Fitness profiling of the Streptococcus mutans genome

描述(申请人提供)：变形链球菌基因组的适合度分析。变形链球菌的龋齿形成需要有机体形成生物膜，成为牙菌斑微生物群落的重要成员，并在菌斑内的压力下生存(Marsh，2003)。龋病的发生涉及变形链球菌发酵糖所产生的有机酸对牙釉质的脱矿作用。变形链球菌在酸性环境中生存的方式是通过上调一些蛋白质或脂肪酸的表达，这一过程被称为酸适应。来自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、多基因的过程，目前只有部分了解。随着变形链球菌基因组序列的获得，1,963个开放阅读框的注释，以及精确删除单个开放阅读框的方法的发展，分析变形链球菌中的每一个非必需基因成为可能。在这个项目中，我们将研究每个基因对变形链球菌在酸性条件下生长能力的影响。将确定生物体中的每个基因对与龋齿形成有关的事件的贡献。具体目标1a。高通量的方法将被量身定做，以重复删除整个变形链球菌基因组中的单个基因。每个缺失将由两个独特的DMA序列(18-20个碱基)的存在来标记，这样每个特定突变株在突变株和野生型群体中的丰度可以使用这些“条形码”来评分。具体目标1b。将构建一组标记缺失的基因组，从而产生一个带有所有非必需基因敲除的变形链球菌菌株文库。通过适应度谱分析基因功能的方法将被用来确定变形链球菌中每个基因的功能。具体目标2。为了寻找可能影响口腔生长的基因，我们将测试突变菌株在体外酸性条件下的生长能力。然后，我们将在确定的条件下将基因敲除文库用于适合性分析实验，其中将包括：1)在酸性环境中生长的突变菌株文库的共培养，包括浮游和生物膜培养；以及2)小鼠感染模型，在该模型中，小鼠被喂以高度致龋性的饮食。这些实验将作为候选菌株的筛选，在小鼠龋齿模型中进行单独检查。具体目标3.我们还将检查突变菌株文库在其他口腔微生物物种存在的情况下生长的能力，目的是识别变形链球菌中那些有助于其竞争对手的基因。从这些研究中，我们希望确定变形链球菌中参与耐酸的基因。与公共卫生相关的是，从这些研究中获得的信息将导致对变形链球菌用来引起人类疾病的特定机制的新见解，并可能产生新的治疗干预目标。

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.