Type IV pili-associated secretion of a biofilm matrix protein from Clostridium perfringens that forms intermolecular isopeptide bonds

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen of humans and animals. C. perfringens also produces type IV pili (T4P) and has two complete sets of T4P-associated genes, one of which has been shown to produce surface pili needed for cell adherence. One hypothesis about the role of the other set of T4P genes is that they could comprise a system analogous to the type II secretion systems (TTSS) found in Gram-negative bacteria, which is used to export folded proteins from the periplasm through the outer membrane to the extracellular environment. Gram-positive bacteria have a similar secretion barrier in the thick peptidoglycan (PG) layer, which blocks secretion of folded proteins >25 kD. To determine if the T4P-associated genes comprise a Gram-positive TTSS, the secretome of mutants lacking type IV pilins were examined and a single protein, a von Willebrand A domain containing protein BsaC (CPE0517) was identified as being dependent on PilA3 for secretion. BsaC is in an operon with a signal peptidase and two putative biofilm matrix proteins with homology to Bacillus subtilis TasA. One of these proteins, BsaA, was shown by another group to produce high mol wt oligomers. We analyzed BsaA monomer interactions with de novo modeling, which projected that the monomers formed isopeptide bonds as part of a donor strand exchange process. Mutations in residues predicted to form the isopeptide bonds led to loss of oligomerization, supporting the predicted bond formation process. Phylogenetic analysis showed the BsaA family of proteins are widespread among bacteria and archaea but only a subset are predicted to form isopeptide bonds.