Structure and catalytic function of Pseudomonas aeruginosa secreted proteins

铜绿假单胞菌分泌蛋白的结构和催化功能

• 批准号: 2594502
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2594502
• 关键词: Structure; catalytic; function; Pseudomonas; aeruginosa

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.

分泌的毒力因子是新型抗菌剂的理想目标：它们很容易被外部施用的化学物质所接触。细菌分泌专门针对并杀死其他细菌的蛋白质（细菌素）：潜在有用的新型抗菌剂。两种类型的蛋白质都可以作为出口蛋白质用于生物技术用途的底盘，从而减少下游纯化。该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、慢性阻塞性肺病和伤口患者的主要机会性病原体，对抗生素具有内在耐药性，并且几乎不可能从医院中根除，部分原因是其能够以生物膜群落的形式生长。Pyocin S3 是由铜绿假单胞菌产生的一种细菌素，可杀死其他种类的铜绿假单胞菌。一种同源免疫蛋白与产生细胞中的化脓菌素结合以防止自杀。我们已经证明pyocin S3是一种DNase，并解析了与其同源免疫蛋白复合物的DNase结构域的晶体结构，揭示了其非典型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DNase HNH排列。此外，还发现了章鱼通过同源免疫蛋白对DNA酶的新掌握。本项目的目的是纯化和结晶： 1．天然形式的 Pyocin S3 DNase 带有受体结合和易位结构域 2。没有同源免疫蛋白的 Pyocin S3 DNase 结构域揭示了活性位点基序上结合金属离子的存在。纯化后的DNA酶将用于研究DNA催化的作用方式。3． Pyocin S3 同源免疫蛋白。定点诱变将用于灭活预计与功能有关的残基，以增加我们对其功能域的了解。