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

基本信息

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
项目类别：
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

项目摘要

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）：革兰氏阳性粘合剂pili由一系列排序酶组装在细胞表面，这些酶将pilin亚基连接起来，然后锚定的聚合物将聚合物与细胞壁锚定。在与分子酶相互作用之前，新生的pilin从细胞质中转移出来，束缚在膜的质质面上并折叠。虽然，已经很好地描述了分子酶介导的菌毛组件的机制，但毛线亚基在类质中折叠的方式不太清楚。该提议的目的是描述这些毒力因子的细胞外折叠途径。最近对FIMA和SPAA的结构研究，分别由放线杆菌的pilus轴蛋白和二甲状腺细菌表达的pilus轴蛋白揭示了对这一生物学问题的见解。两种蛋白质的晶体结构都揭示了与C末端的二硫键。当形成这些突变的半胱氨酸残基被突变时，FIMA和SPAA不再聚合，而是将其分泌为单体和降解产物中的培养基。用预测的硫醇 - 氧化核苷酸域样结构域编码细胞外蛋白的基因的缺失，产生了相似的表型，这表明PILINS需要氧化蛋白折叠。静脉细菌中氧化蛋白折叠的途径尚未阐明。我们假设需要二硫键形成机械才能正确折叠外生质蛋白质和其他毒力因子。使用静脉细菌A. Oris和C. diphtheriae作为实验模型，我们的目的是（1）研究PILUS蛋白质中的二硫键形成及其在Pilus组装中的作用，（2）阐明二硫键键形成的机理，（3）确定二硫键形成是否是折叠折叠机制和折叠式折叠机制和非PILUS的一般折叠机制。这些模型中二硫键形成的研究将为开发抗菌药物提供新的靶标。