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

菌毛蛋白的氧化蛋白折叠和革兰氏阳性菌的毒力

• 批准号: 8734900
• 负责人: Melissa Elizabeth Robinson
• 金额: $ 2.98万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2015-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734900
• 关键词: Actinobacteria class; Actinomyces; Address; Adherence; Adhesives; Affect; Alkylation; Biochemical; Bioinformatics; Biological; Biological Assay; C-terminal; Cavia; Cell Wall; Cell membrane; Cell surface; Corynebacterium diphtheriae; Culture Media; Cysteine; Cytoplasm; Data; Defect; Development; Diphtheria; Diphtheria Toxin; Disease; Enzymes; Epithelium; Escherichia coli; Experimental Models; Extracellular Protein; Face; Fimbriae Proteins; Fluorescence; Gene Deletion; Genes; Genetic; Genome; Goals; Gram-Positive Bacteria; Health; Human; Immunity; Immunoglobulin G; Investigation; Link; Mass Spectrum Analysis; Mediating; Membrane; Microbial Biofilms; Modeling; Molecular Conformation; Mutate; Mutation; Mycobacterium tuberculosis; Natural regeneration; Organism; Outcome Study; Oxidation-Reduction; Oxidoreductase; Pathogenesis; Pathway interactions; Peptidyltransferase; Phenotype; Pilum; Polymers; Process; Propilin; Protein Export Pathway; Protein Precursors; Proteins; Resistance; Role; Septic Toxemia; Series; Structure; Study models; Sulfhydryl Compounds; System; Techniques; Therapeutic; Tissues; Trans-Activators; Virulence; Virulence Factors; antimicrobial; antimicrobial drug; base; catalyst; cell motility; disulfide bond; extracellular; factor C; fimbria; in vivo; insight; monomer; mutant; oral pathogen; pathogenic bacteria; polymerization; polypeptide; prevent; protein folding; public health relevance; single molecule; sortase; vitamin K epoxide reductase

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.

描述（由申请人提供）：革兰氏阳性粘附皮利通过一系列分选酶在细胞表面组装，所述分选酶共价连接菌毛蛋白亚基，然后将所得聚合物锚于细胞壁。在与分选酶相互作用之前，新生的菌毛蛋白被易位出细胞质，拴系到膜的外质面，并折叠。虽然，分选酶介导的菌毛装配的机制已被很好地描述，菌毛蛋白亚基折叠的方式在外质内是不太清楚的。这项提案的目的是 描述这些毒力因子的细胞外折叠途径。最近的结构研究FimA和SpaA，菌毛轴蛋白表达的放线菌口放线菌和白喉棒状杆菌，分别揭示了洞察这个生物学问题。这两种蛋白质的晶体结构显示与C-末端的二硫键。当形成这些的半胱氨酸残基突变时，FimA和SpaA不再聚合，而是作为单体和降解产物分泌到培养基中。预测的巯基氧化还原酶样结构域的编码细胞外蛋白的基因的缺失产生了类似的表型，这表明菌毛蛋白需要氧化蛋白折叠。放线菌内氧化蛋白质折叠的途径尚未阐明。我们推测，二硫键形成机制需要适当折叠菌毛蛋白和其他毒力因子的外质。利用放线菌A. oris和C.白喉杆菌作为实验模型，我们的目的是（1）研究菌毛蛋白中二硫键的形成及其在菌毛组装中的作用，（2）阐明二硫键形成的机制，（3）确定二硫键的形成是否是菌毛和非菌毛毒力因子折叠的一般机制。对这些模型中二硫键形成的研究将为开发抗微生物药物提供新的靶点。