Membranes of the Dental Pathogen Streptococcus mutans

描述(由申请人提供)：在美国，龋齿每年造成250-300亿美元的医疗费用，由致病细菌变形链球菌引起。一个主要的致病因素是通过从饮食碳水化合物中产生乳酸而使牙齿脱矿，以及对酸性产品的细菌耐受性。这在很大程度上是由一个多亚单位F1F0 ATPase介导的，它跨越细胞膜，裂解ATP，并将质子从细胞中泵出。在其他原核和真核细胞中，以及线粒体中，同样的酶作为ATP合成酶反向工作。信号识别颗粒(SRP)途径是一种共翻译的蛋白质转位途径，存在于所有活细胞中，参与蛋白质插入和分泌到细胞质或内质网膜。在变形链球菌中证明这一点之前，SRP途径一直被认为是必不可少的。在变形链球菌中，SRP途径的消除会导致对包括酸、盐和氧化休克在内的许多应激源的脆弱性，但细菌会产生一个功能膜并存活下来。在变形链球菌中已经鉴定出线粒体/细菌/叶绿体中高度保守的膜定位伴侣插入酶家族(OxA/YidC/Alb)的两个类似基因。YidC2的消除导致与SRP途径中断类似的应激敏感表型，在这两种情况下，都观察到膜组成的许多扰动，包括ATPase活性受损。YidC2的消除也干扰了变形链球菌的另一个关键毒力因子粘附素P1的定位和功能。在缺乏SRP途径的情况下，线粒体通过OXa1介导的共翻译机制将关键蛋白插入到细胞膜中，这需要其C端具有核糖体结合功能。变形链球菌YidC2具有相似的序列，YidC2可以取代酵母中的Oxa1，这意味着细菌YidC具有迄今未知的共翻译易位功能，并为缺乏SRP途径的变形链球菌的抗逆性和耐酸性提供了解释。相反，YidC2对P1的影响不依赖于C-末端的尾巴，这表明YidC2也具有翻译后功能，并参与介导表面蛋白向细胞外部转移的一般分泌途径。本研究的目标是：1)利用生化、分子和蛋白质组学的方法，剖析导致变形链球菌毒力的特定底物的膜插入和分泌的共翻译和翻译后途径；2)通过化学交联和免疫沉淀，确定YidC2在共翻译易位中的作用及其与链球菌核糖体的相互作用；以及3)剖析变形链球菌分泌机制组件之间的组成和相互作用。这些目标将促进我们对变形链球菌耐酸性和表面生物发生的理解，为治疗干预提供新的靶点，并代表着膜生物发生和蛋白质分泌研究的新起点，将进一步加深与其他致病链球菌和革兰氏阳性微生物相关的基础知识。 与公共卫生相关：龋齿是一种传染病，在美国每年造成250-300亿美元的医疗费用，由致病的口腔变形链球菌引起。这种有机体会导致牙齿腐烂，因为它会从饮食中的碳水化合物中产生酸，而它之所以能存活下来，是因为它能忍受自己的酸性最终产物和其他恶劣的环境压力。这些特性源于变形链球菌将蛋白质插入其细胞膜的方式。这项研究将解释蛋白质如何进入和穿过这种病原体和其他相关病原体的膜。

DESCRIPTION (provided by applicant): Dental caries results in 25-30 billion dollars in annual health care costs in the US and is caused by the pathogenic bacterium Streptococcus mutans. A major virulence factor is demineralization of teeth by generation of lactic acid from dietary carbohydrates and bacterial tolerance of the acid products. This is largely mediated by a multi-subunit F1F0 ATPase that spans the cytoplasmic membrane, cleaves ATP and pumps protons from the cell. In other prokaryotic and eukaryotic cells, as well as mitochondria, the same enzyme works in reverse as an ATP synthase. The signal recognition particle (SRP) pathway is a co-translational protein translocation pathway conserved in all living cells and involved in insertion and secretion of proteins into and through the cytoplasmic or endoplasmic reticulum membrane. Until it was demonstrated otherwise in S. mutans, the SRP pathway was considered essential. In S. mutans, elimination of the SRP pathway results in vulnerability to numerous stressors including acid, salt, and oxidative shock, but the bacteria generate a functional membrane and survive. Two paralogs of a family (Oxa/YidC/Alb) of highly conserved membrane-localized chaperone- insertases in mitochondria/bacteria/chloroplasts have been identified in S. mutans. Elimination of yidC2 results in a similar stress-sensitive phenotype as disruption of the SRP pathway and in both cases numerous perturbations are observed in membrane composition, including impaired ATPase activity. Elimination of yidC2 also interferes with localization and function of another key virulence factor of S. mutans, adhesin P1. Mitochondria insert key proteins into their membranes in the absence of an SRP pathway via a co-translational mechanism mediated by Oxa1 requiring a ribosome binding function of its C-terminus. S. mutans YidC2 has a similar sequence and YidC2 can replace Oxa1 in yeast implying a heretofore unrecognized co-translational translocation function of a bacterial YidC and providing an explanation for stress and acid tolerance of S. mutans lacking the SRP pathway. Conversely, the influence of YidC2 on P1 is independent of the C-terminal tail suggesting that YidC2 also serves a post-translational function and participates in the general secretion pathway that mediates translocation of surface proteins to the exterior of the cell. The goals of this proposal are 1) to use biochemical, molecular and proteomic approaches to dissect co- and post-translational pathways responsible for membrane insertion and secretion of specific substrates contributing to S. mutans virulence, 2) to establish the role of YidC2 in co-translational translocation and its interaction with streptococcal ribosomes using chemical cross-linking and immunoprecipitation, and 3) to dissect the composition and interactions among the S. mutans secretion machinery components. These objectives will advance our understanding of acid tolerance and surface biogenesis in S. mutans, suggest novel targets of therapeutic intervention, and represent a new departure in studies of membrane biogenesis and protein secretion that will further fundamental knowledge relevant to other pathogenic streptococci and Gram-positive organisms. PUBLIC HEALTH RELEVANCE: Dental caries is an infectious disease that results in 25-30 billion dollars in annual health care costs in the US and is caused by the pathogenic oral bacterium Streptococcus mutans. This organism causes tooth decay because it generates acid from dietary carbohydrates and it survives because it can tolerate its own acid end products and other harsh environmental stressors. These properties stem from the way S. mutans inserts proteins into its cytoplasmic membrane. This research will explain how proteins get into and through the membrane of this and other related pathogens.