The Clostridium difficile Flagellar System Mediates Toxin Synthesis, Pathogenicity, and Activation of Innate Immune Responses

Abstract

Clostridium difficile has emerged as the most common healthcare-associated infection responsible for an estimated annual cost of $6 billion to the US healthcare system. Disease is mediated through the action of two glucosylating toxins which manifests as a fulminant diarrhea. Despite the severity of C. difficile infections, large gaps in knowledge, particularly pertaining to the molecular mechanisms of host colonization and interaction with the innate immune response, have hampered the development of novel targeted therapies. This work explores the molecular mechanisms used by C. difficile, in the context of the flagellar system, to colonize the host and interact with the resident immune system. The flagellin (FliC) protein was shown to not only be a critical mediator of C. difficile motility, but also participated in regulating sporulation, biofilm formation, and the expression of toxin genes. The FliC protein also robustly activated host inflammatory signaling through interaction with TLR5, demonstrating this molecule’s importance as a mediator of both bacterial and host responses to C. difficile infection. The flagellar capping protein (FliD) by contrast was found to have an important role mediating attachment to host tissues as well as to regulate and prevent the secretion of FliC into the extracellular environment. Finally, a novel FliC-specific glycosylation system was characterized. Through the addition of glycans, the reactivity of FliC with TLR5 was dampened, resulting in a weaker inflammatory response to infection. Flagellin glycans were also found to be necessary for C. difficile flagellar assembly, and different strains exhibited significant variation in both the number and structure of glycan moieties attached. The C. difficile flagellar system is a key mediator of bacterial motility, virulence, and host-pathogen interactions during infection with this enigmatic organism.