SummaryEnteritis induced by non-typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella-induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sipdependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.

SummarySalmonella spp. interact with ileal mucosa and disrupt normal intestinal function, which results in an acute inflammatory cell influx, fluid secretion and enteritis. We have recently characterized SopB, a novel secreted effector protein of Salmonella dublin, and presented evidence that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses. Here, we show that sopB is located on a large DNA fragment unique to the Salmonella chromosome. This locus is conserved in Salmonella and maps at approximately 20 centisome of the S. typhimurium chromosome. Sequence analysis revealed that this Salmonella-specific DNA fragment is flanked by DNA sequences with significant sequence similarity to the Escherichia coli K-12 genes, tRNA 1 Ser (serT ) on one side and copS/copR on the other. Thus, this Salmonella-specific DNA fragment has features characteristic of 'pathogenicity islands' and, therefore, it was denoted SPI-5 (Salmonella pathogenicity island-5). SPI-5 was sequenced and was found to contain five novel genes, pipA , pipB, pipC, pipD (pathogenicity island-encoded proteins) and orfX, in addition to sopB. The effect of mutations in pipA , pipB and pipD on the induction of fluid secretion and an acute inflammatory cell influx was assessed in bovine ligated ileal loops. The effect of mutations in SPI-5-encoded genes on systemic salmonellosis was assessed in mice. The results of these experiments suggest that SPI-5-encoded genes contribute to enteric but not to systemic salmonellosis.

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.Over 40 genes are required for the structure, assembly, and function of flagella (25). These genes are categorized into three classes that are temporally expressed in a cascade-like manner. Class 1 genes include the master regulatory genes (flhD and flhC) which are required for the activation of transcription from class 2 promoters. Class 2 genes encode hookbasal body proteins (which make up the flagellar secretory apparatus), as well as the alternate sigma factor (FliA) which transcribes class 3 genes involved in motor and chemotaxis functions and filament structures. Regulation of flagellar synthesis is accomplished through an interaction between FliA and FlgM, an antisigma factor.Previously, we and others demonstrated that flagella are not required for Salmonella enterica serovar Typhimurium virulence in the murine typhoid model (3, 23). Rather, we showed that some aspects of flagellar regulation, namely the FlgMFliA regulatory system, are involved in the in vivo pathogenicity of serovar Typhimurium. Specifically, we found that the flgM gene, which encodes a negative regulator of flagellar synthesis (12), is required for the virulence...

SummarySirA of Salmonella typhimurium is known to regulate the hilA and prgH genes within Salmonella pathogenicity island 1 (SPI1). To identify more members of the SirA regulon, we screened 10 000 random lacZY fusions (chromosomal MudJ insertions) for regulation by SirA and identified 10 positively regulated fusions. Three fusions were within the SPI1 genes hilA (an SPI1 transcriptional regulator), spaS (a component of the SPI1 type III export apparatus) and sipB (a substrate of the SPI1 export apparatus). Two fusions were within the sopB gene (also known as sigD ). sopB is located within SPI5, but encodes a protein that is exported via the SPI1 export apparatus. In addition, five fusions were within genes of unknown function that are located in SPI4. As spaS and sipB were likely to be hilA dependent, we tested all of the fusions (except hilA) for hilA dependence. Surprisingly, we found that all of the fusions require hilA for expression and that plasmid-encoded SirA cannot bypass this requirement. Therefore, SirA regulates hilA, the product of which regulates genes within SPI1, SPI4 and SPI5. Both sirA and hilA mutants are dramatically attenuated in a bovine model of gastroenteritis, but have little or no effect in the mouse model of typhoid fever. This study establishes the SirA /HilA regulatory cascade as the primary regulon controlling enteropathogenic virulence functions in S. typhimurium. Because S. typhimurium causes gastroenteritis in both cattle and humans, we believe that this information may be directly applicable to the human disease.

Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. Salmonella spp. produce diseases that range from a mild enteritis to a severe systemic infection in a variety of animal hosts. Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different major surface proteins, the flagellin subunit proteins FljB and FliC, no biological function has been ascribed to this capacity. Over 40 genes, arranged in 17 operons, are required for the structure, assembly, and function of flagella (34). We have demonstrated that flagellar regulation is linked to virulence of S. enterica serovar Typhimurium (6,7,45,46,57). A region adjacent to flagellar genes (flg), originally named mviS, was shown to be required for virulence in mice (6). Subsequently, that gene was identified as flgM (46). The flgM product is an anti-sigma factor that negatively regulates flagellar synthesis by inhibiting FliA, an alternate sigma factor required for the transcription of late-class flagellar genes (25). A mutation in flgM causes decreased survival of Salmonella in mouse peritoneal macrophages and attenuation of virulence in a mouse model of typhoid fever (46).In S. enterica serovar Typhimurium, expression of flagella is also controlled by phase variation, a mechanism by which the organism alternately expresses two different types of flagellin subunit proteins, FljB and FliC. Flagellar phase variation was first described in Salmonella by Andrewes over 75 years ago (2). Since that time, many studies have focused on the molecular mechanism of switching of flagellin type, and a model has been generated (13, 14, 18, 22, 24, 26, 27, 29, 30, 39, 40, 47-49, 59, 60). Flagellar phase variation involves the inversion of approximately 1 kb of DNA containing the promoter of fljB (49,59,60). In one orientation, the promoter is situated directly upstream of the...