A scalable gut epithelial organoid model reveals the genome-wide colonization landscape of a human-adapted pathogen

Studying the pathogenesis of human-restricted microbes is challenging, since small animal models often fail to recapitulate human physiology and suffer from infection bottlenecks that confound genetic screens. Hence, the comprehensive genetic and regulatory circuits driving the infection process of major human pathogens like Shigella flexneri remain to be defined. We combined large-scale Shigella infections of enteroids and colonoids with Transposon Directed Insertion Sequencing (TraDIS) and Bayesian statistical modelling, thereby establishing the first genome-wide map of Shigella genes required to colonize human intestinal epithelium. Our study revealed the Shigella virulence effectors essential for epithelial cell invasion across geometries and intestinal segments, identified over 100 chromosomal genes involved in the process, and uncovered a post-transcriptional mechanism whereby tRNA modification enzymes exert global control of a bacterial virulence program. Our findings provide a broadly applicable framework for combining advanced organotypic tissue culture with computational tools to map human – microbe interactions at scale. Overall design: Infection of human organoids with a Shigella flexneri M90T Tn5 mutant library containing 10,000 or 30,000 mutants.