerse ScienceDirect Journal of Biotechnology j ournal homepage: www.elsevier.com/locate/jbiotec Autodisplay of enzymes—Molecular basis and perspectives a,∗ b a Joachim Jose , Ruth Maria Maas , Mark George Teese a Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany b Autodisplay Biotech GmbH, Merowingerplatz 1a, D-40225 Düsseldorf, Germany a r t i c l e i n f o a b s t r a c t Article history: To display an enzyme on the surface of a living cell is an important step forward towards a broader use of Received 8 October 2011 biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It Received in revised form 14 February 2012 is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the Accepted 4 April 2012 enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells Available online 30 April 2012 can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has some extra features that could overcome limitations in the industrial applications of enzymes. One major Keywords: advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell Autodisplay Biocatalysis surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay Synthesis enzymes with prosthetic groups can be displayed, expanding the application of surface display to the 5 6 Enzymes industrial important P450 enzymes.
    [Show full text]
  • Polarity and Secretion of Shigella Flexneri Icsa: a Classical Autotransporter
    Polarity and Secretion of Shigella flexneri IcsA: A Classical Autotransporter MATTHEW THOMAS DOYLE, B. SC. (BIOTECHNOLOGY) Submitted for the degree of Doctor of Philosophy Department of Molecular and Cellular Biology School of Biological Sciences The University of Adelaide Adelaide, South Australia, Australia July 2015 Declaration I certify that this Thesis contains no material which has been accepted for the award of any other degree or diploma in my name, in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I certify that no part of this work will, in the future, be used in a submission in my name for any other degree or diploma in any university or other tertiary institution without the prior approval of the University of Adelaide and where applicable, any partner institution responsible for the joint- award of this degree. I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968. I acknowledge that copyright of published works contained within this thesis resides with the copyright holders of those works. I give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library Search and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.
    [Show full text]
  • Complete Dissertation
    VU Research Portal Molecular insight into the pathogenetic synergy between E. coli and B. fragilis in secondary peritonitis Sijbrandi, R. 2006 document version Publisher's PDF, also known as Version of record Link to publication in VU Research Portal citation for published version (APA) Sijbrandi, R. (2006). Molecular insight into the pathogenetic synergy between E. coli and B. fragilis in secondary peritonitis. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. E-mail address: [email protected] Download date: 09. Oct. 2021 MOLECULAR INSIGHT INTO THE PATHOGENIC SYNERGY BETWEEN E. COLI AND B. FRAGILIS IN SECONDARY PERITONITIS This thesis has been reviewed by: dr. W. Bitter, VU medisch centrum, Amsterdam dr. K. Bosscha, Jeroen Bosch Ziekenhuis, 's Hertogenbosch dr. E.N.G. Houben, University of Basel, Basel, Switzerland dr. L. Rutten, Universiteit Utrecht, Utrecht prof.dr. J.R.H. Tame, Yokohama City University, Yokohama, Japan dr.
    [Show full text]
  • Folding Control in the Path of Type 5 Secretion
    toxins Review Folding Control in the Path of Type 5 Secretion Nathalie Dautin 1,2 1 Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, Université de Paris, LBPC-PM, CNRS, UMR7099, 75005 Paris, France; [email protected] 2 Institut de Biologie Physico-Chimique, Fondation Edmond de Rothschild pour le Développement de la Recherche Scientifique, 75005 Paris, France Abstract: The type 5 secretion system (T5SS) is one of the more widespread secretion systems in Gram-negative bacteria. Proteins secreted by the T5SS are functionally diverse (toxins, adhesins, enzymes) and include numerous virulence factors. Mechanistically, the T5SS has long been considered the simplest of secretion systems, due to the paucity of proteins required for its functioning. Still, despite more than two decades of study, the exact process by which T5SS substrates attain their final destination and correct conformation is not totally deciphered. Moreover, the recent addition of new sub-families to the T5SS raises additional questions about this secretion mechanism. Central to the understanding of type 5 secretion is the question of protein folding, which needs to be carefully controlled in each of the bacterial cell compartments these proteins cross. Here, the biogenesis of proteins secreted by the Type 5 secretion system is discussed, with a focus on the various factors preventing or promoting protein folding during biogenesis. Keywords: secretion; folding; autotransporter; type 5 secretion system; intimin; invasin; two-partner secretion; trimeric autotransporter Key Contribution: This short review describes the different factors preventing or promoting folding of proteins secreted by the T5SS. Citation: Dautin, N. Folding Control in the Path of Type 5 Secretion.
    [Show full text]
  • The Autotransporter Secretion System Mickael Desvaux, Nicholas Parham, Ian Henderson
    The autotransporter secretion system Mickael Desvaux, Nicholas Parham, Ian Henderson To cite this version: Mickael Desvaux, Nicholas Parham, Ian Henderson. The autotransporter secretion system. Research in Microbiology, Elsevier, 2004, 155 (2), pp.53 - 60. 10.1016/j.resmic.2003.10.002. hal-02910767 HAL Id: hal-02910767 https://hal.inrae.fr/hal-02910767 Submitted on 3 Aug 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Research in Microbiology 155 (2004) 53–60 www.elsevier.com/locate/resmic Mini-review The autotransporter secretion system Mickaël Desvaux, Nicholas J. Parham, Ian R. Henderson ∗ Bacterial Pathogenesis and Genomics Unit, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK Received 9 September 2003; accepted 3 October 2003 First published online 7 October 2003 Abstract The type V secretion system includes the autotransporter family, the two-partner system and the Oca family. The autotransporter secretion process involving first the translocation of the precursor through the inner membrane and then its translocation through the outer membrane via a pore formed by a β-barrel is reviewed. 2003 Elsevier SAS. All rights reserved. Keywords: Autotransporter; Membrane transport protein; Protein translocation; Virulence factors 1.
