Characterization and membrane-biogenesis of Streptococcus mutans magnesium transporters

变形链球菌镁转运蛋白的表征和膜生物发生

• 批准号: 10353066
• 负责人: Surabhi Mishra
• 金额: $ 15.25万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10353066
• 关键词: Address; Alloys; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Proteins; Binding Proteins; Biochemical; Bioinformatics; Biological Process; Cations; Cells; Charge; Communicable Diseases; Competence; Data; Dental; Dental Enamel; Dental Implants; Dental caries; Dependence; Development; Divalent Cations; Endocarditis; Environment; Enzymes; Escape Mutant; Evaluation; Exposure to; Foundations; Future; Gene Proteins; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Growth; Health Care Costs; Homeostasis; Homologous Gene; Human; Human body; Implant; Infective endocarditis; Ions; Magnesium; Measurement; Membrane; Membrane Proteins; Metals; Microbial Biofilms; Molecular Cloning; Mutagenesis; Mycobacterium tuberculosis; Nucleic Acids; Oral cavity; Pathway interactions; Persons; Phenotype; Play; Predisposition; Protein translocation; Proteins; Proteomics; Quality of life; Regulation; Resistance; Resistance development; Ribosomes; Role; Saliva; Salts; Signal Recognition Particle; Streptococcus mutans; Suppressor Mutations; Surgeon; Therapeutic; Tooth structure; Toothpaste; Toxic effect; Transition Elements; Vertebrates; Virulence; Work; anticaries; bone; cofactor; combinatorial; dental agent; divalent metal; efflux pump; experience; forward genetics; gain of function; genetic approach; membrane biogenesis; mutant; novel; oral bacteria; oral pathogen; oral streptococci; pathogen; protein transport; reverse genetics; skills; tool; uptake

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.

项目概要 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。根据外科医生 (Surgeon General)（2020 年）的数据，每年在以下方面花费约 1,250 亿美元： 仅在美国就有相关治疗。龋齿的主要致病菌是变形链球菌， 这也与感染性心内膜炎有关。变形链球菌的原生环境富含 金属阳离子包括Ca2+、K+和Mg2+。事实上，Mg2+是自然界中最丰富的二价金属阳离子。 细菌，在包括人类在内的脊椎动物中含量第四。总 Mg2+ 含量 人体中，60-70%存在于骨骼和牙齿中。因此，口腔细菌 S. mutans 是 持续暴露于 Mg2+ 盐中。镁是牙膏和牙科材料的重要成分 植入物。尽管 Mg2+ 含量丰富且需要支持细菌生长和毒力，但 尚未对变形链球菌或其他口腔链球菌的体内平衡进行研究。一些孤立的研究 讨论了补充 Mg2+ 盐对于变形链球菌生物膜形成和遗传的重要性 能力，但 Mg2 传输尚不清楚。我们将从小说中探讨 Mg2+ 稳态 我们不仅要描述转运蛋白的特征，还要研究它们插入到 膜。膜定位/插入是所有器官正常运作的关键必要条件 膜蛋白。单独或组合删除假定的转运蛋白，然后 细胞金属含量的测量将确定 Mg2+ 转运蛋白的身份。补偿 其他二价金属阳离子转运蛋白的吸收/流出已被认为会干扰 Mg2+ 其他细菌的体内平衡。因此，Mn2+和Fe2+转运蛋白也包括在本研究中。 假定的 Mg2+ 转运蛋白缺陷的突变体将在转录水平进行评估， 细胞金属含量和插入膜的情况。接下来，我们将使用正向遗传筛选 使用突变体识别 Mg2+ 充足/耗尽条件下的功能增益/抑制突变 Mg2+转运蛋白有缺陷，或缺乏膜生物发生机制组件的突变体 影响 Mg2+ 稳态。我们认识到运输工具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/技能来研究这方面 变形链球菌中的镁稳态。插入途径的表征将涉及 膜生物发生成分组合突变体的构建和表征 与 Mg2+ 转运蛋白，以及用于表征的表型分析 运输突变体。分子克隆、反向和正向遗传学、生物信息学和生物化学 将采用各种方法来实现本提案的目标。最终更好的理解 变形链球菌中的 Mg2+ 稳态有望指导新型防龋策略的未来发展。