This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-α or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.

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...

Thirty-two clinical isolates of Shiga-like toxin (SLT)-producing Escherichia coli associated with single cases or outbreaks of bloody diarrhea, hemorrhagic colitis, the hemolytic uremic syndrome, or edema disease of swine were examined for multiple copies of genes belonging to the slt-I or slt-II toxin families. Five of 19 strains that were known to produce SLT-II or to hybridize to slt-II-specific probes by colony blot were found by Southern hybridization to contain two copies of toxin genes related to slt-IH. The genes for two toxins closely related to slt-II were cloned from one of the isolates, Escherichia coli 0157:H-strain E32511. One copy of the operon was found to be essentially identical to slt-IH; it differed from sit-II by only one nucleotide base. This single nucleotide difference did not affect the predicted amino acid sequence. The predicted amino acid sequence of the A subunit of the second operon was identical to that of SLT-II, but the predicted amino acid sequence of the B subunit was identical to that of the B2F1 toxin VT2ha. We designated this second operon sit-IIc. Neutralization assays using several monoclonal antibodies and polyclonal antiserum prepared against SLT-IH showed that SLT-IIc was antigenically related to but distinct from SLT-II.

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...

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