Post-translocational protein folding in Gram-positive bacteria

革兰氏阳性菌中的易位后蛋白质折叠

基本信息

批准号：
9773401
负责人：
Hung Ton-That
金额：
$ 27.79万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9773401
关键词：
Actinobacteria class Actinomyces Affect Alanine Anabolism Anti-Infective Agents Bacterial Infections Biochemistry Biological Assay Biophysics C-terminal Cell membrane Cell physiology Cells Corynebacterium diphtheriae Coupled Coupling Crystallography Cysteine Defect Dental Dental Models Dental Plaque Dental caries Development Disease Disulfides Ectopic Expression Electron Transport Enzymes Escherichia coli Eukaryota Experimental Models Genes Genetic Genome Gram-Negative Bacteria Gram-Positive Bacteria Homologous Gene In Vitro Knowledge Laboratories Libraries Maps Mass Spectrum Analysis Mediating Membrane Microbial Biofilms Modality Mutation Mycobacterium tuberculosis NADH dehydrogenase (ubiquinone)Organism Oxidation-Reduction Oxides Oxidoreductase Pathogenesis Pathway interactions Play Prevention Prevention strategy Process Protein Precursors Proteins Role Spectrum Analysis Streptococcus oralis Structure System Thiol Disulfide Oxidoreductase Virulence Virulence Factors Vitamin K 2 bacterial fitness base design disulfide bond inhibitor/antagonist interdisciplinary approach mutant oral biofilm pathogen periplasm prevent protein folding public health relevance reconstitution single molecule vitamin K epoxide reductase

Actinobacteria class Actinomyces Affect Alanine Anabolism Anti-Infective Agents Bacterial Infections Biochemistry Biological Assay Biophysics C-terminal Cell membrane Cell physiology Cells Corynebacterium diphtheriae Coupled Coupling Crystallography Cysteine Defect Dental Dental Models Dental Plaque Dental caries Development Disease Disulfides Ectopic Expression Electron Transport Enzymes Escherichia coli Eukaryota Experimental Models Genes Genetic Genome Gram-Negative Bacteria Gram-Positive Bacteria Homologous Gene In Vitro Knowledge Laboratories Libraries Maps Mass Spectrum Analysis Mediating Membrane Microbial Biofilms Modality Mutation Mycobacterium tuberculosis NADH dehydrogenase (ubiquinone)Organism Oxidation-Reduction Oxides Oxidoreductase Pathogenesis Pathway interactions Play Prevention Prevention strategy Process Protein Precursors Proteins Role Spectrum Analysis Streptococcus oralis Structure System Thiol Disulfide Oxidoreductase Virulence Virulence Factors Vitamin K 2 bacterial fitness base design disulfide bond inhibitor/antagonist interdisciplinary approach mutant oral biofilm pathogen periplasm prevent protein folding public health relevance reconstitution single molecule vitamin K epoxide reductase

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项目摘要

DESCRIPTION (provided by applicant): Proper protein folding is critical to cellular function. Disulfide bond-forming machines that facilitate proper protein folding are well recognized in eukaryotes and Gram-negative bacteria. Disulfide bond formation contributes to the overall protein folding process, stabilizing structures and protecting against degradation. In Gram-negative bacteria, this process occurs in the oxidizing periplasmic space and is required a pair of oxidoreductase enzymes DsbA and DsbB. In contrast, little is known about oxidative protein folding in single- membrane Gram-positive bacteria, which are not considered to have periplasms. Specifically, how protein precursors translocated across the cytoplasmic membrane by the general secretion Sec translocon in an unfolded state manage to fold correctly is poorly understood. Recent findings of oxidoreductase-encoding genes in the genome of actinobacteria and Vitamin K epoxide reductase in Mycobacterium tuberculosis, considered as a functional homolog of Escherichia coli DsbB, offer some clue to an oxidative folding mechanism in these organisms. Therefore, our laboratory recently began to investigate this fundamental problem using an experimental model in Actinomyces oris, an actinobacterium known to play an important role in the formation of oral biofilms or dental plaque. By structural analysis, we identified disulfide bonds in FimA of A. oris. FimA is the fimbrial shaft required for biofilm formation and interspecies interactions. We demonstrated that the C-terminal disulfide bond of FimA is essential for fimbrial assembly and biofilm formation. More recently, we revealed that disruption of a disulfide bond in coaggregation factor CafA eliminates A. oris coaggregation with Streptococcus oralis. To find additional factors that affect interspecies interactions, we performed a large-scale screen with a Tn5 transposon mutant library in A. oris and identified coaggregation- defective mutants mapped to genes potentially encoding various components of an oxidative protein folding pathway. By using a multidisciplinary approach that combines genetics, biophysics, biochemistry, crystallography, mass spectrometry, cell-based assays, and models of dental caries and bacterial infection, we aim to elucidate the mechanism of oxidative protein folding in A. oris, to determine the conservation of this pathway in other actinobacteria, and to explore preventive strategies for dental caries and bacterial infections.

描述（适用提供）：正确的蛋白质折叠对细胞功能至关重要。在真核生物和革兰氏阴性细菌中良好地认识到促进蛋白质折叠的二硫键形成的机器。二硫键形成有助于整体蛋白质折叠过程，稳定结构并防止降解。在革兰氏阴性细菌中，此过程发生在氧化的外围空间中，并且需要一对氧化酶DSBA和DSBB。相比之下，对于单膜革兰氏阳性细菌中的氧化蛋白折叠知之甚少，这些蛋白折叠不被认为没有外围。具体而言，对未折叠状态下的大分泌SEC转运跨细胞质膜易位的蛋白质前体如何正确地理解了折叠。在静脉细菌基因组和维生素K环氧化核酸杆菌的基因组中，氧化还原酶 - 编码基因的最新发现被认为是这些生物体中氧化物折叠机制的一些线索，被认为是埃切里希菌大肠杆菌DSBB的功能同源物。因此，我们的实验室最近开始使用放线菌Oris中的实验模型来研究这个基本问题，这是一种已知的放线杆菌，在口腔生物膜或牙斑的形成中起着重要作用。通过结构分析，我们确定了A. Oris Fima中的二硫键。 FIMA是生物膜形成和种间相互作用所需的纤维轴。我们证明了FIMA的C末端二硫键对于纤维化组装和生物膜形成至关重要。最近，我们揭示了凝聚因子CAFA中二硫键的破坏消除了A. oris与Oralis链球菌的凝聚。为了找到影响种间相互作用的其他因素，我们与A. Oris中的TN5转座子突变体库进行了大规模筛选，并确定了映射到潜在地编码氧化蛋白折叠途径的各种成分的基因的凝集有缺陷的突变体。通过使用结合遗传学，生物物理学，生物化学，晶体学，质谱法，基于细胞的质谱，基于细胞的测定法和牙科龋齿和细菌感染模型的多学科方法，我们旨在阐明A. ORIS中氧化蛋白质折叠的机制，以确定该途径在其他ActInobacceria和Actinobaciation中的保存，以探讨其他预防性的策略。