Loss of RpoS results in attenuated Escherichia coli colonization of human intestinal organoids and a competitive disadvantage within the germ-free mouse intestine

Abstract

Pluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a previously uncolonized, naive intestinal epithelium in an in vitro system. We recently demonstrated that microinjection of the non-pathogenic Escherichia coli strain, ECOR2, into HIOs induced morphological and functional maturation of the HIO epithelium, including increased secretion of mucins and cationic antimicrobial peptides. In the current work, we use ECOR2 as a biological probe to investigate the bacterial response to colonization of the HIO lumen. In E. coli and other Gram-negative bacteria, adaptation to environmental stress is regulated by the general stress response sigma factor, RpoS. We generated an isogenic {triangleup}rpoS ECOR2 mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine, which is currently the best available system for studying the initial establishment of bacterial populations within the gut. We demonstrate that loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, though it does not prevent colonization of germ-free mice. Rather, the {triangleup}rpoS ECOR2 exhibits a fitness defect in the germ-free mouse intestine only in the context of microbial competition. These results indicate that HIOs pose a differentially restrictive luminal environment to E. coli during colonization, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses. ImportanceTechnological advancements have and will continue to drive the adoption of organoid-based systems for investigating host-microbe interactions within the human intestinal ecosystem. Using E. coli deficient in the RpoS-mediated general stress response, we demonstrate that the type or severity of microbial stressors within the HIO lumen differ from those of the in vivo environment of the germ-free mouse gut. This study provides important insight into the nature of the HIO microenvironment from a microbiological standpoint.