Targeting the outer membrane protein translocation pathways

性状（由申请人提供）：革兰氏阴性菌是多种重要人类感染性疾病的病原体。由这些病原体中的许多病原体引起的感染的成功治疗受到其内在抗性机制的限制，包括不可渗透的外膜（OM）和各种外排泵的活性。在这个项目中，我们建议开发新型抗生素，这些抗生素不会进入细菌细胞质，而是通过干扰OM的生物发生来发挥作用。由Lol和Bam机制组成的两个途径分别负责脂蛋白和β桶非脂化OM蛋白的运输。这些途径在铜绿假单胞菌中是必不可少的，并且将被小分子抑制剂靶向破坏。对铜绿假单胞菌菌株进行工程改造，使其能够调节Bam和Lol途径的关键组分，并携带响应于Lol和Bam消耗的荧光素酶报告基因构建体。这些铜绿假单胞菌试验菌株将用于筛选化合物文库，并鉴定OM蛋白运输的抑制剂。所述化合物将被表征以鉴定那些具有最大杀伤效力的化合物，所述化合物表现出针对其它革兰氏阴性病原体的广谱，在生物膜、血清和呼吸道粘液中有活性，表现出低细胞毒性并增强细菌的杀菌活性。 其他抗生素这些活性化合物的蛋白质靶标将使用遗传和化学方法来鉴定。将在鼠呼吸道感染模型中测试这些化合物中的每一种单独或与其它抗生素组合在保护小鼠免受铜绿假单胞菌定殖方面的功效。这项工作可能导致开发一类新的广谱抑制剂，适用于治疗由抗生素耐药性革兰氏阴性病原体引起的各种感染。 公共卫生相关性：拟议的项目是针对两个平行的外膜蛋白定位途径的新类别的广谱抗生素的发现。如果成功，这项工作的成果将是开发出能够杀死即使是最耐抗生素的革兰氏阴性病原体的强效抗菌剂。

DESCRIPTION (provided by applicant): Gram-negative bacteria are the causative agents a variety of important human infectious diseases. The successful therapy of infections, caused by many of these pathogens is limited by their intrinsic resistance mechanism including the impermeable outer membrane (OM) and the activities of various efflux pumps. In this project, we propose to develop novel antibiotics that do not enter the bacterial cytoplasm, but instead, they act by interfering with the biogenesis of the OM. Two pathways, consisting of the Lol and Bam machineries, are responsible for trafficking of lipoproteins and beta-barrel non-lipidated OM proteins, respectively. These pathways are essential in Pseudomonas aeruginosa and will be targeted for disruption by small molecule inhibitors. Strains of P. aeruginosa were engineered that allow regulation of the key components of the Bam and Lol pathways and carry a luciferase reporter construct responsive to Lol and Bam depletion. These P. aeruginosa test strains will be used to screen compound libraries and inhibitors of OM protein trafficking will be identified. The compounds will be characterized to identify those with maximal killing potency potent, exhibit a broad spectrum against other Gram-negative pathogens, are active in biofilms, serum and respiratory mucus, exhibit low cytotoxicity and potentiate the bactericidal activities of other antibiotics. The protein targets of these active compounds will be indentified using genetic and chemical approaches. The efficacy of each of these compounds alone, or in combination with other antibiotics, in protecting mice against P. aeruginosa colonization will be tested in a murine respiratory infection model. This work could lead to the development of a new class of broad-spectrum inhibitors suitable for therapy of a variety of infections caused by antibiotic- resistant Gram-negative pathogens. PUBLIC HEALTH RELEVANCE: The proposed project is directed towards the discovery of new classes of broad-spectrum antibiotics targeting two parallel pathways of outer membrane protein localization. If successful, the outcome of this work will be the development of potent antimicrobial agents capable of killing even the most antibiotic resistant Gram-negative pathogens.