We report here the development of a pathogenesis model utilizing
Mycobacterium marinum infection of zebrafish (
Danio rerio) for the study of mycobacterial disease. The zebrafish model mimics certain ...aspects of human tuberculosis, such as the formation of granuloma-like lesions and the ability to establish either an acute or a chronic infection based upon inoculum. This model allows the genetics of mycobacterial disease to be studied in both pathogen and host.
The human pathogen Vibrio cholerae specifically expresses virulence factors within the host, including cholera toxin (CT) and the toxin co‐regulated pilus (TCP), which allow it to colonize the ...intestine and cause disease. V. cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence, yet the exact role of motility in pathogenesis has remained undefined. The two‐component regulatory system FlrB/FlrC is required for polar flagellar synthesis; FlrC is a σ54‐dependent transcriptional activator. We demonstrate that the transcriptional activity of FlrC affects both motility and colonization of V. cholerae. In a purified in vitro reaction, FlrB transfers phosphate to the wild‐type FlrC protein, but not to a mutant form in which the aspartate residue at amino acid position 54 has been changed to alanine (D54A), consistent with this being the site of phosphorylation of FlrC. The wild‐type FlrC protein, but not the D54A protein, activates σ54‐dependent transcription in a heterologous system, demonstrating that phospho‐FlrC is the transcriptionally active form. A V. cholerae strain containing a chromosomal flrCD54A allele did not synthesize a flagellum and had no detectable levels of transcription of the critical σ54‐dependent flagellin gene flaA. The V. cholerae flrCD54A mutant strain was also defective in its ability to colonize the infant mouse small intestine, approximately 50‐fold worse than an isogenic wild‐type strain. Another mutation of FlrC (methionine 114 to isoleucine; M114I) confers constitutive transcriptional activity in the absence of phosphorylation, but a V. cholerae flrCM114I mutant strain, although flagellated and motile, was also defective in its ability to colonize. The strains carrying D54A or M114I mutant FlrC proteins expressed normal levels of CT and TCP under in vitro inducing conditions. Our results show that FlrC ‘locked’ into either an inactive (D54A) or an active (M114I) state results in colonization defects, thereby demonstrating a requirement for modulation of FlrC activity during V. cholerae pathogenesis. Thus, the σ54‐dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.
The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum. Flagellar transcriptional regulatory factors have been demonstrated to contribute to V. cholerae ...virulence, but the role these factors play in the transcription hierarchy controlling flagellar synthesis has been unclear. The flagellar genes revealed by the V. cholerae genome sequence are located in three large clusters, with the exception of the motor genes, which are found in three additional locations. It had previously been demonstrated that the alternative sigma factor σ54 and the σ54‐dependent activators FlrA and FlrC are necessary for flagellar synthesis. The V. cholerae genome sequence revealed the presence of a fliA gene, which is predicted to encode the alternative flagellar sigma factor σ28. A V. choleraeΔfliA mutant strain is non‐motile, and synthesizes a truncated flagellum. Vibrio cholerae FliA complements both V. cholerae and Salmonella typhimurium fliA mutants for motility, consistent with its function as an alternative flagellar sigma factor. Analysis of lacZ transcriptional fusions of the V. cholerae flagellar promoters in both V. cholerae and S. typhimurium identified σ28‐, σ54‐, FlrA‐ and FlrC‐dependent promoters, as well as promoters that were independent of all these factors. Our results support a model of V. cholerae flagellar gene transcription as a novel hierarchy composed of four classes of genes. Class I is composed solely of the gene encoding the σ54‐dependent activator FlrA, which along with the σ54‐holoenzyme form of RNA polymerase activates expression of Class II genes. These genes include structural components of the MS ring, switch and export apparatus, as well as the genes encoding both FliA and FlrC. FlrC, along with σ54‐holoenzyme, activates expression of Class III genes, which include basal body, hook and filament genes. Finally, σ28‐holoenzyme activates expression of Class IV genes, which include additional filament genes as well as motor genes. Thus, this novel V. cholerae flagellar hierarchy has incorporated elements from both the σ54‐dependent Caulobacter crescentus polar flagellar hierarchy and the σ28‐dependent S. typhimurium peritrichous flagellar hierarchy.
ABSTRACT
Vibrio cholerae
has a single polar sheathed flagellum that propels the cells of this bacterium. Flagellar synthesis, motility, and chemotaxis have all been linked to virulence in this human ...pathogen.
V. cholerae
expresses flagellar genes in a hierarchy consisting of σ
54
- and σ
28
-dependent transcription. In other bacteria, σ
28
transcriptional activity is controlled by an anti-σ
28
factor, FlgM. We demonstrate that the
V. cholerae
FlgM homologue (i) physically interacts with σ
28
, (ii) has a repressive effect on some
V. cholerae
σ
28
-dependent flagellar promoters, and (iii) is secreted through the polar sheathed flagellum, consistent with anti-σ
28
activity. Interestingly, FlgM does not have a uniform repressive effect on all σ
28
-dependent promoters, as determined by measurement of σ
28
-dependent transcription in cells either lacking FlgM (Δ
flgM
) or incapable of secretion (Δ
fliF
). Further analysis of a Δ
fliF
strain revealed that this flagellar assembly block causes a decrease in class III (FlrC- and σ
54
-dependent) and class IV (σ
28
-dependent), but not class II (FlrA- and σ
54
-dependent), flagellar transcription.
V. cholerae flgM
and
fliA
(encodes σ
28
) mutants were only modestly affected in their ability to colonize the infant mouse intestine, a measure of virulence. Our results demonstrate that
V. cholerae
FlgM functions as an anti-σ
28
factor and that the sheathed flagellum is competent for secretion of nonstructural proteins.
Vibrio cholerae has a single polar sheathed flagellum that propels the cells of this bacterium. Flagellar synthesis, motility, and chemotaxis have all been linked to virulence in this human pathogen. ...V. cholerae expresses flagellar genes in a hierarchy consisting of sigma54- and sigma28-dependent transcription. In other bacteria, sigma28 transcriptional activity is controlled by an anti-sigma28 factor, FlgM. We demonstrate that the V. cholerae FlgM homologue (i) physically interacts with sigma28, (ii) has a repressive effect on some V. cholerae sigma28-dependent flagellar promoters, and (iii) is secreted through the polar sheathed flagellum, consistent with anti-sigma28 activity. Interestingly, FlgM does not have a uniform repressive effect on all sigma28-dependent promoters, as determined by measurement of sigma28-dependent transcription in cells either lacking FlgM (DeltaflgM) or incapable of secretion (DeltafliF). Further analysis of a DeltafliF strain revealed that this flagellar assembly block causes a decrease in class III (FlrC- and sigma54-dependent) and class IV (sigma28-dependent), but not class II (FlrA- and sigma54-dependent), flagellar transcription. V. cholerae flgM and fliA (encodes sigma28) mutants were only modestly affected in their ability to colonize the infant mouse intestine, a measure of virulence. Our results demonstrate that V. cholerae FlgM functions as an anti-sigma28 factor and that the sheathed flagellum is competent for secretion of nonstructural proteins.
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