Defining functional domains of a P. aeruginosa efflux pump using periplasmic nanobodies

使用周质纳米抗体定义铜绿假单胞菌外排泵的功能域

• 批准号: 10038521
• 负责人: STEPHEN LORY
• 金额: $ 21.19万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-03 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10038521
• 关键词: Affinity; Antibiotics; Antibodies; Bacteria; Binding; Biochemical; Biological; Biological Assay; Biological Process; Cell Separation; Cell physiology; Cells; Chemicals; Chimeric Proteins; Clinical; Combined Modality Therapy; Complex; Cystic Fibrosis; Cytoplasm; Development; Epitope Mapping; Epitopes; Failure; Family; Flow Cytometry; Generations; Genetic; Genome; Goals; Gram-Negative Bacteria; Human; Immunocompromised Host; Individual; Infection; Intrabody; Laboratories; Libraries; Magnetism; Mammalian Cell; Maps; Medical; Membrane Fusion; Membrane Proteins; Multi-Drug Resistance; Nodule; Organism; Outcome; Pharmaceutical Preparations; Phenotype; Plasmids; Proteins; Protons; Pseudomonas aeruginosa; Pump; Reagent; Recombinants; Research; Resistance; Series; Site; Specificity; Structure; Surface; Testing; Therapeutic; Validation; Work; Yeasts; antibiotic efflux; antiporter; base; cell envelope; drug discovery; effective therapy; efflux pump; expression vector; human pathogen; inhibitor/antagonist; microorganism; mutant; nanobodies; novel therapeutics; pathogen; periplasm; preclinical development; protein complex; resistance mechanism; screening; small molecule; small molecule inhibitor; small molecule therapeutics; synthetic antibodies; tool

PROJECT SUMMARY Some of the most important human pathogens includes those that cause difficult to treat infections due to their resistance to commonly used antibiotics. For Gram negative organisms, the main resistance mechanism includes an impermeable cell envelope in conjunction with activities of expulsion (efflux) pumps. This group includes Pseudomonas aeruginosa, a pathogen of immunocompromised individuals and those with cystic fibrosis, with numerous efflux pumps encoded in its genome. This proposal aims at creating new enabling tools for drug discovery, by exploring the feasibility of using periplasmically expressed single domain antibodies (nanobodies) capable of blocking biological processes that take place in this compartment. The work proposed here will target the periplasmic components of the MexXY-OprM efflux pump, with the objective of identifying nanobodies that interfere with antibiotic efflux. In the first Aim, recombinant MexX and the fused periplasmic domains of MexY will be purified and these will be used to select binders from a synthetic library of nanobodies displayed on yeast surface. These will be transferred to bacterial expression vectors and introduced into P. aeruginosa where the secretion of antibodies into the periplasm should interfere with the assembly or function of MexXY-OprM, rendering the bacteria sensitive to antibiotics that are substrates of this efflux pump. A number of genetic and biochemical approaches will be used to map the nanobody epitopes on the Mex proteins. Nanobodies showing weak affinities will be subjected to several round of affinity maturation. The project should generate not only information about functional domains of an efflux pump but also tools suitable for screening and validation of small molecule inhibitors of antibiotic efflux.

项目摘要 一些最重要的人类病原体包括那些因由于 它们对常用抗生素的抵抗力。对于革兰氏负生物，主要抗性 机理包括一个不可渗透的细胞包络，并结合驱逐活动（外排） 泵。该组包括铜绿假单胞菌，一种免疫功能低下个体的病原体， 那些具有囊性纤维化的人，其基因组中编码了许多外排泵。该提议的目的是 通过探索使用周质的可行性来创建新的启用药物发现工具 表达的单域抗体（纳米构造）能够阻止发生的生物过程 在这个隔间中。这里提出的工作将针对mexxy-oprm的周质成分 外排泵，目的是鉴定干扰抗生素外排的纳米剂。在第一个目标中 重组MEXX和Mexy的融合圆锥状域将被纯化，这些域将用于 从酵母表面上显示的纳米构造的合成库中选择粘合剂。这些将被转移 向细菌表达载体，并引入铜绿假单胞菌，其中抗体分泌到 周质应干扰Mexxy-oprm的组装或功能，使细菌敏感 是该外排泵的底物的抗生素。许多遗传和生化方法将 用于绘制MEX蛋白上的纳米幼体表位。表现出微弱亲和力的纳米体将是 经过几轮亲和力成熟。该项目不仅应生成有关的信息 外排泵的功能域，也适合用于筛选和验证小分子的工具 抗生素外排的抑制剂。