Biology subjects

Strijbis, K.

Publications and source records attributed to Strijbis, K..

6 recordsLinked to original sources

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↗

bioRxiv2025

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.

microbiology↗

bioRxiv2024

Prevotella timonensis degrades the vaginal epithelial glycocalyx through high fucosidase and sialidase activities

Bacterial vaginosis (BV) is a polymicrobial infection of the female reproductive tract (FRT). BV is characterized by replacement of health-associated Lactobacillus species by diverse anaerobic bacteria, including the well-known Gardnerella vaginalis. Prevotella timonensis and Prevotella bivia are anaerobes that are found in a significant percentage of BV patients, but their pathogenic properties are yet to be determined. Defining characteristics of anaerobic overgrowth in BV are adherence to the mucosal surface and the increased activity of mucin-degrading enzymes such as sialidases in vaginal secretions. Here, we demonstrate that P. timonensis but not P. bivia strongly adhered to vaginal and endocervical cells to a similar level as G. vaginalis but did not elicit a comparable pro-inflammatory response. The P. timonensis genome uniquely encodes a large set of mucus-degrading enzymes including 4 putative fucosidases and 2 putative sialidases, PtNanH1 and PtNanH2. Enzyme assays demonstrated that fucosidase and sialidase activity in P. timonensis cell-bound and secreted fractions was significantly higher than for other vaginal anaerobes. Infection assays revealed that P. timonensis fucosidases and sialidases efficiently removed fucose and 2,3- and 2,6-linked sialic acid moieties from the epithelial glycocalyx. Recombinantly expressed P. timonensis NanH1 and NanH2 efficiently removed 2,3 and 2,6-linked sialic acids from the epithelial surface and sialic acid removal by P. timonensis could be blocked using inhibitors. This study demonstrates that P. timonensis has distinct virulence properties that include initial adhesion and a high capacity for mucin degradation at the vaginal epithelial mucosal surface. Our results underline the importance of understanding the role of different anaerobic bacteria in BV. Significance statement (Layman)Bacterial vaginosis (BV) is a common vaginal infection that affects a high percentage of women and is associated with reduced fertility and increased risk of secondary infections. Gardnerella vaginalis is the most well-known BV-associated bacterium, but Prevotella species including P. timonensis and P. bivia may also play an important role. We showed that, similar to G. vaginalis, P. timonensis adhered well to the vaginal epithelium, suggesting that both bacteria could be important in the first stage of infection. Compared to the other bacteria, P. timonensis was unique in efficiently removing the protective mucin sugars that cover the vaginal epithelium. These results underscore that vaginal bacteria play different roles in the initiation and development of BV.

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↗

bioRxiv2022

MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

Regulation and adaptation of intestinal epithelial barrier function is essential for human health. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in intestinal epithelial monolayers MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not result in increased TEER. The increased buildup of TJ complexes in {Delta}MUC13 and MUC13-{Delta}CT cells was dependent on PKC, which is in line with a predicted PKC motif in the MUC13 cytoplasmic tail. The responsible PKC member might be PKC{delta} based on elevated protein levels in the absence of MUC13. Our results identify MUC13 as a central player in TJ complex stability and intestinal barrier permeability.

cell biology↗

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↗