PROTEASE SECRETION IN PSEUDOMONAS AERUGINOSA

这次调查的主要目的是增进我们对 铜绿假单胞菌的致病机制。P.的性质。 铜绿假单胞菌在发病机制中起主要作用的是它的 分泌有毒和可降解的外切酶，这些酶会导致肿瘤部位的坏死 感染。然而，人们对其分泌机制知之甚少。 在铜绿假单胞菌的胞外酶中有两种酶，称为弹性蛋白酶。 和拉萨，它们有各种各样的底物，但它们也可以发挥作用 协同降解弹性蛋白。铜绿假单胞菌的降解能力 弹性蛋白被认为是其发病机制中的一个重要因素，因为 它是结缔组织的主要蛋白质，也是肺的重要组成部分。 功能。弹性蛋白酶和LASA蛋白酶最初都形成为 前原酶在它们被加工之前经过两次蛋白质分解 由生物体释放出来。我们最近提供的证据表明 前弹力酶的加工是通过自催化进行的，但LASA的机制 处理过程未知。弹性蛋白酶和LASA蛋白水解酶以及许多 其他外源蛋白由复合体通过外膜分泌。 由XCP基因编码的一组蛋白质。人们对此知之甚少 对通过这种分泌途径引导外源蛋白的信号进行分类。 这项建议的目的是了解 铜绿假单胞菌弹性蛋白酶的加工和分泌。 这一建议结合了遗传和生化方法来比较这些 蛋白酶及其分泌途径。关于这个结构，人们知道得很多。 弹性蛋白酶，但对LASA作为一种蛋白酶知之甚少。我们会 确定LASA的结构/功能关系 生化和免疫学方法。我们将描述 弹性蛋白酶和LASA蛋白的加工和分泌 被其活性部位的取代所改变的。这将使我们能够 评估自动处理的作用并确定单元中的位置 其中分泌被阻断，以更好地了解分泌途径。 弹性蛋白酶和LASA都有长的氨基末端多肽，这些多肽被去除 在分泌过程中，并可能参与转位、折叠和/或 蛋白水解酶的活性。这些可能性将是 通过表征前肽缺失、氨基酸的影响进行评估 酸取代，以及通过前肽结构域转换形成的杂交物。 对分泌蛋白相互作用的机制知之甚少。 通过细胞外转位机制。我们将确定目标 弹性蛋白酶和LASA中的信号，它们可能与XCP相互作用- 分泌系统，并以这些蛋白质为靶点通过 外膜。这将通过删除分析来执行，站点- 定向诱变，以及对P. 为了阐明弹性蛋白酶和LASA中的序列， 含有细胞外分泌的基本靶向信号。这 研究为理解蛋白质提供了一个极好的模型系统 输出革兰氏阴性细菌，应该会产生新的信息 一个重要的发病机制在机会主义者中占有重要地位。

The broad objective of this investigation is to increase our understanding of the pathogenic mechanisms of Pseudomonas aeruginosa. A property of P. aeruginosa which plays a major role in pathogenesis is its ability to secrete toxic and degradative exoenzymes which cause necrosis at sites of infection. However, little is known about the mechanism of secretion. Among the exoenzymes of P. aeruginosa are two proteases, called elastase and LasA, which have a variety of substrates, but they also can act cooperatively to degrade elastin. The ability of P. aeruginosa to degrade elastin is believed to be a significant factor in its pathogenesis because it is a major protein of connective tissue and a vital component for lung function. Both elastase and LasA protease are initially formed as preproenzymes which are proteolytically processed twice before they are released by the organisms. We have recently provided evidence that proelastase processing occurs by autocatalysis, but the mechanism of LasA processing is unknown. Both elastase and LasA protease, as well as many other exoproteins, are secreted through the outer membrane by a complex set of proteins encoded by the xcp genes. Little is known about the sorting signals which channel exoproteins through this secretion pathway. The objective of this proposal is to understand the mechanisms of processing and secretion of the elastolytic proteases in P. aeruginosa. This proposal combines genetic and biochemical approaches to compare these proteases and their secretion pathways. Much is known about the structure of elastase, but little is known about LasA as a protease. We will determine the structure/function relationships of LasA using genetic, biochemical, and immunological approaches. We will characterize the processing and secretion of elastase and LasA proteins that have been altered by substitutions in their active sites. This will allow us to evaluate the role of autoprocessing and identify the location in the cell where secretion is blocked to better understand the pathway of Secretion. Both elastase and LasA have long amino terminal peptides which are removed during secretion, and may be involved in translocation, folding, and/or the enzymatic activity of the proteases. These possibilities will be evaluated by characterizing the effects of propeptide deletions, amino acid substitutions, and hybrids formed by propeptide domain switching. Little is known about the mechanisms by which secreted proteins interact with the extracellular-translocation machinery. We will identify targeting signals in elastase and LasA, which presumably interact with the Xcp- secretion system, and target these proteins for secretion through the outer membrane. This will be performed through a deletion analysis, site- directed mutagenesis, and a study of the behavior of protein fusions in P. aeruginosa in order to elucidate the sequences in elastase and LasA that contain essential targeting signals for extracellular Secretion. This research provides an excellent model system for understanding protein export in gram-negative bacteria and should result in new information on a significant mechanism of pathogenesis in an important opportunist.