STRUCTURAL AND FUNCTION STUDIES OF QUINOLONE SIGNALING IN PSEUDOMONAS AERUGINOSA

这个子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 本研究的目的是确定调节假单胞菌喹诺酮信号（PQS）的生物合成和信号活性的分子机制。 细菌病原体铜绿假单胞菌引起慢性肺部感染，这是患有囊性纤维化的患者死亡的主要原因。 此外，铜绿假单胞菌是最常见的医院病原体之一，占所有医院获得性感染的10%。 假单胞菌的致病性是由一组细胞外分子控制的，这些分子统称为群体感应信号，它们激活细胞外毒力因子的产生。 由于群体感应在铜绿假单胞菌致病性中的重要作用，参与群体感应的蛋白已被认为是新的抗菌治疗开发的靶点。 虽然PQS在假单胞菌毒力中的重要性已经得到很好的确立，但PQS生物合成中的一些关键步骤还没有被表征。 PQS在毒力调节中的功能也没有完全了解。使用PQS信号传导缺陷的铜绿假单胞菌突变株的遗传研究已经确定需要由pqs基因操纵子编码的蛋白质。 具体而言，PqsA、PqsB、PqsC和PqsD参与PQS合成;而PqsE是操纵子的最后一个基因的产物，是下游毒力因子的PQS依赖性激活所必需的。 然而，在分子水平上对这些蛋白质的功能的研究非常有限。 我们有初步的数据表明，PqsD通过丙二酰-ACP（或CoA）与邻氨基苯酰-CoA的克莱森缩合催化形成DHQ，DHQ是铜绿假单胞菌分泌的一种未酰化的喹诺酮。 本研究的目的是：1）确定PqsB、PqsC和PqsD在PQS合成中催化的确切顺序和反应; 2）确定PqsE在PQS信号网络和假单胞菌毒力中的作用。 我们建议使用综合的方法来研究这些蛋白质的功能和结构与生化分析和X射线晶体学。拟议研究的结果不仅使我们能够更好地了解PQS群体感应系统，而且还为通过抑制毒力发现抗菌新靶点提供了新的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this research is to determine the molecular mechanisms that regulate the biosynthesis and signaling activity of the Pseudomonas quinolone signal (PQS). Opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infections which are the major causes of mortality in patients suffering from cystic fibrosis. Moreover, P. aeruginosa is one of the most common nosocomial pathogens that accounts for 10% of all hospital-acquired infections. The pathogenicity of Pseudomonas is controlled by a set of extracellular molecules collectively known as the quorum sensing signals, which activate the production of extracellular virulence factors. Because of the essential roles of quorum sensing in the pathogenicity of P. aeruginosa, proteins involved in quorum-sensing have been recognized as targets for new antibacterial therapeutic development. Although the importance of PQS in Pseudomonas virulence is well established, some key steps in PQS biosynthesis are not characterized. Nor are the functions of PQS in the regulation of virulence fully understood. Genetic studies using P. aeruginosa mutant strains, which are defective in PQS signaling, have identified that proteins encoded by the pqs gene operon are required. Specifically, PqsA, PqsB, PqsC and PqsD are involved in PQS synthesis; whereas PqsE, which is the product of the last gene of the operon, is required for the PQS-dependent activation of the downstream virulence factors. However, there is very limited research on the functions of these proteins at the molecular level. We have preliminary data that PqsD catalyzes the formation of DHQ, a unacylated quinolone secreted by P. aeruginosa, by the Claisen condensation of malonyl-ACP (or CoA) with anthraniloyl-CoA. The aims of this proposal are: 1) to determine the exact order and reactions catalyzed by PqsB, PqsC and PqsD in the synthesis of PQS; 2) to determine the role(s) of PqsE in the PQS signaling network and Pseudomonas virulence. We propose to use integrated approaches to study the functions and structures of these proteins with biochemical analyses and X-ray crystallography. The findings of the proposed research will not only allow us to better understand the PQS quorum sensing system, but also provide novel insights on new targets for antibacterial discovery by the inhibition of virulence.