Zebrafish-Mycobacterium marinum model for mycobacterial pathogenesis.

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.

Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC.

The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence. It has previously been demonstrated that the sigma(54)-dependent activator FlrC is necessary for both flagellar synthesis and for enhanced intestinal colonization. In order to characterize FlrC binding, we analyzed two FlrC-dependent promoters, the highly transcribed flaA promoter and the weakly transcribed flgK promoter, utilizing transcriptional lacZ fusions, mobility shift assays, and DNase I footprinting. Promoter fusion studies showed that the smallest fragment with wild-type transcriptional activity for flaAp was from positions -54 to +137 with respect to the start site, and from -63 to +144 for flgKp. Gel mobility shift assays indicated that FlrC binds to a fragment containing the region from positions +24 to +95 in the flaAp, and DNase I footprinting identified a protected region between positions +24 and +85. Mobility shift and DNase I footprinting indicated weak binding of FlrC to a region downstream of the flgKp transcription start site. These results demonstrate a relatively novel sigma(54)-dependent promoter architecture, with the activator FlrC binding downstream of the sigma(54)-dependent transcription start sites. When the FlrC binding site(s) in the flaA promoter was moved a large distance (285 bp) upstream of the transcription start site of either flaAp or flgKp, high levels of FlrC-dependent transcription resulted, indicating that this binding region functions as an enhancer element. In contrast, the relatively weak FlrC binding site(s) in the flgK promoter failed to function as an enhancer element at either promoter, suggesting that FlrC binding strength contributes to enhancer activity. Our results suggest that the differences in FlrC binding to various flagellar promoters results in the differences in transcription levels that mirror the relative requirement for the flagellar components within the flagellum.

Roles of the regulatory proteins FlhF and FlhG in the Vibrio cholerae flagellar transcription hierarchy.

Vibrio cholerae, the causative agent of the human diarrheal disease cholera, is a motile bacterium with a single polar flagellum, and motility has been inferred to be an important aspect of virulence. The V. cholerae flagellar hierarchy is organized into four classes of genes. The expression of each class of genes within a flagellar hierarchy is generally tightly regulated in other bacteria by both positive and negative regulatory elements. To further elucidate flagellar biogenesis in V. cholerae, we characterized the roles of the three putative regulatory genes, flhF, flhG, and VC2061. V. cholerae flhF and flhG mutants appeared nonmotile in a soft agar assay. Electron microscopy revealed that the flhF mutant lacked a polar flagellum, while interestingly, the flhG mutant possessed multiple (8 to 10) polar flagella per cell. The transcriptional activity of class III and class IV gene promoters in the flhF mutant was decreased, suggesting that FlhF acts as a positive regulator of class III gene transcription. The transcription of all four classes of flagellar promoters was increased in the flhG mutant, suggesting that FlhG acts as a negative regulator of class I gene transcription. Additionally, the ability to colonize the infant mouse intestine was reduced for the flhG mutant (approximately 10-fold), indicating that the negative regulation of class I flagellar genes enhances virulence. The V. cholerae VC2061 mutant was motile and produced a polar flagellum indistinguishable from that of the wild type, and the transcriptional activities of the four classes of flagellar promoters were similar to that of the wild type. Our results indicate that FlhG and FlhF regulate class I and class III flagellar transcription, respectively, while VC2061 plays no detectable role in V. cholerae flagellar biogenesis.

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

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.

The sodium-driven flagellar motor controls exopolysaccharide expression in Vibrio cholerae.

Vibrio cholerae causes the life-threatening diarrheal disease cholera. This organism persists in aquatic environments in areas of endemicity, and it is believed that the ability of the bacteria to form biofilms in the environment contributes to their persistence. Expression of an exopolysaccharide (EPS), encoded by two vps gene clusters, is essential for biofilm formation and causes a rugose colonial phenotype. We previously reported that the lack of a flagellum induces V. cholerae EPS expression. To uncover the signaling pathway that links the lack of a flagellum to EPS expression, we introduced into a rugose flaA strain second-site mutations that would cause reversion back to the smooth phenotype. Interestingly, mutation of the genes encoding the sodium-driven motor (mot) in a nonflagellated strain reduces EPS expression, biofilm formation, and vps gene transcription, as does the addition of phenamil, which specifically inhibits the sodium-driven motor. Mutation of vpsR, which encodes a response regulator, also reduces EPS expression, biofilm formation, and vps gene transcription in nonflagellated cells. Complementation of a vpsR strain with a constitutive vpsR allele likely to mimic the phosphorylated state (D59E) restores EPS expression and biofilm formation, while complementation with an allele predicted to remain unphosphorylated (D59A) does not. Our results demonstrate the involvement of the sodium-driven motor and suggest the involvement of phospho-VpsR in the signaling cascade that induces EPS expression. A nonflagellated strain expressing EPS is defective for intestinal colonization in the suckling mouse model of cholera and expresses reduced amounts of cholera toxin and toxin-coregulated pili in vitro. Wild-type levels of virulence factor expression and colonization could be restored by a second mutation within the vps gene cluster that eliminated EPS biosynthesis. These results demonstrate a complex relationship between the flagellum-dependent EPS signaling cascade and virulence.