Maturation of human intestinal epithelial cell layers fortifies the apical surface against Salmonella attack

Pathogen invasion of intestinal epithelial cells (IECs) is a key gut infection event. The dynamics of this process in intact epithelia and the factors governing IEC susceptibility remain underexplored. We present a resource for live-cell infection imaging in human enteroid-/colonoid-derived IEC layers across maturation states and in the presence or absence of soluble mucus production. Utilization of this resource shows how human IEC maturation fortifies the apical surface against invasion by Salmonella Typhimurium (S.Tm). Immature IEC layers appear permissive to S.Tm invasion, but maturation toward an enterocyte/colonocyte phenotype reduces the susceptibility by up to 10-fold. This shift couples to downregulated expression of actin regulatory proteins exploited by the pathogen, an increased dependence on the S.Tm effector SipA, and the build-up of the apical IEC glycocalyx linked to surface-attached mucins like MUC13 and nullifiable by StcE-enzyme treatment. This underscores how the maturation state of human IECs governs susceptibility to bacterial invasion.