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.

Analysis of the role of 13 major fimbrial subunits in colonisation of the chicken intestines by Salmonella enterica serovar Enteritidis reveals a role for a novel locus.

BACKGROUND: Salmonella enterica is a facultative intracellular pathogen of worldwide importance. Over 2,500 serovars exist and infections in humans and animals may produce a spectrum of symptoms from enteritis to typhoid depending on serovar- and host-specific factors. S. Enteritidis is the most prevalent non-typhoidal serovar isolated from humans with acute diarrhoeal illness in many countries. Human infections are frequently associated with direct or indirect contact with contaminated poultry meat or eggs owing to the ability of the organism to persist in the avian intestinal and reproductive tract. The molecular mechanisms underlying colonisation of poultry by S. Enteritidis are ill-defined. Targeted and genome-wide mutagenesis of S. Typhimurium has revealed conserved and host-specific roles for selected fimbriae in intestinal colonisation of different hosts. Here we report the first systematic analysis of each chromosomally-encoded major fimbrial subunit of S. Enteritidis in intestinal colonisation of chickens. RESULTS: The repertoire, organisation and sequence of the fimbrial operons within members of S. enterica were compared. No single fimbrial locus could be correlated with the differential virulence and host range of serovars by comparison of available genome sequences. Fimbrial operons were highly conserved among serovars in respect of gene number, order and sequence, with the exception of safA. Thirteen predicted major fimbrial subunit genes were separately inactivated by lambda Red recombinase-mediated linear recombination followed by P22/int transduction. The magnitude and duration of intestinal colonisation by mutant and parent strains was measured after oral inoculation of out-bred chickens. Whilst the majority of S. Enteritidis major fimbrial subunit genes played no significant role in colonisation of the avian intestines, mutations affecting pegA in two different S. Enteritidis strains produced statistically significant attenuation. Plasmid-mediated trans-complementation partially restored the colonisation phenotype. CONCLUSION: We describe the fimbrial gene repertoire of the predominant non-typhoidal S. enterica serovar affecting humans and the role played by each predicted major fimbrial subunit in intestinal colonisation of the primary reservoir. Our data support a role for PegA in the colonisation of poultry by S. Enteritidis and aid the design of improved vaccines.

Systemic translocation of Salmonella enterica serovar Dublin in cattle occurs predominantly via efferent lymphatics in a cell-free niche and requires type III secretion system 1 (T3SS-1) but not T3SS-2.

Salmonella enterica is an important diarrheal pathogen, and infections may involve severe systemic sequelae depending on serovar- and host-specific factors. The molecular mechanisms underlying translocation of host-restricted and -specific serovars of S. enterica from the intestines to distal organs are ill defined. By surgical cannulation of lymph and blood vessels draining the distal ileum in cattle, S. enterica serovar Dublin was observed to translocate predominantly via mesenteric lymph nodes to efferent lymphatics in a manner that correlates with systemic virulence, since the fowl typhoid-associated serovar Gallinarum translocated at a significantly lower level. While both S. enterica serovars Dublin and Gallinarum were intracellular while in the intestinal mucosa and associated with major histocompatibility complex class II-positive cells, the bacteria were predominantly extracellular within efferent lymph. Screening of a library of signature-tagged serovar Dublin mutants following oral inoculation of calves defined the role of 36 virulence-associated loci in enteric and systemic phases of infection. The number and proportion of tagged clones reaching the liver and spleen early after oral infection were identical to the values in efferent lymph, implying that this may be a relevant mode of dissemination. Coinfection studies confirmed that lymphatic translocation requires the function of type III secretion system 1 (T3SS-1) but, remarkably, not T3SS-2. This is the first description of the mode and genetics of systemic translocation of serovar Dublin in its natural host.

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.

Role in virulence and protective efficacy in pigs of Salmonella enterica serovar Typhimurium secreted components identified by signature-tagged mutagenesis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic enteric pathogen of worldwide importance and pigs are a significant reservoir of human infection. Signature-tagged transposon mutagenesis (STM) was used to identify genes required by S. Typhimurium to colonize porcine intestines. A library of 1045 signature-tagged mutants of S. Typhimurium ST4/74 Nal(R) was screened following oral inoculation of pigs in duplicate. A total of 119 attenuating mutations were identified in 95 different genes, many of which encode known or putative secreted or surface-anchored molecules. A large number of attenuating mutations were located within Salmonella pathogenicity islands (SPI)-1 and -2, confirming important roles for type III secretion systems (T3SS)-1 and -2 in intestinal colonization of pigs. Roles for genes encoded in other pathogenicity islands and islets, including the SPI-6-encoded Saf atypical fimbriae, were also identified. Given the role of secreted factors and the protection conferred against other pathogens by vaccination with extracellular and type III secreted proteins, the efficacy of a secreted protein vaccine from wild-type S. Typhimurium following intramuscular vaccination of pigs was evaluated. Serum IgG responses against type III secreted proteins were induced following vaccination and a significant reduction in faecal excretion of S. Typhimurium was observed in the acute phase of infection compared to mock-vaccinated animals. Vaccination with secreted proteins from an isogenic S. Typhimurium prgH mutant produced comparable levels of protection to vaccination with the preparation from the parent strain, indicating that protection was not reliant on T3SS-1 secreted proteins. The data provide valuable information for the control of Salmonella in pigs.

SiiE is secreted by the Salmonella enterica serovar Typhimurium pathogenicity island 4-encoded secretion system and contributes to intestinal colonization in cattle.

Here we report that Salmonella enterica serovar Typhimurium pathogenicity island 4 carries a type I secretion system (siiCDF) which secretes an approximately 600-kDa protein (encoded by siiE). SiiE is surface expressed, and its production is regulated by HilA. SiiE and SiiF influence colonization in cattle and the invasion of bovine enterocytes.

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium.

The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature-tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI-4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI-4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many 'housekeeping' genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.