Intestinal epithelial Casd1 influences mucus sialic acid O- acetylation and tissue damage susceptibility toward large-intestinal mucosal insults

ABSTRACT The intestinal mucus network, primarily composed of O -glycosylated MUC2 mucin polymers, is essential for protecting the gastrointestinal tract from microbial threats. Sialic acid (Sia), a terminal monosaccharide on complex O -glycans, plays a key role in maintaining mucus integrity and is frequently modified by Casd1-dependent O -acetylation ( O Ac). Despite its prevalence, the biological significance of sialic acid O Ac ( O Ac-Sia) modifications in human and murine mucus remains unclear. We hypothesized that O Ac-Sia variants on mucus interact with the microbiota and are required for optimal mucus barrier function and host-microbe homeostasis in the colon. To test this, we profiled O Ac-Sia on human and mouse MUC2 in situ using viral-derived probes with bacterial FISH and confocal microscopy; generated intestinal epithelial cell (IEC)-specific Casd1 null mice (IEC Casd1 -/- ); performed sialylomic and O -glycomic HPLC-MS analyses; assessed microbial communities by 16S rRNA sequencing with quantitative microbial profiling (QMP); and evaluated disease susceptibility using DSS colitis and Citrobacter rodentium infection models. Results revealed that both human and murine mucins are extensively O -acetylated and interact with the microbiota, suggesting biological relevance. IEC Casd1 -/- mice were viable and displayed a complete loss of mucin O Ac-Sia, indicating Casd1 is the sole contributor to O Ac-status. Unexpectedly, mucus function was intact in IEC Casd1 -/- mice, with no difference in structure or quality vs. WT co-housed littermates.16S rRNA analysis showed a modest but significant sex-specific reduction of microbial loads in male IEC Casd1 -/- mice, and a clear trend toward reduced Turicibacter spp. vs. WT mice in both male and females, without impacting overall short-chain fatty acid (SCFA) production. DSS treatment led to more severe and extensive tissue damage in IEC Casd1 -/- mice. C. rodentium infection led to increased damage in the cecum and distal colon of IEC Casd1 -/- mice without affecting pathogen load, suggesting that O Ac-Sia status has a role in tolerance defense. These findings establish intestinal epithelial Sia O -acetylation as a component dispensable for mucus and host-microbe homeostasis at baseline, but important in limiting damage to mucosal inflammatory insults.