Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function.

Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.

Leptin acts independently of food intake to modulate gut microbial composition in male mice.

Shifts in the composition of gut bacterial populations can alter host metabolism and may contribute to the pathogenesis of metabolic disorders, including obesity. Mice deficient in leptin action are obese with altered microbiota and increased susceptibility to certain intestinal pathogens. Because antimicrobial peptides (AMPs) secreted by Paneth cells represent a major mechanism by which the host influences the gut microbiome, we examined the mRNA expression of gut AMPs, several of which were decreased in leptin receptor (LepR)-deficient db/db mice, suggesting a potential role for AMP modulation of microbiota composition. To address the extent to which the alterations in gut microbiota and AMP mRNA expression in db/db mice result from increased food intake vs other defects in leptin action, we examined the effects of pair feeding and gut epithelial LepRb ablation on AMP mRNA expression and microbiota composition. We found that the phylum-level changes in fecal microbial content and AMP gene expression persist in pair-fed db/db mice, suggesting that these differences do not stem from hyperphagia alone. In addition, despite recent evidence to support a role for intestinal epithelial LepRb signaling in pathogen susceptibility, ablation of LepRb from the intestinal epithelium fails to alter body weight, composition of the microbiota, or AMP expression, suggesting a role for LepRb elsewhere for this regulation. Indeed, gut LepRb cells are not epithelial but rather constitute a previously uncharacterized population of perivascular cells within the intestinal submucosa. Overall, our data reveal a role for LepRb signaling extrinsic to the intestinal epithelium and independent of food intake in the control of the gut microbiome.