Targeting the outer membrane protein translocation pathways

描述(申请人提供)：革兰氏阴性菌是多种重要的人类传染病的病原体。许多由这些病原体引起的感染的成功治疗受到其内在耐药机制的限制，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，我们建议开发不进入细菌细胞质的新型抗生素，但它们通过干扰OM的生物发生来发挥作用。两条途径，包括Lol和Bam机制，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，并将成为小分子抑制剂的靶点。铜绿假单胞菌的菌株被设计成允许调节Bam和Lol途径的关键成分，并携带响应Lol和Bam耗尽的荧光素酶报告结构。这些铜绿假单胞菌测试菌株将用于筛选化合物文库，并将识别OM蛋白运输的抑制剂。这些化合物将被用来鉴定那些具有最大杀伤力的化合物，对其他革兰氏阴性病原体表现出广泛的杀灭能力，在生物被膜、血清和呼吸道粘液中具有活性，表现出低细胞毒性，并增强 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进行鉴定。这些化合物中的每一种单独或与其他抗生素联合使用，在保护小鼠免受铜绿假单胞菌侵袭方面的有效性将在小鼠呼吸道感染模型中进行测试。这项工作可能会导致开发一类新的广谱抑制剂，适用于治疗由抗生素耐药的革兰氏阴性病原体引起的各种感染。 与公共卫生相关：拟议的项目旨在发现新类别的广谱抗生素，目标是两条平行的外膜蛋白定位途径。如果成功，这项工作的结果将是开发出有效的抗菌剂，即使是最具抗药性的革兰氏阴性病原体也能被杀死。

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.