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Salmonella enterica serotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgK mutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljB mutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3␣ and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.Salmonella enterica serotype Typhimurium is a major cause of gastroenteritis in humans, which is characterized by acute intestinal inflammation and diarrhea (11,36). One of the serotype Typhimurium virulence factors contributing to intestinal inflammation are flagella. Nonflagellated serotype Typhimurium mutants have been shown to cause less inflammation than their isogenic parents do after infection of bovine ligated ileal loops (59), streptomycin-pretreated mice (65, 74), and chickens (24).Several possible mechanisms by which flagella may contribute to eliciting proinflammatory responses have been proposed. Flagella are surface appendages of serotype Typhimurium that are required for motility and chemotaxis. Motility contributes to serotype Typhimurium invasion of intestinal epithelial cell lines by increasing bacterial contact with host cells (26,27). The invasion-associated type III secretion system (T3SS-1) is important for inducing intestinal inflammation in animal models (1,20,70,81). Nonmotile serotype Typhimurium mutants may thus cause reduced intestinal inflammation in vivo because the efficiency of T3SS-1-mediated invasion is reduced.In addition to its role in motility and invasion, the proteinaceous monomer of the flagellar filament, flagellin, has been shown to be a potent activator of the innate immune response in tissue culture models. Flagellin is an agonist of Toll-like ...

SummarySalmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogenactivated protein kinases (MAPK) through b-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an antiinflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.

Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteraemia, by many pathogenic bacteria. Salmonella enterica serovar Choleraesuis is an important enteric pathogen that causes serious systemic infections in swine and humans. Here, it was found that, when introduced into Escherichia coli, a recombinant plasmid carrying the pagC gene from a plasmid-based genomic library of S. enterica serovar Choleraesuis conferred a high-level resistance to the bactericidal activity of pooled normal swine serum. The resistance was equal to the level conferred by rck, a gene encoding a 17 kDa outer-membrane protein which promotes the serum resistance phenotype in S. enterica serovar Typhimurium. Insertional mutagenesis of the cloned pagC gene generated a mutation that resulted in the loss of the serum resistance phenotype in E. coli. When this mutation was introduced into the chromosome of S. enterica serovar Choleraesuis by homology recombination with the wild-type allele, the resulting strain could not produce PagC, and it showed a decreased level of resistance to complement-mediated killing. The mutation could be restored by introduction of the intact pagC gene on a plasmid, but not by introduction of the point-mutated pagC gene. In addition, PagC was able to promote serum resistance in the S. enterica serovar Choleraesuis LPS mutant strain, which is highly sensitive to serum killing. Although PagC is not thought to confer serum resistance directly, these results strongly suggest that PagC is an important outer-membrane protein that plays an important role in the serum resistance of S. enterica serovar Choleraesuis.

Salmonella pathogenicity islands are inserted into the genome by horizontal gene transfer and are required for expression of full virulence. Here, we performed tRNA scanning of the genome of Salmonella enterica serovar Typhimurium and compared it with that of nonpathogenic Escherichia coli in order to identify genomic islands that contribute to Salmonella virulence. Using deletion analysis, we identified four genomic islands that are required for virulence in the mouse infection model. One of the newly identified pathogenicity islands was the pheV-tRNA-located genomic island, which is comprised of 26 126 bp, and encodes 22 putative genes, including STM3117-STM3138. We also showed that the pheV tRNA-located genomic island is widely distributed among different nontyphoid Salmonella serovars. Furthermore, genes including STM3118-STM3121 were identified as novel virulence-associated genes within the pheV-tRNA-located genomic island. These results indicate that a Salmonella-specific pheV-tRNA genomic island is involved in Salmonella pathogenesis among the nontyphoid Salmonella serovars.

The complete nucleotide sequence of pKDSC50, a large virulence plasmid from Salmonella enterica serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the spv operon, which is composed of genes essential for systemic infection by nontyphoidal Salmonella. Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the spvRABCD genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the pef (plasmid-encoded fimbria) operon and rck (resistance to complement killing) gene, which has been identified on the virulence plasmid of S. enterica serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic Escherichia coli O157:H7 and the enteropathogenic E. coli ( Plasmid-encoded gene products are required for full expression of virulence in many enteropathogenic bacteria, including those of the genera Shigella (53, 54) and Yersina (17,20), as well as Salmonella (12,33,35,47,56). Nontyphoidal Salmonella serovars are important agents of gastroenteritis and can cause systemic infection, such as bacteremia (septicemia), in animals and humans. Many of these serotypes typically carry large plasmids which are essential to the production of systemic infection in animal models (21,23). Although the virulence plasmids of these Salmonella strains are variable in size, ranging from 50 to 94 kb, their distribution is dependent on the serotype. For example, S. enterica serovar Choleraesuis, S. enterica serovar Enteritidis, S. enterica serovar Dublin, S. enterica serovars Gallinarum and Pullorum, and S. enterica serovar Typhimurium harbor the 50-, 60-, 80-, 90-, and 94-kb virulence plasmids, respectively.Strains of serovar Typhimurium cured of the virulence plasmid are strongly attenuated in their subsequent spreading infection to the mesenteric lymph nodes, spleen, and liver (23), while the presence of the virulence plasmid of Salmonella does not appear to be required for bacterial adherence to and invasion of cultured eukaryotic cells or for colonization of the cecum or invasion of Peyer's patches in the mouse (24, 42). All of these virulence plasmids contain a highly conserved 8-kb region, which contains the spv (Salmonella plasmid virulence) locus that can confer complete virulence on a strain of serovar Typhimurium cured of the plasmid (25).The spv region consists of spvR, a gene that encodes a transcriptional factor of the LysR family, and the spvABCD operon of structural genes (1,2,22,25,37). The spv operon is required for the systemic phase of disease in specific hosts, i.e., serovar Choleraesuis in pigs (15), serovar Dublin in cattle (39, 61), serovars Gallinarum and Pullorum in fowl (5, 6), and serova...