Abstract
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.
Highlights
Vibrio cholerae, the causal organism of the diarrheal disease cholera, is a Gram-negative motile bacterium acquired via intake of contaminated food or water
Permeability was assessed as transepithelial electrical resistance (TER) and the average expression levels/cell of mRNA for the pro-inflammatory cytokines IL-8, IL-6, and tumor necrosis factor-a (TNF-a) and the down-regulatory, wound-healing, and ‘‘epithelium-sealing’’ cytokine transforming growth factor-b1 (TGF-b1) were determined
Culture supernatants from DprtV caused a significant drop in TER, i.e., 12.561.1% of that in the LB broth control, suggesting a significant increase in permeability (Fig. 1a). mRNA expression levels in LB broth controls were low for IL-6 and TNF-a, intermediate for IL-8 (4.664.3 mRNA copies/18S rRNA unit, n = 3) and high for TGF-b1 (33.665.2 mRNA copies/18S rRNA unit, n = 3)
Summary
The causal organism of the diarrheal disease cholera, is a Gram-negative motile bacterium acquired via intake of contaminated food or water. Secreted cholera toxin (CTX) is the major factor that causes diarrhea. Proposed candidates are the Hap protease [6,7], the multifunctional autoprocessing RTX toxin [8], and Vibrio cholera cytolysin (VCC), called hemolysin A and El Tor hemolysin [9]. 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
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