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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.

The enteropathogen Shigella flexneri employs a Type Three Secretion System (T3SS) to colonize intestinal epithelial cells. Genes encoding the T3SS are located on a large IncFII virulence plasmid, pINV. T3SS expression comes at the expense of slowed Shigella growth and is therefore strictly controlled by both transcriptional and post-transcriptional mechanisms. Following up on a recent genome-wide screen, we here show that the chromosomal gene pcnB, encoding the poly-A polymerase I (PAP-I), slows Shigella growth at 37°C, while it at the same time promotes early colonization of a human epithelial enteroid model. Proteomic profiling revealed that pcnB drives global increase of the Shigella T3SS virulence program. Accordingly, pcnB upholds pINV replication to a level favourable for Shigella virulence. This is achieved through increased degradation of the antisense RNA CopA, involved in plasmid replication control. The pcnB effect on pINV replication was found to also ensure longer-term intraepithelial expansion of Shigella following human intestinal epithelium invasion. Our findings exemplify how an adequate pINV level, sustained by pcnB, underpins the successful execution of Shigella´s infection cycle.