The Vibrio cholerae FlgM homologue is an anti-sigma28 factor that is secreted through the sheathed polar flagellum

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 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.

FIG. 1.

Interaction of V. cholerae FlgM with σ²⁸. His-σ²⁸ and FLAG-FlgM were expressed separately and together in E. coli BL21(DE3), which contains T7 polymerase, from T7 promoters in plasmids pKEK462 and pKEK470, respectively, and magnetic anti-His beads (Dynal Biotech) were used to capture His-tagged protein from cell lysates as described in Materials and Methods. In the upper panel, samples were separated on Coomassie-stained polyacrylamide gel, and the lower panels show Western immunoblots of these same samples probed with anti-His tag and anti-FLAG tag antibodies. Masses of molecular size markers are noted on the left in kilodaltons, and arrows depict migration of His-σ²⁸ and FLAG-FlgM. Lane numbers correspond to BL21(DE3) cells expressing His-σ²⁸ (lane 1, whole-cell lysate; lane 2, anti-His bead eluate), FLAG-FlgM (lane 3, whole-cell lysate, lane 4, anti-His bead eluate), and His-σ²⁸ and FLAG-FlgM (lane 5, whole-cell lysate, lane 6, anti-His bead eluate).

FIG. 2.

FlgM is secreted through the V. cholerae flagellum. FLAG-FlgM was expressed from plasmid pKEK474 in V. cholerae strains KKV598 (wild type [WT]) and KKV1247 (fliF mutant). Bacterial pellets (P) and supernatants (S) were processed for Western immunoblot analysis with anti-FLAG antibodies as described in Materials and Methods.

FIG. 3.

Motility phenotype and electron micrograph of a V. cholerae flgM strain. (A) V. cholerae strains KKV598 (wild type [WT]), KKV1114 (fliA mutant), KKV1461 (flgM mutant), and KKV1247 (fliF mutant) were inoculated into motility agar and incubated at 30°C. Electron micrographs of V. cholerae strains KKV598 (wild type) (B) and KKV1461 (flgM) (C) are also shown. Bars are equivalent to 2 μm.

FIG. 4.

Expression of flagellin promoters in fliA and flgM mutant V. cholerae strains. V. cholerae strains KKV598 (wild type), KKV113 (fliA mutant), KKV1461 (flgM mutant), and KKV1384 (fliA flgM mutant) carrying plasmids pKEK80 (flaAp-lacZ), pKEK79 (flaBp-lacZ), pKEK76 (flaCp-lacZ), pKEK77 (flaDp-lacZ), and pKEK81 (flaEp-lacZ) were assayed for β-galactosidase activity during logarithmic growth in LB. Assays were performed in triplicate, and standard deviations are shown.

FIG. 5.

Expression of class II, III, and IV flagellar promoters in a fliF mutant V. cholerae strain. V. cholerae strains KKV598 (wild type) and KKV1247 (fliF mutant) carrying plasmids pKEK72 (flrBp-lacZ), pKEK327 (fliEp-lacZ), pKEK329 (flhAp-lacZ), pKEK332 (flgBp-lacZ), pKEK331 (flgKp-lacZ), pKEK80 (flaAp-lacZ), pKEK415 (flaGp-lacZ), pKEK79 (flaBp-lacZ), pKEK76 (flaCp-lacZ), pKEK77 (flaDp-lacZ), and pKEK81 (flaEp-lacZ) were assayed for β-galactosidase activity during logarithmic growth in LB. Assays were performed in triplicate, and standard deviations are shown.

FIG. 6.

Intestinal colonization of fliA and flgM mutant V. cholerae strains. Strains KKV1113 (fliA mutant), KKV1461 (flgM mutant), and KKV1384 (fliA flgM mutant) were coinoculated with O395 perorally into infant mice at a ratio of ∼1:1; intestinal homogenates were recovered at 24 h postinoculation, and CFU of wild-type and mutant strains were counted. The CI is given as the output mutant/wild-type ratio divided by the input mutant/wild-type ratio; each value shown is from an individual mouse.