Alkaline stress tolerance in Streptococcus mutans

变形链球菌的碱性胁迫耐受性

基本信息

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
项目类别：
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

项目摘要

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.

龋齿是人类最常见的慢性感染之一，它是由酸的产生引起的。摄入可发酵碳宿主促进酸的产生，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引发的，产生生物膜发育所需的粘附蛋白和葡聚糖，尽管它会影响 pH 值。减少，变形链球菌对极端 pH 值很敏感，这种极端值在口腔中很少遇到。正常唾液的 pH 值范围为 6.0 至 7.8，刺激唾液流的 pH 值范围为 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出乎意料地删除了 adhC 基因编码乙偶姻脱氢酶 (Adh) 的硫酰化 E2 亚基，赋予急性敏感性 pH 值约为 7.5，这是人类唾液中常见的突变，导致唾液产生缺陷。其他 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 adh/lplA 突变体在 pH 值 (pH 7.2-7.6) 中也表现出碳水化合物摄取和/或消耗方面的缺陷。当蔗糖存在时，adhD 无效突变会导致生物膜形成严重缺陷。靶向变形链球菌的特定功能以使细菌对环境 pH 值敏感的可能性人类口腔的同时也损害口腔病原体内的代谢运作。本文提出的探索性、假设生成项目将确定与 Adh-相关的因素克服 adhC 的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，这样的突变将识别出那些基因。 Adh 可能在网络中发挥作用，使细胞对 pH 值升高产生抵抗力。 adhC 和 adhD 中编码的辅因子附着位点将发生突变失活，从而影响将测试碱性敏感性，以评估 Adh 氧化还原化学在碱性耐受性中的重要性。抑制 2-含氧酸脱氢酶的微摩尔 Zn2+ 浓度将用于检查 Adh 测试 pH 耐受性的催化要求将进行基因组 Tn-seq 实验来遗传鉴定基因座。这些突变将与 adhC 抑制突变结合以发挥碱性耐受性。确定已识别基因的功能是否与 Adh 依赖性耐碱性相关。突变体和突变体筛选中确定的突变体将在与共生体的混合培养物中进行适应性测试，精氨酸分解竞争物种，S. gordonii 或 S. sanguinis，已知会导致菌斑 pH 值升高这些实验将使用浮游培养物和混合物种生物膜进行。将产生干扰变形链球菌在人类口腔环境中增殖的潜在目标。