The MUC1 extracellular domain and cytoplasmic tail play distinct roles during Salmonella invasion of enterocytes

The intestinal mucus layer consists of secreted and transmembrane (TM) mucins that are expressed on the apical surface of enterocytes and goblet cells. The TM mucin has a complex domain structure consisting of an extended highly O-glycosylated extracellular domain (ED) and a cytoplasmic tail (CT) with signaling potential. The enteropathogenic bacterium Salmonella expresses a giant adhesin called SiiE that interacts with the MUC1, resulting in invasion at the apical surface of the intestinal epithelium. Here, we determined the contributions of the MUC1 ED and CT to Salmonella invasion and subsequent immune responses. To investigate the role of the MUC1 ED in Salmonella invasion, the mucin-selective protease StcE was used to remove the glycosylated ED from the surface of intestinal HT29-MTX cultures. StcE-mediated removal of the MUC1 ED blocked Salmonella invasion to levels comparable to MUC1 knockout cells. To study the contribution of the MUC1 CT, a targeted CRISPR/Cas9 approach was used that resulted in the removal of the CT but left the ED and transmembrane domain intact. Salmonella invasion into these MUC1-ΔCT cultures was comparable to wild type cells. These results demonstrate that MUC1 ED, but not the MUC1 CT, is essential for apical Salmonella invasion. To determine the contributions of MUC1 domains, we performed a transcriptomics analysis of uninfected MUC1-WT, MUC1-ΔCT, and ΔMUC1 cultures and cultures that were infected with Salmonella for 6-hour. Deletion of the full MUC1 protein led to a large number of differentially expressed genes, while a smaller group of 132 genes was differentially expressed in infected MUC1-WT cultures compared to MUC1-ΔCT cultures. Amongst these genes were many NFκB-regulated genes that showed reduced expression in the absence of the MUC1 CT. Immunoblot analysis demonstrated that expression of the NFκB inhibitory cytoplasmic subunits p100, p105, and IκBα was significantly reduced in MUC1-WT cultures compared to MUC1-ΔCT and ΔMUC1 cultures. We conclude that the MUC1 ED is essential for Salmonella invasion, and that the MUC1 CT plays a role in activation of the NFκB pathway.