Abstract
ABSTRACTIntestinal mucus secretion is critical in maintaining mucosal host defense against a myriad of pathogens by preventing direct association with the epithelium. Entamoeba histolytica specifically binds colonic MUC2 mucin and also induces potent hypersecretion from goblet cells; however, characterization of the nature of the mechanisms controlling mucus release remains elusive. In this report, we identify vesicle SNARE vesicle-associated membrane protein 8 (VAMP8) present on mucin granules as orchestrating regulated exocytosis in human goblet cells in response to the presence of E. histolytica. VAMP8 was specifically activated during E. histolytica infection, and ablation of VAMP8 led to impaired mucin secretion. As a consequence, loss of VAMP8 increased E. histolytica adherence to epithelial cells associated with enhanced cell death through apoptosis characterized by caspase 3 and 9 cleavages and DNA fragmentation. With the mucosal barrier compromised in Vamp8−/− animals, E. histolytica induced an aggressive proinflammatory response with elevated levels of interleukin-1 alpha (IL-1α), IL-1β, and tumor necrosis factor alpha (TNF-α) secretion. This report is the first to characterize regulated mucin exocytosis in intestinal goblet cells in response to a pathogen and the downstream consequences of improper mucin secretion in mucosal barrier defense.
Highlights
Intestinal mucus secretion is critical in maintaining mucosal host defense against a myriad of pathogens by preventing direct association with the epithelium
The other SNARE proteins that we suspected of participating in exocytosis, SNAP23, syntaxin 3, and Munc18b, were not affected by VAMP8KD (Fig. 1C); aberrant expression of VAMP2 was often seen in VAMP8KD cells
This study focused on the primary R-SNARE vesicle-associated membrane protein 8 (VAMP8) present on mucin granules that facilitated mucin exocytosis
Summary
Intestinal mucus secretion is critical in maintaining mucosal host defense against a myriad of pathogens by preventing direct association with the epithelium. Entamoeba histolytica binds colonic MUC2 mucin and induces potent hypersecretion from goblet cells; characterization of the nature of the mechanisms controlling mucus release remains elusive. E. histolytica possesses specific cysteine proteases (CP5) that target MUC2 for degradation, allowing penetration of the mucus layer [16] This virulence factor is critical in inducing mucus hypersecretion and depletion by binding to host integrins and inducing a signaling cascade involving focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/PKC␦ [15]. Breakdown of these protective mechanisms is fundamental in E. histolytica pathogenesis and leads to direct cytolysis of epithelial cells. This led to apoptosis of host cells and a subsequent proinflammatory response, exacerbating E. histolytica pathogenesis
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