Disulfide bond formation in Streptococcus gordonii

戈登链球菌中二硫键的形成

• 批准号: RGPIN-2019-04880
• 负责人: Lee, Song
• 金额: $ 2.33万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743702
• 关键词: Disulfide; bond; formation; Streptococcus; gordonii

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.

二硫键对许多蛋白质的正确折叠是重要的，并由硫醇-二硫键氧化还原酶(TDORs)催化。这些胞外TDOR参与不同的生理过程。在革兰氏阳性细菌中，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。所取得的进展包括确定了多种表型所需的主要TDOR(SDBA)，以及确定了SDBA的氧化还原伙伴(SDBB和CcdA2)。我的研究计划的长期目标是阐明革兰氏阳性细菌中二硫键的形成机制。短期目标将集中在戈登氏链霉菌中二硫键形成和异构化途径的表征上。这三个目标是：(1)进一步研究SDBA的氧化途径；(2)鉴定SDBA的其他天然底物；(3)表征二硫键异构化途径。对于目标(1)，将通过转座子突变来鉴定最终的电子受体(S)和SDBA-SDBB-CCDA2途径的其他成分。将使用标准技术来研究已识别的基因(S)及其在该途径中的作用。对于目标(2)，将使用蛋白质组学和蛋白质组学相结合的方法来鉴定SDBA的天然底物。含有2个或2个以上半胱氨酸的分泌型和膜相关蛋白将从戈尔登氏链霉菌蛋白质组中鉴定出来。同时，细胞外和细胞表面的蛋白质将通过分离得到单半胱氨酸SdbAC89A点突变体，它产生了许多混合的二硫键复合体。在亲和层析中使用抗SDBA抗体来分离这些配合物，并用质谱仪进行鉴定。与上述表型相关的蛋白质将被进一步分析，以证实它们是SDBA的底物。对于目标(3)，我们证明了SDBB具有二硫键异构酶活性。这项工作是为了确定SDBB的伴侣和天然底物以及异构化途径在细胞过程中的作用。突变将被用来研究SDBB及其合作伙伴的功能。将使用免疫亲和层析和类似于目标(2)的蛋白质组学方法来鉴定SDBB底物。这项工作的意义。这些结果将为理解口腔共生戈登氏链球菌中二硫键的形成机制和细胞过程提供依据。所获得的知识对于了解口腔细菌的适应和生存是至关重要的。从长远来看，这一知识将为了解革兰氏阳性病原体中形成了多少含有二硫键的毒力因子(如超抗原)奠定了基础。