Contribution of the Streptococcus mutans cid and lrg Murein Hydrolase Regulator H

描述（由申请人提供）：变形链球菌是龋齿的主要病原体，龋齿是儿童中最常见的慢性疾病，在35岁以上的成年人中，有多达30%的人未得到治疗。当日常的牙齿卫生习惯（如刷牙和使用牙线）将这种细菌短暂地引入血液中时，这种细菌也会在高危患者中引起潜在致命的心内膜炎感染。变形链球菌的毒力与其形成生物膜的能力密切相关，但促进其最初附着在牙齿表面以及生物膜成熟和扩散的分子机制尚不清楚。在这方面，细胞死亡和裂解是细菌生理学的一个方面，与变形链球菌生物膜的形成有关。虽然在其他微生物中已经有了很好的研究，但在变形链球菌中调节细胞死亡和自溶的分子机制还没有明确定义。为此，我们提出了一个发展研究项目，开始研究变形链球菌的cid和lrg基因，因为这些基因已被证明是金黄色葡萄球菌细胞死亡、自溶和生物膜发育的重要调节因子。该应用程序的具体目的是：(1)确定cid和lrg基因在生物膜发育过程中调节变形链球菌细胞死亡和裂解中的作用，以及(2)检查控制变形链球菌中cid和lrg基因表达的遗传和代谢因素。在实验室和临床突变链球菌菌株中，cid、lrg和lytS基因都会产生等位基因替换突变。这些突变对细胞死亡和裂解的影响将通过各种表型测定来测量。共聚焦显微镜将用于评估它们对流动细胞和静态模型中生物膜粘附和发育的影响，以及监测cid和lrg GFP启动子融合的荧光。代谢信号和lytS基因对cid和lrg表达的作用也将被评估。预计这项研究将描述变形链球菌生理和发病机制的一个关键方面，这将有助于改善龋齿和感染性心内膜炎的预防和治疗策略。这项研究也是实现长期目标的重要的第一步，即确定口腔链球菌控制细胞死亡和自溶的机制，以及这些过程如何促进它们在牙菌斑中生长和存活的能力，以及它们在心内膜炎期间定植心脏瓣膜的能力。

DESCRIPTION (provided by applicant): Streptococcus mutans is the primary causative agent of dental caries, which remains the most common chronic disease among children and is untreated in up to 30% of adults over 35 yrs-old. When transiently introduced into the bloodstream following daily dental hygienic practices such as tooth brushing and flossing, this bacterium can also cause potentially lethal endocarditis infections in atrisk patients. S. mutans virulence is intimately related to its ability to form biofilm, but the molecular mechanisms facilitating its initial attachment to the tooth surface, as well as biofilm maturation and dispersal, are not well understood. In this respect, cell death and lysis are aspects of bacterial physiology that have been implicated in S. mutans biofilm formation. Although well-studied in other microorganisms, the molecular mechanisms that regulate cell death and autolysis in S. mutans have not been clearly defined. To this end, a developmental research project is proposed that initiates study of the cid and lrg genes in S. mutans, as these genes have been shown to be important regulators of cell death, autolysis and biofilm development in the pathogen Staphylococcus aureus. The specific aims of this application are (1) to ascertain the role of the cid and lrg genes in regulating S. mutans cell death and lysis during biofilm development, and (2) to examine the genetic and metabolic factors that control expression of the cid and lrg genes in S. mutans. Allele replacement mutations will be created in the cid, lrg, and lytS genes in both laboratory and clinical S. mutans strains. The effect of these mutations on cell death and lysis will be measured by a variety of phenotypic assays. Confocal microscopy will be used to assess their effect on biofilm adherence and development in flow cell and static models, as well as to monitor fluorescence of cid and lrg GFP promoter fusions. The role of metabolic signals and the lytS gene on cid and lrg expression will also be assessed. It is anticipated that this research will delineate a critical aspect of S. mutans physiology and pathogenesis that will help improve strategies of prevention and treatment of dental caries, as well as infective endocarditis. This research is also an important first step in achieving the long-term goal of defining the mechanisms that control cell death and autolysis in the oral streptococci, and how these processes contribute to their ability to grow and survive in dental plaque as well as their ability to colonize heart valves during endocarditis. PUBLIC HEALTH RELEVANCE: Despite wide-spread implementation of preventative measures such as community water fluoridation and school-based dental sealant programs, dental caries remains the most common chronic disease among children, and is also prevalent among adults. Streptococcus mutans, the bacteria that causes dental caries, can also cause infective endocarditis in at-risk patients. Therefore, research that contributes to our understanding of how this pathogen is able to colonize and grow in dental plaque will facilitate the development of novel preventative and/or treatment strategies for both of these diseases.