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 expressed on the apical surface of enterocytes. The TM mucin MUC1 has a highly O-glycosylated extracellular domain (ED) and a cytoplasmic tail (CT) with signaling potential. MUC1 is a target for the Salmonella adhesin SiiE, which mediates apical invasion of the bacterium into enterocytes. Here, we determined the contributions of the MUC1 ED and CT to Salmonella invasion and subsequent host immune responses. Enzymatic removal of the MUC1 ED from HT29-MTX intestinal cultures blocked Salmonella invasion to levels comparable to MUC1 knockout cells. CRISPR-mediated targeted deletion of the MUC1 CT (MUC1-{Delta}CT) did not quantitatively affect Salmonella invasion. To investigate downstream host responses, RNAseq transcriptomics analysis of uninfected and Salmonella-infected MUC1-WT, MUC1-{Delta}CT, and {Delta}MUC1 cultures was performed. Deletion of full-length MUC1 greatly altered the transcriptome, while only a small group of 132 genes was differentially expressed in MUC1-{Delta}CT cultures during infection. Several of these CT-dependent genes are related to the NF{kappa}B pathway. Immunoblot analysis demonstrates that under uninfected conditions, expression of NF{kappa}B subunits RelB, NfkB1-p105, NfkB2-p100, and I{kappa}B was significantly lower in MUC1-WT compared to MUC1-{Delta}CT and {Delta}MUC1 cultures. Secretion of cytokines and immune factors was severely reduced in {Delta}MUC1 cultures, coinciding with reduced Salmonella invasion. In MUC1-{Delta}CT cultures, only galectin-3 and IL-18 secretion were significantly reduced. We conclude that the MUC1 ED is essential for Salmonella invasion, while the CT modulates the canonical and non-canonical NF{kappa}B pathway, pointing at distinct roles for MUC1 domains in microbe-host interactions and signaling. ImportanceThe intestinal mucus layer plays an important role in separating commensal and pathogenic microbes from the underlying epithelium. The transmembrane mucin MUC1 is expressed by different types of intestinal epithelial cells and is thought to have important protective and signaling functions. However, enteropathogenic Salmonella bacteria can hijack MUC1 through engagement with the SiiE adhesin which leads to bacterial invasion of enterocytes at the apical surface. In this study, we determined how the different MUC1 domains contributed to Salmonella invasion and subsequent host responses. We found that the glycosylated MUC1 extracellular domain, but not the cytoplasmic tail, is essential for bacterial invasion. In infected and uninfected intestinal cultures, the MUC1 cytoplasmic tail modulates immune responses including NF{kappa}B activation and cytokine secretion. Our study contributes to our understanding of the diverse functions of transmembrane mucins at the intestinal microbe-host interface.