PROTEASE SECRETION IN PSEUDOMONAS AERUGINOSA

这项调查的主要目的是增加我们对 铜绿假单胞菌的致病机制P的一个性质。 在发病机制中起主要作用的铜绿假单胞菌是其能够 分泌有毒的和降解的外切酶， 感染然而，对分泌的机制知之甚少。 在铜绿假单胞菌的胞外酶中有两种蛋白酶，称为弹性蛋白酶 和LasA，它们有多种底物，但它们也可以 协同降解弹性蛋白。铜绿假单胞菌降解 弹性蛋白被认为是其发病机制中的重要因素， 它是结缔组织的主要蛋白质，也是肺的重要成分 功能弹性蛋白酶和LasA蛋白酶最初形成为 前酶原，其在被蛋白水解之前被蛋白水解处理两次， 由有机体释放。我们最近提供的证据表明， 弹性蛋白酶前体的加工是通过自催化进行的，但LasA的机制 处理是未知的。弹性蛋白酶和LasA蛋白酶，以及许多 其他外蛋白，通过外膜分泌的复合物 由xcp基因编码的一组蛋白质。很少有人知道的 分选通过该分泌途径引导外蛋白的信号。 本提案的目的是了解 铜绿假单胞菌中弹性蛋白酶的加工和分泌。 这项建议结合了遗传和生物化学的方法来比较这些 蛋白酶及其分泌途径。我们对它的结构了解很多 弹性蛋白酶，但很少有人知道LasA作为一种蛋白酶。我们将 使用遗传学方法确定LasA的结构/功能关系， 生物化学和免疫学方法。我们将描述 弹性蛋白酶和LasA蛋白的加工和分泌， 通过活性位点的取代而改变。这将使我们能够 评估自动处理的作用并确定单元格中的位置 以更好地了解分泌途径。 弹性蛋白酶和LasA都具有长的氨基末端肽， 在分泌过程中，并且可能涉及易位、折叠和/或 蛋白酶的酶活性。这些可能性将是 通过表征前肽缺失、氨基 酸取代和通过前肽结构域转换形成的杂合体。 对于分泌蛋白相互作用的机制知之甚少 与细胞外易位机制的结合我们将确定目标 弹性蛋白酶和LasA中的信号，可能与Xcp-相互作用 分泌系统，并靶向这些蛋白质通过分泌系统分泌， 外膜这将通过删除分析、研究中心- 定向诱变，并在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 出口革兰氏阴性细菌，并应导致新的信息， 一个重要的发病机制中的一个重要的机会主义者。

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.