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: 10544751
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10544751
• 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; 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.

项目摘要 龋齿是一种无处不在的传染病，影响了数十亿人的生活质量 全世界。根据外科医生（2020年）的说法，每年花费1.25亿美元 仅在美国相关治疗。龋齿的主要病因是链球菌突变， 这也与感染性心内膜炎有关。 S. mutans的本地环境丰富 金属阳离子在内，包括Ca2+，K+和Mg2+。实际上，MG2+是最丰富的二价金属阳离子 细菌和包括人类在内的脊椎动物中最丰富的第四大。 MG2+内容的 人体在骨骼和牙齿中发现60-70％。因此，口腔细菌，链球菌是 不断暴露于MG2+沙拉。镁是牙膏和牙齿的重要组成部分 植入物。尽管它丰富及其支持细菌生长和病毒的要求，但MG2+ 尚未在S. mutans或其他链球菌中研究稳态。一些孤立的研究 讨论了补充MG2+销售对链球菌生物膜形成和遗传的重要性 能力，但是MG2运输尚不清楚。我们将从小说中解决MG2+稳态 透视图，我们不仅会表征转运蛋白，而且还研究了他们的插入 膜。膜定位/插入是所有人正确运作的关键必需品 膜蛋白。删除推定运输商的删除，然后结合起来 细胞金属含量的测量将建立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/排出已被认为干扰Mg2+ 其他细菌中的稳态。因此，本研究还包括MN2+和Fe2+转运蛋白。 假定MG2+转运蛋白（S）中有缺陷的突变体将在转录水平上进行评估， 细胞金属含量，并插入膜。接下来，我们将使用前遗传屏幕 使用突变体确定在MG2+重复/耗尽条件下的功能/抑制突变的增益 MG2+转运蛋白或缺乏膜生物发生机制成分的突变体中有缺陷 这会影响MG2+稳态。我们赞赏转运蛋白适当定位到的重要性 他们的活动；因此，我们将运用实验室的经验/工具/技能来研究 S. mutans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结构和表征 使用MG2+转运蛋白和表型分析，用于表征 运输突变体。分子克隆，反向和正向遗传学，生物信息学和生化 方法将用于解决该提案的目的。最终对 预计MG2+稳态有望指导新型反卡里斯策略的未来发展。