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© 2020 Background: Colombians in coastal Tumaco have a lower incidence of Helicobacter pylori-associated gastric cancer compared to individuals from Tuquerres in the high Andes. This is despite nearly universal prevalence of H. pylori infection and chronic gastritis. Methods: H. pylori infection was confirmed by Steiner stain and serology using African and European-origin strains. Gastric histology and serum inflammatory biomarkers in dyspeptic Tumaco or Tuquerres patients were evaluated to predict progression of gastric lesions. Results: H. pylori infection was nearly universal by Steiner stain and serology. IgG response to European-origin H. pylori strains were greater than African-origin. High gastric cancer-risk Tuquerres patients, compared to low-risk Tumaco, had significant odds ratios for lesion progression associated with serum IL-5, trefoil factor 3 (TFF3), and low pepsinogen I/II ratio. Sensitivity and specificity for these parameters was 63.8% and 67.9%, respectively, with correctly classifying patients at 66.7%. Most odds ratios for 26 other biomarkers were significant for the town of residency, indicating an environmental impact on Tumaco patients associated with decreased lesion progression. Conclusion: An IL-5 association with progression of gastric lesions is novel and could be evaluated in addition to TFF3 and pepsinogen I/II ratio as a non-invasive prognostic screen. Results suggest Tumaco patients were exposed to infectious diseases beyond H. pylori such as the documented high incidence of helminthiasis and toxoplasmosis. Impact: Results support a prior recommendation to evaluate TFF3 and pepsinogen I/II together to predict aggressive gastric histology. Our data indicate IL-5 should be further evaluated as prognostic parameter.

© 2020 Background: Colombians in coastal Tumaco have a lower incidence of Helicobacter pylori-associated gastric cancer compared to individuals from Tuquerres in the high Andes. This is despite nearly universal prevalence of H. pylori infection and chronic gastritis. Methods: H. pylori infection was confirmed by Steiner stain and serology using African and European-origin strains. Gastric histology and serum inflammatory biomarkers in dyspeptic Tumaco or Tuquerres patients were evaluated to predict progression of gastric lesions. Results: H. pylori infection was nearly universal by Steiner stain and serology. IgG response to European-origin H. pylori strains were greater than African-origin. High gastric cancer-risk Tuquerres patients, compared to low-risk Tumaco, had significant odds ratios for lesion progression associated with serum IL-5, trefoil factor 3 (TFF3), and low pepsinogen I/II ratio. Sensitivity and specificity for these parameters was 63.8% and 67.9%, respectively, with correctly classifying patients at 66.7%. Most odds ratios for 26 other biomarkers were significant for the town of residency, indicating an environmental impact on Tumaco patients associated with decreased lesion progression. Conclusion: An IL-5 association with progression of gastric lesions is novel and could be evaluated in addition to TFF3 and pepsinogen I/II ratio as a non-invasive prognostic screen. Results suggest Tumaco patients were exposed to infectious diseases beyond H. pylori such as the documented high incidence of helminthiasis and toxoplasmosis. Impact: Results support a prior recommendation to evaluate TFF3 and pepsinogen I/II together to predict aggressive gastric histology. Our data indicate IL-5 should be further evaluated as prognostic parameter.

GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis. It has recently been demonstrated that activation of GPR4 by acidosis increases the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and also augments EC-leukocyte adhesion. Inhibition of GPR4 by siRNA or small molecule inhibitors reduces endothelial cell inflammation. As acidotic tissue microenvironments exist in many types of inflammatory disorders, including inflammatory bowel disease (IBD), we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for 7 days to induce acute colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. Collectively, our results suggest GPR4 provides a pro-inflammatory role in the inflamed gut as the absence of GPR4 ameliorates intestinal inflammation in the acute experimental colitis mouse model. Keywords: GPR4; Inflammatory bowel disease (IBD); Acidosis; Endothelial cell; Inflammation

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomark, National Institutes of Health (U.S.) (NIH grant T32-RR007036), National Institutes of Health (U.S.) (NIH grant P30-ES002109)

The recently identified type VI secretion system (T6SS) of proteobacteria has been shown to promote pathogenicity, competitive advantage over competing microorganisms, and adaptation to environmental perturbation. By detailed phenotypic characterization of loss-of-function mutants, in silico, in vitro and in vivo analyses, we provide evidence that the enteric pathogen, Campylobacter jejuni, possesses a functional T6SS and that the secretion system exerts pleiotropic effects on two crucial processes – survival in a bile salt, deoxycholic acid (DCA), and host cell adherence and invasion. The expression of T6SS during initial exposure to the upper range of physiological levels of DCA (0.075%–0.2%) was detrimental to C. jejuni proliferation, whereas down-regulation or inactivation of T6SS enabled C. jejuni to resist this effect. The C. jejuni multidrug efflux transporter gene, cmeA, was significantly up-regulated during the initial exposure to DCA in the wild type C. jejuni relative to the T6SS-deficient strains, suggesting that inhibition of proliferation is the consequence of T6SS-mediated DCA influx. A sequential modulation of the efflux transporter activity and the T6SS represents, in part, an adaptive mechanism for C. jejuni to overcome this inhibitory effect, thereby ensuring its survival. C. jejuni T6SS plays important roles in host cell adhesion and invasion as T6SS inactivation resulted in a reduction of adherence to and invasion of in vitro cell lines, while over-expression of a hemolysin co-regulated protein, which encodes a secreted T6SS component, greatly enhanced these processes. When inoculated into B6.129P2-IL-10[superscript tm1Cgn] mice, the T6SS-deficient C. jejuni strains did not effectively establish persistent colonization, indicating that T6SS contributes to colonization in vivo. Taken together, our data demonstrate the importance of bacterial T6SS in host cell adhesion, invasion, colonization and, for the first time to our knowledge, adaptation to