Disulfide bond formation in Streptococcus gordonii

二硫键对于许多蛋白质的正确折叠是重要的，并且由硫醇-二硫键氧化还原酶（TDORs）催化。这些细胞质外的TDORs参与不同的生理过程。在革兰氏阳性菌中，二硫键的形成知之甚少。我实验室的研究表明，口腔细菌戈登链球菌是研究二硫键形成的模式生物。所取得的进展包括鉴定了多种表型所需的主要TDOR（SdbA）和鉴定了SdbA的氧化还原伴侣（SdbB和CcdA 2）。我的研究计划的长期目标是阐明革兰氏阳性菌中二硫键形成的机制。短期目标将重点关注S中二硫键形成和异构化途径的表征。gordonii。这三个目标是：（1）SdbA氧化途径的进一步研究;（2）SdbA的其他天然底物的鉴定;（3）二硫键异构化途径的表征。对于目的（1），将通过转座子诱变鉴定SdbA-SdbB-CcdA 2途径的最终电子受体和其他组分。标准技术将用于研究鉴定的基因及其在途径中的作用。 对于目标（2），将使用计算机模拟和蛋白质组学方法的组合鉴定SdbA的天然底物。分泌的和膜相关的蛋白质与2个或更多的半胱氨酸将被确定从S。戈登氏菌蛋白质组平行地，细胞外和细胞表面蛋白将通过从单个半胱氨酸SdbAC 89 A点突变体（其产生许多混合二硫键复合物）分级分离获得。将使用抗SdbA抗体在亲和色谱中分离复合物，并通过质谱法鉴定。将进一步分析与上述表型相关的蛋白质，以验证它们是SdbA的底物。对于目标（3），我们表明SdbB具有二硫键异构酶活性。这项工作是确定的合作伙伴和天然底物的SdbB和异构化途径在细胞过程中的作用。采用诱变方法研究SdbB及其配偶体的功能。将使用与目标（2）相似的免疫亲和层析和蛋白质组学方法鉴定SdbB底物。工作的意义。本研究结果将为深入了解口腔黏膜S细胞二硫键形成机制和细胞过程提供理论依据。gordonii。所获得的知识对于理解口腔细菌的适应和生存具有根本的重要性。从长远来看，这些知识将成为了解革兰氏阳性病原体中形成多少含二硫键的毒力因子（例如超抗原）的基础。

A disulfide bond is important for the correct folding of many proteins and is catalyzed by thiol-disulfide oxidoreductases (TDORs). These extracytoplasmic TDORs participate in diverse physiological processes. In Gram-positive bacteria, disulfide bond formation is poorly understood. Research in my laboratory has indicated that the oral bacterium Streptococcus gordonii is a model organism to study disulfide bond formation. Progress made include identification of a major TDOR (SdbA) that is needed for multiple phenotypes and identification of the redox partners (SdbB and CcdA2) of SdbA. The LONG-TERM GOAL of my research program is to elucidate the mechanisms of disulfide bond formation in Gram-positive bacteria. The SHORT-TERM GOALS will focus on the characterization of the disulfide bond formation and isomerization pathways in S. gordonii. The 3 objectives are: (1) Further investigations of the SdbA oxidative pathway; (2) Identification of additional natural substrates of SdbA; (3) Characterization of the disulfide isomerization pathway. For objective (1), the final electron acceptor(s) and other components of the SdbA-SdbB-CcdA2 pathway will be identified via transposon mutagenesis. Standard techniques will be used to study the identified gene(s) and its role in the pathway. For objective (2), the natural substrates of SdbA will be identified using a combination of in silico and proteomic approaches. Secreted and membrane-associated proteins with 2 or more cysteines will be identified from the S. gordonii proteome. In parallel, extracellular and cell surface proteins will be obtained via fractionation from the single cysteine SdbAC89A point mutant, which produced a number of mixed disulfide complexes. The complexes will be isolated using anti-SdbA antibody in affinity chromatography and identified by mass spectrometry. Proteins related to phenotypes mentioned above will be further analyzed to verify that they are substrates of SdbA. For objective (3), we showed that SdbB has disulfide isomerase activity. The work is to identify the partner and natural substrates of SdbB and the role of the isomerization pathway in cellular processes. Mutagenesis will be employed to investigate the functions of SdbB and its partner. SdbB substrates will be identified using immuno-affinity chromatography and proteomic approach similar to objective (2). SIGNIFICANCE of the work. The results will provide understanding to the mechanism of disulfide bond formation and cellular processes in the oral commensal S. gordonii. The knowledge gained is of fundamental importance to the understanding of adaptation and survival of oral bacteria. In the long-term, the knowledge will form the foundation of understanding how many of the disulfide bond-containing virulence factors (e.g. superantigens) are formed in Gram-positive pathogens.