The interaction of Salmonella Typhimurium with host derived lipids by Jessica Louise Rooke A thesis submitted to the University of Birmingham and the University of Melbourne for the jointly awarded degree of DOCTOR OF PHILOSOPHY Department of Microbiology & Immunology Institute of Microbiology & Infection The Peter Doherty Institute College of Medical & Dental sciences University of Melbourne University of Birmingham September 2018 Abstract Autotransporters are some of the most abundantly secreted proteins in Gram-negative bacteria. Many of these proteins function as virulence factors. S. enterica serovar Typhimurium has five autotransporter proteins: SadA; ApeE; MisL; YaiU; and ShdA. Here, the outer membrane biogenesis and virulence properties of the S. Typhimurium autotransporters have been investigated. The outer membrane biogenesis of the multimeric autotransporter SadA was shown to be dependent upon BamA and BamD, which are essential components of the β-barrel assembly machinery complex, but the non-essential components, BamB, C and E, are not required. Contrary to a report in the literature, the translocation assembly module complex is also not required for SadA secretion. ApeE is a classical autotransporter that has a GDSL lipase motif. ApeE was shown to have phospholipase B activity, with the ability to cleave acyl chains of phospholipids. The phospholipase activity of ApeE was inhibited by the serine lipase inhibitor methyl arachidonyl fluorophosphonate. Using an in vivo murine infection model, bacterial survival at 21 days post-infection in gallbladders of mice, and efficient faecal shedding, required a functional ApeE protein. ApeE was also required for S. Typhimurium to utilise lyso-phosphatidylcholine as a sole carbon source for growth. It is proposed that Salmonella survival in the mouse gallbladder depends on the ability of ApeE to hydrolyse bile phospholipids for use as a nutrient source in vivo. i Acknowledgements First, I would like to thank my supervisor Professor Ian Henderson for all his help and guidance throughout these last 6 years, from undergraduate placement all the way through to the end of my PhD. I am looking forward to the new adventure in Brisbane with you and the Henderson lab! I would also like to thank Professor Jeffrey Cole for all his advice and guidance on writing, presentations and experiments. I don’t think I will ever forget the difference between “may and might”. I would also like to thank my University of Melbourne supervisor Professor Dick Strugnell. I am very grateful for my time in Melbourne, both experimentally and personally, and I thank you for all of your help. I would like to thank Professor Dan Slade for hosting me at Virginia Tech for a month, and teaching me how to become an enzyme Biochemist. Thank you for making me feel so welcome, for days out to go hiking and bouldering and for helpful scientific suggestions. I would like to Dr Amanda Rossiter, my mini boss for all of her help with the project and also Bio21 for all of the lipidomics analysis and to Dr Alan McNally for the phylogenetic analysis. To Chris Icke and Emily Goodall, thank you for all of the fantastic times we have had over the last 3-4 years. To Chris for all of the out of tune singing, terrible jokes and general foolishness, I want to thank you for being an awesome friend (and housemate) and excellent colleague. To Emily, thank you for frequently housing me, for the time we stayed up late writing thesis with cups of tea and a fish finger sandwich! Thank you both, I can’t wait for the new adventure in Brisbane! I’d also like to thank the rest of the Sweden crew (Charles Moore-Kelly and Richard Meek) for always being up for cake, pints and for providing joy, even during the most stressful times. I would like to say a huge thank you to Jack Bryant and Tamar Cranford-Smith for pushing me to go spinning, even on days when I didn’t feel like it. Especially to Jack, who is always up for a coffee break. Thank you for all of your suggestion in the lab, for teaching me how to do TLCs and for generally being indispensable to the Henderson lab. I would also like to thank Georgia Isom, another long standing member of the grease crew, former housemate and long-time bestie. Thank you for all of your support scientifically (protein clustering analysis), and with my personal life, throughout all of these years that I have known you. Thank you to Irene Beriotto (and Georgia) for all of our trips over the years, and for being incredible friends. Both of you, and the original members of the magic rainbow 5, truly made the first years of my time in the Henderson lab unforgettable. Finally, I would like to thank my Partner Karthik Pullela, who has supported me through this process from the opposite side of the world. Thank you for all of your patience, and kind words at the end of a very stressful day. I’d like to thank my family for their continued love and support. To Mom, Kev, Joe and Kate, thank you for all of the cups of tea (Kev), for the cupcakes (Kate), for sharing you “man cave” (Joe) and for gin (Mom). Thank you to Nan and Grandad for all of your support over the years (and for the recent trip to Spain!) I would also like to thank my Dad and Shirley for the many carveries that have sustained me over these few years. ii Table of contents CHAPTER 1 General Introduction ....................................................................... 1 1.1 Salmonella enterica ........................................................................................ 2 1.1.1 Salmonella enterica. ...................................................................................... 2 1.1.2 S. enterica lifecycle ....................................................................................... 4 1.1.3 S. enterica acute infections ........................................................................... 7 1.1.4 Invasive systemic disease of S. enterica ....................................................... 7 1.1.5 Host immune responses to S. enterica infections. ......................................... 8 1.1.6 Asymptomatic chronic carriage of systemic Salmonella. ............................. 11 1.2 Pathogen interaction with host phospholipids .......................................... 16 1.2.1 Host Phospholipids. ..................................................................................... 16 1.2.2 Intracellular pathogens and host lipids ........................................................ 18 1.3 Salmonella virulence factors ....................................................................... 22 1.3.1 The Salmonella Outer Membrane. .............................................................. 22 1.4 Protein secretion in Gram negative bacteria................................................... 24 1.4.1 Inner membrane translocation ..................................................................... 24 1.4.2 Protein secretion overview .......................................................................... 25 1.5 Type 5 secretion. .......................................................................................... 27 1.5.1 Type 5 protein overview and functions. ....................................................... 27 1.5.2 Classes of Type 5 proteins .......................................................................... 28 1.5.3 Type 5 mode of secretion ............................................................................ 32 1.5.4 The macromolecular complexes important for Type 5 outer membrane biogenesis. ................................................................................................... 34 1.6 Salmonella enterica autotransporter proteins. .......................................... 37 1.6.1 The Salmonella and Yersinia trimeric autotransporters, SadA and YadA. .. 37 1.7 GDSL lipase autotransporters ........................................................................ 38 1.7.1 Overview of GDSL lipase/esterases. ........................................................... 38 1.7.2 The Salmonella GDSL lipase autotransporter, ApeE. ................................. 42 1.8 Aims and objectives ..................................................................................... 43 CHAPTER 2 Materials and Methods ................................................................. 45 2.1 Culture media, growth conditions and strains .......................................... 46 2.1.1 Bacterial strains and plasmids. .................................................................... 46 2.1.2 Standard laboratory growth conditions. ....................................................... 46 2.1.3 M9 minimal medium preparation. ................................................................ 46 2.1.4. MOPS minimal medium preparation ........................................................... 50 2.1.5 Microtitre plate growth assays. .................................................................... 50 2.1.6 Spot plating of bacterial dilution series onto agar plates.............................. 50 iii 2.1.7 Repression of gene expression. .................................................................. 52 2.2 Bacterial transformation .............................................................................. 52 2.2.1 Chemical competent transformations. ......................................................... 52 2.2.2 Electroporation ............................................................................................ 53 2.3 Molecular biology techniques ....................................................................