Role of the Spx Regulator in Streptococcus mutans

描述(申请人提供)：来自临床和基础研究的数据表明，变形链球菌是龋齿的主要微生物病原体，龋齿是困扰人类的最常见的传染病之一。变形链球菌的毒力存在于三个核心属性：在牙齿表面附着和形成生物膜的能力，从各种碳水化合物中产生大量有机酸的能力，以及耐受环境压力，特别是低pH值的能力。由于胁迫耐受性与变形链球菌的毒力相互交织，因此剖析这些细菌在应激条件下在口腔生物膜中生长的机制对于全面了解龋病的发病机制至关重要。在革兰氏阳性细菌中，能量依赖的蛋白酶，特别是ClpXP系统，是压力耐受的核心，并与细菌的发病机制有关。然而，CLP蛋白水解酶在发病机制中的生物学作用还没有一个清晰的图景。我们最近的工作表明，ClpXP在变形链球菌的关键毒力属性的表达中发挥着重要作用，这些属性包括生物膜形成、细胞活力和耐酸性。在枯草芽孢杆菌中，全局调节子Spx是ClpXP的底物，Spx的积累与ClpXP菌株的表型相关。我们已经证明，spx基因(这里称为spxA和spxB)的失活恢复了变形链球菌ClpXP菌株中观察到的许多表型。因此，ClpXP影响变形链球菌毒力特性的潜在机制与Spx的积累密切相关。尽管CLP蛋白降解与细菌毒力有关，Spx的积累与ClpP和clpX突变所产生的表型密切相关，但Spx对病原菌的整体调控作用仍有待探索。我们的工作假设是，CLP对Spx调节因子的蛋白水解性控制在变形链球菌致病过程中起着关键作用。因此，本研究的重点在于SpxAB全球调节子的特性，以及它们在控制变形链球菌毒力表达中的作用。为了证明我们的假设，我们提出了三个具体目标。在目标1中，我们将剖析调控Spx水平的转录和翻译后机制，这将为CLP蛋白酶和Spx之间相互作用的分子和生理意义提供更多的见解。在目标2中，将对ClpXP和SPx突变株进行全面的体外和体内鉴定，以揭示Spx调控在致病过程中的意义。最后，在AIM 3中，完整的基因组微阵列将被用来识别Spx控制下的遗传网络，并在与AIM 1和2一起分析时，识别参与毒力表达的新基因。最终，这项提议的进展将有助于识别可用于开发预防龋齿的新型治疗剂的蛋白质和信号通路。 公共卫生相关性：变形链球菌被认为是人类龋齿的主要病原体，通常与心内膜炎有关，心内膜炎是一种危及生命的心脏瓣膜炎症。这项研究的目的是了解变形链球菌黏附在牙齿上、产酸和在口腔酸性条件下生存的机制。我们的研究将有助于发现可用于减少或消除龋齿的新产品。

DESCRIPTION (provided by applicant): Data from clinical and basic research have implicated Streptococcus mutans as the primary microbial etiologic agent of dental caries, one of the most common infectious diseases afflicting humans. The virulence of S. mutans resides in three core attributes; its abilities to adhere and form biofilms on tooth surfaces, to produce large quantities of organic acids from a wide range of carbohydrates, and to tolerate environmental stresses, particularly low pH. Because stress tolerance is intertwined with S. mutans virulence, the dissection of the mechanisms that allow these bacteria to thrive in oral biofilms during stressful conditions is central for a complete understanding of the pathogenesis of dental caries. In Gram-positive bacteria, energy-dependent proteases, in particular the ClpXP system, are central for stress tolerance and have been implicated in bacterial pathogenesis. However, a clear picture of the biological role of Clp proteases in pathogenesis has yet to emerge. Our recent work revealed that ClpXP plays an important role in the expression of key virulence attributes of S. mutans, including biofilm formation, cell viability and acid tolerance. In Bacillus subtilis, the global regulator Spx is a substrate of ClpXP, and accumulation of Spx has been correlated to phenotypes of ?clpXP strains. We have demonstrated that inactivation of spx genes (herein designated spxA and spxB) restored many phenotypes observed in the S. mutans ?clpXP strains. Thus, the underlying mechanisms by which ClpXP affects virulence traits in S. mutans are intimately associated with accumulation of Spx. Despite the linkage of Clp proteolysis with bacterial virulence, and the close association of Spx accumulation with phenotypes resulting from clpP and clpX mutations, the effects of global regulation by Spx in pathogenic bacteria remains to be explored. Our working hypothesis is that Clp proteolytic control of the Spx regulator plays a critical role in processes underlying S. mutans pathogenesis. Therefore, this application focuses on the characterization of the SpxAB global regulators, and their role in controlling virulence expression in S. mutans. To prove our hypothesis, three Specific Aims are proposed. In Aim 1, we will dissect the transcriptional and post-translational mechanisms regulating Spx levels, which will provide additional insights into the molecular and physiologic significance of the interactions between Clp proteases and Spx. In Aim 2, a thorough in vitro and in vivo characterization of the ?clpXP and ?spx mutant strains will be conducted in order to disclose the significance of Spx regulation in processes underlying pathogenesis. Finally, in Aim 3 whole genomic microarrays will be used to identify the genetic network under Spx control and, when analyzed in conjunction with Aims 1 and 2, to identify new genes that participate in the expression of virulence. Ultimately, the advances from this proposal will help the identification of proteins and signaling pathways that could be used for the development of novel therapeutic agents to prevent dental caries. PUBLIC HEALTH RELEVANCE: Streptococcus mutans is considered the primary causative agent of human dental caries, and is often implicated in cases of endocarditis, a life-threatening inflammation of the heart valves. The objective of this study is to understand the mechanisms used by S. mutans to adhere to the tooth, produce acids, and survive acidic conditions in the oral cavity. Our studies will facilitate the discovery of new products that could be used to reduce or eliminate caries.