MEMBRANES OF THE DENTAL PATHOGEN STREPTOCOCCUS MUTANS

项目摘要。变形链球菌是一种普遍存在的口腔细菌， 人类龋齿某些菌株还可引起细菌性心内膜炎和侵入人体冠状动脉 上皮细胞美国每年仅与蛀牙相关的医疗费用就超过640亿美元。这 项目解决了S.变异人一种半透性脂质双层膜 包围着所有的细胞，并决定细胞的功能， 在不同的脂质环境中。S.变形杆菌与其他细菌膜蛋白插入有共同的特征 系统，以及真核内质网和细胞器，但也表现出独特的性质。 S.变形杆菌正在成为研究革兰氏阳性细菌中蛋白质转运的既定模型。所有已知 调控电路和多重毒力属性。变形菌源于其膜蛋白组成。 生物膜是约50%质量的蛋白质，膜蛋白代表了大多数已知的药物靶标。 理解S。变形蛋白转运和插入途径将有助于针对 以及相关的病原体。本项目主要研究包括YidC插入酶在内的蛋白质共翻译转运 （细菌，线粒体和叶绿体），信号识别颗粒（SRP）途径在所有细胞中都是保守的。 活细胞我们确定了各自的S。变形杆菌YidC 1、YidC 2和SRP底物，并鉴定了四种途径 框架：典型的SRP途径，自主YidC途径，协调YidC 2-SRP途径，和一个 YidC 1与SecYEG易位子相互作用的SRP非依赖性途径和一个未表征的 一种叫Jag的蛋白质我们还描述了S.变形菌膜和 显示了脂质成分对转运的特定组分的膜分配的影响 机械.在这个更新的应用程序中，我们将确定相互作用的蛋白质组分的运输 机械与阴离子脂质使用Martini 22粗粒分子动力学模拟结合 圆二色性、溶液NMR和生物层干涉测量法（SA 1）。我们还将评估蛋白质-蛋白质 通过分子动力学模拟在不同脂质环境下的相互作用，蓝色-天然聚丙烯酰胺 凝胶电泳，并将利用固态核磁共振来评估蛋白质-蛋白质接触和结构， 蛋白质对和低聚物。将使用表位标记的底物在体内的插入来验证结果，并且 用定制的脂蛋白体（SA 2）进行体外转录-翻译-插入测定。最后，膜- 定位的Jag是预测的RNA结合蛋白（RBP）。线粒体膜定位的RBP具有 最近被证明是伴侣蛋白特异性mRNA的偶联阻断-插入。jag和rnpA编码 核糖核酸酶P与yidC 1在同一操纵子中。因此，我们还将评估Jag和RnpA在以下方面的作用： yidC 1、yidC 2和编码相关底物的基因的转录后调节（SA 3）。

Project Summary. Streptococccus mutans is a ubiquitous oral bacterium and prominent etiologic agent of human dental caries. Certain strains can also cause bacterial endocarditis and invade human coronary epithelial cells. US health care costs associated with dental decay alone are over $64 billion annually. This project addresses membrane protein insertion in S. mutans. A semi-permeable lipid bilayer membrane encloses all cells and determines cellular function by dictating what can cross by virtue of proteins embedded within differing lipid milieus. S. mutans shares features common to other bacterial membrane protein insertion systems, and those of eukaryotic endoplasmic reticulum and organelles, but also exhibits unique properties. S. mutans is becoming an established model for studying protein transport in Gram+ bacteria. All known regulatory circuits and multiple virulence attributes of S. mutans stem from its membrane protein composition. Biological membranes are ~50% protein by mass and membrane proteins represent most known drug targets. Thus understanding S. mutans protein transport and insertion pathways will facilitate targeted therapy against this and related pathogens. This project focuses on co-translational protein transport including YidC insertases (bacteria, mitochondria, and chloroplasts), and the signal recognition particle (SRP) pathway conserved in all living cells. We identified respective S. mutans YidC1, YidC2, and SRP substrates and identified four pathway frameworks: canonical SRP pathway, autonomous YidC pathway, coordinated YidC2-SRP pathway, and an SRP-independent pathway in which YidC1 interacts with the SecYEG translocon and an uncharacterized protein called Jag. We also characterized the cardiolipin-rich lipidome of the S. mutans membrane and showed the influence of lipid composition on membrane partitioning of specific components of the transport machinery. In this renewal application we will identify interactions of protein components of the transport machinery with anionic lipids using Martini22 coarse-grain molecular dynamic simulation in conjunction with circular dichroism, solution NMR, and biolayer interferometry (SA1). We will also evaluate protein-protein interactions in the context of varying lipid milieus by molecular dynamic simulation, blue-native polyacrylamide gel electrophoresis, and will utilize solid state NMR to evaluate protein-protein contacts and structures of protein pairs and oligomers. Findings will be validated using insertion of epitope-tagged substrates in vivo, and in vitro transcription-translation-insertion assays with customized proteoliposomes (SA2). Lastly, membrane- localized Jag is a predicted RNA binding protein (RBP). Mitochondrial membrane-localized RBPs have recently been shown to chaperone specific mRNAs for coupled translation-insertion. jag and rnpA encoding ribonuclease P are in the same operon as yidC1. Therefore we will also evaluate the roles of Jag and RnpA in post-transcriptional regulation of yidC1, yidC2, and genes encoding relevant substrates (SA3).