Colonic MUC2 Mucin Is Stabilized By FCGBP To Resist <i>Salmonella Enterica</i> Invasion In Goblet Cells

MUC2 mucin produced by goblet cells form the colonic mucus bilayer as the first line of innate defense against pathogen invasion by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. Goblet cells also produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we elucidated the putative functions of FCGBP embedded in MUC2 mucin in response to Salmonella enterica adherence and invasion in human colonic goblet cells. We hypothesis that FCGBP is coordinately produced with MUC2 mucin to play an integral role in forming the protective mucus layer in innate host defense. The aims of the study are: 1) to determine if FCGBP alters the structural integrity of the mucus layer and 2) to quantify structural alterations of the mucus layer in S. enterica adherence and invasion of goblet cells. To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: Wildtype LS174T (WT) MUC2 mucus‐producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS). To determine if FCGBP MS led to loss in barrier function in the mucus layer, S. enterica adherence and invasion, and 0.2, 1, and 2 µM fluorescent bead penetrability inoculated on WT and FCGBP MS monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by S. enterica in WT and FCGBP MS goblet cells were analysed by RT‐PCR and Western blotting. Cell killing by S. enterica was determined by LDH assay. S. enterica induced pro‐inflammatory cytokines responses were analyzed by RT‐PCR and human focus 15‐plex cytokine and chemokine array. FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetrability further towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of live S. enterica was significantly increased in FCGBP MS but not WT cells and induced robust MUC2 mRNA expression and mucus secretion in a time‐dependent manner. Similarly, S. enterica elicited high expression of pro‐inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by S. enterica that resulted in increased cell death as compared to WT cells. More S. enterica adhered, invaded, and killed FCGBP MS as compared to WT cells. Fluorescent beads penetrated the mucus layer closer to the cell surface in FCGBP MS cells. In WT goblet cells, FCGBP and MUC2 were coordinately upregulated in response to S. enterica . The concurrent increase in pro‐inflammatory cytokine expression in FCGBP MS cells in response to S. enterica suggests that bacteria directly interacted with the cell surface suggesting an impaired protective mucus layer. The overall trend for increased bacterial invasion, pro‐inflammatory response, and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity of the mucus layer.

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