In vivo essential genome of Salmonella

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SUMMARY Defining genome-wide genetic requirements for fitness of a bacterial pathogen in the host is a fundamental goal of pathogenomics. Transposon sequencing (Tn-seq) is a power functional genomics tool for genome-wide identification of essential and conditionally essential genes in bacteria. When applied to a bacterial pathogen with an appropriate animal infection model, Tn-seq can accelerate the discovery of the genetic determinants of a bacterial pathogen for in vivo fitness. However, the full potential of this powerful approach to understand bacterial virulence determinants is often hampered by the bottlenecks associated with animal infection models. The bottlenecks cause stochastic loss of the mutants without contribution of their genotypes, which can lead to false positive discoveries. In this application, we will develop a novel strategy to overcome this limitation by employing a transposon system that generates a genome-saturating random mutant library from the bacterial cells multiplied from a small seeder population established in the target host tissue upon induction, circumventing the bottleneck problem. The Tn-seq profile obtained from the genome-saturating mutant population recovered from the host tissue would allow genome-wide identification of in vivo essential genes by the genes lacking insertions in the same manner by which in vitro essential genes are identified. This new approach will be fully developed and evaluated (Aim1) and applied for genome-wide identification of the gut colonization factors of Salmonella Typhimurium 14028 during infection in mice (Aim 2). Once established and validated, this approach can be easily extended to other bacterial pathogens to fully explore in vivo essential genomes.