Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.cholera ͉ host interactions ͉ environmental persistence C holera continues to be a major public and individual health problem, especially in those regions of the world where it is endemic. Colwell (1) first hypothesized that coastal waters were an important reservoir of Vibrio cholerae. Huq et al. (2) reported that V. cholerae O1 cells could be observed to be attached to a variety of phytoplankton and zooplankton species. The incidence and severity of epidemics have been linked to salinity, water temperature, turbidity, and plankton blooms (3, 4). Cholera epidemics occur in a regular seasonal pattern. It has been suggested that during interepidemic periods V. cholerae exists in an unexplained ecological association with aquatic organisms (5). During the environmental phase, V. cholerae resides in diverse aquatic environments, often in association with marine plankton (6). The association of V. cholerae with zooplankton has proven to be a key factor in deciphering the global nature of cholera epidemics (7). In such natural bacterioplankton communities V. cholerae and other bacteria are also at the base of the pelagic microbial food web (8). Bacterial growth and survival are subject to constraint by bacteriovorous predators, e.g., protozoa such as ciliates and flagellates (9, 10). Little has been known about mechanisms and adaptations of bacteria to reduce grazing mortality compared with adaptations toward abiotic factors (substrate, temperature, pH, etc.) (11).V. cholerae expresses well characterized factors to establish and cause disease in the mammalian host, including cholera toxin (CT) and toxin-coregulated pili (Tcp). It has been shown that quorum sensing (QS) plays a role in the regulation of virulence in V. cholerae (12). At least three autoinducer signaling circuits function through the action of LuxO, leading to the repression of...

Rod-shaped bacteria, probably of the indicated species, constituted a significant fraction of the proximal small intestine microbiota in children born during the Swedish CD epidemic and may have been an important risk factor for CD contributing to the fourfold increase in disease incidence in children below 2 years of age during that time.

The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature‐ and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non‐pathogenic Gram+ and Gram− bacteria from the apical side and the proinflammatory cytokines interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin‐2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA‐related cell adhesion molecule‐1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human β‐defensin‐1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL‐8 and the cytokines IL‐6 and TNF‐α. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF‐α levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF‐α induced significantly increased levels of IL‐8 and TNF‐α itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1‐L and CEACAM1‐S. In T84 cells, IFN‐γ was selective for CEACAM1‐L while TNF‐α upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.

Background Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV.Methodology/Principal FindingsCulture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-α (TNF-α) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis.Conclusions/SignificanceWe suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-α in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.

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