Pathogenic & functional studies of Gram-positive pilins

Many Gram-positive pathogens polymerize hairlike structures on their cell surfaces called pili that allow for a specific interaction with host target cells. Airway pathogens, Corynebacterium diphtheriae and Streptococcus agalactiae (GBS), construct multiple types of pili through an elaborate mechanism involving pilin-specific sortases. Through genetic and biochemical experiments we prove that C. diphtheriae assemble SpaDEF pili, which are immunologically and biochemically distinct from the two other C. diphtheriae pili. Previous work has highlighted how SrtA from S. aureus catalyzes surface protein anchoring whereby SrtA cleaves the C-terminal cell wall sorting signal (CWSS) between the threonine (T) and glycine (G) residues in the LPXTG motif. A resulting thioester enzyme intermediate is formed between the sortase sulfhydryl of cysteine in the catalytic domain and the carboxyl group of threonine. Surface protein anchoring to the cell surface occurs through an amide bond formation between the carboxyl group of threonine and a free amino group in the cell wall cross-bridge. We set out to determine if the pilin-specific sortase A in C. diphtheriae uses the same mechanism. Point mutations in the conserved cysteine residue within the SrtA catalytic domain, threonine within the SpaA CWSS, and a previously characterized conserved lysine in the pilin motif, all abolished the formation of SpaABC pili, suggesting that SrtA links the threonine of the CWSS of one pilus subunit and the lysine side chain amino group in the pilin motif of another pilus subunit. After polymerization, pili are anchored to the bacterial surface and serve as potential virulence factors. To uncover the factors necessary for bacterial colonization we analyzed the contribution of GBS PI-1 pili in bacterial adherence and pathogenesis. PI-1 minor pilins GBS52 and GBS104 were discovered to be GBS pilus adhesins and recombinant GBS52 bound specifically to human lung epithelial cells. Interestingly, similar to other bacterial adhesins, the crystal structure of GBS52 uncovered a two domain structure, each forming an IgG-like fold. Taken together, these studies illuminate the molecular mechanism of pilus formation and its pathogenic function in Gram-positive organisms.