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.

项目摘要 革兰氏阴性外膜是针对众多化学物质的有效渗透性屏障 攻击，包括抗生素。外膜的固有屏障容量至少部分是由于 具有不对称性质；内部小叶由磷脂组成，而外部小叶由 脂多糖（LPS）。外膜充当渗透性屏障的能力取决于 相邻LPS分子之间的强横向相互作用被存在 外部小叶中的磷脂。另外，负责LP和的生物合成途径 磷脂生产利用共享的生物合成前体池，这使得通量至关重要 通过这两种途径都平衡，可以公平访问前体池。几个外部 将磷脂限制在内部传单或平衡LP的膜稳态因素和 磷脂生物合成的特征在肠杆菌中（例如大肠杆菌），但 关于人类病原体假单胞菌中的外膜稳态知之甚少。 此外，多药抗铜绿假单胞菌的增加速率突出了需要更深层次 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中的外膜稳态。在AIM 1中，我们将 研究如何控制通过LPS生物合成途径的通量以满足细胞的需求。我们的 初步数据表明，LPXC在LPS中催化第二个步骤 生物合成途径受到铜绿假单胞菌的新机制的调节，该机制将在遗传上和 在团契期间以生化为特征。在AIM 2中，我们将识别和表征小说 铜绿假单胞菌中外膜稳态所需的因素，包括负责 维持外膜不对称以及参与调节LP和磷脂的膜的维护 生物合成。总体而言，这项工作将揭示对药物抗药性重要的基本机制。 铜绿疾病有可能指导未来的抗生素发展。拟议的研究是 旨在建立在申请人的先前技能的基础上，同时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养的协作环境中完成 哈佛医学院微生物学系。此外，申请人将定期 参加科学会议，还参加涵盖主题的职业发展研讨会 例如科学沟通和实验室管理。因此，申请人将获得全面的 培训成功职业生涯所需的技能。