Roles of Streptococcus mutans YidC1 and YidC2 in membrane biogenesis

描述（由申请人提供）：变形链球菌是龋齿的病原体，每年在相关卫生保健中花费数十亿美元。其产酸和耐酸（耐酸）特性导致发病。在导致疾病的生理特性中，细胞质膜组成起着不可或缺的作用。酸耐受性在很大程度上由膜相关的F1 Fo ATP酶介导，该膜相关的F1 Fo ATP酶将质子泵出细胞质。我们在S.信号识别颗粒（SRP）共翻译蛋白易位途径的可免除性，以前认为在每个真核、古细菌和原核细胞中是必需的，这意味着在该生物体中存在膜生物发生和胁迫耐受性的后备机制。细菌YidC是普遍保守的Oxa 1/YidC/Alb 3（线粒体/细菌/叶绿体）家族的膜相关伴侣和插入酶的成员。我们在S.变异人酵母线粒体Oxa 1具有带电荷的胞质尾区，并表现出核糖体结合和共翻译易位功能。S.变形杆菌YidC 2也有类似的带电尾巴。与SRP破坏类似，YidC 2的消除导致应激（酸、渗透、氧化）敏感性，包括S.变异人有趣的是，S。变形杆菌YidC 2和YidC 1均能在功能上替代E. coliYidC，包括F_1F_0ATPase膜插入;变异体消除YidC 1对酸或应激耐受性没有影响，表明在同源分泌机制的背景下生物体特异性差异。到目前为止，同时消除YidC 2和SRP组分，或YidC 1和YidC 2，从S。没有实现mutans。将开发使用菊粉诱导型、葡萄糖阻遏型fruA启动子的条件表达系统，以评估YidC 1和YidC 2在S.变形菌膜生物发生还将进行结构域交换实验以开始剖析两种蛋白质的各自作用。F1 Fo ATP酶的生物发生将通过优化用于S.变形杆菌倒置膜囊泡和9-氨基-6-氯-2-甲氧基吖啶（ACMA）淬灭的测量。将使用特异性抑制剂（P型原钒酸盐和F型ATP酶的寡霉素）确认F1 Fo ATP酶活性。最后，免疫共沉淀和化学交联将评估YidC 1和YidC 2彼此之间的相互作用以及与蛋白分泌装置的其他组分（包括含SecY的膜易位子孔）的相互作用。相关性：本研究将进一步加深我们对S。变异株毒力机制，并提出新的治疗靶点。

DESCRIPTION (provided by applicant): Streptococcus mutans is the etiological agent of dental caries costing billions of dollars annually in associated health care. Its acidogenic and aciduric (acid tolerant) properties result in pathogenesis. Of the physiological characteristics leading to disease, cytoplasmic membrane composition plays an integral role. Acid tolerance is mediated in large part by a membrane associated F1 Fo ATPase that pumps protons out of the cytoplasm. We demonstrated in S. mutans the dispensability of the signal recognition particle (SRP) co-translational protein translocation pathway, previously believed to be essential in every eukaryotic, archaeal and prokaryotic cell implying the existence of a back-up mechanism for membrane biogenesis and stress tolerance in this organism. Bacterial YidC is a member of the universally conserved Oxa1 /YidC/Alb3 (mitochondria/ bacterial/ chloroplast) family of membrane-associated chaperones and insertases. We identified two YidC orthologs in S. mutans. Yeast mitochondrial Oxa1 has a charged cytoplasmic tail and demonstrated ribosome binding and co-translational translocation functions. S. mutans YidC2 has a similar charged tail. Like SRP disruption, elimination of YidC2 results in stress (acid,osmotic, oxidative) sensitivity including decreased membrane associated ATPase activity in S. mutans. Interestingly, S. mutans YidC2 and YidC1 both can functionally replace E. coli YidC, including F1 Fo ATPase membrane insertion; however, in S. mutans elimination of YidC1 has no effect on acid or stress tolerance indicating organism specific differences in the context of the homologous secretion machinery. To date simultaneous elimination of YidC2 and SRP components, or YidC1 and YidC2, from S. mutans has not been achieved. A conditional expression system using the inulin inducible, glucose repressible fruA promoter will be developed to assess the relative roles of YidC1 and YidC2 in S. mutans membrane biogenesis. Domain swapping experiments also will be performed to begin to dissect the respective roles of the two proteins. Biogenesis of the F1 Fo ATPase will be investigated specifically by optimizing a PMF (proton motive force) assay for use with S. mutans inverted membrane vesicles and measurement of quenching of 9-amino-6- chloro-2-methoxyacridne (ACMA). F1Fo ATPase activity will be confirmed using specific inhibitors, orthovanadate for P-type, and oligomycin to F-type ATPases. Lastly, co-immunoprecipitation and chemical crosslinking will evaluate YidC1 and YidC2 interactions with each other and other components of the protein secretion apparatus including the SecY-containing membrane translocon pore. RELEVANCE: this research will further our understanding of S. mutans virulence mechanisms and suggest novel therapeutic targets.