Role of the Spx Regulator in Streptococcus mutans

描述（由申请人提供）：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感染性疾病之一。对S.变形链球菌具有三个核心属性：在牙齿表面粘附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，以及耐受环境应力，特别是低pH的能力。变形杆菌的毒力，解剖的机制，使这些细菌在口腔生物膜中茁壮成长的压力条件下是一个完整的理解龋齿的发病机制的核心。在革兰氏阳性细菌中，能量依赖性蛋白酶，特别是ClpXP系统，是应激耐受性的核心，并与细菌发病机制有关。然而，一个清晰的图片的生物学作用的Clp蛋白酶的发病机制尚未出现。我们最近的工作揭示了ClpXP在S.变异，包括生物膜形成，细胞活力和耐酸性。在枯草芽孢杆菌中，全球调节Spx是ClpXP的底物，Spx的积累与表型相关？clpXP菌株。我们已经证明，spx基因（本文指定spxA和spxB）的失活恢复了在S.变种人？clpXP菌株。因此，ClpXP影响S.变异体与Spx的积累密切相关。尽管Clp蛋白水解与细菌毒力的联系，以及Spx积累与clpP和clpX突变引起的表型的密切关联，但Spx在致病菌中的全局调节作用仍有待探索。我们的工作假设是，Clp蛋白水解控制的Spx调节发挥了关键作用的过程中，潜在的S。变形菌致病机理因此，本申请集中于SpxAB全局调节子的表征，以及它们在控制S.变异人为了证明我们的假设，提出了三个具体目标。在目标1中，我们将剖析转录和翻译后机制调节Spx水平，这将提供更多的见解Clp蛋白酶和Spx之间的相互作用的分子和生理意义。在目的2，一个彻底的体外和体内表征？clpXP和？将进行spx突变株的研究，以揭示Spx调节在发病机制基础过程中的重要性。最后，在目标3中，全基因组微阵列将用于鉴定Spx控制下的遗传网络，并且当与目标1和2结合分析时，鉴定参与毒力表达的新基因。最终，该提案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。

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.