Fitness profiling of Streptococcus gordonii in oral microenvironments

口腔微环境中戈登链球菌的适应度分析

• 批准号: 9375169
• 负责人: M Margaret VICKERMAN
• 金额: $ 26.58万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375169
• 关键词: Actinomyces; Address; Alleles; Animal Model; Automobile Driving; Bacteria; Bacterial Adhesins; Base Sequence; Bioinformatics; Biological; Cardiac; Cells; Coculture Techniques; Communities; Community Developments; Complement; DNA; Data; Development; Dinucleoside Phosphates; Disease; Endocarditis; Environment; Equilibrium; Essential Genes; Failure; Foundations; Frequencies; Future; Genes; Genetic; Genetic study; Genome; Goals; Growth; Health; High-Throughput Nucleotide Sequencing; Human; Infective endocarditis; Interruption; Investigation; Lead; Libraries; Liquid substance; Maintenance; Metabolic; Methodology; Methods; Microbial Biofilms; Modeling; Molecular; Mouth Diseases; Oral; Oral Microbiology; Oral cavity; Oral health; Organism; Pathogenesis; Pathogenicity; Pathway interactions; Play; Population; Recovery; Role; Saliva; Serum; Signal Transduction; Site; Streptococcus gordonii; Surface; Testing; Virulence; bacterial adhesin receptor; cardiovascular health; experimental study; extracellular; fitness; functional genomics; genome-wide; in vitro Model; in vivo; insight; microbial; microbial community; microbiota; microorganism; mutant; novel; oral biofilm; oral commensal; oral condition; oral microbiome; oral streptococci; prevent; receptor; response; success; tool; tooth surface

Streptococcus gordonii is an initial colonizer of the biofilm that forms on the saliva-coated tooth surface. These pioneer colonizers and their extracellular products form a substratum with host oral components to which subsequent colonizing bacteria can attach. Accordingly, S. gordonii plays a pivotal role in the establishment of the mature oral biofilm through mechanisms such as host and bacterial adhesin-receptor, metabolic, and intercellular signaling interactions. The establishment and maintenance of a healthy oral microbiome requires a favorable balance of these ecological interactions. The working hypothesis of the proposed studies is that identification of S. gordonii genes essential for species survival and proliferation under synergistic or antagonistic conditions will provide essential insights into community dynamics that favor or disfavor a state of oral health. The goal of this exploratory project is to use a genome-wide approach to identify and examine genes involved in S. gordonii fitness under selected in vitro model conditions relevant to those in the oral cavity. We propose a state-of-the-art, Tn-Seq approach to construct a saturated library of transposon genome insertions in S. gordonii strain Challis, a widely-used model of commensal oral streptococci. The usefulness of the library will be confirmed by growth under test conditions that will allow identification of mutants with insertions in genes that are essential for growth and fitness. Mutants with disruptions of genes conferring fitness will decrease in frequency in the population during the course of the experiment; in contrast, mutants in the library with insertions in nonessential genes will be maintained at an equal or increased frequency. Recovered DNA from the entire population will be collected at the end of experiment and compared to the input library via a high throughput sequencing of the chromosomal DNA flanking the integrated transposon. Interrupted and hence, less or non- functional genes will be identified by comparison of the transposon-flanking nucleotide sequence to the reference S. gordonii genome sequence. The frequency of recovery of each mutant will be used to calculate the fitness of each gene under the experimental conditions. Conditions to be studied will include biofilm formation and planktonic conditions important for colonization in presence of whole human saliva, serum, and dual species cultures with Actinomyces oris and Filifactor alocis, representing bacterial species that have been shown to promote or impede, respectively, S. gordonii colonization in biofilms. The roles of selected genes identified as playing potential roles in fitness will be further examined by construction of specific mutant strains by allelic exchange and their complements to verify their roles in the survival and proliferation of S. gordonii during biofilm development. These studies will provide an important foundation for future bacterial interaction and survival studies. Understanding the roles of genes that favor the commensal oral microbiota will provide essential insights into preventing the microbial dysbiosis that can lead to oral disease.

戈登链球菌是唾液覆盖的牙齿表面上形成的生物膜的初始定居者。 这些先驱殖民者及其胞外产物与宿主口腔成分形成基质， 随后定居的细菌可以附着在上面。因此，S。戈登二世在 通过宿主和细菌粘附素受体等机制建立成熟的口腔生物膜， 代谢和细胞间信号相互作用。建立和维护健康的口腔 微生物组需要这些生态相互作用的有利平衡。的工作假设 建议的研究是鉴定S.戈登氏菌对物种生存和增殖至关重要的基因 在协同或拮抗条件下，将提供对社区动态的重要见解，有利于 或不利于口腔健康的状态。这个探索性项目的目标是使用全基因组方法， 鉴定和检测与S.在选定的体外模型条件下的戈登适合度， 那些在口腔里的。我们提出了一种最先进的Tn-Seq方法来构建一个饱和的 转座子基因组插入S. gordonii菌株查利斯，一种广泛使用的口腔粘膜模型， 链球菌。该文库的有用性将通过在测试条件下的生长来证实， 鉴定在对生长和适应性至关重要的基因中插入突变体。突变体 赋予适应性的基因的破坏在种群中的频率将在进化过程中降低。 相反，文库中具有非必需基因插入的突变体将保持在 频率相等或增加。从整个人口中提取的DNA将在年底收集。 通过染色体DNA的高通量测序进行实验并与输入文库进行比较 整合转座子的侧翼中断，因此，较少或无功能的基因将被确定， 转座子侧翼核苷酸序列与参考S.戈登氏菌基因组序列 每个突变体的恢复频率将用于计算每个基因在 实验条件待研究的条件将包括生物膜形成和渗透性条件 在全人唾液、血清和双种培养物存在下， 口腔放线菌和非丝状菌，代表已显示促进或 分别阻碍S.戈登氏菌在生物膜中的定植选定的基因的作用被确定为发挥 通过等位基因交换构建特定的突变株，将进一步研究在适应性中的潜在作用 及其互补物，以验证它们在S.戈登氏菌在生物膜期间 发展这些研究将为未来细菌的相互作用和生存提供重要基础 问题研究了解有利于口腔微生物群的基因的作用将提供必要的 对预防可能导致口腔疾病的微生物生态失调的见解。