Comprehensive assignment of roles for Salmonella typhimurium genes in intestinal colonization of food-producing animals.

Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype-genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry.

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium DeltaaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4log(10) colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium DeltaaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to DeltaspaS or DeltassaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the DeltaaroA mutant. Expression in the DeltaaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry.

Salicylidene acylhydrazide-mediated inhibition of type III secretion system-1 in Salmonella enterica serovar Typhimurium is associated with iron restriction and can be reversed by free iron.

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. Intestinal colonization, induction of enteritis and systemic translocation by this bacterium requires type III protein secretion. Strategies that target this process have the potential to control infection, pathology and transmission. We defined the global transcriptional response of S. Typhimurium to INP0403, a member of a family of salicylidene acylhydrazides that inhibit type III secretion (T3S). INP0403 treatment was associated with reduced transcription of genes involved in T3S, but also increased transcription of genes associated with iron acquisition. We show that INP0403 restricts iron availability to Salmonella, and that inhibition of T3S system-1 by INP0403 is, at least in part, reversible by exogenous iron and independent of the iron response regulator Fur.

Inhibition of type III secretion in Salmonella enterica serovar Typhimurium by small-molecule inhibitors.

Type III secretion systems (T3SS) are conserved in many pathogenic gram-negative bacteria. Small molecules that specifically target T3SS in Yersinia and Chlamydia spp. have recently been identified. Here we show that two such compounds inhibit Salmonella T3SS-1, preventing secretion of T3SS-1 effectors, invasion of cultured epithelial cells, and enteritis in vivo.