Giesbers, K. C. A. P. | 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↗

bioRxiv2023

Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-based de novo sequencing

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at different mucosal surfaces including breast and intestine. In the gastrointestinal tract, MUC1 has a barrier function against bacterial invasion, but can also serve as an entry receptor for pathogenic Salmonella bacteria. Moreover, MUC1 is well known for its aberrant expression and glycosylation in adenocarcinomas The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated.. Monoclonal antibodies against the MUC1 VNTR can be powerful tools because of their multiplicity of binding and possible applications in the diagnosis and treatment of MUC1-expressing cancers. One such antibody is the hybridoma mouse monoclonal 139H2, which is also widely used as a research tool to study non-cancer MUC1. Here we report direct mass spectrometry-based sequencing of hybridoma-derived 139H2 IgG, which enabled reverse engineering of a recombinant 139H2. The performance of the reverse engineered 139H2 IgG and its Fab fragment were validated by comparison to the hybridoma-derived product in Western blot and immunofluorescence microscopy. The reverse engineering of 139H2 allowed us to characterize binding to the VNTR peptide epitope by surface plasmon resonance (SPR) and solve the crystal structure of the 139H2 Fab fragment in complex with the MUC1 VNTR peptide. These analyses reveal the molecular basis for 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostics, targeting and treatment strategies.

biochemistry↗