Structure and catalytic function of Pseudomonas aeruginosa secreted proteins

分泌的毒力因子是新型抗菌剂的理想靶点：它们很容易被外部应用的化学物质所接近。细菌分泌的蛋白质，专门针对和杀死其他细菌（细菌素）：潜在的有用的新型抗菌剂。这两种类型的蛋白质都可以作为输出蛋白质用于生物技术用途的底盘，从而减少下游纯化。本项目旨在表征铜绿假单胞菌毒力因子的结构和功能。铜绿假单胞菌是CF、支气管扩张症、COPD和伤口患者的主要机会致病菌，对抗生素具有内在耐药性，并且几乎不可能从医院中根除，部分原因是其作为生物膜群落生长的能力。绿脓菌素S3是由铜绿假单胞菌产生的细菌素，其杀死其他物种的铜绿假单胞菌。一种同源免疫蛋白与产生细胞中的绿脓菌素结合以防止自杀。我们已经证明绿脓菌素S3是一种DNA酶，并且已经解决了与其同源免疫蛋白复合的DNA酶结构域的晶体结构，揭示了其非特征性的一级序列仍然保持活性位点β-β-α二级结构基序，而没有典型的DNA酶HNH排列。另外，还发现了一种新的章鱼对DNA酶的免疫原性蛋白的捕获。绿脓菌素S3 DNA酶以其天然形式与受体结合和易位结构域连接2。绿脓菌素S3脱氧核糖核酸酶结构域没有其同源免疫蛋白，以揭示在活性位点基序处存在结合的金属离子。然后将纯化的DNA酶用于研究DNA催化的作用方式。3.绿脓菌素S3同源免疫蛋白。将使用定点诱变来消除预测与功能有关的残基，以增加我们对其功能结构域的理解。

Secreted virulence factors are ideal targets for novel antimicrobials: they are readily accessible to externally applied chemicals. Bacteria secrete proteins that specifically target and kill other bacteria (Bacteriocins): potentially useful novel antimicrobials. Both types of protein could serve as a chassis to export proteins for biotechnological uses thereby reducing downstream purification. This project seeks to characterize the structure and function of Pseudomonas aeruginosa virulence factors. P. aeruginosa is a major opportunistic pathogen for CF, bronchiectasis, COPD and wound patients, is intrinsically resistant to antibiotics, and almost impossible to eradicate from hospitals in part due to its ability to grow as biofilm communities.Pyocin S3 is a bacteriocin produced by P. aeruginosa that kills other species of P. aeruginosa. A cognate immunity protein binds to the pyocin in the producing cell to prevent suicide. We have shown that pyocin S3 is a DNase, and have solved the crystal structure of the DNase domain in complex with its cognate immunity protein, revealing its uncharacteristic primary sequence still maintains an active site beta-beta-alpha secondary structure motif without the typical DNase HNH arrangement. In addition, a novel octopus grasp of the DNAse by the cognate immunity protein was discovered.This project aims to purify and crystallize:1. Pyocin S3 DNase in its native form with the receptor binding and translocation domains attached2. Pyocin S3 DNase domain without its cognate Immunity protein to reveal the presence of bound metal ion at the active site motif. The purified DNase will then be used to investigate the mode of action of DNA catalysis.3. The Pyocin S3 cognate Immunity protein.Site directed mutagenesis will be used to inactivate residues predicted to be implicated in the function to increase our understanding of their functional domains.