Characterization and membrane-biogenesis of Streptococcus mutans magnesium transporters

项目摘要 龋齿是一种普遍存在的传染病，影响着数十亿人的生活质量 全世界。根据卫生局局长(2020)的数据，每年在以下方面花费约1250亿美元 仅在美国就有相关治疗。一种主要的龋病病原体是变形链球菌， 这也与感染性心内膜炎有关。变形链球菌的原生环境中含有丰富的 金属阳离子包括钙离子、钾离子和镁离子。事实上，镁是水中含量最丰富的二价金属离子。 细菌，在包括人类在内的脊椎动物中含量第四多。所含镁离子总量的百分比 人体内，60%-70%存在于骨骼和牙齿中。因此，口腔细菌，变形链球菌，是 经常暴露在镁盐中。镁是牙膏和牙科的重要成分。 植入物。尽管镁离子含量丰富，而且需要支持细菌生长和毒力，但镁离子 变形链球菌或其他口腔链球菌体内的动态平衡尚未被研究过。几个孤立的研究 探讨了添加镁盐对变形链球菌生物膜形成和遗传的意义。 能力，但不理解mg2传输。我们将从一本小说中解决镁离子的动态平衡问题 视角，我们不仅将描述转运体的特征，而且还将研究它们插入到 膜。膜定位/插入是ALL功能正常的关键要素 膜蛋白。删除单个和组合的假定转运体，然后 细胞金属含量的测量将确定镁离子转运体的身份。补偿性的 其他二价金属阳离子转运体的摄取/外排已被认为干扰镁离子 其他细菌的动态平衡。因此，Mn2+和Fe2+转运蛋白也包括在本研究中。 在推测的镁离子转运体(S)中存在缺陷的突变体将在转录水平上进行评估， 细胞内金属含量，并插入细胞膜。接下来，我们将使用正向基因筛查来 利用突变体鉴定镁离子充足/枯竭条件下功能/抑制基因突变的增益 镁离子转运体缺陷，或缺乏膜生物发生机制组件的突变体 这会影响镁离子的动态平衡。我们认识到运输商适当本地化的重要性 他们的活动；因此，我们将应用我们实验室的经验/工具/技能来研究 变形链球菌的镁动态平衡。插入途径的特征将涉及 膜生物发生组分组合突变体的构建与鉴定 与镁离子转运体，以及表型分析之后，用于表征 运输变种人。分子克隆、正反向遗传学、生物信息学和生化 将利用各种方法来解决这项提案的目标。最终更好地理解 变形链球菌中的镁离子动态平衡有望指导未来新的防龋策略的发展。

Project Summary Dental caries is a ubiquitous infectious disease that impacts the quality of life of billions of people worldwide. According to the Surgeon General (2020), ~USD 125 billion dollars are spent annually on related treatments in the USA alone. A major causative agent of dental caries is Streptococcus mutans, which is also associated with infectious endocarditis. The native environment of S. mutans is rich in metal cations including Ca2+, K+, and Mg2+. In fact, Mg2+ is the most abundant divalent metal cation in bacteria and fourth most abundant in vertebrates including humans. Of the total Mg2+ content in the human body, 60‐70% is found in bones and teeth. Therefore, the oral bacterium, S. mutans, is constantly exposed to Mg2+ salts. Magnesium is an important component of toothpastes and dental implants. Despite its abundance and its requirement to support bacterial growth and virulence , Mg2+ homeostasis has not yet been studied in S. mutans, or other oral streptococci. A few isolated studies discussed the significance of supplemental Mg2+ salts for S. mutans biofilm formation and genetic competence, but Mg2 transport is not understood. We will address Mg2+ homeostasis from a novel perspective, where we will not only characterize the transporters, but also study their insertion into the membrane. Membrane localization/insertion is a key requisite for the proper functioning of all membrane proteins. Deletion of putative transporters singly and in combination, followed by measurement of cellular metal content will establish identity of Mg2+ transporters. Compensatory uptake/efflux by other divalent metal cation transporters have been recognized to interfere with Mg2+ homeostasis in other bacteria. Therefore, Mn2+ and Fe2+ transporters are also included in this study. Mutants defective in putative Mg2+ transporter(s) will be evaluated at the level of transcription, cellular metal content, and insertion into membrane. Next, we will use a forward genetic screen to identify gain of function/suppressor mutations in Mg2+‐replete/deplete conditions using mutants defective in Mg2+ transporters, or in mutants lacking membrane biogenesis machinery components that impact Mg2+ homeostasis. We appreciate the significance of proper localization of transporters to their activity; therefore, we will apply the experience/tools/skills of our lab to study that aspect of magnesium homeostasis in S. mutans. Characterization of insertion pathways will involve construction and characterization of combinatorial mutants of membrane biogenesis components with Mg2+ transporters, and phenotypic analysis following that used for characterization of the transport mutants. Molecular cloning, reverse and forward genetics, bioinformatics, and biochemical approaches will be utilized to address the aims of this proposal. Ultimately a better understanding of Mg2+ homeostasis in S. mutans is expected to guide future development of novel anti‐caries strategies.