Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease.

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 FINDINGS: Culture 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-alpha (TNF-alpha) 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/SIGNIFICANCE: We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha 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.

Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease.

OBJECTIVES: Alterations in the composition of the microbiota in the intestine may promote development of celiac disease (CD). Using scanning electron microscopy (SEM) we previously demonstrated that rod-shaped bacteria were present on the epithelium of proximal small intestine in children with CD but not in controls. In this study we characterize the microbiota of proximal small intestine in children with CD and controls and identify CD-associated rod-shaped bacteria. METHODS: Proximal small intestine biopsies from 45 children with CD and 18 clinical controls were studied. Bacteria were identified by 16S rDNA sequencing in DNA extracted from biopsies washed with buffer containing dithiothreitol to enrich bacteria adhering to the epithelial lining, by culture-based methods and by SEM and transmission electron microscopy. RESULTS: The normal, mucosa-associated microbiota of proximal small intestine was limited. It was dominated by the genera Streptococcus and Neisseria, and also contained Veillonella, Gemella, Actinomyces, Rothia, and Haemophilus. The proximal small intestine microbiota in biopsies from CD patients collected during 2004-2007 differed only marginally from that of controls, and only one biopsy (4%) had rod-shaped bacteria by SEM (SEM+). In nine frozen SEM+ CD biopsies from the previous study, microbiotas were significantly enriched in Clostridium, Prevotella, and Actinomyces compared with SEM- biopsies. Bacteria of all three genera were isolated from children born during the Swedish CD epidemic. New Clostridium and Prevotella species and Actinomyces graevenitzii were tentatively identified. CONCLUSIONS: 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.

A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing.

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.

Ascorbic acid secretion in the human stomach and the effect of gastrin.

AIM:To investigate the changes of gastric mucosal ascorbic acid secretion in patients with nonulcer dyspepsia and the effect of gastrin on it, and to relate any observed changes to H.pylori infection and mucosal histology.METHODS:Ascorbic acid secretions in patients were examined by collecting continuously gastric juice for one hour after having aspirated and discarded fasting gastric juice. Using the clearance rate (mL/min) of ascorbic acid from blood to gastric juice represented ascorbic acid secretion in the gastric mucosa.Ascorbic acid concentrations in plasma and juice were measured by ferric reduced method.RESULTS:Gastric ascorbic acid secretions in H.pylori-positive patients (1.46mL/min,range 0.27-3.78) did not significantly differ from those in H.pylori-negative patients(1.25mL/min, 0.47-3.14)(P>0.05). There were no significant differences in ascorbic acid secretions between patients with mild (1.56mL/min, 0.50-3.30), moderate (1.34mL/min, 0.27-2.93) and severe (1.36mL/min, 0.47-3.78) inflammation (P >0.05). There were no significant differences in ascorbic acid secretions between patients without activity (1.45mL/min, 0.27-3.14) and with mild (1.32mL/min, 0.61-2.93), moderate (1.49mL/min, 0.50-3.78) and severe (1.43mL/min, 0.51-3.26) activity of chronic gastritis either (P>0.05). Ascorbic acid secretions in patients with severe atrophy (0.56mL/min, 0.27-1.20) were markedly lower than those in patients without atrophy (1.51mL/min, 0.59-3.30) and with mild (1.43mL/min, 0.53-3.78) and moderate (1.31mL/min,0.47-3.16)atrophy(P<0.005). There was a significant negative correlation between ascorbic acid secretion and severity of atrophy (correlation coefficient =-0.43, P<0.005). After administration of pentagastrin, ascorbic acid secretions were markedly elevated (from 1.39mL/min, 0.36-2.96 to 3.53mL/min, 0.84-5.91) (P <0.001).CONCLUSION:Ascorbic acid secretion in gastric mucosa is not affected by H.pylori infection. Gastric ascorbic acid secretion is markedly related to the severity of atrophy, whereas not related to the severity of inflammation and activity. Gastrin may stimulate gastric ascorbic acid secretion. A decreased ascorbic acid secretion may be an important factor in the link between atrophic gastritis and gastric carcinogenesis.