Pseudomonas detection and metabolism of sphingosine

假单胞菌检测和鞘氨醇代谢

• 批准号: 8417470
• 负责人: MATTHEW J WARGO
• 金额: $ 27.45万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-11-15 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8417470
• 关键词: Acinetobacter; Address; Alcaligenes; Antibiotic Resistance; Area; B-Lymphocytes; Bacteria; Binding; Biological Assay; Biology; Burkholderia; Cells; Chronic Obstructive Airway Disease; Cystic Fibrosis; DNA Binding; Data; Degradation Pathway; Detection; Development; Disease; Enzymes; Escherichia coli; Gene Expression; Gene Expression Regulation; Genes; Genetic Screening; Genetic Transcription; Goals; Health; Healthcare; Human; Immune; Immune response; Immune system; In Vitro; Infection; Inflammatory; Knowledge; Lead; Life; Ligand Binding; Ligands; Link; Lung; Maps; Mechanical ventilation; Metabolic; Metabolic Pathway; Metabolism; Microarray Analysis; Mission; Mus; Mycobacterium tuberculosis; Pathogenesis; Pathway interactions; Patients; Phosphoric Monoester Hydrolases; Physiological; Play; Pseudomonas; Pseudomonas aeruginosa; Regulation; Regulon; Reporter; Reporting; Research; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Specificity; Sphingolipids; Sphingosine; Stenotrophomonas maltophilia; Surveys; System; T-Cell Activation; Testing; Therapeutic Agents; Tight Junctions; United States National Institutes of Health; Virulence; antimicrobial; care burden; fitness; improved; in vivo; innovation; insight; interest; macrophage; migration; mouse model; mutant; new therapeutic target; novel; novel therapeutics; pathogen; promoter; public health relevance; response; screening; sphingosine 1-phosphate; sphingosine-1-phosphate phosphatase; therapeutic target; transcription factor

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.

描述（由申请人提供）：铜绿假单胞菌肺部感染危及生命并构成巨大的医疗负担。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范例。初步数据表明，铜绿假单胞菌可以降解有效的免疫信号分子 1-磷酸鞘氨醇 (S1P) 及其前体鞘氨醇。 S1P 降解将使铜绿假单胞菌改变宿主免疫反应，这可能对感染的进展和疾病的发病机制产生深远的影响。该提案中描述的是一种先前未表征的转录因子，它可响应 S1P 和鞘氨醇调节铜绿假单胞菌基因表达，并且是其降解所必需的。该转录因子的缺失降低了体内铜绿假单胞菌的毒力，凸显了该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的表征将为理解细菌生物学和发病机制中鞘氨醇相关化合物做出具体贡献。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。为了实现这一长期目标，该应用程序的总体目标是表征识别 铜绿假单胞菌用于响应 S1P 的降解系统，并确定 S1P 降解对肺内铜绿假单胞菌毒力的影响。核心假设是，铜绿假单胞菌检测到 S1P 会诱导 S1P 降解所需的基因，从而增强毒力。将通过追求三个具体目标来检验中心假设：（1）表征铜绿假单胞菌中的 S1P 和鞘氨醇依赖性转录〜（2）识别 S1P 降解途径的组成部分〜（3）测试 S1P 降解对铜绿假单胞菌感染的影响。目标 1 的目标是通过启动子作图、DNA 结合和报告基因分析确定鞘氨醇响应转录因子的配体和启动子结合特异性，并使用微阵列分析确定其对鞘氨醇调节子的贡献。目标 2 的目标是通过遗传筛选、代谢物追踪和体外酶测定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 的目标是使用申请人实验室建立的小鼠肺部感染模型来测试以下假设：转录因子突变体的毒力降低是由于缺乏 S1P 降解，而不是与降解无关的基因的调节。该提案具有创新性，因为它将促进对先前未表征的鞘氨醇检测和代谢途径的理解，铜绿假单胞菌可能会利用该途径来检测宿主并干扰有效的宿主信号传导途径。拟议的研究意义重大，因为它将扩大对细菌病原体与宿主之间代谢联系的理解，或许会带来新的抗菌或抗毒力疗法。