Investigating the regulation of outer membrane homeostasis in Pseudomonas aeruginosa

项目总结 革兰氏阴性外膜是抵抗多种化学物质的有效渗透性屏障 袭击，包括抗生素。外膜的本征阻隔能力至少部分是由于 由于其非对称性，内叶由磷脂组成，而外叶由磷脂组成 脂多糖(LPS)。外膜作为通透性屏障的能力取决于 邻近的内毒素分子之间存在强烈的横向相互作用，这种相互作用会被 外层小叶中的磷脂。此外，内毒素和内毒素的生物合成途径 磷脂生产利用一个共享的生物合成前体池，因此至关重要的是， 通过这两条途径都是平衡的，以便公平地进入前体储存库。几个外部 膜动态平衡因子将磷脂限制在叶内，或平衡内毒素和 磷脂的生物合成在肠杆菌如大肠杆菌中得到了很好的表征，但 对人类致病菌铜绿假单胞菌的外膜动态平衡知之甚少。 此外，多重耐药铜绿假单胞菌的比率不断上升，突显了需要更深层次的 了解维持其外膜屏障能力所需的因素。这个 拟议的研究旨在表征铜绿假单胞菌的外膜动态平衡。在目标1中，我们将 研究如何控制通过内毒素生物合成途径的流量以适应细胞的需要。我们的 初步数据表明，催化内毒素第二步和承诺步骤的LpxC 在铜绿假单胞菌中，生物合成途径受到一种新的机制的调节，这将是遗传和 在团契期间的生化特征。在目标2中，我们将识别和描述小说 铜绿假单胞菌外膜动态平衡所需的因素，包括那些与 维持外膜的不对称性以及参与调节内毒素和磷脂的那些 生物合成。总体而言，这项工作将揭示P。 铜绿假单胞菌具有指导未来抗生素开发的潜力。拟议的研究一直是 旨在以申请人已有的技能为基础，同时在以下方面提供充足的培训机会 新技术。这项工作将在赞助商实验室培育的协作环境中完成 以及哈佛医学院微生物学系。此外，申请者将定期 参加科学会议并参加涵盖主题的职业发展研讨会 例如科学交流和实验室管理。因此，申请人将获得全面的 培训成为一名成功的独立研究人员所需的技能。

PROJECT SUMMARY The Gram-negative outer membrane is an effective permeability barrier against numerous chemical assaults, including antibiotics. The intrinsic barrier capacity of the outer membrane is at least in part due to its asymmetric nature; the inner leaflet consists of phospholipids while the outer leaflet is composed of lipopolysaccharide (LPS). The ability of the outer membrane to serve as a permeability barrier relies on strong lateral interactions between neighboring LPS molecules, which are disrupted by the presence of phospholipids in the outer leaflet. In addition, the biosynthetic pathways responsible for LPS and phospholipid production utilize a shared pool of biosynthetic precursors, making it critical that the flux through both pathways is balanced to allow equitable access to the precursor pool. Several outer membrane homeostasis factors that confine phospholipids to the inner leaflet or balance LPS and phospholipid biosynthesis have been well characterized in enterobacteria such as Escherichia coli, but little is known about outer membrane homeostasis in the human pathogen Pseudomonas aeruginosa. Further, the increasing rates of multidrug resistant P. aeruginosa highlight the need for a deeper understanding of the factors required to maintain the barrier capacity of its outer membrane. The proposed research aims to characterize outer membrane homeostasis in P. aeruginosa. In Aim 1, we will investigate how flux through the LPS biosynthetic pathway is controlled to fit the needs of the cell. Our preliminary data suggest that LpxC, which catalyzes the second and committed step in the LPS biosynthetic pathway, is regulated by a novel mechanism in P. aeruginosa, which will be genetically and biochemically characterized during the fellowship period. In Aim 2, we will identify and characterize novel factors required for outer membrane homeostasis in P. aeruginosa, including those responsible for the maintenance of outer membrane asymmetry as well as those involved in regulating LPS and phospholipid biosynthesis. Overall, this work will reveal fundamental mechanisms important for drug-resistance in P. aeruginosa with the potential to guide future antibiotic development. The proposed research has been designed to build on the applicant's pre-existing skillset while providing ample training opportunities in new techniques. The work will be completed in a collaborative environment fostered by the sponsor's lab and the Harvard Medical School Department of Microbiology. In addition, the applicant will regularly attend scientific conferences and also participate in career development workshops that cover topics such as scientific communication and lab management. Thus, the applicant will receive comprehensive training in the skills needed for a successful career as an independent researcher.