Characterization of pseudoverdine, a novel virulence-related quinolone molecule p

新型毒力相关喹诺酮分子pseudoverdine的表征

• 批准号: 7953444
• 负责人: Abdul N Hamood
• 金额: $ 18.56万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7953444
• 关键词: Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biological Assay; Blood; Burn injury; Cell Communication; Cells; Code; Communication; Complement; Defect; Electrophoretic Mobility Shift Assay; Environment; Exotoxins; Floor; Future; Gel; Gene Targeting; Genes; Goals; Hospitals; Immune response; Immunocompromised Host; Individual; Infection; Inflammatory; Learning; Lung; Measures; Microbe; Modeling; Multi-Drug Resistance; Mus; N-butyrylhomoserine lactone; Operon; Organ; Organism; Patients; Plasmids; Production; Pseudomonas; Pseudomonas aeruginosa; Quinolones; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Subcutaneous Injections; System; Systemic infection; Tissues; Virulence; Virulence Factors; cytokine; design; in vivo; injured; mutant; novel; pathogen; pathogenic bacteria; public health relevance; quorum sensing; research study; resistant strain; response

DESCRIPTION (provided by applicant): The emergence of antibiotic resistance among different pathogenic bacteria is a major global problem. Within the last two decades, antibiotic-resistant strains emerged at an alarming rate; yet, only a few new antibiotics were developed. The gram-negative opportunistic pathogen Pseudomonas aeruginosa is an example of this problem. Multidrug resistant clones of P. aeruginosa may persist within the hospital environment for several years by transferring among patients. As a result, research efforts are directed at developing alternative P. aeruginosa therapies that would weaken the bacteria but would not induce the production of resistant mutants. One target for these therapies is the cell-to-cell communication system, or quorum sensing (QS) system, through which P. aeruginosa synchronizes the production of numerous virulence factors including exotoxins. This occurs through secondary metabolites or small communication molecules termed autoinducers. Due to their importance, P. aeruginosa carries multiple autoinducers. Therefore, identifying and fully characterizing these autoinducers is essential for designing future therapies that block their actions. At present, three P. aeruginosa autoinducers have been fully characterized: 3OC12-HSL, C4-HSL, and PQS - the Pseudomonas quinolone signal. Recently, we and others have identified pseudoverdine, a novel quinolone molecule which is synthesized from a precursor molecule termed paerucumarin. These molecules are synthesized by the pvc operon which is located adjacent to ptxR. The ptxR gene codes for the P. aeruginosa global regulator PtxR, which regulates the expression of numerous P. aeruginosa virulence genes. Evidence suggests that pseudoverdine and/or paerucumarin activates PtxR. Additional analysis, using the murine model of systemic infection, revealed that specific deletions within the pvc operon reduced the in vivo virulence of P. aeruginosa significantly. We have synthesized paerucumarin and we are currently synthesizing pseudoverdine. We hypothesize that pseudoverdine is a novel communication molecule that P. aeruginosa utilizes to coordinate the production of different virulence factors. The specific aims of the application are: 1) to examine the influence of synthesized pseudoverdine and/or paerucumarin on the production of virulence factors as well as the expression of different P. aeruginosa genes, 2) to determine if pseudoverdine and/or paerucumarin enhance PtxR binding to its target genes, and 3) to determine the effect of exogenously added pseudoverdine and/or paerucumarin on the in vivo virulence of P. aeruginosa using the murine model of systemic infection. These experiments will be done using a specific assay for each virulence factor examined, quantitative RT-PCR, transcriptional fusion studies, and microarray experiments. Additionally, we will determine if pseudoverdine and/or paerucumarin induce a conformational change in PtxR using DNA/gel shift assays and other assays to detect conformational changes. We will examine the effect of these molecules on the spread P. aeruginosa both locally within the infected tissues and systemically within the blood and internal organs of thermally-injured mice. In addition, we will assess the effect of the molecules themselves on the host immune response by measuring the level of expression of pro- inflammatory cytokines in response to subcutaneous injection of pseudoverdine and/or paerucumarin. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a microbe that produces serious illness in people with weakened resistance, such as severely burned patients and patients with lung infections. The organism causes this harmful effect by producing numerous factors that damage the host. This research is designed to study a system that controls production of these factors. The final aim from learning this is to stop the organism from producing such factors.

描述（由申请人提供）：不同病原菌之间出现抗生素耐药性是一个重大的全球性问题。在过去的二十年里，抗生素耐药菌株以惊人的速度出现；然而，只有少数新的抗生素被开发出来。革兰氏阴性机会致病菌铜绿假单胞菌就是这个问题的一个例子。铜绿假单胞菌的多重耐药克隆可能通过在患者之间转移而在医院环境中持续存在数年。因此，研究努力的方向是开发铜绿假单胞菌的替代疗法，这种疗法可以削弱细菌，但不会诱导产生耐药突变体。这些疗法的一个目标是细胞间通信系统，或群体感应（QS）系统，通过该系统，铜绿假单胞菌同步产生包括外毒素在内的许多毒力因子。这是通过次级代谢物或称为自诱导剂的小通讯分子发生的。由于它们的重要性，铜绿假单胞菌携带多种自诱导剂。因此，识别和充分表征这些自诱导剂对于设计未来阻断其作用的治疗方法至关重要。目前，铜绿假单胞菌的三种自诱导剂已被充分表征：3OC12-HSL、C4-HSL和PQS -喹诺酮假单胞菌信号。最近，我们和其他人已经鉴定了伪绿草碱，这是一种新的喹诺酮分子，它是由一种叫做绿草素的前体分子合成的。这些分子是由位于ptxR附近的pvc操纵子合成的。ptxR基因编码铜绿假单胞菌全球调控因子ptxR，调控铜绿假单胞菌众多毒力基因的表达。有证据表明，伪绿草碱和/或黄柏素可激活PtxR。另外，利用小鼠全身感染模型进行的分析显示，pvc操纵子的特异性缺失显著降低了铜绿假单胞菌的体内毒力。我们已经合成了绿毛藻素，目前正在合成伪绿毛藻碱。我们假设伪绿草碱是铜绿假单胞菌利用的一种新的通讯分子，以协调不同毒力因子的产生。该申请的具体目的是：1)检验合成的伪绿草碱和/或paucumarin对毒力因子的产生以及不同铜绿假单胞菌基因表达的影响；2)确定伪绿草碱和/或paucumarin是否增强PtxR与其靶基因的结合；3)利用小鼠全身感染模型确定外源添加的伪绿草碱和/或paucumarin对铜绿假单胞菌体内毒力的影响。这些实验将使用对每个被检查的毒力因子的特定分析、定量RT-PCR、转录融合研究和微阵列实验来完成。此外，我们将使用DNA/凝胶移位法和其他检测构象变化的方法来确定伪绿草碱和/或白桦素是否会诱导PtxR的构象变化。我们将研究这些分子对铜绿假单胞菌在感染组织中的局部传播和在热损伤小鼠的血液和内脏中的全身传播的影响。此外，我们将评估分子本身对宿主免疫反应的影响，通过测量对皮下注射伪绿草碱和/或paucumarin的反应中促炎细胞因子的表达水平。