Alkaline stress tolerance in Streptococcus mutans

变形链球菌的碱性胁迫耐受性

• 批准号: 10117222
• 负责人: PETER ZUBER
• 金额: $ 23.1万
• 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/10117222
• 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.

龋齿是人类最普遍的慢性感染之一。它是由酸的产生引起的， 由定居在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，导致牙齿脱矿质，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启动的， 产生生物膜发育所需的粘附蛋白和葡聚糖。虽然它有助于pH值 还原，S.变形杆菌对极端pH值敏感。这种极端pH值在口腔中很少遇到。 环境中，正常唾液的pH范围为6.0至7.8，刺激唾液流的pH范围为7.4至7.8。 7.8.虽然许多研究致力于了解S。变形杆菌能承受低pH值， 针对S.变形菌适应碱性条件。出乎意料的是，删除 adhC基因编码乙偶姻脱氢酶（Adh）的脂酰化E2亚单位， 至pH ~7.5，这是人类唾液中常见的pH值。突变导致产生缺陷的 其它adh操纵子产物（E1、E3和LplA，硫辛酰连接酶）也赋予对适度升高的敏感性 在pH（pH 7.2-7.6）中。adh/lplA突变体还表现出碳水化合物摄取和/或消耗的缺陷， 当存在蔗糖时，adhD无效突变赋予生物膜形成严重缺陷。这一发现提高了 靶向S.以使细菌对环境pH敏感 同时也损害了口腔病原体内的代谢操作。的 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碱性敏感性的抑制突变 突变已经被分离出来，并将继续被发现。这些突变将识别出 可能在Adh的网络中起作用，使细胞对升高的pH具有抗性。 在adhC和adhD中编码的辅因子附着位点将被突变失活， 将测试碱性敏感性以评估Adh氧化还原化学在碱性耐受性中的重要性。 微摩尔Zn 2+浓度，抑制2-氧代酸脱氢酶，将被用来检查测试Adh pH耐受性的催化要求。将进行基因组Tn-seq实验以鉴定遗传位点 在碱性耐受中起作用。这些突变将与adhC抑制基因突变组合， 确定所鉴定的基因的功能是否与Adh依赖的耐碱性相关。的adh 突变体和在突变体筛选中鉴定的那些将在与海藻酸钠的混合培养物中测试适合性， 溶藻竞争种S. gordonii或S.血，这是已知的导致pH值升高的斑块 生物膜这些实验将使用培养物和混合物种生物膜进行。项目 将产生干扰S的潜在目标。在人类口腔环境中的变形菌增殖。