Pseudomonas detection and metabolism of sphingosine

描述(申请人提供)：铜绿假单胞菌肺部感染危及生命，构成巨大的卫生保健负担。宿主免疫反应的调节是病原体用来启动和延长感染的一种模式。初步数据表明，铜绿假单胞菌能够降解免疫信号分子鞘氨醇-1-磷酸(S1P)及其前体鞘氨醇。S1P的降解可使铜绿假单胞菌改变宿主的免疫反应，从而对感染的进展和疾病的发病机制产生深远的影响。本提案中描述的是一种以前未知的转录因子，它调节铜绿假单胞菌基因的表达，以响应S1P和鞘氨醇，并且是它们降解所必需的。该转录因子的缺失降低了体内铜绿假单胞菌的毒力，突显了这一途径在感染过程中的重要性。铜绿假单胞菌对鞘氨醇的检测和代谢特征将对了解鞘氨醇相关化合物在细菌生物学和致病机制中的作用具有重要意义。长期目标是了解铜绿假单胞菌S1P和鞘氨醇代谢在肺部感染过程中的作用。为了追求这一长期目标，本应用程序的总体目标是表征识别 以及铜绿假单胞菌用来响应S1P的降解系统，确定S1P降解对铜绿假单胞菌在肺内毒力的影响。中心假设是，铜绿假单胞菌检测到S1P诱导S1P降解所需的基因，从而促进毒力。中心假设将通过追求三个特定的目标来验证：(1)表征S1P和鞘氨醇依赖的转录在铜绿假单胞菌中~(2)确定S1P降解途径的组成部分~和(3)测试S1P降解对铜绿假单胞菌感染的影响。目标1的目的是通过启动子定位、DNA结合和报告分析来确定鞘氨醇反应转录因子的配体和启动子结合的特异性，并使用微阵列分析来确定其对鞘氨醇调节子的贡献。目标2的目的是通过遗传筛选、代谢物跟踪和体外酶分析来鉴定铜绿假单胞菌S1P磷酸酶和降解鞘氨醇的酶。最后，目标3的目的是利用申请人实验室建立的小鼠肺部感染模型，验证转录因子突变的毒力降低是由于缺乏S1P降解，而不是调节与降解无关的基因这一假设。这一建议具有创新性，因为它将促进对以前未描述的鞘氨醇检测和代谢途径的理解，铜绿假单胞菌可能利用这一途径来检测宿主并干扰有效的宿主信号途径。这项拟议的研究意义重大，因为它将扩大对细菌病原体和宿主之间代谢联系的理解，可能会导致新的抗菌或抗毒力疗法。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa lung infections are life threatening and constitute a large health-care burden. Modulation of host immune responses is one paradigm used by pathogens to initiate and prolong infections. Preliminary data demonstrate that P. aeruginosa can degrade the potent immune signaling molecule sphingosine-1-phosphate (S1P) and its precursor sphingosine. S1P degradation would allow P. aeruginosa to alter the host immune response, which could have profound effects on progression of infection and the pathogenesis of disease. Described in this proposal is a previously uncharacterized transcription factor that regulates P. aeruginosa gene expression in response to S1P and sphingosine and is required for their degradation. Deletion of this transcription factor reduces P. aeruginosa virulence in vivo, highlighting the importance of this pathway during infection. Characterization of the detection and metabolism of sphingosine by P. aeruginosa will be a concrete contribution to understanding of sphingosine-related compounds in bacterial biology and pathogenesis. The long term goal is to understand the role of P. aeruginosa S1P and sphingosine metabolism during the course of lung infection. In pursuit of this long-term goal, the overall objectives of this application are to characterize the recognition and degradation system used by P. aeruginosa to respond to S1P and determine the impact of S1P degradation on P. aeruginosa virulence in the lung. The central hypothesis is that P. aeruginosa detection of S1P induces genes necessary for S1P degradation, which promotes virulence. The central hypothesis will be tested by pursuing three specific aims: (1) Characterize S1P and sphingosine-dependent transcription in P. aeruginosa~ (2) Identify the components of the S1P degradation pathway~ and (3) Test the impact of S1P degradation on P. aeruginosa infection. The goals of Aim 1 are to determine the ligand and promoter-binding specificities of the sphingosine-responsive transcription factor by promoter mapping, DNA binding, and reporter assays, and determine its contribution to the sphingosine regulon using microarray analysis. The goal of Aim 2 is to identify the P. aeruginosa S1P phosphatase and the enzymes that degrade sphingosine using genetic screens, metabolite tracking, and in vitro enzymatic assays. Finally, the goal of Aim 3 is to test the hypothesis that the reduced virulence of the transcription factor mutant is due to lack of S1P degradation, versus regulation of genes unrelated to degradation, using a mouse model of lung infection established in the applicant's lab. This proposal is innovative because it will advance understanding of a previously uncharacterized sphingosine detection and metabolic pathway, which may be used by P. aeruginosa to detect the host and interfere with a potent host signaling pathway. The proposed research is significant because it will expand understanding of the metabolic links between bacterial pathogens and the host, perhaps leading to novel antimicrobial or anti-virulence therapies.