The flagellin FliC of Clostridium difficile is responsible for pleiotropic gene regulation during in vivo infection.

Clostridium difficile is the main agent responsible for hospital acquired antibiotic associated diarrhoea. In recent years, epidemic strains have emerged causing more severe infections. Whilst C. difficile has two major virulence factors, toxins TcdA and TcdB, it is generally accepted that other virulence components of the bacterium contribute to disease. Previously, it has been suggested that flagella expression from pathogenic bacteria might be implicated in virulence. In a recent study, we observed an increased mortality in a gnotobiotic mouse model when animals were colonized with an isogenic fliC mutant constructed in the PCR-ribotype 027 (B1/NAP1) strain R20291, while animals survived when colonized by the parental strain or after colonization by other high-toxin-producing C. difficile strains. To understand the reasons for this increased virulence, we compared the global gene expression profiles between the fliC-R20291 mutant and its parental strain using an in vitro and in vivo transcriptomic approach. The latter made use of the gnotobiotic mouse model. Interestingly, in the fliC mutant, we observed considerable up-regulation of genes involved in mobility, membrane transport systems (PTS, ABC transporters), carbon metabolism, known virulence factors and sporulation. A smaller but significant up-regulation of genes involved in cell growth, fermentation, metabolism, stress and antibiotic resistance was also apparent. All of these genes may be associated with the increased virulence of the fliC-R20921 mutant. We confirmed that the fliC mutation is solely responsible for the observed changes in gene expression in the mutant strain since expression profiles were restored to that of the wild-type strain in the fliC-complemented strain. Thus, the absence of FliC is directly or indirectly involved in the high mortality observed in the fliC mutant infected animals. Therefore, we provide the first evidence that when the major structural component of the flagellum is neutralized, deregulation of gene expression can occur during infection.

Role of fibronectin-binding protein A in Clostridium difficile intestinal colonization.

Clostridium difficile is a frequent cause of severe, recurrent, post-antibiotic diarrhoea and pseudomembranous colitis. Its pathogenicity is mediated mainly by two toxins, TcdA and TcdB. However, different adhesins have also been described as important colonization factors which are implicated in the first step of the intestinal infection. In this study, we focused our interest on one of these adhesins, fibronectin-binding protein A (FbpA), and on its role in the intestinal colonization process. A mutant of FbpA (CDΔFbpA) was constructed in C. difficile strain 630Δerm by using ClosTron technology. This mutant was characterized in vitro and in vivo and compared to the isogenic wild-type strain. Adhesion of the CDΔFbpA mutant to the human colonic epithelial cell line Caco-2 and to mucus-secreting HT29-MTX cells was examined. Surprisingly, the CDΔFbpA mutant adhered more than the wild-type parental strain. The CDΔFbpA mutant was also analysed in three different mouse models by following the intestinal implantation kinetics (faecal shedding) and caecal colonization (7 days post-challenge). We showed that in monoxenic mice, CDΔFbpA shed C. difficile in faeces at the same rate as that of the isogenic wild-type strain but its colonization of the caecal wall was significantly reduced. In dixenic mice, the shedding rate was slower for the CDΔFbpA mutant than for the isogenic wild-type strain during the first days of infection, but no significant difference was observed in caecal colonization. Similar rates of intestinal implantation and caecal colonization were observed for both strains in assays performed in human microbiota-associated mice. Taken together, our data suggest that FbpA plays a role in intestinal colonization by C. difficile.