The Gram-positive anaerobe Clostridium difficile is the major cause of nosocomial diarrhea; manifestations of infection include diarrhea, pseudomembranous colitis, and death. Genes for type IV pili, a bacterial nanofiber often involved in colonization and until relatively recently described only in Gram-negatives, are present in all members of the Clostridiales. We hypothesized that any pilins encoded in the C. difficile genome would be immunogenic, as has been shown with pilins from Gram-negative organisms. We describe nine pilin or pilin-like protein genes, for which we introduce a coherent nomenclature, in the C. difficile R20291 genome. The nine predicted pilin or pilin-like proteins have relatively conserved N-terminal hydrophobic regions, but diverge at their C-termini. Analysis of synonymous and nonsynonymous substitutions revealed evidence of diversifying selective pressure in two pilin genes. Six of the nine identified proteins were purified and used to immunize mice. Immunization of mice with each individual protein generated antibody responses that varied in titer and cross-reactivity, a notable result given the low amino acid sequence identity among the pilins. Further studies in other small mammals mirrored our results in mice. Our results illuminate components of the C. difficile type IV pilus and help identify targets for an anti-C. difficile vaccine.
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