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。它是由酸产生引起的 通过酸性细菌进行牙齿表面的发酵。摄入可发酵碳 宿主促进酸产生，导致牙齿脱矿化，这是龋齿的症状。 建立导致龋齿的产生酸性生物膜由链球菌突变引发 产生生物膜发育所需的粘合蛋白和麸质。虽然有助于PH 还原，S。变形物对pH的极端敏感。这样的极端很少在口腔中遇到 环境正常唾液的pH值范围为6.0至7.8，刺激的唾液流量为pH值为7.4-- 7.8。尽管许多研究专门用于了解S. mutans如何承受低pH值，但很少 针对葡萄糖链球菌对饮酒条件的反应的研究。非常出乎意料的是，删除了 ADHC基因编码乙酰脱氢酶（ADH）的Lipoypated E2亚基，带来了急性敏感性 至〜7.5的pH值，在人类唾液中通常观察到的pH值。导致产生有缺陷的突变 其他ADH操纵子产品（E1，E3和LPLA，Lipoyl连接酶）也赋予对适中海拔的敏感性 在pH（pH 7.2-7.6）中。 ADH/LPLA突变体也存在于碳水化合物摄取和/或消耗中的缺陷， 当存在蔗糖时，多动症无效突变承认生物膜形成的严重缺陷。这一发现提高了 为了将细菌感知到环境pH值 人类口腔的同时也损害了口腔病原体内的代谢作用。这 本文提出的探索性，假设生成的项目将确定与ADH-相关的因素 依赖性酒精耐受性。克服ADHC的酒精敏感性的抑制突变 突变已被隔离，并将继续被发现。这样的突变将确定基因 可能在网络中运行ADH功能使细胞具有抗升高的pH值。 Lipoyl ADHC和ADHD中编码的辅因子附着位点将在突变中被灭活，对 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。 抑制2-氧气酸脱氢酶的微摩尔Zn2+浓度将用于检查ADH 对pH耐受性的催化需求。将进行基因组TN-SEQ实验以鉴定遗传基因座 该功能在酗酒的耐受性中。这些突变将与ADHC抑制突变结合到 确定已鉴定的基因的功能是否与ADH依赖性碱抗性有关。 突变体和突变体筛选中鉴定的突变体将在具有共识的混合培养物中测试适应性 精氨酸水解竞争物种，戈尔多尼（S. gordonii）或sanguinis，已知会导致斑块中的pH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。项目 将产生潜在的靶标，以干扰人类口腔环境中的突变。