Function of MVFR in Pseudomonas aeruginosa virulence

描述(申请人提供)：由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脓毒症、囊性纤维化患者持续肺部感染和死亡、艾滋病毒和其他免疫抑制患者医院感染以及医院爆发有害的多重耐药感染的主要病原体。这项建议的长期目标是提供有效和选择性的治疗方法，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途径激活的药物来实现，而这种抗感染化合物的开发是这一应用的直接目标。这一建议是基于这样的假设，即MvfR是铜绿假单胞菌的转录调控因子，是抗感染药物的候选靶点，因为它在调节许多QS控制的毒力相关因子的表达方面发挥着核心作用；它的激活是通过与特定的配体结合介导的，这是其功能所必需的。为了识别MvfR途径的抑制剂，并证明它们在体内的抗感染活性，本研究提出了三个具体的目标：1)确认MvfR配体的身份，确定其结合部位，并确定其作用机制；2)寻找抑制MvfR/HAQ途径的化合物；3)确定这些抑制剂在体内限制铜绿假单胞菌感染的有效性和潜在的可行性。这些目标将通过三组实验来实现。首先，生化、质谱学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配体结合域，并确定MvfR和MvfR-配体复合体识别和结合的pqsA启动子序列。其次，生化和质谱学分析将确定通过限制其配体的合成和/或结合来阻止其配体介导的MvfR激活的化合物，以及在铜绿假单胞菌中代谢稳定的化合物。第三，每个已确定的抑制剂将在果蝇、小鼠全层皮肤烫伤和小鼠新生儿呼吸模型中进行测试，以确定它们的毒性，它们在体内限制铜绿假单胞菌感染的有效性，以及它们对细菌耐药性发展的免疫力。与包括大多数抗生素在内的降低细菌生存能力的药物相比，对直接介导毒力的途径的特定抑制不太可能产生对抑制剂产生抗药性的选择压力。这种靶向抑制物可以显著改善烧伤、囊性纤维化和HIV患者的长期预后。为此，这里的结果应该能够使新的治疗方法能够治疗和/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.