Characterization of Slam-mediated Surface Lipoprotein Translocation across the Bacterial Outer Membrane by Yogesh Hooda A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Biochemistry University of Toronto © Copyright by Yogesh Hooda 2019 i Characterization of Slam-mediated surface lipoprotein translocation across the bacterial outer membrane Yogesh Hooda Doctor of Philosophy Department of Biochemistry University of Toronto 2019 Abstract Surfaces of many Gram-negative bacteria are decorated by peripheral membrane proteins that are anchored in the membrane by a lipid group, commonly referred to as surface lipoproteins or SLPs. SLPs play key role in nutrient acquisition, immune evasion and have been proposed as excellent vaccine antigens. Previously our lab had shown that the proper display of host transferrin binding SLP TbpB in Neisseria meningitidis required an outer membrane protein called Slam. The aim of the present study was to investigate the role Slam plays in SLP biogenesis. Using bioinformatic analysis, we show that Slams are present in a number of Gram-negative bacteria. Putative Slam genes are often found adjacent to their putative SLP substrates. In N. meningitidis, we discovered two Slam paralogs, Slam1 and Slam2, that are specific for SLPs TbpB and HpuA respectively. All putative Slam-dependent SLPs contain a C-terminal 8-stranded soluble barrel domain. The last two strands of the SLP were found to be essential for TbpB translocation and the C-terminal 8-stranded barrel domain mediated Slam specificity. Using GST-fused TbpB, we showed that the Slam-dependent translocation occurs from the C-terminus to the N-terminus. To investigate Slam mechanism, we developed an in vitro translocation system. Upon the addition of the periplasmic chaperone LolA, SLPs can be released from spheroplasts into the supernatant. ii We discovered that Slam containing proteoliposomes can successfully translocate spheroplast released SLPs into the liposomal lumen. Addition of other outer membrane factors such as the Bam complex did not increase the efficiency of SLP insertion and Slam1&2 retained their specificity in the assay. Interestingly, Slam1 proteoliposomes were also able to translocate purified unfolded TbpB into the lumen. Collectively, these findings show that Slams are both necessary and sufficient for the translocation of SLPs across the outer membrane, indicating that they act as translocons. iii Acknowledgments I am more interested in research now than I was before starting graduate school. I might be more competent at it too. Being in school for so long, I realize how rare that is and I have a lot of people to thank for it. First and foremost, I would like to thank my supervisor Dr. Trevor Moraes. Trevor has been an excellent mentor and adviser. He always highlighted the importance of designing sound experiments with proper controls and being respectful to other people and equipment in the lab. I have learnt a lot under his supervision, and I hope to have him as my mentor for the rest of my scientific career. Apart from Trevor, I would also like to thank a number of people in the Moraes lab who worked on this project with me. Christine identified Slam as one of the hits in a genetic screen 7 years ago now. Since then, she has been instrumental in the development of tools and assays I describe in this thesis. She manages the lab with an iron fist and lots of cat pictures, which kept me going. I am also grateful to Andrew and Sang for being my co-collaborators on the Slam project. For those who know them, they are two very different people to work with and yet they were instrumental in some of the most important results presented in this study. My favorite part of working in a lab is arguing with other people over why a given experiment has failed, especially given that everyone involved is generally wrong. During my time in the Moraes lab, I have interacted with a lot of intelligent and enthusiastic people who never hesitated to hypothesize. I would personally like to thank current and past Moraes lab members: Ana, Megha, Charles, Esther, Tom, Nick, Chuxi, Epshita, Jaime, Steven and Maciej for all their constructive feedback. During my time here, I have also had the opportunity to interact with and mentor highly intelligent and motivated undergraduates. I am happy to report that most of them still intend to pursue a PhD, or at least that is what they tell me. I would also like to thank our collaborators Drs. Scott Gray-Owen and Tony Schryvvers for their support. I had a very fruitful time being at the University of Toronto. My committee members Drs. Roman Melnyk and Will Navarre were very helpful, and I always came out of committee meetings smarter. I also interacted with a number of other faculty members in the department of Biochemistry who were always supportive of my work. Apart from academics, I also had a lot of fun being part of the iv Biochemistry Graduate Student Union. I helped in organizing departmental social events and the experience was always rewarding and often more successful than scientific experiments. Being part of UofT has provided me with numerous opportunities of growth in my personal and professional life. While I actively tried to avoid as many of these events as possible, the vast size of UofT made sure that I end up attending a few stochastic events, for which I am now very grateful. Starting graduate school in Toronto was one of the best decisions I made, especially given that it was the only choice I had. None of the work described here would have been possible without the constant support of my family. My parents might not understand what I do, but they always knew in their heart that I could be doing something worse. I also want to thank my brother who has aged considerably waiting in earnest for a PhD brother; his watch is ending soon. During graduate school I acquired two parents and a younger brother through marriage. Having done their PhDs many moons ago, my parent-in- laws always accepted that I will never be rich. Same goes from my brother-in-law, who is on his own path of doing a PhD but in the much poorer social sciences. This brings me to the most important person I want to acknowledge, my cat’s legal guardian and my wife Dr. Senjuti Saha. A lot has happened in our lives in the past 8 years. We got a cat named Ava Fontaine, started traveling the world, got married, moved into a beautiful apartment and survived graduate school together. I am excited and curious about what the future holds. v Table of Contents Acknowledgments ...................................................................................................................... iv Table of Contents ...................................................................................................................... vi List of Figures ............................................................................................................................x List of Appendices .................................................................................................................. xiii List of Abbreviations ............................................................................................................... xiv Chapter 1 Introduction ...............................................................................................................1 1.1 Overview ............................................................................................................................... 1 1.2 Cell envelope of Gram-negative bacteria ............................................................................. 1 1.2.1 Inner membrane proteins.................................................................................................................. 3 1.2.2 Outer membrane proteins ................................................................................................................. 3 1.2.3 Lipoproteins .................................................................................................................................... 4 1.3 Surface lipoproteins ............................................................................................................. 6 1.3.1 Experimental methods for identifying SLPs...................................................................................... 7 1.3.2 Prevalence of SLPs .......................................................................................................................... 8 1.3.3 Translocation pathways for SLPs ..................................................................................................... 9 1.4 Surface lipoproteins in Neisseria .........................................................................................11 1.4.1 Iron acquisition systems: TbpB, LbpB and HpuA ........................................................................... 12 1.4.2 Vaccine antigens: fHbp and NHBA ................................................................................................ 13 1.4.3 Partially surface-exposed proteins: AniA, NalP .............................................................................. 13 1.4.4 Other SLPs: MIP, SliC and FrpD ................................................................................................... 15 1.5 Study of neisserial SLP biogenesis and discovery of Slam .................................................16 1.5.1 Role of Slam in N. meningitidis pathogenicity ...............................................................................