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人类传染病的病原体。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，包括不渗透外膜（OM）和各种外排泵的活动。在这个项目中，我们建议开发一种新的抗生素，这种抗生素不进入细菌的细胞质，而是通过干扰OM的生物发生来起作用。由Lol和Bam机制组成的两条通路分别负责脂蛋白和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了工程设计，使其能够调节Bam和Lol通路的关键组分，并携带对Lol和Bam缺失敏感的荧光素酶报告结构。这些铜绿假单胞菌试验菌株将用于筛选化合物文库和OM蛋白运输抑制剂。这些化合物将被鉴定为具有最大杀伤效力，对其他革兰氏阴性病原体具有广谱性，在生物膜，血清和呼吸道粘液中具有活性，具有低细胞毒性和增强细菌的杀菌活性

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.