Citrobacter rodentium infection disrupts colonic mucus barrier function via ablation of intercrypt goblet cells

The colonic mucus barrier, composed of distinct layers maintained by specialized goblet cell (GC) subtypes, forms the first line of defense against enteric pathogens. However, the individual contributions of inner (IML) and fecal (FML) mucus layers to barrier integrity, and how these layers and the GC responses are dynamically modulated during infection remain incompletely understood. Using Citrobacter rodentium infection in mice, we show that early colonization disrupts the inner mucus layer, increases mucus penetrability and drives alterations in faecal mucus layer thickness. Spatial imaging revealed progressive crypt infiltration and concurrent accumulation of pathogen clusters within the FML. Transcriptomic and functional analyses identified Spdef-dependent intercrypt goblet cells (icGCs) as essential for sustaining IML integrity and limiting epithelial access. Spdef-deficient mice exhibited delayed clearance and enhanced crypt invasion, highlighting the protective role of icGCs. Infection selectively depleted icGCs, collapsing the intercrypt mucus and creating permissive niches for colonization. Mechanistically, C. rodentium leveraged its type III secretion system, specifically the EspF effector to reprogram GC transcriptional responses, inducing metabolic stress and promoting apoptotic signatures in GCs specifically. These findings reveal a previously unrecognized virulence strategy in which EspF-driven GC remodeling compromises intercrypt mucus defenses, potentially facilitating pathogen persistence and transmission.