Investigating the regulation of outer membrane homeostasis in Pseudomonas aeruginosa

研究铜绿假单胞菌外膜稳态的调节

• 批准号: 10725108
• 负责人: Katherine Ruth Hummels
• 金额: $ 6.42万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-07-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10725108
• 关键词: Acylation; Affinity Chromatography; Anabolism; Antibiotic Resistance; Antibiotics; Architecture; Bacteremia; Bacteria; Biochemical; Biogenesis; Biological Assay; Cell Wall; Cells; Charge; Chemicals; Communication; Cystic Fibrosis; Data; Development; Disaccharides; Disease; Drug resistance; ESKAPE pathogens; Educational workshop; Enterobacteriaceae; Enzymes; Equilibrium; Equipment and supply inventories; Equity; Escherichia coli; Exhibits; Fellowship; Fostering; Future; Genes; Genetic Techniques; Gram-Negative Bacteria; Growth; Health; Homeostasis; Human; Immunocompromised Host; Individual; Lateral; Lipid A; Lipids; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Lung diseases; Maintenance; Mediating; Membrane; Membrane Biology; Microbiology; Mutate; Nature; Nosocomial Infections; Organism; Pathway interactions; Peptide Hydrolases; Permeability; Phospholipase; Phospholipids; Polysaccharides; Production; Proteins; Proteolysis; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Regulation; Research; Research Personnel; Role; Site; System; Techniques; Training; Variant; Work; Wound Infection; assault; blind; burn wound; candidate identification; career; career development; cell envelope; collaborative environment; cystic fibrosis patients; design; experimental study; human pathogen; insight; lipid biosynthesis; medical schools; member; multidrug-resistant Pseudomonas aeruginosa; mutant; novel; opportunistic pathogen; pathogen; reverse genetics; skills; symposium; targeted treatment; therapeutic development; training opportunity; transposon sequencing

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.

项目概要 革兰氏阴性外膜是针对多种化学物质的有效渗透屏障 攻击，包括抗生素。外膜的内在屏障能力至少部分归因于 其不对称性质；内层小叶由磷脂组成，而外层小叶由 脂多糖（LPS）。外膜作为渗透屏障的能力依赖于 相邻 LPS 分子之间存在强烈的横向相互作用，这种相互作用会因存在 外层有磷脂。此外，负责 LPS 和 磷脂的生产利用生物合成前体的共享池，因此通量至关重要 通过这两种途径实现平衡，以便公平地获得前体池。几个外 将磷脂限制在内部小叶或平衡 LPS 的膜稳态因子 磷脂生物合成已在大肠杆菌等肠细菌中得到很好的表征，但是 关于人类病原体铜绿假单胞菌的外膜稳态知之甚少。 此外，多重耐药铜绿假单胞菌的发病率不断增加，凸显了更深入研究的必要性。 了解维持其外膜屏障能力所需的因素。这 拟议的研究旨在表征铜绿假单胞菌的外膜稳态。在目标 1 中，我们将 研究如何控制脂多糖生物合成途径的通量以满足细胞的需要。我们的 初步数据表明 LpxC 催化 LPS 的第二步和关键步骤 生物合成途径，由铜绿假单胞菌中的一种新机制调节，该机制将在遗传和 在研究期间进行了生化表征。在目标 2 中，我们将识别并描述小说的特征 铜绿假单胞菌外膜稳态所需的因素，包括那些负责 维持外膜不对称性以及参与调节 LPS 和磷脂 生物合成。总体而言，这项工作将揭示对疟原虫耐药性重要的基本机制。 铜绿假单胞菌有潜力指导未来抗生素的开发。拟议的研究已 旨在以申请人现有的技能为基础，同时提供充足的培训机会 新技术。这项工作将在申办者实验室营造的协作环境中完成 和哈佛医学院微生物学系。此外，申请人将定期 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 作为独立研究员取得成功职业生涯所需的技能培训。