Function of PilJ in the Clostridium difficile Type IV Pilus

Abstract

Clostridium difficile, a Gram-positive obligate anaerobe, is the leading cause of nosocomial infections in the United States. C. difficile express type IV pili, extracellular appendages that, in Gram-negatives, are involved in adhesion, colonization, and twitching motility, as well as in phage attachment and DNA uptake. T4P structural subunits are called pilins; other proteins with similar structures to those of pilins, termed pilin-like proteins or minor pilins, are involved in pilus biogenesis and dynamics. Here, we demonstrate that the C. difficile genome contains all genes necessary for T4P biogenesis, including nine separate genes for pilins or pilin-like proteins. We hypothesize that the pilin gene pilJ encodes a minor pilin that functions as an adhesin, and that pilA1 encodes the major pilin for the pili expressed under our standard growth conditions. Analysis of pilin gene sequences from different C. difficile strains demonstrated that pilA1 sequences are the most diverse of all pilin genes. PilA1 is far more highly expressed than PilJ under our growth conditions, with an average molar ratio of PilA1 to PilJ over 900. These results lead us to conclude that PilJ is a minor pilin in a pilus composed primarily of PilA1. The unique two-domain structure of PilJ led us to hypothesize that the protein is an adhesin. Binding experiments in colonic epithelial cells demonstrate that PilJ binds specifically and saturably to a cell-surface receptor. Scatchard analysis of those binding data suggests that PilJ binds to a high-affinity and a low-affinity receptor, with estimated dissociation constants of 1.35 x 10-11 ± 4.81 x 10-11 and 3.44 x 10-9 ± 2.16 x 10-8, respectively. Preliminary pulldown experiments demonstrate the presence of three possible receptors or receptor subunits, with estimated masses of 45 kDa, 55 kDa, and 75 kDa. Mass spectrometry allowed identification of possible receptor candidates. The work presented here delineates the genes for the C. difficile T4P and identifies the major pilin of at least one pilus. We also define a role for the minor pilin PilJ, the first Gram-positive minor pilin for which this has been done.