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Investigating Phagosome Dynamics of Microbial Pathogens

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Investigating Phagosome Dynamics of Microbial Pathogens Investigating phagosome dynamics of microbial pathogens By Leanne May Smith A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences University of Birmingham September 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Many miCrobial pathogens are able to evade Killing by phagoCytes of the innate immune system. This thesis focuses on two pathogens: the fungal pathogen Cryptococcus neoformans and the bacterial pathogen Streptococcus agalactiae. C. neoformans causes severe cryptocoCCal meningitis in mostly immunoCompromised hosts, suCh as those with HIV infeCtion. In contrast, S. agalactiae is the leading cause of neonatal sepsis and meningitis. The interaCtion between maCrophages and these pathogens is likely to be CritiCal in determining dissemination and outCome of disease in both instanCes. A ColleCtion of S. agalactiae CliniCal isolates, ranging in origin from Colonisation Cases to severe infection cases, were tested for their ability to persist with a maCrophage Cell line. Surprisingly, persistenCe within maCrophages was a charaCteristiC shared by all of the isolates tested. Furthermore, by investigating the Streptococcus-Containing phagosome, it was revealed that streptoCoCCi are able to manipulate the aCidifiCation of maCrophage phagosomes. Similarly, the maturation of phagosomes Containing the fungal pathogen C. neoformans was explored. Cryptococci are shown to be able to manipulate the phagosome they reside within. This is driven by modified acquisition of Rab GTPases to the phagosome, as well as altered acidification and cathepsin activity within Cryptococcus-containing phagosomes. Acknowledgements A PhD is a qualifiCation awarded to an individual. However, no PhD is truly aCComplished by an individual alone without the help and support of those around them. I therefore use this seCtion of the thesis to thanK some of the people that helped me along my way over the last four years (well… aCtually 8 years in total, so far!) at the University of Birmingham. Research itself is often an expensive business so there are a few funding bodies to be thanKed for helping to pay for all the fun times. Firstly, the University of Birmingham for the sCholarship that Kept a roof over my head. SeCondly, the funding bodies that paid for the researCh itself, the WellCome Trust, Lister Institute, MediCal ResearCh CounCil (MRC) and National Institute for Health Research (NIHR). Finally, the scientific societies that payed for my attendanCe at various ConferenCes during my PhD studies, providing me with the opportunity to present my worK to experts in the field (and also see a little more of the world than I otherwise would!), the SoCiety for General MiCrobiology (SGM), the Federation of European MiCrobiological Societies (FEMS) and the Society of Experimental Biology (SEB). I would also liKe to thanK some people who provided various reagents and miCrobial strains that made this worK possible: Arturo Casadevall, Julian Rutherford, MarK Anthony, Marie-Cécile Lamy and Howard JenKinson. And, of Course, MarK Webber and Bernhard Hube for examining this thesis and maKing the entire proCess surprisingly not too sCary! Thank you both. I also owe a great deal of gratitude to Julia Lodge. As my undergraduate tutor it was you who mentored me through my first years at university and taught me many of the sKills I would require for this PhD, such as: at least knowing what end of a pipette is whiCh! Quite CruCial really! ThanKs for giving me the basiCs in sCientifiC writing and praCtiCal sKills. I’m so pleased we ended up moving offiCes and lab spaCe; hearing your booming laughter from down the Corridor always maKes the residents of N110 laugh along too! Next up, my supervisors, Robin May and Anne-Marie Krachler. Both of you have been wonderful and inspiring people to worK with and be mentored by. Robin, without a doubt you are the most enthusiastiC, optimistiC, limb-flailing person anyone could Know! These qualities maKe you one of the best people to Come to with any worries, professional or personal. You care a great deal about all of us and we all appreciate that so muCh. Thanks for maKing suCh an amazing environment to sCientifiCally ‘grow up’ in. Now for the people in the lab. Firstly: NiCola you were the one to initially show me the ropes in the lab, thanK you for you’re patienCe. Simon, you showed me how to use our (slightly