Alkaline stress tolerance in Streptococcus mutans

变形链球菌的碱性胁迫耐受性

• 批准号: 9977562
• 负责人: PETER ZUBER
• 金额: $ 19.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977562
• 关键词: Acids; Actinobacteria class; Acute; Adhesives; Adult; Affect; Alkalies; Alkalinization; Alleles; Ammonia; Arginine; Bacteria; Carbohydrates; Carbon; Carbon Dioxide; Cells; Chemistry; Child; Chronic Disease; Codon Nucleotides; Communities; Complement; Consumption; Defect; Deletion Mutation; Dental Enamel; Dental Hygiene; Dental caries; Development; Dietary Carbohydrates; Environment; Enzymes; Exhibits; Fermentation; Generations; Genes; Genetic; Genomics; Glucans; Glucose; Human; Intake; Investigation; Lactobacillus; Ligase; Lysine; Measures; Mediating; Metabolic; Metabolic dysfunction; Microbial Biofilms; Mutagenesis; Mutation; Operon; Oral; Oral cavity; Oxidation-Reduction; Oxidoreductase; Parents; Phenotype; Production; Proteins; Research; Resistance; Saliva; Site; Source; Specific qualifier value; Streptococcus; Streptococcus gordonii; Streptococcus mutans; Streptococcus sanguis; Stress; Sucrose; Suppressor Mutations; Surface; Symptoms; Testing; Tooth Demineralization; Tooth root structure; United States; Urea; Ursidae Family; alkalinity; base; biological adaptation to stress; chronic infection; cofactor; commensal bacteria; dental biofilm; dihydrolipoamide dehydrogenase; dihydrolipoyllysine-residue acetyltransferase; dysbiosis; experimental study; fitness; fitness test; gene function; genome sequencing; genomic locus; insight; lipoate; mutant; null mutation; operation; oral commensal; oral microbial community; oral pathogen; polymicrobial biofilm; response; stress tolerance; sugar; tooth surface; transposon sequencing; uptake; whole genome

Dental caries is one of the most prevalent chronic infections in humans. It is caused by acid production from fermentation conducted by acidogenic bacteria that colonize the tooth surface. Intake of fermentable carbon by the host promotes acid generation, resulting in tooth demineralization, which is symptomatic of dental caries. Establishment of the acid-producing biofilm that leads to caries is initiated by Streptococcus mutans, which produces adhesive proteins and glucans required for biofilm development. Although it contributes to pH reduction, S. mutans is sensitive to extremes of pH. Such extremes are rarely encountered in the oral environment, as normal saliva has a pH range from 6.0 to 7.8, with stimulated saliva flow having a pH from 7.4- 7.8. While much research has been devoted to understanding how S. mutans withstands low pH, there are few studies that have targeted the response of S. mutans to alkaline conditions. Quite unexpectedly, a deletion of the adhC gene, encoding the lipoylated E2 subunit of acetoin dehydrogenase (Adh), confers acute sensitivity to pH of ~7.5, a pH value commonly observed in human saliva. Mutations that render defective production of the other adh operon products (E1, E3, and LplA, the lipoyl ligase) also confer sensitivity to modest elevations in pH (pH 7.2-7.6). The adh/lplA mutants also exhibit defects in carbohydrate uptake and/or consumption and the adhD null mutation confers a severe defect in biofilm formation when sucrose is present. The finding raises the possibility of targeting specific functions in S. mutans in order to sensitize the bacterium to the ambient pH of the human oral cavity while also compromising metabolic operations within the oral pathogen. The exploratory, hypothesis-generating project proposed herein will identify the factors associated with Adh- dependent alkaline tolerance. Suppressor mutations that overcome the alkaline sensitivity of the adhC mutation have been isolated and will continue to be uncovered. Such mutations will identify genes that potentially operate within the network that Adh functions to render cells resistant to elevated pH. The lipoyl cofactor attachment sites of encoded in adhC and adhD will be mutationally inactivated and the effect on alkaline sensitivity will be tested to assess the importance of Adh redox chemistry in alkaline tolerance. Micromolar Zn2+ concentrations, which inhibit 2-oxo acid dehydrogenases, will be used to examine test the Adh catalytic requirement for pH tolerance. A genomic Tn-seq experiment will be undertaken to identify genetic loci that function in alkaline tolerance. These mutations will be combined with the adhC suppressor mutations to determine if the identified genes’ functions are related to that of Adh-dependent alkali resistance. The adh mutants and those identified in the mutant screens will be tested for fitness in mixed cultures with commensal, arginolytic competing species, S. gordonii or S. sanguinis, which are known to cause pH elevation in plaque biofilms. These experiments will be performed with planktonic cultures and mixed-species biofilms. The project will generate potential targets for interfering with S. mutans proliferation in the human oral environment.

龋病是人类最常见的慢性感染之一。它是由酸的产生引起的 由定植于牙齿表面的产酸细菌进行的发酵。可发酵碳的摄入量 宿主促进酸的产生，导致牙齿脱矿，这是龋齿的症状。 导致龋齿的产酸生物膜的建立是由变形链球菌发起的，它 产生生物膜形成所需的黏附蛋白和葡聚糖。尽管它对PH值有贡献 在还原过程中，变形链球菌对极端的pH很敏感。这样的极端在口语中很少遇到。 环境，因为正常唾液的PH值范围为6.0至7.8，刺激唾液流出的PH值为7.4- 7.8.虽然许多研究致力于了解变形链球菌如何耐受低pH值，但很少有研究 针对变形链球菌对碱性条件的反应的研究。非常出人意料的是，删除了 ADHC基因编码乙酰脱氢酶(ADH)的脂基化E2亚单位，具有急性敏感性。 至~7.5的pH值，这是人类唾液中普遍观察到的pH值。导致产品有缺陷的突变 其他adh操纵子产物(e1、e3和lplA，硫酰连接酶)也使人对中等海拔高度敏感。 在pH为7.2-7.6的条件下。Adh/lplA突变体还表现出碳水化合物摄取和/或消耗方面的缺陷和 当蔗糖存在时，ADHD零突变导致生物膜形成的严重缺陷。这一发现提高了 以变形链球菌的特定功能为靶点以使细菌对环境pH敏感的可能性 同时也损害了口腔病原体的代谢操作。这个 本文提出的探索性假设生成项目将确定与ADH相关的因素。 依赖碱性耐受性。克服ADHC碱性敏感性的抑制子突变 突变已经被分离出来，并将继续被发现。这样的突变将识别出 潜在地在ADH发挥功能的网络内工作，使细胞对升高的pH具有抵抗力。硫辛基 在ADHC和ADHD中编码的辅因子连接位点将突变失活，并且对 将进行碱性敏感性测试，以评估ADH氧化还原化学在耐碱性中的重要性。 抑制2-氧代酸脱氢酶的微摩尔锌离子浓度将被用来检测ADH。 对pH耐受性的催化要求。将进行基因组Tn-seq实验以确定遗传位点。 在耐碱性方面起作用。这些突变将与ADHC抑制子突变结合起来 确定已鉴定基因的功能是否与ADH依赖的耐碱性有关。《广告》 突变株和突变筛选中确定的突变株将在具有共生关系的混合培养物中进行适应性测试， 精氨酸分解竞争种，戈登链霉菌或血链球菌，已知会导致菌斑pH值升高 生物膜。这些实验将使用浮游培养物和混合物种生物膜进行。该项目 将产生潜在的靶点来干扰变形链球菌在人类口腔环境中的增殖。