PROTEASE SECRETION IN PSEUDOMONAS AERUGINOSA

描述（改编自申请人的摘要）：铜绿假单胞菌是 一种主要的机会致病菌，本研究将阐明其致病性 机制等铜绿假单胞菌分泌多种有毒和 降解酶在其发病机制中起主要作用。其中 外切酶是LasB蛋白酶（弹性蛋白酶）和LasA蛋白酶（葡萄球菌分解酶 蛋白酶），其协同作用以降解弹性蛋白和许多其它 临床相关蛋白LasA和LasB蛋白酶最初都是在 作为具有三个结构域的前酶原：信号序列、前肽和 成熟序列前肽结构域作为分子内伴侣， 是酶的活性及其细胞外定位所必需的。 本提案的目的是了解LasA和LasB分子 分子伴侣辅助折叠的机制， 铜绿假单胞菌的分泌和临床相关性。该提案结合了 分子、生物化学和生物物理方法来比较处理和 分泌这两种重要的蛋白酶。调查人员将分析 LasA和LasB外蛋白的遗传学和晶体学结构 方法.他们将确定前肽（分子伴侣）介导的 LasA和LasB蛋白的折叠和分泌。他们将决定 proLasA和proLasB与II型分泌相互作用的机制 释放到环境中的装置。他们还将确定临床 LasA和LasB在发病机制中的相关性，并测试LasA作为 特异性抗葡萄球菌治疗。这项研究将导致 在一个重要的发病机制的新信息， 机会生物它还提供了一个模型系统， 革兰氏阴性菌中蛋白质输出的主要途径。

DESCRIPTION (Adapted from the Applicant's Abstract): Pseudomonas aeruginosa is a major opportunistic pathogen, and this study will elucidate its pathogenic mechanisms. The ability of P. aeruginosa to secrete numerous toxic and degradative enzymes plays a major role in its pathogenesis. Among these exoenzymes are LasB protease (elastase) and LasA protease (staphylolytic protease) which act synergistically to degrade elastin and many other clinically relevant proteins. Both LasA and LasB proteases are initially formed as pre-proenzymes with three domains: a signal sequence, a propeptide and the mature sequence. The propeptide domain acts as an intramolecular chaperone that is required for both the enzyme's activity and its extracellular localization. The objective of this proposal is to understand LasA and LasB molecular structures, the mechanisms of their chaperone-assisted folding, pathways of secretion in P. aeruginosa, and clinical relevance. This proposal combines molecular, biochemical and biophysical approaches to compare the processing and secretion of these two important proteases. The investigators will analyze the structures of LasA and LasB exoproteins by genetic and crystallographic methods. They will determine the mechanisms of propeptide (chaperone)-mediated folding and secretion of LasA and LasB proteins. They will determine the mechanism by which proLasA and proLasB interact with the type II secretion apparatus for release to the environment. They will also determine the clinical relevance of LasA and LasB in pathogenesis, and test the efficacy of LasA as a specific anti-staphylococcal therapeutic treatment. This research will result in new information on a significant mechanism of pathogenesis in an important opportunistic organism. It also provides a model system for understanding a major pathway of protein export in gram-negative bacteria.