Gram-positive adhesive pili are assembled at the cell surface by a series of sortase enzymes which covalently link pilin subunits and then anchor resulting polymers to the cell wall. Prior to interacting with sortase, nascent pilins are translocated out of the cytoplasm, tethered to the exoplasmic face of the membrane, and folded. Although, the mechanism for sortase-mediated pilus assembly has been well described, the manner in which pilin subunits fold within the exoplasm is less clear. The goal of this proposal is to describe an extracellular folding pathway for these virulence factors. Recently structural studies of FimA and SpaA, the pilus shaft proteins expressed by the Actinobacteria Actinomyces oris and Corynebacterium diphtheriae, respectively, revealed insight into this biological problem. The crystal structures for both proteins revealed disulfide bonds with the C-termini. When cysteine residues forming these were mutated, FimA and SpaA were no longer polymerized, but secreted into culture media as monomers and degradation products. Deletion of genes encoding extracellular proteins with predicted thiol-oxidoreductase-like domains produced similar phenotypes suggesting that the pilins require oxidative protein folding. A pathway for oxidative protein folding within Actinobacteria has yet to be elucidated. We hypothesize that disulfide bond forming machinery is required to properly fold pilus proteins and other virulence factors within the exoplasm. Using Actinobacteria A. oris and C. diphtheriae as experimental models, we aim to (1) investigate disulfide bond formation within pilus proteins and their role in pilus assembly, (2) elucidate the mechanism of disulfide bond formation, and (3) determine whether disulfide bond formation is a general folding mechanism to fold both pilus and non-pilus virulence factors. The study of disulfide bond formation within these models will provide new targets to develop antimicrobial drugs.
To infect hosts pathogenic bacteria secrete an arsenal of virulence factors including adhesive pili, which facilitate bacterial adherence to host tissues, cultivate biofilm formation, and modulate host immunity. This study aims to elucidate the pathways that Gram-positive bacteria maintain proper folding of these virulence factors, thus providing targets for the development of new antimicrobial therapeutics which can serve to prevent disease.
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