Function of MVFR in Pseudomonas aeruginosa virulence

描述（由申请方提供）：铜绿假单胞菌引起的感染是人类医疗保健中的一个重大问题。这种细菌病原体是烧伤患者中败血症的主要病原体;囊性纤维化患者中持续性肺部感染和死亡率的主要病原体; HIV和其他免疫抑制患者中的医院感染的主要病原体;以及医院中有害的多药耐药性感染的爆发的主要病原体。该提案的长期目标是提供有效和选择性的治疗方法，以减少人类铜绿假单胞菌感染的发生率和并发症。这项研究提出，这一目标可以通过阻止或限制MvfR/HAQ通路激活的药物来实现，开发这种抗感染化合物是这一应用的直接目标。该提议基于以下假设：MvfR（一种铜绿假单胞菌转录调节因子）是抗感染药物的候选靶标，因为它在调节许多QS控制的毒力相关因子的表达中起核心作用;并且其活化通过其与对其功能至关重要的特定配体的结合来介导。为了鉴定MvfR途径抑制剂，并证明其体内抗感染活性，本研究提出了三个具体目标：1）确认MvfR配体的身份，鉴定其结合位点，并确定其作用机制; 2）鉴定抑制MvfR/HAQ途径的化合物;和3）确定这些抑制剂限制哺乳动物中铜绿假单胞菌感染的体内功效和潜在可行性。这些目标将通过三组实验来实现。首先，生物化学、质谱和分子遗传学分析将确认铜绿假单胞菌MvfR-配体的身份;定义MvfR配体结合结构域;并确定MvfR和MvfR-配体复合物识别和结合的pqsA启动子序列。其次，生物化学和质谱分析将鉴定通过限制其配体的合成和/或结合来防止配体介导的MvfR活化的化合物，并且这些化合物在铜绿假单胞菌中代谢稳定。第三，将在果蝇、小鼠全层皮肤热损伤和小鼠新生儿呼吸模型中测试每种鉴定的抑制剂，以确定其毒性、其限制铜绿假单胞菌感染的体内功效;以及其对细菌耐药性发展的“免疫力”。与降低细菌活力的药物（包括大多数抗生素）相比，直接介导毒力的途径的特异性抑制不太可能产生选择性压力，从而产生对抑制剂的耐药性。这种靶向抑制剂可以显着提高烧伤，囊性纤维化和艾滋病毒患者的长期预后。为此，本文的结果应该能够实现治疗和/或预防铜绿假单胞菌-人感染的新疗法。

DESCRIPTION (provided by applicant): Infections caused by Pseudomonas aeruginosa are a significant problem in human healthcare. This bacterial pathogen is the principle agent of sepsis in burn patients; of persistent lung infections and mortality in cystic fibrosis patients; of nosocomial infections in HIV and other immune-suppressed patients; and of the outbreak of deleterious multi-drug resistant infections in hospitals. The long-term goal of this proposal is to provide effective and selective therapies that reduce the incidence and complications of human P. aeruginosa infections. This study proposes this goal can be achieved by drugs that prevent or limit the activation of the MvfR/HAQ pathway, and the development of such anti-infective compounds is the immediate goal of this application. This proposal is based on the hypothesis that MvfR, a P. aeruginosa transcriptional regulator, is a candidate target for anti-infective drugs, as it plays a central role in modulating the expression of many QS-controlled virulence-associated factors; and its activation is mediated by its binding to a specific ligand, which is essential for its function. To identify MvfR-pathway inhibitors, and demonstrate their in vivo anti-infective activity, this study proposes three Specific Aims: 1) to confirm the identity of the MvfR ligand, identify its binding site, and determine its mechanism of action; 2) to identify compounds that inhibit the MvfR/HAQ pathway; and 3) to determine the in vivo efficacy and potential feasibility of these inhibitors to limit P. aeruginosa infection in mammals. These aims will be accomplished via three sets of experiments. First, biochemical, mass spectrometric, and molecular genetic analyses will confirm the identity of the P. aeruginosa MvfR-ligand; define the MvfR ligand binding domain; and determine the pqsA promoter sequence recognized and bound by MvfR and the MvfR-ligand complex. Second, biochemical and mass spectrometric analyses will identify compounds that prevent ligand-mediated MvfR activation by limiting the synthesis and/or binding of its ligand, and that are metabolically stable in P. aeruginosa. Third, each identified inhibitor will be tested in the Drosophila melanogaster, the mouse full-thickness skin thermal injury, and the mouse neonatal respiratory model, to determine their toxicity, their in vivo efficacy to limit P. aeruginosa infection; and their "immunity" to the development of bacterial resistance. Specific inhibition of a pathway that directly mediates virulence is less likely to generate selective pressure to develop resistance to the inhibitor, than for drugs, including most antibiotics, that reduce bacterial viability. Such targeted inhibitors could significantly enhance the long-term prognosis of burn, cystic fibrosis, and HIV patients. To this end, the results here should enable novel therapies to treat and/or prevent P. aeruginosa-human infections.