Global assignment of the function of Salmonella genes in livestock

Bacteria of the species Salmonella enterica are a threat to public health and sustainable agriculture. Over 2460 distinct variants of the organism have been described and these can be divided into two groups; serovars restricted or adapted to a given host that cause systemic illness (e.g. S. Typhi, which causes Typhoid fever in humans) and serovars causing gastroenteritis in a wide range of hosts (e.g. S. Typhimurium). Typhoid fever is a largely water-borne disease perpetuated by unsanitary conditions and 21 million human illnesses and 216,510 deaths are estimated to occur each year worldwide. Gastroenteritis caused by non-typhoidal Salmonella strains is also common, with an estimated 1.4 million cases and 600 deaths per annum in the United States alone. These infections are often associated with the consumption of foods derived from chickens, pigs and cattle owing to the ability of the bacteria to colonise the intestines of such animals. Strategies to reduce the carriage of S. enterica in food-producing animals are expected to lower the incidence of human infections and improve animal health. Whilst vaccines exist for the control of Salmonella in poultry, they confer weak and variable protection against different serovars and it is widely agreed that effective vaccines or treatments are needed for use in other livestock species. Furthermore, not all S. enterica serovars found in animals pose a threat to humans and tools are needed to predict the epidemic potential of such strains. The development of novel vaccines, treatments and diagnostics for Salmonella requires an understanding of the molecular events underlying intestinal colonisation. Our laboratories have identified some S. Typhimurium factors needed for colonisation of chickens, pigs and calves, but the function of less than one fifth of all Salmonella genes has so far been examined. S. Typhimurium infections in calves and pigs result in acute gastroenteritis whilst carriage in chickens tends to be asymptomatic. The reasons why Salmonella causes disease in one host but not another are not understood. Furthermore, it is not known why some S. enterica serovars cause systemic disease in a given host whereas others are restricted to the alimentary tract. We have invented a new method that permits the simultaneous screening of thousands of individual Salmonella mutants, each lacking a particular character, during infection of animals. The method (transposon-mediated differential hybridisation, TMDH) can rapidly describe the role of virtually all Salmonella genes during infection and relies on detecting signals from each mutant on a chip on which all the bacterial genes are arrayed. Bacterial genes are mutated at random then the mutants are assembled into large pools that can be inoculated into animals. The use of large pools dramatically reduces the number of animals that are needed. If a mutant is represented in the inoculum, but not in a pool recovered after the bacteria have passed through the animal, it can be inferred that the character disrupted in that mutant is important for colonisation. TMDH has been validated in mice, but it now needs to be used in food-producing animals. The method supersedes existing approaches, since it can inform the researcher when a gene is, or is not, required during infection without the need to isolate and examine the mutated gene. We propose to screen 10,000 S. Typhimurium mutants by this method for their ability to colonise the intestines of chickens, pigs and cattle. This will provide insights that cannot be obtained using surrogate rodent or cell-based assays and relies on the unique expertise and facilities at the applicants' laboratories. TMDH will greatly accelerate the identification of factors needed for Salmonella pathogenesis, some of which may be suitable as targets for drugs or included in a vaccine. Treatments developed for use in livestock may also be suitable for use in humans against typhoid fever.

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. 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. 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. 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.