Membranes of the Dental Pathogen Streptococcus mutans

描述（由申请人提供）：龋齿在美国每年造成250 - 300亿美元的卫生保健费用，是由致病菌变形链球菌引起的。一个主要的毒力因素是由饮食碳水化合物产生的乳酸和细菌对酸产物的耐受性导致牙齿脱矿。这在很大程度上是由一个多亚基F1F0 ATP酶介导的，该酶跨越细胞质膜，切割ATP并从细胞中泵出质子。在其他原核和真核细胞以及线粒体中，同样的酶作为ATP合酶反过来起作用。信号识别颗粒（signal recognition particle， SRP）通路是一种在所有活细胞中保守的共翻译蛋白易位通路，参与蛋白质插入和分泌进入或通过细胞质或内质网膜。直到在变形链球菌中被证明不同，SRP途径被认为是必不可少的。在变形链球菌中，SRP通路的消除导致了对酸、盐和氧化休克等多种应激源的脆弱性，但细菌产生了功能膜并存活下来。在变形链球菌的线粒体/细菌/叶绿体中发现了两个高度保守的膜定位伴侣插入酶家族（Oxa/YidC/Alb）的类似物。yidC2的消除导致与SRP通路破坏相似的应激敏感表型，并且在这两种情况下，在膜组成中观察到许多扰动，包括atp酶活性受损。yidC2的消除也会干扰变形链球菌另一个关键毒力因子粘连素P1的定位和功能。在没有SRP通路的情况下，线粒体通过Oxa1介导的共翻译机制将关键蛋白插入其膜中，这需要其c端具有核糖体结合功能。变形链球菌YidC2具有类似的序列，并且YidC2可以取代酵母中的Oxa1，这意味着细菌YidC具有迄今为止未被认识到的共翻译易位功能，并为缺乏SRP途径的变形链球菌的胁迫和耐酸性提供了解释。相反，YidC2对P1的影响是独立于c端尾部的，这表明YidC2还具有翻译后功能，并参与介导表面蛋白向细胞外易位的一般分泌途径。本研究的目标是：1)利用生物化学、分子和蛋白质组学的方法来解剖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通路；2)利用化学交联和免疫沉淀来确定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。3)剖析变形链球菌分泌机制各组分的组成及相互作用。这些目标将促进我们对变形链球菌的酸耐受性和表面生物发生的理解，提出新的治疗干预靶点，并代表了膜生物发生和蛋白质分泌研究的新起点，这将进一步了解与其他致病性链球菌和革兰氏阳性菌相关的基础知识。

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.