Probing host-anaerobe interactions in innovative human gut cellular models

Interactions of anaerobic gut bacteria with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Gut colonization by the anaerobic pathogen Clostridium difficile , a leading cause of hospital-acquired diarrhea, is a key determinant of disease outcome. However, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut cells co-cultured within controlled dual environment chambers, we describe the dynamics of C. difficile infection. We demonstrate that C. difficile adheres and multiplies as micro-communities when grown on a mucin-producing Caco-2/HT29-MTX gut epithelial cell layer. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, and interestingly C. difficile filamentation, a potentially new mechanism of C. difficile persistence. Intriguingly, we find that even in the absence of major C. difficile toxins, the bacterium is able to disrupt epithelial barrier function. To examine C. difficile -commensal interactions, we co-cultured C. difficile with the anaerobic gut commensal Bacteroides dorei . B. dorei adheres to gut cells and inhibits C. difficile multiplication. Furthermore, we have developed novel multilayer and three-dimensional gut models containing gut myofibroblasts. C. difficile adheres more efficiently to epithelial cells in these models compared to the monolayer systems, inducing a stronger chemokine response, indicating a role for myofibroblasts in early host responses to C. difficile . Our study describes new human gut models which effectively recreate the C. difficile infection process, and enable host-anaerobe and pathogen-commensal interaction studies in vitro .