Barron, M. R. | Search bioRxiv

bioRxiv2020

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

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.

microbiology↗

bioRxiv2020

Intestinal inflammation and altered gut microbiota associated with inflammatory bowel disease renders mice susceptible to Clostridioides difficile colonization and infection

Clostridioides difficile has emerged as a noteworthy pathogen in patients with inflammatory bowel disease (IBD). Concurrent IBD and CDI is associated with increased morbidity and mortality compared to CDI alone. IBD is associated with alterations of the gut microbiota, an important mediator of colonization resistance to C. difficile. Here, we describe and utilize a mouse model to explore the role of intestinal inflammation in susceptibility to C. difficile colonization and subsequent disease severity in animals with underlying IBD. Helicobacter hepaticus, a normal member of the mouse gut microbiota, was used to trigger inflammation in the distal intestine akin to human IBD in mice that lack intact IL-10 signaling. Development of IBD resulted in a distinct intestinal microbiota community compared to non-IBD controls. We demonstrate that in this murine model, IBD was sufficient to render mice susceptible to C. difficile colonization. Mice with IBD were persistently colonized by C. difficile, while genetically identical non-IBD controls were resistant to C. difficile colonization. Concomitant IBD and CDI was associated with significantly worse disease than unaccompanied IBD. IL-10-deficient mice maintained gut microbial diversity and colonization resistance to C. difficile in experiments utilizing an isogenic mutant of H. hepaticus that does not trigger intestinal inflammation. These studies in mice demonstrate that the IBD-induced microbiota is sufficient for C. difficile colonization and that this mouse model requires intestinal inflammation for inducing susceptibility to CDI in the absence of other perturbations, such as antibiotic treatment. IMPORTANCEThe incidence of CDI continues to increase significantly among patients with IBD, independent of antibiotic use, yet the relationship between IBD and increased risk for CDI remains to be understood. However, antibiotic-induced perturbations of the gut microbiota may mask mechanisms specific to IBD-induced C. difficile susceptibility and infection. Our study sought to describe and utilize a mouse model to specifically explore the relationship between the IBD-induced gut microbial community and susceptibility to C. difficile colonization and CDI development. We demonstrate that IBD is sufficient for C. difficile colonization and infection in mice and results in significantly worse disease than IBD alone, representing a murine model that recapitulates human IBD and CDI comorbidity. Furthermore, this model requires IBD-induced inflammation to sculpt a microbiota permissible to C. difficile colonization. Use of this model will aid in developing new clinical approaches to predict, diagnose, and treat C. difficile infection in the IBD population.

microbiology↗