STRUCTURAL AND FUNCTION STUDIES OF QUINOLONE SIGNALING IN PSEUDOMONAS AERUGINOSA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本研究的目的是确定调控假单胞菌喹诺酮信号(PQS)生物合成和信号活性的分子机制。条件致病菌铜绿假单胞菌可引起慢性肺部感染，是囊性纤维化患者死亡的主要原因。此外，铜绿假单胞菌是最常见的医院内病原体之一，占所有医院感染的10%。假单胞菌的致病性由一组细胞外分子控制，这些分子统称为群体感应信号，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中的重要作用，参与群体感应的蛋白质已被认为是新的抗菌治疗开发的靶点。尽管PQS在假单胞菌毒力中的重要性已得到证实，但PQS生物合成的一些关键步骤尚未确定。PQS在毒力调节中的作用也不完全清楚。利用PQS信号缺陷的铜绿假单胞菌突变株进行的遗传学研究发现，PQS基因操纵子编码的蛋白质是必需的。具体来说，PqsA、PqsB、PqsC和PqsD参与了PQS的合成，而PqsE是操纵子最后一个基因的产物，是依赖PQS激活下游毒力因子所必需的。然而，在分子水平上对这些蛋白质的功能的研究非常有限。我们已有初步数据表明，PqsD通过丙二酰-ACP(或CoA)与邻氨基甲酰-CoA的Claisen缩合，催化由铜绿假单胞菌分泌的一种非酰基喹诺酮类化合物DHQ的形成。本建议的目的是：1)确定PqsB、PqsC和PqsD在PQS合成中的确切顺序和反应；2)确定PqsE在PQS信号网络和假单胞菌毒力中的作用(S)。我们建议用生化分析和X射线结晶学的综合方法来研究这些蛋白质的功能和结构。这项研究的结果不仅将使我们更好地了解PQS群体感应系统，还将为通过抑制毒力发现抗菌药物的新靶点提供新的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.