SNARE-mediated exocytosis of MUC2 from intestinal goblet cells
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AbstractThe GI tract faces countless luminal threats, be it noxious substances, commensal bacteria or pathogenic in nature. The first line of innate host defense in the intestine is the mucus layer that spatially separates these threats from the single layer of epithelial cells. Replenishment and maintenance of the mucus layer is facilitated by goblet cells that produce and store MUC2 mucin within secretory granules. Release of mucin occurs constitutively to maintain the barrier and is inducibly when goblet cells sense a threat such as an invading pathogen. Given the immense importance of the mucus layer in maintaining homeostasis, surprisingly little is known on the mechanisms of mucin release from intestinal goblet cells. Herein we describe for the first time that the SNARE complex in goblet cells mediates mucin exocytosis and degradation of these regulatory pathways exacerbates pathogenesis of the intestinal pathogen Entamoeba histolytica. The SNARE complex is the classical exocytosis machinery as the effector mechanism governing mucin secretion. In this model, exocytosis is carried out by specialized members of the Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein SNARE family that facilitates vesicle-membrane fusion events. R-SNAREs, predominantly vesicle-associated membrane proteins (VAMP) present on vesicles bind to Qabc-SNARE complexes present on the plasma membrane following a stimulatory signal allowing for exocytosis of vesicle content. We have interrogated the mechanisms of how the colonic protozoan pathogen, E. histolytica (Eh), evokes mucin secretion. In particular, how the virulence factor Eh cysteine proteinase 5 (EhCP5) ligates αvβ3 integrin on goblet cells to stimulate a signal transduction cascade and the first characterization of how a pathogen directly evokes mucin secretion in the colon. Additionally, ligation of integrins by EhCP5 resulted in downstream activation of PKCδ, a well-known inducer of mucin secretion in goblet cells. We hypothesized that this signal transduction pathway would culminate in the vesicle SNARE Vamp8 being activated in goblet cells following Eh contact. In support of this, we have shown that VAMP8 is specifically activated following Eh contact to orchestrate protein-protein interactions critical in mucin granule exocytosis. In cells lacking VAMP8, there was increased epithelial killing due to loss of the protective mucus layer. This held true in a mouse model of Eh infection where loss of Vamp8 resulted in increased apoptosis and pro-inflammatory cytokine release. Additionally, we describe that mucus exocytosis in the gut solely utilizes the vesicle SNARE VAMP8 under basal conditions to maintain homeostasis. Vamp8-/- mice exhibit a mild pro-inflammatory state in the colon basally due to an altered mucus layer and increased encounters with microbial antigens. Microbial diversity skews to a detrimental flora with pathogenic and mucolytic bacteria increasing in abundance. To alleviate the heavy microbial burden and inflammatory state basally, Vamp8-/- skew T cell phenotypes towards tolerance. Despite this, Vamp8-/- is highly susceptible to both chemical and infectious colitis demonstrating the fragility of the intestinal mucosa without proper mucus exocytosis mechanisms.
CitationCornick, S. (2018). SNARE-mediated exocytosis of MUC2 from intestinal goblet cells (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/5422
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