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Investigation of Host Factors Involved in Legionella Pneumophila Virulence

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Investigation of Host Factors Involved in Legionella Pneumophila Virulence Investigation of host factors involved in Legionella pneumophila virulence Sze Ying Ong Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy October 2017 Department of Microbiology and Immunology The University of Melbourne at the Peter Doherty Institute of Infection and Immunity Produced on archival quality paper ABSTRACT Legionella pneumophila is an environmental organism that can become accidental bacterial pathogen when inhaled into the lungs of humans. On entering the lungs, L. pneumophila is phagocytosed by alveolar macrophages. However, instead of being removed accordingly by the host immune system, the bacteria rapidly establish a Legionella-containing vacuole (LCV) and replicate intracellularly. The ability to establish the LCV relies on a type IV secretion system, also known as the Dot/Icm system. The Dot/Icm system is essential for virulence and delivers a large repertoire (> 300) of effector proteins into infected host cells. These effector proteins modulate a wide range of host processes such as vesicle trafficking, host protein translation, regulation of GTPases and apoptosis. Despite the phenomenal number (one of the highest among known bacterial pathogens) of effector proteins translocated by L. pneumophila into host cells, effector secretion is not detected until the bacterium contacts a host cell. This is in contrast to other bacterial pathogens such as enteropathogenic Escherichia coli or Salmonella sp., which can be induced to secrete effector proteins via a type III secretion system into liquid bacteriological cultures. The interactions that occur between the host cells and L. pneumophila in order to activate the Dot/Icm system are poorly understood. In this study, we used an RNAi approach to screen for host factors that contribute to Dot/Icm effector protein translocation. A genome-wide siRNA screen was performed that individually silenced each protein-coding gene in the human genome (~ 18 000 genes) and monitored for any changes in translocation of the prototypic effector protein, RalF, after L. pneumophila infection. This screen identified 119 genes whose silencing resulted in increased translocation of RalF while knockdown of 321 genes resulted in decreased translocation. Strikingly, we found that genes in the ubiquitination pathway were significantly over-represented among the factors that contribute to the efficiency of RalF translocation during L. pneumophila infection. We further showed these ubiquitination factors also influence translocation of another effector protein, SidB. This suggested that the host ubiquitination pathway was important in mediating Dot/Icm effector protein translocation. Interestingly, we also found that two of the host ubiquitination factors that facilitated effector protein translocation were also important for L. pneumophila intracellular replication. These i were UBE2E1 and CUL7; an E2-conjugating enzyme and E3 ligase respectively. On top of host ubiquitination factors, this screen also identified many other host factors that were not previously appreciated as having possible roles in Dot/Icm mediated effector protein translocation. Given the finding that ubiquitination is important for Dot/Icm effector protein translocation and that the LCV is an ER-like compartment, we hypothesised that this process occurred in a manner equivalent to retrotranslocation, a process that moves ubiquitinated protein substrates from within the ER into the cytoplasm. Unfortunately, we were not able to conclude this as silencing gene expression of the ATPase, p97, which drives this process, resulted in significant host cell death. To this end, the significance of ubiquitination pathways on Dot/Icm mediated effector protein translocation is yet to be elucidated. Nevertheless, this study has contributed significantly to our understanding of host-Legionella interactions and provided much new information about the plausible interplay between the host cell and the Dot/Icm system. Finally, we demonstrated the utility of genome-wide high-throughput screening in human cells to study L. pneumophila pathogenesis. ii DECLARATION This is to certify that: i. This thesis comprises only my original work towards the Doctor of Philosophy except where indicated in the preface ii. Due acknowledgement has been made in the text to all other material used iii. This thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices Sze Ying Ong Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity iii PREFACE In accordance with the regulations of The University of Melbourne, I acknowledge that some of the work presented in this thesis was collaborative. Specifically: