Global Assignment of the Function of Salmonella Genes in Livestock

The molecular basis of intestinal colonisation, induction of enteritis and systemic translocation by Salmonella is incompletely understood. Human infections are frequently acquired via consumption of products derived from poultry, pigs and cattle and effective vaccines or treatments for control of Salmonella in food-producing animals are lacking. <P>
We have invented a powerful new method to study the function of virtually all transposable genes in vivo. Transposon- mediated differential hybridisation (TMDH) uses Tn5 and Mu- based transposons containing T7 and SP6 promoters enabling the composition of mutant pools and location of insertion sites to be simultaneously determined by hybridisation of run-off transcripts to high-density oligonucleotide arrays. <P>The method has been successfully used to screen 10,000 S. Typhimurium mutants in a murine typhoid model, providing insights into the role of every mutated gene during infection. The method supersedes existing approaches since it informs the researcher when a gene is, or is not, required during infection without the need for cloning and sequencing of Tn-ends and allows many more mutants to be screened. Screening of signature-tagged mutants at IAH and Cambridge has indicated that S. Typhimurium uses both conserved and host-specific factors to colonise the intestines of chickens, pigs and cattle. However, the function of less than a fifth of the Salmonella genome has been probed. Furthermore, analysis of mutants in streptomycin-pretreated mice has indicated that the mouse is often incapable of detecting factors needed in livestock species. <P>Having validated TMDH in mice, we propose to screen the S. Typhimurium mutant libraries in chickens, pigs and calves. These screens will be the most relevant and exhaustive yet described and will enable us to ascribe an attenuation index for every transposed gene in each host. The role of selected genes will be confirmed by construction and characterisation of defined mutants.