Structural and Evolutionary Analyses Show Unique Stabilization Strategies in the Type IV Pili of Clostridium difficile

Summary Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion, and horizontal gene transfer. However, many Gram-positive species, including Clostridium difficile , also produce type IV pili. Here, we identify the major subunit of the type IV pili of C. difficile , PilA1, and describe multiple 3D structures of PilA1, demonstrating the diversity found in three strains of C. difficile . We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the type IVb pilins from Gram-negative bacteria that suggest that the type IV pili of C. difficile are involved in microcolony formation.