Probing Clostridium difficile infection in innovative human gut cellular models

Abstract

33Interactions of anaerobic gut bacteria with the intestinal mucosa have been poorly 34 studied due to challenges in culturing anaerobes with the oxygen-requiring gut 35 epithelium. Gut colonization by the anaerobic pathogen Clostridium difficile, a leading 36 cause of hospital-acquired diarrhea, is a key determinant of disease outcome. However, 37 precise mechanisms of mucosal attachment and spread remain unclear. Here, using 38 human gut cells co-cultured within controlled dual environment chambers, we describe 39 the dynamics of C. difficile infection. We demonstrate that C. difficile adheres and 40 multiplies as micro-communities when grown on a mucin-producing Caco-2/HT29-MTX 41 gut epithelial cell layer. Prolonged infection causes redistribution of actin and loss of 42 epithelial integrity, accompanied by production of C. difficile spores, and interestingly C. 43 difficile filamentation, a potentially new mechanism of C. difficile persistence. 44Intriguingly, we find that even in the absence of major C. difficile toxins, the bacterium is 45 able to disrupt epithelial barrier function. To examine C. difficile-commensal interactions, 46 we co-cultured C. difficile with the anaerobic gut commensal Bacteroides dorei. B. dorei 47 adheres to gut cells and inhibits C. difficile multiplication. Furthermore, we have 48 developed novel multilayer and three-dimensional gut models containing gut 49 myofibroblasts. C. difficile adheres more efficiently to epithelial cells in these models 50 compared to the monolayer systems, inducing a stronger chemokine response, 51 indicating a role for myofibroblasts in early host responses to C. difficile. Our study 52 describes new human gut models which effectively recreate the C. difficile infection 53 process, and enable host-anaerobe and pathogen-commensal interaction studies in 54 vitro. 55 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/269035 doi: bioRxiv preprint first posted online Feb. 21, 2018; 3 56 57 IMPORTANCE 58 59Understanding the host responses to anaerobic gut pathogens or commensals, and 60 gaining mechanistic insights into the three-way interaction between pathogens, 61 commensals and the gut epithelium is fundamental to advancing our knowledge of gut 62 microbiome function. Host-pathogen interaction studies of the human anaerobic 63 pathogen, C. difficile and other gut-associated anaerobes is challenging due to the lack 64 of appropriate in vitro systems. In this study, employing novel two and three-65 dimensional models, we have tracked C. difficile infection over longer time scales, 66 examining the attachment and invasion of the gut epithelium and the effects of key 67 virulence factors. Furthermore, we demonstrate interactions of other important gut 68 commensals like Bacteroides with the epithelium and with C. difficile. This study 69 provides new insight into the molecular and cellular events that occur at the ...