In chapter 3, optimisation of the genome-wide RNAi screen was performed with the assistance of Ralf Schülen. In chapter 3, the pXDC61:LseA and pXDC61:LseB constructs were made by Ralf Schülen. In chapters 3, 4 and 5, the pXDC61:RalF construct was made by Ralf Schülen. In chapter 4, the genome-wide RNAi screen was performed with the assistance of Ralf Schülen. Operation of transfection robot was performed by Daniel Thomas while data was analysed with assistance from Piyush Madhamshettiwar (Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre). In chapter 5, the L. pneumophila 130b ΔicmSW was constructed by Ralf Schülen. The remainder of this thesis comprises only my original work. iv LIST OF PUBLICATIONS ARISING FROM THIS THESIS This thesis contains material that is published or is currently in preparation for publication: Ong SY, Schülen R, Thomas D, Madhamshettiwar P, Luu J, Handoko Y, Freytag S, Bahlo M, Simpson KJ, Hartland EL (2017). Genome-wide RNAi screen identified host ubiquitination as important for Legionella pneumophila Dot/Icm mediated effector translocation. v ACKNOWLEDGEMENTS Completion of this PhD will not be possible without the help, support and guidance of many people, all of whom needs to be properly acknowledged. Firstly, thank you Liz for taking me on as a research assistant in 2010. Back then, I really appreciated the opportunity you gave me even though I came fresh out of Honours without any real experience in Microbiology. Even more surprising for me is when you fully supported and encouraged me to complete a PhD in your lab 2 years later. I really appreciate the confidence you had in me and I am extremely grateful that you allowed me to take on such a big and crazy project in my PhD. Thank you for all your guidance and encouragement over the years, for giving me confidence to do and take on so many things that I otherwise will have been too scared to do. Your little timely push always does the trick in making me get over myself and get on with the task. I have grown and learnt so much under your supervision during my PhD, be it how to think and run experiments as a scientist to enthusiasm for science. Till now, I still think that I am extremely lucky that we crossed paths many years ago and you graciously took me on. So once again, a big THANK YOU! Thank you also to my second supervisor, Ralf. You have taught me all that I ever know about molecular biology and Legionella. You are an incredibly patient man who always had time for me and answered all my naive questions. Your meticulous approach to science is something that I truly admire and I am indebted to you for all the guidance you gave me. Without you, the RNAi screen will have been a lot harder, both physically and mentally! The daily trips to Peter Mac were more enjoyable with your company and those sneaky lunches that we squeeze in are something I looked forward to. I have learnt a lot from you and you had a significant role in making me the scientist (and person) I am today. I really couldn’t have asked for a better lab supervisor. Finally, thank you also for your friendship. It helped me get through PhD without going insane. Thank you also to Shivani for the guidance towards the end of my PhD. You made sure my thesis drafts were not an embarrassment before they were submitted to Liz. vi They could have been torn to shreds if you hadn’t so generously given up some of your time to help me. To my PhD buddies in the Hartland lab – Cristina, Tania and Ying Z, thank you for ‘being in it’ together! It was fantastic having company and people who are going through the same thing together. You girls motivate me to work harder and push me to do things which I would not have done if I did not have you guys doing it with me together. Being the last in the gang to write up, I also probably had the easiest job putting my thesis together as I had all your theses to look at as a guide/template. Thank you for all the thesis help! Cristina, thank you for making me sit down and submit my 80-word summary. I would still have been umm-ing and ahh-ing about it if you hadn’t put your foot down and got me to submit it. Also, thank you for all the free rides I get at the end of the day so that I don’t have to go onto the dreaded tram to get home! Finally, our birthday celebrations, dinners and drinking sessions were critical in helping me make it to the end of my PhD, so thank you for all the fun over the years too! We might have gone on different paths now, but this experience together is something that will stay with us forever. Ka Yee, thank you for being my listening ear in the lab. You are the best lab bench buddy and I can truly say that no one else had been able to replace you since you left the lab. You had the wisdom of making it through a PhD and always give me intelligent opinions in my scientific dilemmas.
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