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

项目摘要 一些最重要的人类病原体包括那些由于以下原因而导致难以治疗的感染的病原体： 它们对常用抗生素的耐药性。对于革兰氏阴性菌，主要耐药菌 机制包括一个不可渗透的细胞包膜与活动的驱逐（流出） 泵.该组包括铜绿假单胞菌，免疫功能低下个体的病原体， 那些患有囊性纤维化的人，其基因组中编码有许多外排泵。这项建议旨在 通过探索使用周质的可行性，为药物发现创造新的使能工具， 表达的单结构域抗体（纳米抗体）能够阻断发生的生物过程， 在这个隔间里本文提出的工作将针对MexXY-OprM的周质组分 外排泵，目的是鉴定干扰抗生素外排的纳米抗体。在第一个目标中， 将纯化重组MexX和MexY的融合周质结构域， 从酵母表面展示的纳米抗体的合成文库中选择结合剂。这些将被转移到 细菌表达载体，并引入铜绿假单胞菌，其中将抗体分泌到 周质应干扰MexXY-OprM的组装或功能，使细菌敏感 抗生素是这种外排泵的底物。一些遗传和生物化学方法将 用于绘制Mex蛋白上的纳米抗体表位。显示弱亲和力的纳米抗体将是 进行几轮亲和力成熟。该项目不仅应产生关于 外排泵的功能结构域，而且也是适用于筛选和验证小分子的工具 抗生素外排抑制剂。

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.