Phenotypes of Salmonella sdiA in Mice and Pigs DISSERTATION Presented in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Graduate School of The Ohio State University By Matthew Charles Swearingen, M.S. The Ohio State University Graduate Program in Microbiology The Ohio State University 2013 Dissertation Committee: Associate Professor Brian Ahmer, Ph.D., Thesis Advisor Professor John Gunn, Ph.D. Professor Daniel Wozniak. Ph.D. Assistant Professor Sheryl Justice, Ph.D. Copyright by Matthew Charles Swearingen 2013 Abstract Bacteria of the Salmonella genera encode a solo LuxR homolog, SdiA, capable of detecting N-acylhomoserine lactone molecules (acyl-HSLs) produced by other bacteria. In response to acyl-HSLs, SdiA activates seven putative host- interaction genes, however, it is unclear if acyl-HSLs exist in mammalian intestines. Previously, we demonstrated that Salmonella enterica serovar Typhimurium could detect Yersinia enterocolitica acyl-HSLs through SdiA in the mouse gut, but acyl-HSL detection did not result in an sdiA fitness phenotype. We attempted to study sdiA phenotypes modeling S. Typhimurium – Y. enterocolitica co-infections in conventional pigs because pigs are a known reservoir of both Salmonella and Yersinia. We measured SdiA activity using an S. Typhimurium in vivo reporter system (RIVET) during an S. Typhimurium – Y. enterocolitica co-infection, and observed SdiA activity occurring in pig mesenteric lymph node samples (MLN). We did not, however, observe an sdiA fitness phenotype. We probed the Human Microbiome Project (HMP) gene catalog and ii reviewed the literature for gut-commensal organisms with the potential to produce acyl-HSLs. We found that Hafnia alvei, Edwardsiella tarda, a Ralstonia species (HMP), and Acinetobacter baumannii, Citrobacter rodentium, Pseudomonas aeruginosa, and Serratia odorifera (literature) all encode LuxI homologs. The human gut microbiome is biologically diverse and densely populated, but the Proteobacteria comprise ≤ 4% of the community. Disturbances in the gut community, however, result in a shift in the community that favors a bloom of proteobacterial species. Indeed, Salmonella-induced gut inflammation results in a proteobacterial bloom. Conventional mice are useful for studying invasive salmonellosis, but do not develop gastroenteritis after S. Typhimurium infection. However, germ-free mice colonized with human gut flora are susceptible to Salmonella infection, and do develop cecal inflammation. Thus, we hypothesized that SdiA detects the commensal proteobacteria bloom that occurs during Salmonella -induced inflammation. To test this hypothesis, we orally gavaged germ-free Swiss Webster mice with the feces of a healthy human donor, and infected them with the S. Typhimurium RIVET reporter. We did not detect SdiA activity in humanized mice, suggesting that acyl-HSLs were not present in the humanized mouse gut. We are currently investigating SdiA activity in mice during Salmonella-induced inflammation in a long-term persistence model in CBA/J mice. We tested the hypothesis that Salmonella-induced inflammation, which causes a proteobacterial bloom, would lead to the overgrowth of proteobacterial members that synthesize acyl-HSLs, and that SdiA would detect the quorum sensing molecules. Using a srgE-tnpR RIVET, we have iii indeed observed SdiA activity for WT Salmonella recovered in the feces of CBA/J mice in the third week of infection, but no activity was observed for the sdiA mutant. Thus far, we can conclude that SdiA is active during long-term persistence in the CBA/J mouse gut, but more work is needed to determine I.) when inflammation begins and ends II.) the mouse gut microbial composition before, during, and after Salmonella-induced inflammation III.) how SdiA activity corresponds to inflammation, and IV.) which member(s) of the gut flora are responsible for acyl-HSL production. iv Dedication This research was conducted whole-heartedly and in the name of science, however, this work is dedicated to the hundreds of mice and pigs that died by my hands. v Acknowledgements I would like to thank my advisor, Dr. Brian M. M. Ahmer, for his guidance and support. Dr. Ahmer encouraged me to think critically & independently, and allowed me to be creative. I would also like to thank my esteemed committee members, Dr. John Gunn, Dr. Dan Wozniak, and Dr. Sheryl Justice for their guidance and support throughout this dissertation. Never before have I met a wittier assemblage of scientists. I would like to sincerely thank my parents Glenn and Rebecca, who have devoted their lives to the success of all of their children, and I am no exception. I would like to thank my siblings Susan, Annie, Mike and Charlie for their love and support. Finally, I thank my best friend and wife, Nikki, who has stood by me through challenging times and shared the joy of the good times. vi Vita October 1984 Born in Steubenville, Ohio May 2007 B.S., Biology, Bethany College Bethany, West Virginia June 2008 - Present. M.S., Ph.D. candidate, The Ohio State University Columbus, Ohio Publications Swearingen MC and Ahmer BMM. Are there acyl-homoserine lactones within mammalian intestines? J. Bacteriol. 2012 Jan 195(2): 173-9. Swearingen MC, Porwollik S, Desai PT, McClelland M, Ahmer BMM. Mouse Virulence of 32 Strains of Salmonella . PLoS ONE 2012 7(4): e36043. doi:10.1371/journal.pone.0036043 Dyszel JL, Smith JN, Lucas DE, Soares JA, Swearingen MC, Vross MA, Young GM, Ahmer BM. Salmonella enterica serovar Typhimurium can detect acyl homoserine lactone production by Yersinia enterocolitica in mice. J Bacteriol. 2010 Jan;192(1):29-37. Dyszel JL, Soares JA, Swearingen MC, Lindsay A, Smith JN, Ahmer BM. E. coli K-12 and EHEC genes regulated by SdiA. PLoS One. 2010 Jan 28;5(1):e8946. Fields of Study: Microbiology vii Table of Contents Abstract ................................................................................................................ ii Dedication ............................................................................................................ v Acknowledgements ............................................................................................ vi Vita ...................................................................................................................... vii Table of Contents ............................................................................................. viii List of Figures ...................................................................................................... x List of Tables ...................................................................................................... xii List of Abbreviations ........................................................................................ xiii 1. Introduction ...................................................................................................... 1 1.1 Significance ................................................................................................................. 1 1.2 Bacterial Quorum Sensing Phenomena ...................................................................... 2 1.3 Gram Negative Bacterial Quorum Sensing ................................................................. 3 1.3.1 LuxI-LuxR type quorum sensing systems .................................................... 3 1.3.2 LuxI-type enzymes synthesize HSLs ........................................................... 5 1.3.3 LuxR homologous transcription factors ........................................................ 7 1.4 Quorum Sensing Mediated Pathogenicity ................................................................. 10 1.5 Escherichia coli sdiA ................................................................................................. 12 1.6 Salmonella sdiA ........................................................................................................ 15 1.3.1 Salmonella eavesdropping ......................................................................... 19 2. Virulence of 32 Salmonella strains in mice ................................................. 24 2.1 Abstract ..................................................................................................................... 25 2.3 Results ...................................................................................................................... 28 2.3.1 Mouse virulence assays ............................................................................. 28 2.3.2 Fecal shedding and assessment of cross-protection ................................. 28 2.4 Discussion ................................................................................................................. 30 2.5 Methods .................................................................................................................... 33 2.5.1 Ethics statement ......................................................................................... 33 2.5.2 Bacterial strains and media ........................................................................ 33 2.5.3 Mouse virulence and fecal shedding .......................................................... 33 viii 2.5.4 LD50 determinations .................................................................................... 34 3. The contribution of sdiA to Salmonella fitness in pigs and mice ............. 45 3.1 Abstract ..................................................................................................................... 46