Regulation of the ppGpp-dependent virulence gene programmes of S. Typhimurium

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Many Salmonella virulence genes are clustered together within pathogenicity islands. Salmonella pathogenicity islands 1 and 2 (SPI1; SPI2) encode systems required for the uptake of Salmonella and survival within macrophages respectively. The Salmonella transcriptional networks involved in invasion and survival (which include SPI1 and SPI2) will be referred to as the Salmonella extra and intracellular virulence gene expression programmes (STEX and STIN respectively). Guanosine tetraphosphate (ppGpp) is a small signalling molecule that is present in almost all bacteria. The RelA and SpoT enzymes produce ppGpp, with RelA being activated under conditions of amino acid limitation (the 'stringent response'). SpoT produces ppGpp throughout growth and is involved in mediating the response to other stressors. ppGpp acts by binding to the beta subunit of RNA polymerase (RNAP) causing the re-allocation of RNAP from stable RNA promoters to the promoters of genes encoding biosynthetic or stress related functions. We recently demonstrated that ppGpp is required for invasion and survival of Salmonella in the host. We showed that the low-oxygen activation of SPI1 requires ppGpp. The growth phase-dependent activation of SPI2 also requires ppGpp, but only under aerated growth conditions. ppGpp therefore plays a clear role in mediating the environmentally induced regulation of both the STEX and the STIN programmes. This proposal aims to identify unknown regulators of the STEX and STIN virulence gene programmes.

We will:-1) Determine the binding sites and specificity of RNAP/ppGpp under STEX and STIN inducing conditions 2) Determine the role of DksA in recruiting RNAP/ppGpp to specific STEX and STIN virulence gene promoters 3) Use a novel assay to identify putative regulators required for the ppGpp-dependent STEX and STIN virulence gene expression programme. 4) Determine the ability of putative regulators to modulate the ability of Salmonella to infect mammalian cells.

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