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...

Immunologic tolerance was originally defined as specific hyporesponsiveness to an antigen which results from prior contact with that antigen (1, 2). The concept of natural tolerance to self-antigens was perceived by Burnet (3) as the deletion of clones reactive to autologous antigens after contact of these antigens with the reactive cells' specific antigen receptors during the development of the cell's immunologic competence. If such a theory were to have physiological relevance, then several predictions should be realized: (a) developing cells should be significantly more susceptible to tolerance induction than their mature counterparts; (b) tolerance should be inducible over a wide range of antigen concentrations, since the concentrations of different self-antigens present presumably vary; (c) since the spectrum of self-antigens is diverse, the development of unresponsiveness should be plausible with a wide variety of antigenic structures; (d) since bone marrow-derived precursors to antibody-forming cells (B cells) presumably mature independently of other cell types, induction of B-cell tolerance should occur in the absence of antigen-specific thymus-derived lymphocytes (T cells); (e) the discrimination between self and nonself would be absolute, and therefore, tolerance should exhibit exquisite determinant specificity.

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.

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...

The study of the effects of osmolarity and growth phase on Salmonella typhi adherence to and invasion of Henle 407 epithelial cells provides the first evidence of a clear overlap between these two environmental stimuli. High-osmolarity conditions are required in the late-log phase for optimum induction of the adherent and invasive phenotypes.