Pseudomonas detection and metabolism of sphingosine

假单胞菌检测和鞘氨醇代谢

• 批准号: 9170956
• 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/9170956
• 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 response; Immune signaling; Immune system; In Vitro; Infection; Inflammatory; Knowledge; Life; Ligand Binding; Ligands; Link; Lung; 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; bacterial metabolism; care burden; co-infection; 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; 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 response; Immune signaling; Immune system; In Vitro; Infection; Inflammatory; Knowledge; Life; Ligand Binding; Ligands; Link; Lung; 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; bacterial metabolism; care burden; co-infection; 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正在威胁生命，构成了巨大的医疗保健负担。宿主免疫反应的调节是病原体使用的一种范式来启动和延长感染。初步数据表明，铜绿假单胞菌可以降解有效的免疫信号传导分子1-磷酸盐（S1P）及其前体鞘氨酸。 S1P降解将使铜绿假单胞菌能够改变宿主免疫反应，这可能会对感染的进展和疾病的发病机理产生深远影响。该提案中描述的是先前未表征的转录因子，该因子对S1P和鞘氨酸的响应调节铜绿假单胞菌基因表达，这是其降解所必需的。该转录因子的缺失减少了体内铜绿假单胞菌的毒力，突出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是对细菌生物学和发病机理中与鞘氨酸相关化合物的理解的具体贡献。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，本申请的总体目标是表征识别 铜绿假单胞菌使用的降解系统对S1P做出反应，并确定S1P降解对肺中铜绿假单胞菌毒力的影响。中心假设是铜绿假单胞菌的检测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目的来检验：（1）在铜绿假单胞菌〜（2）中表征S1P和鞘氨酸依赖性转录，确定S1P降解途径的成分〜和（3）测试S1P降解对铜绿假单胞菌感染的影响。 AIM 1的目标是通过启动子映射，DNA结合和报告基因测定法确定鞘氨酸反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氨酸调节剂的贡献。 AIM 2的目的是鉴定铜绿假单胞菌S1P磷酸酶和使用遗传筛选，代谢物跟踪和体外酶试验降解鞘氨酸的酶。最后，目标3的目的是检验以下假设：转录因子突变体的毒力降低是由于缺乏S1P降解，而不是使用申请人实验室中建立的肺部感染的小鼠模型的基因与与降解无关的基因的调节。该建议具有创新性，因为它将提高对先前未表征的鞘氨醇检测和代谢途径的理解，铜绿假单胞菌可以将其用于检测宿主并干扰有效的宿主信号传导途径。拟议的研究之所以重要，是因为它将扩大对细菌病原体与宿主之间代谢联系的理解，这可能导致新型抗菌或抗病毒疗法。