Function of MVFR in Pseudomonas aeruginosa virulence

描述（由申请人提供）：铜绿假单胞菌引起的感染是人类医疗保健中的一个重要问题。这种细菌病原体是烧伤患者脓毒症的主要病原体；囊性纤维化患者持续性肺部感染与死亡率的关系HIV和其他免疫抑制患者的医院感染；以及医院中有害的多重耐药性感染的爆发。本建议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病率和并发症。本研究提出，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，而开发这种抗感染化合物是这一应用的直接目标。这一建议是基于以下假设：铜绿假单胞菌转录调节剂MvfR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着核心作用；它的激活是通过与特定配体的结合来介导的，这对它的功能至关重要。为了鉴定MvfR通路抑制剂，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，确定其作用机制；2)鉴定抑制MvfR/HAQ通路的化合物；3)确定这些抑制剂在哺乳动物体内限制铜绿假单胞菌感染的有效性和潜在可行性。这些目标将通过三组实验来实现。首先，生化、质谱和分子遗传学分析将确认铜绿假单胞菌mvrr配体的身份；定义MvfR配体结合域；确定被MvfR和MvfR-配体复合物识别和结合的pqsA启动子序列。其次，生化和质谱分析将确定通过限制配体的合成和/或结合来阻止配体介导的MvfR激活的化合物，并且在铜绿假单胞菌中代谢稳定。第三，将鉴定出的每一种抑制剂分别在黑腹果蝇、小鼠全层皮肤热损伤和小鼠新生呼吸道模型中进行测试，以确定它们的毒性，以及它们在体内限制铜绿假单胞菌感染的功效；以及它们对细菌耐药性的“免疫力”。对直接介导毒力的途径进行特异性抑制，与降低细菌生存能力的药物（包括大多数抗生素）相比，不太可能产生对抑制剂产生耐药性的选择性压力。这些靶向抑制剂可以显著改善烧伤、囊性纤维化和HIV患者的长期预后。为此，这里的结果应该使新的疗法来治疗和/或预防铜绿假单胞菌-人类感染。

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.