PROTEASE SECRETION IN PSEUDOMONAS AERUGINOSA

这项调查的广泛目标是增加我们的 对假单胞菌致病机制的认识 铜绿假单胞菌是一种机会致病菌，它能引起各种 人类受到严重而致命的感染。铜绿假单胞菌是值得注意的 因为它有能力向环境中分泌一些有毒物质 和降解酶，这一特性已被证明 在生物体的致病过程中起主导作用。 人们对这些酶的分泌途径知之甚少。 在铜绿假单胞菌。拟议的研究将审查合成， 铜绿假单胞菌中两种蛋白水解酶的激活和分泌。这 细菌分泌两种不同的蛋白酶，称为弹性蛋白酶和 碱性蛋白水解酶，这些胞外酶负责 大部分坏死和一般侵袭性可见于 感染。我们最近发现，基因lasA编码一种 31kD蛋白，激活细胞的弹性溶解能力 前体，前弹性蛋白酶。我们将调查这一事件的性质 利用遗传和生化技术的激活步骤。 弹性蛋白酶结构基因(LasB)将被克隆， 具有特征性，并进行了排序。弹性蛋白酶前体的加工 将在分子水平上确定，并在 将对分泌途径进行表征。已知的突变 阻断一些胞外酶的分泌， 包括弹性淀粉酶在内，将以它们在 弹性酶中间体的加工。基因融合将被用于 比较LasA和LasB在铜绿假单胞菌中的调节作用。LasB 通过克隆的体外操作将构建突变体 DNA，然后是基因替换，然后表征 蛋白酶的产生。LASA蛋白也在体内起辅助作用。 将被调查和定义的弹性淀粉酶的分泌。我们 还将克隆和鉴定碱性结构基因 蛋白水解酶(ALPA)，一种具有分泌途径的分泌酶 这与弹性蛋白酶明显不同。ALPA突变体 将通过基因替换来构建并进行表征。这个 LasA和LasB突变对碱性蛋白酶分泌的影响 将会被检查。

The broad objective of this investigation is to increase our understanding of the pathogenic mechanisms of Pseudomonas aeruginosa, an opportunistic pathogen which causes a variety of severe and lethal infections in humans. P. aeruginosa is notable for its ability to secrete into its environment a number of toxic and degradative enzymes, a property which has been shown to play a dominant role in the organism's pathogenic processes. Little is known about the pathway of secretion of these enzymes in P. aeruginosa. The proposed study will examine the synthesis, activation, and secretin of two proteases in P. aeruginosa. This bacterium secretes two distinct proteases called elastase and alkaline protease, and these extracellular enzymes are responsible for much of the necrosis and general invasiveness seen at sites of infection. We have recently shown that the gene lasA encodes a 31 Kd protein which activates the elastolytic potential of the precursor, pro-elastase. We will investigate the nature of this activation step employing genetic and biochemical techniques. The structural gene for elastase (lasB) will be cloned, characterized, and sequenced. Processing of elastase precursors will be determined at the molecular level, and intermedicates in the secretion pathway will be characterized. Mutations known to block the secretion of a number of extracellular enzymes, including elastase, will be characterized for their role in the processing of elastase intermediates. Gene fusions will be used to compare the regulation of lasA and lasB in P. aeruginosa. lasB mutants will be constructed by in vitro manipulation of cloned DNA followed by gene replacement and then characterized for protease production. lasA protein also has a secondary role in the secretion of elastase which will be investigated and defined. We will also clone and characterize the structural gene for alkaline protease (alpA), a secreted enzyme with a pathway of secretion which is apparently distinct from that of elastase. alpA mutants will be constructed by gene replacement and characterized. The effect of lasA and lasB mutations on alkaline protease secretion will be examined.