A Mechanistic Study of Biofilm Formation in Clostridioides Difficile

Clostridioides difficile is a leading cause of nosocomial infections in the United States, with an estimated healthcare burden of over $1 billion. One of the chief difficulties in treating C. difficile infections is their recurrence after treatment with antimicrobials, which occurs in 20-30% of patients. The mechanism for C. difficile persistence and reoccurrence remains unclear though recently, biofilm formation has been hypothesized to be a significant contributing factor. Our group and others have identified type IV pili (T4P) as essential for in vitro biofilm formation by C. difficile. T4P are extracellular helical fibers composed of protein subunits (pilins). These appendages have diverse functions in various bacterial species, including surface (twitching) motility, cellular adhesion, horizontal gene transfer, and biofilm formation. Using gene-interruption mutants of T4P subunits, we found that pilA1 (the major subunit) resulted in a total loss of T4P assembly and a significant reduction in biofilm formation. To probe the mechanism of biofilm formation through T4P, we measured biofilm formation by two mutants, pilJ and pilW (minor subunits), which showed an intermediate phenotype with significantly less biofilm than the wild type. We hypothesized that interactions between T4P and extracellular DNA (eDNA) contributed to biofilm stability, as eDNA is a substantial component of the biofilm extracellular matrix (ECM). We found that PilJ and PilW (but not PilA1) could bind to dsDNA using Electrophoretic Mobility Shift Assays, with PilJ having stronger affinity and less specificity than PilW. We are investigating potential DNA binding sites using our recently determined x-ray crystal structure of PilW and our previously determined structure of PilJ. These results suggest that interactions between C. difficile T4P and eDNA stabilize biofilm, mediating the attachment of bacterial cells to the extracellular matrix and that minor pilin subunits (those incorporated at low abundance) are potential targets for biofilm dispersal