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 革兰氏阴性菌外膜是抵抗多种化学物质的有效渗透屏障， 攻击，包括抗生素。外膜的固有屏障能力至少部分是由于 由于其不对称性质，内瓣叶由磷脂组成，而外瓣叶由 脂多糖（LPS）。外膜作为渗透屏障的能力依赖于 相邻LPS分子之间的强横向相互作用，其被存在的 磷脂在外面的小叶。此外，负责LPS的生物合成途径和 磷脂生产利用生物合成前体的共享池，这使得流量 通过这两种途径进行的贸易是平衡的，以允许公平地获得前体库。几个外部 将磷脂限制在内叶或平衡LPS的膜稳态因子， 磷脂生物合成在肠细菌如大肠杆菌中已得到很好的表征，但 对人类病原体绿脓杆菌的外膜稳态知之甚少。 此外，多药耐药铜绿假单胞菌的增加率突出了对更深入的研究的需要。 了解维持其外膜屏障能力所需的因素。的 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 研究如何控制通过LPS生物合成途径的流量以适应细胞的需要。我们 初步的数据表明，LpxC，它催化的第二个和承诺的步骤，在LPS 生物合成途径，是由一种新的机制调节铜绿假单胞菌，这将是遗传和 在研究期间的生化特征。在目标2中，我们将识别和表征新的 铜绿假单胞菌外膜稳态所需的因素，包括那些负责 维持外膜的不对称性以及参与调节LPS和磷脂 生物合成总之，这项工作将揭示重要的耐药机制在P。 具有指导未来抗生素开发的潜力。拟议的研究已被 旨在建立在申请人现有的技能，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，并参加职业发展研讨会， 如科学交流和实验室管理。申请人将获得全面的 培训作为一名独立研究人员成功的职业生涯所需的技能。