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Identifying Human Host Cell Protein Targets of the Bartonella Effector Protein (Bep) Fic Domains

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Identifying Human Host Cell Protein Targets of the Bartonella Effector Protein (Bep) Fic Domains Identifying Human Host Cell Protein Targets of the Bartonella Effector Protein (Bep) Fic Domains Vaughan Trounson A thesis submitted to Victoria University of Wellington in fulfilment of the requirements for the degree of Masters of Biomedical Science Victoria University of Wellington 2016 Abstract Vaughan Trounson Abstract The genus Bartonellae represents an increasing number of emerging bacterial pathogens that utilises an unusual infection strategy, parasitising the red blood cells of their mammalian host. The most common species to infect humans are B. henselae and B. quintana. B. henselae is transmitted between cats by the cat flea, although occasionally infects humans via cat scratches or bites, causing cat-scratch disease (CSD). CSD is characterised by enlarged tender lymph nodes and fever. B. henselae also infects the endothelial cells of both its hosts; likely a factor in disease progression. B. quintana, the cause of trench fever during WWI, is spread between people by the body louse. Trench fever is characterised by relapsing fever, headache, and bone pain. B. quintana is also able to infect human endothelial cells. These bacteria secrete a range of Bartonella effector proteins (Beps) via a Type IV secretion system, directly into endothelial cells, subverting host cell processes and resulting in internalisation of the bacteria. Beps have a range of functions, many of which are not fully characterised. B. henselae secretes three Beps (BepA-C) that contain a filamentation induced by cAMP (Fic) domain and a Bartonella Intracellular Delivery (BID) domain, with BepA being the best studied. BepA’s BID domain is responsible for intracellular delivery as well as inhibition of apoptosis by the host cell, however the exact function of the Fic domain remains unknown. Fic-containing bacterial toxins catalyse the transfer of an AMP moiety from ATP onto a host cell protein. This AMPylation frequently inactivates these proteins resulting in disrupted host cell processes and cytotoxicity. BepA has previously been shown to possess AMPylation activity, although the host target protein(s) are unknown. Evidence suggests that these proteins are novel targets. The aim of this study was to create protein constructs containing these Fic domains, and to develop techniques to identify the host cell target proteins post AMPylation. To this end, both a fluorescent ATP analogue and a fluorescent click chemistry based approach were utilised. While no target protein was identified, a basic methodology was developed for protein production and target protein identification that could be further developed. i Acknowledgements Vaughan Trounson Acknowledgements Firstly, I would like to thank my supervisor, Dr Joanna MacKichan for giving me the opportunity to work on this project. Thank you for all your support throughout my Masters, in particular for always being available to provide help and feedback, even as late as midnight. It was your lectures on Microbiology in BMSC 301 that really helped me settle on an area of molecular biology I wanted to pursue further. Thank you for all your help with setting up funding so that I’m able to attend the conference in Montana. I couldn’t have picked a better supervisor. I would also like to thank Raphael, Alistair, and Varun for helping me get to grips with my initial protein experiments, as well as protein work in general. To the Ackerley lab, you guys were always so helpful in answering my various questions, as well as always providing friendly faces in the lab to talk to while I was waiting for an experiment to complete. Thanks also to Dr Lifeng Peng, Sven, and Richard from AM for all your help running samples on the Mass Spec, as well as a thank you to Bhumi for your help in teaching me how to do western blots properly. I’d also like to thank Dr John Miller and Dr Jane Koehler for supplying the HeLa cells and B. quintana gDNA, respectively. I’d also like to thank the American Society for Microbiology for permission to reproduce the Bartonella Lifecycle diagram illustrated in Figure 1.1. To all the staff and students of SBS, in particular my office mates: James, Jasmine, Jacob, Jen, Jack, Kate, Sonja, Melz and Rory. You guys made doing a Masters less stressful than it otherwise could’ve been. In particular, a special mention to my lab mate Jacob, for always telling me on a rough day “you got this”. I’d also like to thank my flatmate Colin for all your help reading my final draft. Finally, a huge thank you to all my friends and family, in particular Mum, Dad, and Jess for accommodating my busy schedule during both my undergraduate and postgraduate years at university. I couldn’t have done it without all your support. Finally, thank you to both the Victoria University of Wellington Science Faculty, and the American Society of Rickettsiology for providing the funding that enabled me to attend the conference in Montana. ii Table of Contents Vaughan Trounson Table of Contents Abstract .................................................................................................................... i Acknowledgements .................................................................................................. ii Table of Contents .................................................................................................... iii List of Tables and Figures ....................................................................................... viii Abbreviations ........................................................................................................... x 1 Introduction ...................................................................................................... 1 1.1 Bartonella Species and Associated Disease .................................................................... 1 1.1.1 Bartonella ........................................................................................................................ 1 1.1.2 Bartonella quintana ........................................................................................................ 3 1.1.3 Bartonella henselae......................................................................................................... 4 1.1.4 The Bartonella Genome .................................................................................................. 5 1.1.5 Bartonella Lifecycle ......................................................................................................... 6 1.1.6 Bartonella and Endothelial Cells ..................................................................................... 7 1.2 Bartonella Virulence Factors .......................................................................................... 8 1.2.1 VirB/D4 T4SS and Secreted Effector Proteins ................................................................. 9 1.3 Bartonella VirB/D4 Secreted Effector Proteins ............................................................. 10 1.3.1 Bep Protein Structure ................................................................................................... 10 1.3.2 Bep Protein Functions ................................................................................................... 11 1.3.3 BepA .............................................................................................................................. 12 1.4 The Fic Domain ........................................................................................................... 13 1.4.1 Vibrio parahaemolyticus and VopS ............................................................................... 13 1.4.2 Histophilus somni and IbpA ........................................................................................... 14 1.4.3 Fic Domain Motif ........................................................................................................... 14 1.4.4 Catalytic Mechanisms ................................................................................................... 15 1.4.5 Toxin/Antitoxin Modules .............................................................................................. 16 1.4.6 Other Fic domain activities ........................................................................................... 17 1.4.7 Role of Bartonella henselae BepA Fic Domain .............................................................. 17 1.5 Research Aims ............................................................................................................. 18 2 General Methods ............................................................................................. 19 2.1 Media Recipes ............................................................................................................. 19 2.1.1 Liquid Media.................................................................................................................. 19 2.1.2 Solid Media ................................................................................................................... 19 iii Table of Contents Vaughan Trounson 2.1.3 Supplement Solutions ................................................................................................... 20 2.2 Bacterial Strains and Growth Conditions: ..................................................................... 20 2.2.1 Bacterial Strains ............................................................................................................ 20 2.2.2 Bartonella Growth Conditions .....................................................................................
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