Estrellas P S, Alionte L G, Hobden J A
Department of Immunology/Microbiology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
Curr Eye Res. 2000 Mar;20(3):157-65.
Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence.
Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis.
In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue.
These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.
铜绿假单胞菌蛋白酶被认为是角膜疾病发病机制中的重要毒力因子。本研究检测了两株导致两种截然不同临床疾病的铜绿假单胞菌的蛋白酶产生情况：从接触镜诱发的急性红眼病（CLARE）中分离出的菌株Paer1，以及从角膜溃疡中分离出的菌株KEI 1025。将这些菌株与已知在实验感染小鼠中会引发严重角膜炎的实验室菌株（ATCC 19660）进行蛋白酶产生情况和眼部毒力的比较。
采用比色法、明胶酶谱法和蛋白质印迹法检测蛋白酶产生情况。用葡萄球菌溶解试验定量弹性蛋白酶A的活性。使用小鼠角膜划痕模型检测眼部毒力。
与菌株KEI 1025或ATCC 19660不同，Paer1无法产生具有酶活性的弹性蛋白酶A、弹性蛋白酶和蛋白酶IV。所有三株菌株均产生活性碱性蛋白酶。菌株KEI 1025和ATCC 19660在小鼠中引发暴发性角膜炎，而Paer1引发轻度短暂感染。通过基因互补恢复Paer1中的弹性蛋白酶活性并未导致毒力表型。将菌株Paer1和ATCC 19660共同感染小鼠眼睛，最终导致Paer1从角膜组织中消失。
这些研究表明，铜绿假单胞菌弹性蛋白酶A和/或蛋白酶IV而非碱性蛋白酶或弹性蛋白酶，有助于该生物体的眼部毒力。
