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...

Vibrio cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this colonization were investigated. Successful colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX), and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. cholerae encounters barriers to infection including an acidic environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause cholera disease. This adult mouse model of intestinal V. cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.

Cholera is classically considered a noninflammatory diarrheal disease, in comparison to invasive enteric organisms, although there is a low-level proinflammatory response during early infection with Vibrio cholerae and a strong proinflammatory reaction to live attenuated vaccine strains. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host defense to infection. Nontoxigenic El Tor O1 V. cholerae infection is characterized by the upregulation of interleukin-6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha in the intestine, indicating an acute innate immune response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to decreased survival of mice. The role of neutrophils in protection of the host is to limit the infection to the intestine and control bacterial spread to extraintestinal organs. In the absence of neutrophils, the infection spread to the spleen and led to increased systemic levels of IL-1␤ and tumor necrosis factor alpha, suggesting the decreased survival in neutropenic mice is due to systemic shock. Neutrophils were found not to contribute to either clearance of colonizing bacteria or to alter the local immune response. However, when genes for secreted accessory toxins were deleted, the colonizing bacteria were cleared from the intestine, and this clearance is dependent upon neutrophils. Thus, the requirement for accessory toxins in virulence is negated in neutropenic mice, which is consistent with a role of accessory toxins in the evasion of innate immune cells in the intestine. Overall, these data support that neutrophils impact disease progression and suggest that neutrophil effectiveness can be manipulated through the deletion of accessory toxins. G lobal pandemics of the diarrheal disease cholera have occurred throughout human history. The disease is caused by ingestion of the Gram-negative bacterium Vibrio cholerae in contaminated water. Cholera is typified by symptoms of severe, watery diarrhea that leads to life-threatening dehydration and loss of electrolytes (48). There have been seven cholera pandemics in recorded history. The first six were caused by V. cholerae O1 of the classical biotype. The seventh and currently ongoing pandemic began in 1961, when V. cholerae O1 strains of the El Tor biotype began to predominate and spread throughout the world (22). Recent whole-genome analysis of single-nucleotide polymorphisms revealed that the seventh pandemic consists of three distinct waves of transmission from the pandemic center in South Asia. Strain diversification has resulted from continuous local evolution punctuated by sporadic long-range transmission to countries where this pathogen is not endemic, such as in the case of Haiti in 2010 (34). It is estimated that 1.4 billion people worldwide are at risk for cholera, with approximately 2.8 million cholera cases occurring in areas of endemicity each year (1).The innate immune response to V. cholerae infection is poorly understood. Recent studies i...

bVibrio cholerae O1 El Tor strains have been responsible for pandemic cholera since 1961. These strains have evolved over time, spreading globally in three separate waves. Wave 3 is caused by altered El Tor (AET) variant strains, which include the strain with the signature ctxB7 allele that was introduced in 2010 into Haiti, where it caused a devastating epidemic. In this study, we used phenotypic analysis to compare an early isolate from the Haiti epidemic to wave 1 El Tor isolates commonly used for research. It is demonstrated that the Haiti isolate has increased production of cholera toxin (CT) and hemolysin, increased motility, and a reduced ability to form biofilms. This strain also outcompetes common wave 1 El Tor isolates for colonization of infant mice, indicating that it has increased virulence. Monitoring of CT production and motility in additional wave 3 isolates revealed that this phenotypic variation likely evolved over time rather than in a single genetic event. Analysis of available whole-genome sequences and phylogenetic analyses suggested that increased virulence arose from positive selection for mutations found in known and putative regulatory genes, including hns and vieA, diguanylate cyclase genes, and genes belonging to the lysR and gntR regulatory families. Overall, the studies presented here revealed that V. cholerae virulence potential can evolve and that the currently prevalent wave 3 AET strains are both phenotypically distinct from and more virulent than many El Tor isolates.

Colon cancer is a leading cause of cancer morbidity and mortality, and it is hypothesized that dysbiosis in the gut microbiota contributes to colon tumorigenesis. Fusobacterium nucleatum , a member of the oropharyngeal microbiome, is enriched in a subset of human colon tumors. However, it is unclear whether this genetically varied species directly promotes tumor formation, modulates mucosal immune responses, or merely colonizes the tumor microenvironment.