tempermental at times) miCrosCopes. ThanKs so muCh for getting me hooKed! Kerstin and Elizabeth, thanK you both for maKing those first Couple of years that little more hilarious. BeCKy, although you joined the lab later, it seems as if you’ve been here the whole time too! ThanKs to all of you for being my mini mentors and for being my friends. The Current lab: Robbie, Peter, Andy, Emily, Lea and Dan and the whole HAPI group – thanKs for maKing the ‘job’ so fantastiCally ComiCal. WorKing with you all maKes everything so muCh easier. Finally, to my Family – thanKs for doing your best to understand what was going on! And, of Course, BeCKi – thanK you for supporting me and putting up with all the paniC moments that no one else saw. TABLE OF CONTENTS CHAPTER I. INTRODUCTION 1 PHAGOSOME MATURATION 2 STRATEGIES OF DIVERSION AND MODIFICATION OF PHAGOSOME MATURATION – MAKING THE ENVIRONMENT MORE COMFORTABLE. 5 NEISSERIA GONORRHOEAE 7 YERSINIA PESTIS 8 LEGIONELLA PNEUMOPHILA 10 STREPTOCOCCUS PYOGENES (GAS) 12 HISTOPLASMA CAPSULATUM 12 LEISHMANIA SPP. 14 COXIELLA BURNETII 16 RHODOCOCCUS 17 COCCIDIOIDES SPP. 18 BRUCELLA SPP. 19 ASPERGILLUS SPP. 20 STRATEGIES OF ESCAPING PHAGOSOMES AND PHAGOCYTES – KNOWING WHEN TO BAIL OUT 22 CRYPTOCOCCUS NEOFORMANS 22 LISTERIA MONOCYTOGENES 27 SHIGELLA FLEXNERI 29 RICKETTSIA 29 BURKHOLDERIA PSEUDOMALLEI 30 FRANCISELLA TULARENSIS 31 TOXOPLASMA GONDII 32 TRYPANOSOMA CRUZI 34 CANDIDA SPP. 35 STRATEGIES TO PERSIST WITHIN PHAGOSOMES – MAKING THE BEST OF A BAD SITUATION 39 STREPTOCOCCUS AGALACTIAE 39 HELICOBACTER PYLORI 43 SUMMARY 45 THESIS OUTLINE 46 CHAPTER II. CRYPTOCOCCUS NEOFORMANS ALTERS PHAGOSOME MATURATION 49 INTRODUCTION 49 HOST ASSOCIATED RISK FACTORS FOR CRYPTOCOCCOSIS 50 CRYPTOCOCCAL MECHANISMS OF IMMUNE EVASION 54 CRYPTOCOCCAL INTERACTIONS WITH MACROPHAGES 61 EXPERIMENTAL PROCEDURES 71 CRYPTOCOCCAL STRAINS, GROWTH CONDITIONS AND OPSONISATION 71 MACROPHAGE CELL LINE CULTURE 71 HUMAN MONOCYTE DERIVED MACROPHAGES ISOLATION AND DIFFERENTIATION 72 INFECTION OF MACROPHAGES WITH CRYPTOCOCCUS 72 LABELING AND IMAGING OF FIXED CELLS 73 LIVE CELL IMAGING 74 STATISTICS 74 RESULTS 75 ACQUISITION OF RAB GTPASES IS ALTERED ON CRYPTOCOCCUS-CONTAINING PHAGOSOMES 75 LIVE CRYPTOCOCCI ARE ABLE TO BLOCK ACIDIFICATION OF THE PHAGOSOME 81 CRYPTOCOCCUS-CONTAINING PHAGOSOMES DO NOT HARBOUR CATHEPSIN ACTIVITY 86 A VARIETY OF CRYPTOCOCCAL MUTANTS ARE STILL ABLE TO BLOCK ACIDIFICATION 91 THE CRYPTOCOCCAL-CONTAINING PHAGOSOME RAPIDLY PERMEABILISES 93 DISCUSSION 97 CHAPTER III. INTRACELLULAR PERSISTENCE OF STREPTOCOCCUS AGALACTIAE 102 INTRODUCTION 102 NEONATAL GROUP B STREPTOCOCCUS TRANSMISSION AND INFECTION 103 INFECTION INITIATION: HOST CONTRIBUTING FACTORS 106 GROUP B STREPTOCOCCUS CONTRIBUTING FACTORS: ADHERENCE AND COLONISATION 109 GROUP B STREPTOCOCCUS CONTRIBUTING FACTORS: EVASION OF THE IMMUNE SYSTEM 112 GROUP B STREPTOCOCCUS CONTRIBUTING FACTORS: HOST CELL INVASION 115 GROUP B STREPTOCOCCUS CONTRIBUTING FACTORS: INTRACELLULAR SURVIVAL 117 EXPERIMENTAL PROCEDURES 125 STREPTOCOCCAL STRAINS AND GROWTH CONDITIONS 125 MACROPHAGE CELL LINE CULTURE 125 GBS INTRACELLULAR SURVIVAL ASSAY 127 LABELING AND IMAGING OF FIXED CELLS 129 RESULTS 131 INITIAL UPTAKE OF GBS BY MACROPHAGES 131 CLINICAL ISOLATES OF GBS ARE ABLE TO PERSIST WITHIN MACROPHAGES 133 GBS PHAGOSOME MATURATION – ACIDIFICATION 137 DISCUSSION 140 CHAPTER IV. CREATING A TRANSPOSON MUTANT LIBRARY OF STREPTOCOCCUS AGALACTIAE 145 INTRODUCTION 145 EXPERIMENTAL PROCEDURES 147 GENERATION OF COMPETENT GBS STRAIN COH1 147 CONSTRUCTION OF TRANSPOSOMES 148 ELECTROPORATION OF GBS STRAIN COH1 148 EXTRACTION OF GBS GENOMIC DNA 150 CONFIRMING INSERTION SITES 153 RESULTS 154 PRODUCTION OF COMPETENT GBS STRAIN COH1 154 MOLECULAR CONSTRUCTION OF TRANSPOSOMES 155 TRANSFORMATION OF GBS STRAIN COH1 157 DISCUSSION 159 THESIS SUMMARY 161 LIST OF REFERENCES 162 APPENDIX 194 LIST OF FIGURES FIGURE 1. PHAGOSOME MATURATION. 4 FIGURE 2. STRATEGIES USED BY PATHOGENS TO MODIFY PHAGOSOME MATURATION. 6 FIGURE 3. STRATEGIES USED BY PATHOGENS TO ESCAPE THE PHAGOSOME AND PHAGOCYTE. 23 FIGURE 4. PATHOGENS THAT PERSIST WITHIN THE PHAGOSOME. 40 FIGURE 5. CRYPTOCOCCUS NEOFORMANS INTERACTIONS WITH MACROPHAGES. 65 FIGURE 6. ACQUISITION OF RAB GTPASES ONTO PHAGOSOMES AT 15 AND 120 MIN POST PHAGOCYTOSIS. 77 FIGURE 7. ACQUISITION OF RAB5 GTPASE ONTO PHAGOSOMES AT 5, 15 AND 120 MIN POST PHAGOCYTOSIS 78 FIGURE 8. ACQUISITION OF RAB5 GTPASE TO C. NEOFORMANS B3501 – CONTAINING PHAGOSOMES 79 FIGURE 9. SERUM OPSONISATION AND RAB5 GTPASE ACQUISITION TO CCPS 80
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