Oxidative protein folding of pilins and virulence in Gram-positive bacteria

描述（由申请人提供）：革兰氏阳性粘附菌毛通过一系列的分选酶在细胞表面组装，这些分选酶将菌毛亚基共价连接，然后将所得聚合物锚定在细胞壁上。在与分选酶相互作用之前，新生的蛋白柱被移出细胞质，拴在膜的外质表面并折叠。虽然，排序酶介导的菌毛组装机制已经被很好地描述，但菌毛亚基在外质内折叠的方式尚不清楚。本提案的目标是

DESCRIPTION (provided by applicant): 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.