A24 FCGBP MAINTAINS MUC2 MUCUS STRUCTURAL INTEGRITY BY STABILIZING THE MUCUS LAYER IN RESPONSE TO THE COLONIC PATHOGEN, ENTAMOEBA HISTOLYTICA
Abstract Background MUC2 mucin is the major component of the colonic mucus bilayer that serves as the first line of innate host defense against pathogens while supporting a healthy microbiota and regulating epithelial barrier function. Proteomic studies of colonic mucus have identified various mucus-associated proteins. One of the most abundant is FCGBP, similar to MUC2 mucin, but its interaction with MUC2 or function is not known. Here, we elucidated FCGBP functional role in stabilizing MUC2 mucus and in innate host defence against Entamoeba histolytica (Eh). Aims Hypothesis: MUC2 mucin and FCGBP are coordinately produced and play an important role in innate host defense. The specific aims are: 1. To determine if FCGBP alters the structural integrity of the mucus layer 2. To determine the role of FCGBP in Eh infection Methods FCGBP mRNA and protein expression induced by Eh, in WT and FCGBP CRISPR/Cas9 LS174T goblet cells were analysed by RT-PCR and Western blotting. To compare integrity of the mucus layer, fluorescent Eh and 1μM fluorescent beads were inoculated on WT and KO monolayers and adherent Eh and bead penetrability analyzed. To quantify MUC2 and FCGBP degradation by Eh, purified MUC2 and recombinant FCGBP were incubated with Eh proteases (SPs) and Western blotted using highly specific antibodies against various regions of the proteins. Results In response to live Eh, FCGBP and MUC2 mRNA and protein expressions were significantly increased in a time-dependent manner. Surprisingly, FCGBP KO cells elicited robust expression of pro-inflammatory cytokine mRNA and protein as compared to WT cells. More fluorescent Eh were attached to the mucus layer of FCGBP KO cells as compared to WT or MUC2 KO cells. Fluorescent beads penetrated further towards the epithelial cell surface in KO as compared to WT cells. Interestingly, while both MUC2 and FCGBP from purified polymeric mucins were degraded by Eh SPs, FCGBP cleavage occurred at a faster rate than MUC2. Degradation of FCGBP and MUC2 was mediated by EhCP-A5 cysteine proteinase using purified MUC2 and recombinant FCGBP. Conclusions In WT goblet cells, FCGBP and MUC2 were upregulated temporally in response to Eh. The increase in pro-inflammatory cytokine expression in FCGBP KO cells in response to Eh suggests that Eh directly interacted with the cell surface suggesting an impaired protective mucus layer. In support of this, fluorescent beads penetrated the mucus layer close to the cell surface and more Eh were attached to FCGBP KO mucus demonstrating that FCGBP was critical in providing structural integrity of the mucus layer. In response to Eh, FCGBP degradation was a prerequisite for MUC2 cleavage, providing direct evidence that FCGBP and MUC2 interactions conferred biophysical properties of the protective functions of the mucus gel. Funding Agencies CIHR