    [Show full text]
  • Recent Advances in the Understanding of Trimeric Autotransporter Adhesins
    Medical Microbiology and Immunology (2020) 209:233–242 https://doi.org/10.1007/s00430-019-00652-3 REVIEW Recent advances in the understanding of trimeric autotransporter adhesins Andreas R. Kiessling1 · Anchal Malik1 · Adrian Goldman1,2 Received: 12 August 2019 / Accepted: 30 November 2019 / Published online: 21 December 2019 © The Author(s) 2019 Abstract Adhesion is the initial step in the infection process of gram-negative bacteria. It is usually followed by the formation of bioflms that serve as a hub for further spread of the infection. Type V secretion systems engage in this process by binding to components of the extracellular matrix, which is the frst step in the infection process. At the same time they provide protec- tion from the immune system by either binding components of the innate immune system or by establishing a physical layer against aggressors. Trimeric autotransporter adhesins (TAAs) are of particular interest in this family of proteins as they pos- sess a unique structural composition which arises from constraints during translocation. The sequence of individual domains can vary dramatically while the overall structure can be very similar to one another. This patchwork approach allows research- ers to draw conclusions of the underlying function of a specifc domain in a structure-based approach which underscores the importance of solving structures of yet uncharacterized TAAs and their individual domains to estimate the full extent of functions of the protein a priori. Here, we describe recent advances in understanding the translocation process of TAAs and give an overview of structural motifs that are unique to this class of proteins.
    [Show full text]
  • Reevaluating the Fusobacterium Virulence Factor Landscape ​ ​ 1* 1* 1 1 2 Ariana Umana ,​ Blake E
    bioRxiv preprint doi: https://doi.org/10.1101/534297; this version posted January 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Reevaluating the Fusobacterium Virulence Factor Landscape ​ ​ 1* 1* 1 1 2 Ariana Umana ,​ Blake E. Sanders ,​ Chris C. Yoo ,​ Michael A. Casasanta ,​ Barath Udayasuryan ,​ Scott 2 ​ 1,# ​ ​ ​ ​ S. Verbridge ,​ Daniel J. Slade ​ ​ 1 Department​ of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA. 2 ​ Laboratory​ of Integrative Tumor Ecology, and Virginia Tech – Wake Forest School of Biomedical Engineering and Sciences, Blacksburg, VA, USA *These authors contributed equally # To whom correspondence should be addressed: Dr. Daniel J. Slade, Department of Biochemistry, Virginia Polytechnic Institute and ​ State University, Blacksburg, VA 24061. Telephone: +1 (540) 231-2842. Email: [email protected] ​ ________________________________________________________________________________________ ABSTRACT Fusobacterium are Gram-negative, anaerobic, opportunistic pathogens involved in ​ multiple diseases, including the oral pathogen Fusobacterium nucleatum being linked to the ​ progression and severity of colorectal cancer. The global identification of virulence factors in Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium, we report the ​ ​ identification and correction of virulence factors from the Type 5 secreted autotransporter and FadA protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC ​ ​ ​ 23726 and the classic typed strain F. nucleatum subsp.
    [Show full text]
  • Structural and Functional Analysis of Moraxella Catarrhalis Adhesins MCAP and OMPCD
    Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences Structural and Functional Analysis of Moraxella catarrhalis Adhesins MCAP and OMPCD Submitted by: Chritine Akimana In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Examination Committee Major Advisor: Eric Lafontaine, Ph.D. Academic Robert Blumenthal, Ph.D. Advisory Committee: R. Mark Wooten, Ph.D. Ray Larsen, Ph.D. Venkatesha Basrur, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 16, 2007 Structural and Functional Analysis of Moraxella catarrhalis Adhesins McaP and OMPCD Christine Akimana University of Toledo Health Science Campus 2007 DEDICATION This dissertation is dedicated to my son, Bennett Kiza, who has filled my life with untold blessings. I would like to thank my husband, Nicolas Kiza, and my mother-in-law, whose support and encouragement has enabled me to complete this work. I also would like to thank my parents for their love, inspiration, and support, especially for their hard work that has enabled me to achieve so much in life. There are several people, family members, friends, and mentors, whose support and inspiration has helped me throughout my academic years. For those as well, I offer my full gratitude. ii ACKNOWLEDGEMENTS I would like to thank my major advisor Dr. Eric Lafontaine for giving me the opportunity to work on this very exciting project. I thank him for training me, for sharing his expertise, and for providing me with the opportunity to attend several scientific conferences. I also thank him for his kindness, support, and patience.
    [Show full text]