Fitness profiling of the Streptococcus mutans genome

变形链球菌基因组的适应性分析

基本信息

批准号：
7251212
负责人：
Robert G Quivey
金额：
$ 71.77万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7251212
关键词：
Acids Actinomyces naeslundii Affect Bar Codes Cariogenic Diet Coculture Techniques Communities Complex Condition Defect Dental Enamel Dental Plaque Dental caries Diet Disease Environment Essential Genes Event Exhibits Fatty Acids Fermentation Fusobacterium nucleatum Future Gene Mutation Gene Targeting Genes Genome Genomics Growth Homologous Gene Human In Vitro Individual Infection Knock-out Laboratories Lead Learning Libraries Methods Microbe Microbial Biofilms Modeling Mus Mutagenesis Open Reading Frames Oral Oral cavity Organism Population Process Processed Genes Production Protocols documentation Public Health Publishing Research Personnel Resistance Resources Score Streptococcus Streptococcus mutans Streptococcus sanguis Stress Testing Therapeutic Intervention Transcriptional Activation Up-Regulation Yeasts base demineralization feeding fitness gene function genetic analysis genome sequencing indexing insight member method development microbial community mouse model mutant numb protein oral bacteria organic acid programs research study response success sugar trait

项目摘要

DESCRIPTION (provided by applicant): Fitness profiling of the S. mutans genome. Caries formation by S. mutans requires the organism to form a biofilm, become a prominent member of the microbial community of dental plaque and to survive stresses within plaque (Marsh, 2003). The initiation of caries involves demineralization of tooth enamel by organic acids produced by S. mutans fermentation of sugars. S. mutans survives acid environments by the up- regulation of a number of proteins, or, fatty acids in a process referred to as acid-adaptation. Evidence from our laboratory, and others, shows that acid-adaptation is a complex, multi-gene process that is only partially understood at present. With the availability of the S.mutans genome sequence, the annotation of 1,963 open reading frames, and the development of methods precisely to delete individual open reading frames, it is possible to analyze every nonessential gene in S. mutans. In this project, we will examine the effect of each gene on the ability of S. mutans to grow in acidic conditions. The contribution of each gene in the organism to events implicated in caries formation will be determined. Specific Aim 1a. High throughput methods will be tailored to reproducibly delete individual genes in the entire S. mutans genome. Each deletion will be marked by the presence of two unique DMA sequences (18-20 bp) such that abundance of each particular mutant strain in a population of mutants and wild type can be scored using these "bar codes." Specific Aim 1b. A genomic set of marked deletions will be constructed, resulting in a library of S. mutans strains bearing knockouts of all non-essential genes. Methods to analyze gene function by fitness profiling will be used to determine a function for each gene in S. mutans. Specific Aim 2. To find genes that may affect growth in the oral cavity, we will test mutant strains for their ability to grow, in vitro, in acidic conditions. Then, we will use the knockout library in fitness profiling experiments under defined conditions, which will include the following: 1) co-culture of the library of mutant strains growing in acidic environments, including planktonic and biofilm cultures; and, 2) a mouse infection model, in which the mice are fed a highly cariogenic diet. These experiments will be done as screens for candidate strains to be examined individually in the mouse caries model. Specific Aim 3. We will also examine the ability of the library of mutant strains to grow in the presence of additional species of oral microbes, with the aim of identifying those genes in S. mutans that contribute to its ability to out-compete other oral organisms. From these studies, we expect to identify genes in S. mutans that participate in acid-resistance. The relevance to public health is that the information derived from these studies will lead to new insights, and potentially new targets for therapeutic intervention, into specific mechanisms that S. mutans uses to cause disease in humans.

描述（由申请人提供）：S。mutans基因组的健身分析。 S. mutans的龋齿形成要求生物体形成生物膜，成为牙齿斑块微生物群落的重要成员，并在斑块内生存胁迫（Marsh，2003）。龋齿的启动涉及通过糖链球菌发酵产生的有机酸将牙齿搪瓷脱矿化。链球菌通过在称为酸适应的过程中对多种蛋白质或脂肪酸的加强来生存酸环境。我们实验室和其他实验室的证据表明，酸适应是一个复杂的多基因过程，目前仅部分理解。随着S.Mutans基因组序列的可用性，1,963个开放式阅读框的注释以及精确删除单个开放式阅读框的方法的开发，可以分析S. mutans中的每个非必需基因。在这个项目中，我们将研究每个基因在酸性条件下生长的能力的影响。将确定每个基因在生物体中对与龋齿形成有关的事件的贡献。特定目标1a。高吞吐量方法将被量身定制，以可重复地删除整个S. mutans基因组中的单个基因。每个缺失将以两个独特的DMA序列（18-20 bp）的存在为标志，以便可以使用这些“条形码”对突变体群体中每个特定的突变菌株的丰度进行评分。特定目标1B。将构建一组基因组缺失的基因组，从而产生一个包含所有非必需基因的敲除的突变链球菌菌株。通过适应性分析分析基因功能的方法将用于确定链球菌中每个基因的功能。具体目的2。要找到可能影响口腔生长的基因，我们将在酸性条件下测试突变菌株的体外生长能力。然后，我们将在定义的条件下使用敲除图书馆进行健身谱分析实验，其中包括以下条件：1）在酸性环境中生长的突变菌株文库的共文化，包括浮游生物和生物膜培养； 2）小鼠感染模型，其中小鼠被高度致癌的饮食喂食。这些实验将作为筛选进行候选菌株的筛选，以在小鼠龋齿模型中进行单独检查。具体目的3。我们还将检查突变菌株库在存在其他口服微生物的情况下生长的能力，目的是鉴定出突变链球菌中的那些基因，有助于其兼容其他口腔兼容有机体。从这些研究中，我们期望鉴定出参与酸性抗酸的链球菌中的基因。与公共卫生的相关性是，从这些研究中得出的信息将导致新见解，并可能针对治疗干预的新目标，用于链球菌用来引起人类疾病的特定机制。