Post-translocational protein folding in Gram-positive bacteria

 描述(申请人提供)：正确的蛋白质折叠对细胞功能至关重要。促进蛋白质正确折叠的二硫键形成机制在真核生物和革兰氏阴性细菌中得到了很好的识别。二硫键的形成有助于蛋白质的整个折叠过程，稳定结构并防止降解。在革兰氏阴性菌中，这一过程发生在氧化的周质空间，需要一对氧化还原酶DsbA和DsbB。相比之下，人们对单膜革兰氏阳性细菌中的氧化蛋白质折叠知之甚少，因为这种细菌不被认为有周质。具体地说，蛋白质前体如何在未折叠状态下通过一般分泌物SEC转位跨细胞膜转运而正确折叠，目前还知之甚少。最近在放线杆菌基因组中发现的氧化还原酶编码基因和结核分枝杆菌中的维生素K环氧化物还原酶，被认为是大肠杆菌DsbB的功能同源物，为这些生物的氧化折叠机制提供了一些线索。因此，我们实验室最近开始使用口腔放线菌的实验模型来研究这一基本问题。口腔放线菌是一种已知在口腔生物膜或牙菌斑的形成中发挥重要作用的放线杆菌。通过结构分析，我们确定了黄曲霉FIMA中的二硫键。FIMA是生物膜形成和种间相互作用所需的菌毛轴。我们证明了FIMA的C-末端二硫键是菌毛组装和生物膜形成所必需的。最近，我们发现共聚集因子CAFA中二硫键的破坏可以消除口蹄疫杆菌与口腔链球菌的共聚集。为了找到影响物种间相互作用的其他因素，我们用A.oris中的TN5转座子突变体文库进行了大规模筛选，并鉴定了定位于潜在编码氧化蛋白折叠途径各种成分的基因的共聚集缺陷突变体。通过结合遗传学、生物物理学、生物化学、结晶学、质谱学、细胞分析以及龋齿和细菌感染模型的多学科方法，我们旨在阐明口腔不动杆菌氧化蛋白折叠的机制，确定这一途径在其他放线菌中的保守性，并探索预防龋齿和细菌感染的策略。

 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.