Engel L S, Hobden J A, Moreau J M, Callegan M C, Hill J M, O'Callaghan R J
Department of Microbiology, LSU Eye Center, Louisiana State University Medical Center, School of Medicine, New Orleans, USA.
Invest Ophthalmol Vis Sci. 1997 Jul;38(8):1535-42.
The role of protease IV in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated by comparing a mutant strain completely deficient in protease IV activity with its protease IV activity-producing parent.
A protease IV-deficient Pseudomonas strain PA103-29::Tn9 was generated by mutagenesis of strain PA103-29, which produces protease IV, through transposon insertion. Protease IV activity was determined by a casein agar assay, zymography, and cleavage of the chromogenic substrate, Chromozym PL. Corneal virulence was evaluated by slit lamp examination and bacterial cultures in both a rabbit intrastromal model and a mouse topical model of keratitis.
The protease IV-deficient strain PA103-29::Tn9 had significantly reduced corneal virulence relative to its parent strain PA103-29 in both a rabbit intrastromal model and a mouse topical model of infection. In the rabbit model, ocular damage (slit lamp examination score) mediated by the parent strain was severe at 32 hours after infection, whereas damage mediated by the mutant was minimal at both 32 and 55 hours after infection. This difference in virulence was not a result of differences in growth in vivo, because both strains grew equally. In the mouse model, eyes inoculated with the protease IV-producing parent strain had significant corneal damage as early as 24 hours after infection, whereas the protease IV-deficient mutant strain produced no significant corneal damage during 6 days of infection.
The ability to produce active protease IV was the determining factor in the severity of corneal virulence. Protease IV appears to mediate corneal virulence and should be considered as a target in the development of medications designed to minimize corneal damage during Pseudomonas keratitis.
通过将一株完全缺乏蛋白酶IV活性的突变菌株与其产生蛋白酶IV活性的亲本进行比较，研究蛋白酶IV在铜绿假单胞菌角膜炎发病机制中的作用。
通过转座子插入对产生蛋白酶IV的PA103 - 29菌株进行诱变，产生了一株缺乏蛋白酶IV的假单胞菌菌株PA103 - 29::Tn9。通过酪蛋白琼脂试验、酶谱分析和生色底物Chromozym PL的裂解来测定蛋白酶IV活性。在兔基质内模型和小鼠角膜炎局部模型中，通过裂隙灯检查和细菌培养来评估角膜毒力。
在兔基质内模型和小鼠感染局部模型中，与亲本菌株PA103 - 29相比，缺乏蛋白酶IV的菌株PA103 - 29::Tn9的角膜毒力显著降低。在兔模型中，感染后32小时亲本菌株介导的眼部损伤（裂隙灯检查评分）严重，而突变体在感染后32小时和55小时介导的损伤最小。这种毒力差异不是体内生长差异的结果，因为两种菌株生长情况相同。在小鼠模型中，接种产生蛋白酶IV的亲本菌株的眼睛在感染后24小时就出现了明显的角膜损伤，而缺乏蛋白酶IV的突变菌株在感染6天内未产生明显的角膜损伤。
产生活性蛋白酶IV的能力是角膜毒力严重程度的决定因素。蛋白酶IV似乎介导角膜毒力，应被视为开发旨在使铜绿假单胞菌角膜炎期间角膜损伤最小化药物的靶点。
