Huang, L. Z. X. | Search bioRxiv

bioRxiv2026

Salmonella SiiE-mediated apical invasion into colonocytes depends on MUC1 α2,3-linked sialic acids

MUC1 is a highly O-glycosylated cell-bound mucin that plays key roles in intestinal mucosal maintenance and microbe-host interactions. The enteropathogen Salmonella enterica expresses a giant adhesin SiiE, which mediates interaction with MUC1 and apical invasion of epithelial cells in a sialic acid-dependent manner. Here, we investigated the glycan specificity of the SiiE-MUC1 interaction and the expression of glycosylated MUC1 receptor in advanced intestinal epithelial models. Expression of the SiiE adhesin by Salmonella was highest in late logarithmic growth, could be induced by aerobic shock, and was detectable on the bacterial surface and in culture supernatant. Purified SiiE bound multiple O-glycan structures in a MUC1 glycopeptide array, including those bearing terminal sialic acids. Single-cell RNA sequencing of human intestinal epithelium showed that high MUC1 expression correlated with expression of ST3GAL and ST6GALNAC sialyltransferases, indicating the potential presence of both 2,3- and 2,6-linked sialylation in vivo. In HT29-MTX intestinal cultures, both 2,3- and 2,6-linked sialic acids could be detected on the apical surface and 2,3-sialic acid staining colocalized with MUC1. Mass spectrometry-based O-glycomics demonstrated that MUC1 carried predominantly core 1 and core 2 O-glycans decorated with 2,3-linked sialylation. Removal or blocking of 2,3-linked sialic acids abolished Salmonella invasion through the SiiE-MUC1 route. In advanced ex vivo cultures of human ileum and colon, MUC1 was detected in the colon, where regions showed positive staining for 2,3-linked sialic acids, but not in the ileum. After infection of the ex vivo tissues, Salmonella was found in close proximity to 2,3-sialylated colonic MUC1. Together, these findings demonstrate that Salmonella SiiE-mediated apical invasion of colonocytes depends on 2,3-sialylated O-glycans on MUC1. In humans, this pathway might be most relevant during Salmonella invasion in the colon.

microbiology↗

bioRxiv2021

The glycosylated extracellular domain of MUC1 protects against SARS-CoV-2 infection at the respiratory surface

Mucins play an essential role in protecting the respiratory tract against microbial infections but can also serve as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on SARS-CoV-2 epithelial entry. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. Both MUC1 and MUC16 are expressed on the surface of human air-liquid interface (ALI) differentiated airway organoids and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 entry and replication. On the surface of Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are often co-expressed and StcE treatment results in enhanced binding of purified spike protein and SARS-CoV-2 pseudovirus. This study points at an important role for glycosylated mucin domains as components of the host defense that can restrict SARS-CoV-2 infection. Author summarySARS-CoV-2, the virus that has caused the devastating COVID-19 pandemic, causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A fundamental understanding of host factors influencing viral entry is critical to elucidate SARS-CoV-2-host interactions and identify novel therapeutic targets. In this study, we investigated the role of host mucins and mucin glycans on SARS-CoV-2 entry into the airway epithelial cells. Mucins are a family of high molecular weight O-glycosylated proteins that play an essential role in protecting the respiratory tract against viral and bacterial infections. The gel-forming mucins MUC5AC and MUC5B clear pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 can restrict or facilitate microbial invasion at the apical surface of the epithelium. The mucin-selective protease StcE specifically cleaves the glycosylated extracellular part of the mucins without perturbing the underlying domains. We show that removal of mucins from the surface of Calu-3 cells and primary airway epithelial cultures with StcE mucinase increases binding of the SARS-CoV-2 spike protein to the respiratory surface and greatly enhances infection. This study demonstrates the important role of glycosylated extracellular mucin domains as a host defense mechanism during SARS-CoV-2 entry. Future efforts should be focused on characterizing the role of specific soluble and transmembrane mucins during the different stages of SARS-CoV-2 infection.

microbiology↗