Understanding the dynamic interactions between Salmonella and the intestinal microbiota

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Background: Salmonella is a dangerous bacterium that causes unacceptable levels of food poisoning, and more hospital admissions in the UK than any other food-borne pathogen (about 2,500 each year). This project addresses the BBSRC Strategic research priority 1 "Agriculture and food security".Hypothesis: The competitive interactions between Salmonella and the intestinal microbiota involve intra-bacterial cell-to-cell contact and chemical signaling.Research Plan: The foodborne pathogen Salmonella faces fierce competition from the resident intestinal microbiota. However Salmonella Typhimurium (Salmonella) has developed strategies enabling them to undergo a rapid growth-burst resulting in disease. The studentship will characterize the Salmonella-microbiota interactions, identifying molecular mechanisms mediating growth-bursts and pathogenesis. We will focus on Bacteroides initially as a representative component of the intestinal microbiota. 1. The in vitro growth conditions will be refined to mimic the gastro-intestinal environment including varying media components. We will use mono- and co-cultures of Salmonella with Bacteroides, and observe any changes to growth kinetics during co-culture. 2. Salmonella expresses three distinct secretion systems required for virulence: SPI-1 T3SS, SPI-2 T3SS, and SPI-6 T6SS. Using a panel of mutants, reporter strains, and antibodies, we will examine the importance of the three secretion systems to direct cell-to-cell interactions using mono- and co-culture growth kinetics, and examining changes in secreted protein profiles. 3. Important signalling molecules and/or metabolites generated during co-culture of Bacteroides and Salmonella will be identified. Using a variety of bioassays/reporters we will screen culture supernatants for known bacterial cell-cell signalling molecules, and for effects on the expression of a panel of key Salmonella virulence genes. LC MS/MS-based metabolomics will be used to identify differences in the extracellular small molecule/metabolite profiles of supernatants (Paul Williams, Nottingham). Putative signal molecules will be purified allowing examination of their activity in growth stimulation/reporter bioassays. 4. We will globally identify genes regulated during co-culture of Bacteroides-Salmonella using deep RNA sequencing (Jay Hinton, Liverpool). These transcriptional profiles will generate a set of candidate genes regulated by direct interactions between Salmonella and Bacteroides. These will form the basis of testable hypotheses to investigate further using gene knockout mutants and phenotypic screens.5. Depending on progress, we will investigate in vivo colonization dynamics using mono-associated gnotobiotic mice colonized by Bacteroides, and orally challenged with wild-type Salmonella and selected gene knock-out mutants (Lindsay Hall, Quadram Institute, Norwich). Bacteria will be enumerated in the intestine, mesenteric lymph nodes, and organs using standard methods.