SummaryThe 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.

Vibrio cholerae is a motile bacterium responsible for the disease cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates colonization within the intestine and repression of virulence factors maximally induced following colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. cholerae colonization and disease progression.Vibrio cholerae causes the human diarrheal disease cholera. The bacteria are natural inhabitants of aquatic environments and are introduced into the human population through the ingestion of contaminated food or water. Within the human host, V. cholerae expresses virulence factors that facilitate colonization of the intestine (e.g., toxin-coregulated pilus [TCP]) and that stimulate dramatic fluid loss from host tissues (cholera toxin [CT]) (5, 61). A regulatory cascade consisting of a number of different proteins, including ToxR, TcpP, and ToxT, induces the coordinated expression of CT and TCP maximally within the intestine and under specific in vitro growth conditions (for a review, see reference 7).V. cholerae is a highly motile organism by virtue of its single polar flagellum. Flagellar genes are transcribed in a four-tiered transcriptional hierarchy (51). The single class I gene product FlrA activates 54 -dependent transcription of class II genes, which encode components of the MS ring-switch-export apparatus as well as the two-component system FlrBC (31). Phosphorylated FlrC activates 54 -dependent transcription of class III genes, which encode the basal body-hook and the flagellin FlaA (10, 11). Finally, the antisigma factor FlgM is secreted through the basal body-hook to allow 28 -dependent transcription of class IV genes, which encode four additional flagellins and some of the motor components (9, 30). Motility has been linked to the virulence of V. cholerae. Spontaneous nonmot...

No abstract

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.Vibrio cholerae causes the diarrheal disease cholera. This organism is introduced into human populations through the ingestion of contaminated food or water. Within the human, it colonizes the small intestine through the action of a type IV pilus (TCP) and expresses cholera toxin (CT), which causes the electrolyte imbalance and profuse watery diarrhea that is characteristic of this disease. The expression of TCP and CT is coordinated through a complex regulatory cascade that is referred to frequently as the ToxR regulon (for reviews, see references 24 and 31).V. cholerae is a natural inhabitant of the aquatic environment. Epidemic strains can be found in both fresh-and saltwater locations in areas of endemicity and are the cause for the initiation of new cholera epidemics. V. cholerae can form biofilms in the laboratory, and it is believed that this is a likely persistent form of the bacteria within the environment, since biofilms are more resistant to environmental stresses, e.g., chlorine and antibiotics (41,44). A great de...

SummaryThe 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 s 54 -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 wildtype FlrC protein, but not the D54A protein, activates s 54 -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 s 54 -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 wildtype 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 s 54 -dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.

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 granulomalike 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.

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 54 Studies of bacterial flagellar assembly have revealed that the flagellum is assembled in a stepwise fashion that begins by insertion of a type III export apparatus into the cytoplasmic membrane (reviewed in reference 27). Flagellar components are then secreted through this export machinery to be added to the growing tip of the flagellum in the specific order in which they are assembled (reviewed in reference 42). The bulk of the flagellum is composed of flagellin subunits, which are only added to the flagellum after the basal-body-hook structure is completed. Transcription of flagellar genes generally occurs in a hierarchical fashion that mirrors assembly of the nascent flagellum; i.e., the genes encoding early flagellar components are transcribed prior to the genes encoding late flagellar components, such as flagellin subunits (24; reviewed in reference 41).In Salmonella enterica serovar Typhimurium, transcription of the flagellin gene is repressed until the basal-body-hook structure is completed, through the action of an anti-sigma factor, FlgM. The flagellin gene is transcribed by RNA polymerase (RNAP) containing the alternate sigma factor 28 (encoded by fliA) (34). FlgM binds to 28 and prevents its association with RNAP, preventing flagellin gene transcription (23). However, once the basal-body-hook structure is assembled, FlgM is secreted through the flagellar export apparatus to the extracellular milieu, which allows 28 to associate with RNAP and transcribe the flagellin gene (15). Thus, the function of FlgM is to couple flagellar assembly to appropriate temporal flagellar gene transcription.Flagellar gene transcription in Vibrio cholerae, which possesses a single polar sheathed flagellum, is also organized into a transcription hierarchy (35). However, the four-tiered transcription hierarchy has notable differences from the threetiered hierarchy of S. enterica serovar Typhimurium, which possess multiple peritrichous flagella. In V. cholerae, the genes encoding the early structural components of the flagellum (basal-body-hook) are transcribed in two distinct temporal classes (II and III) by RNAP containing the alternate sigma factor 54 ; in contrast, transcription of the equivalent genes in S. enterica serovar Typhimurium occurs within the single "early" temporal class (II) by RNAP containing the housekeeping 70 subunit. However, the last temporal class of flagellar genes in V. cholerae (class IV), as in S. enterica serovar Typhimurium (class III), is transcribed by RNAP containing 28 . The main 28 -dependent genes are four distinct flagellin genes, flaB, flaC, flaD, and flaE (20, 35).54 -dependent transcription of a fifth flagellin gene, flaA, precedes transcription of the other four flagellin genes (20). The four-tiered 54 -and 28 -depend...

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 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