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Studying the pathogenesis of human-adapted microorganisms is challenging, since small animal models often fail to recapitulate human physiology. Hence, the comprehensive genetic and regulatory circuits driving the infection process of principal human pathogens such as Shigella flexneri remain to be defined. We combined large-scale Shigella infections of enteroids and colonoids with transposon-directed insertion sequencing and Bayesian statistical modeling to address infection bottlenecks, thereby establishing the comprehensive genome-wide map of Shigella genes required to infect human intestinal epithelium. This revealed the Shigella virulence effectors essential for epithelial cell colonization across geometries and intestinal segments, identified over 100 chromosomal genes involved in the process and uncovered a post-transcriptional mechanism whereby tRNA-modification enzymes and differential codon usage exert global control of a bacterial virulence program. Our findings provide a broadly applicable framework for combining advanced organotypic tissue culture with functional genomics and computational tools to map human–microorganism interactions at scale.

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.