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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.

Despite their relatedness, Salmonella and Shigella enteropathogens differ in infectious dose, pathogenesis, and disease kinetics. The prototype strains Salmonella enterica serovar Typhimurium (Salmonella) and Shigella flexneri (Shigella) use Type-3-secretion-systems (T3SSs) to colonize intestinal epithelial cells (IECs). However, they have evolved unique sets of T3SS effectors and accessory virulence factors. A synthesis of how these differences impact the temporal progression of infection in non-transformed human epithelia is missing. Here, we followed Salmonella and Shigella infections of human enteroids and colonoids by time-lapse imaging to pinpoint virulence factor modules that shape the divergent epithelial colonization strategies. By an apical targeting module that integrates flagella and the SPI-4-encoded adhesin system with T3SS-1, Salmonella accomplishes appreciable numbers of apical IEC invasion events. These are promptly counteracted by IEC death, thus fostering a polyclonal iterative epithelial colonization strategy. The lack of a corresponding apical targeting module in Shigella makes this pathogen reliant on external factors, for example, preexisting damage, for rare apical access to the intraepithelial environment. However, Shigella compensates for this ineptness by an intraepithelial expansion module reliant on the tight coupling of OspC3-dependent temporal delay of cell death and IcsA-mediated mobility. This module enables intraepithelial Shigella to evade the IEC death response just long enough to expand laterally, thus fostering an essentially monoclonal colonization strategy. Taken together, the study reveals how a small set of virulence factors shapes divergent epithelial colonization strategies by related enterobacteria.

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.