Type IV pili promote Clostridium difficile adherence and persistence in a mouse model of infection

Cyclic diguanylate (c-di-GMP) is a second messenger that regulates the transition from motile to sessile lifestyles in numerous bacteria and controls virulence factor production in a variety of pathogens. In Clostridium difficile, c-di-GMP negatively regulates flagellum biosynthesis and swimming motility and promotes the production of type IV pili (TFP), biofilm formation, and surface motility in vitro. Flagella have been identified as colonization factors in C. difficile, but the role of TFP in adherence to host cells and in colonization of the mammalian gut is unknown. Here we show that c-di-GMP promotes adherence to epithelial cells in vitro, which can be partly attributed to the loss of flagella. Using TFP-null mutants, we demonstrate that adherence to epithelial cells is partially mediated by TFP and that this TFP-mediated adherence requires c-di-GMP regulation. In a mouse model of colonization, the TFP-null mutants initially colonized the intestine as well as the parental strain but were cleared more quickly. Moreover, compared to the parent strain, C. difficile strains lacking TFP were particularly deficient in association with the cecal mucosa. Together these data indicate that TFP and their positive regulation by c-di- GMP promote attachment of C. difficile to the intestinal epithelium and contribute to persistence of C. difficile in the host intestine.