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

描述(申请人提供)：不同病原菌之间出现的抗生素耐药性是一个主要的全球性问题。在过去的二十年里，抗药性菌株以惊人的速度出现；然而，只开发了几种新的抗生素。革兰氏阴性条件致病菌铜绿假单胞菌就是这个问题的一个例子。铜绿假单胞菌的多药耐药克隆可能通过在患者之间转移而在医院环境中持续数年。因此，研究工作的重点是开发替代的铜绿假单胞菌疗法，这种疗法将削弱细菌，但不会诱导产生耐药突变株。这些疗法的一个靶点是细胞间通信系统，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。这是通过次生代谢物或称为自体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个自体诱导物。因此，识别和充分描述这些自动诱导剂对于设计未来阻止其作用的治疗方法是至关重要的。目前，已有3种铜绿假单胞菌自身诱导子被完全鉴定：3OC12-HSL、C4-HSL和PQS--喹诺酮类假单胞菌信号。最近，我们和其他人发现了一种新的喹诺酮分子，它是由一种名为白藜芦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的全局调节因子PtxR，它调节许多铜绿假单胞菌毒力基因的表达。有证据表明，伪甲肾上腺素和/或白藜芦素能激活PtxR。另外，利用小鼠全身感染模型进行的分析显示，pvc操纵子内的特定缺失显著降低了铜绿假单胞菌的体内毒力。我们已经合成了白藜芦素，目前我们正在合成伪奥弗定。我们推测，伪复氨酸是一种新的通讯分子，铜绿假单胞菌利用它来协调不同毒力因子的产生。应用的具体目的是：1)检测合成的伪覆盖碱和/或白菜素对铜绿假单胞菌毒力因子的产生和不同基因表达的影响；2)确定伪覆盖碱和/或白菜素是否增强了PtxR与其靶基因的结合；3)利用系统感染的小鼠模型，确定外源添加的伪覆盖碱和/或白菜素对铜绿假单胞菌体内毒力的影响。这些实验将使用针对所检测的每个毒力因子的特定检测、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/凝胶位移分析和其他检测构象变化的方法来确定伪覆盖和/或白藜芦素是否会诱导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局部传播以及在热损伤小鼠的血液和内脏中系统传播的影响。此外，我们还将通过测量皮下注射伪复定和/或白藜芦素后促炎细胞因子的表达水平，来评估这些分子本身对宿主免疫反应的影响。 公共卫生相关性：铜绿假单胞菌是一种微生物，它会在抵抗力较弱的人中产生严重疾病，如严重烧伤患者和肺部感染患者。生物体通过产生大量损害宿主的因素来造成这种有害影响。这项研究旨在研究一种控制这些因素产生的系统。学习这一点的最终目的是阻止有机体产生这样的因子。

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.