Nagano T, Hao J L, Nakamura M, Kumagai N, Abe M, Nakazawa T, Nishida T
Department of Ophthalmology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan.
Invest Ophthalmol Vis Sci. 2001 May;42(6):1247-53.
The pathobiology of corneal ulceration induced by Pseudomonas aeruginosa was investigated by characterization of the pseudomonal pathogenic factors responsible for degradation of the collagen matrix.
Three-dimensional gels of type I collagen containing (or not) rabbit keratocytes were incubated in the presence of either culture supernatant of P. aeruginosa strain PAO1 or pseudomonal pathogenic factors (elastase, lipopolysaccharide, or exotoxin A), and the extent of collagen degradation was assessed after 24 hours by measurement of released hydroxyproline. Activation of matrix metalloproteinases (MMPs) produced by keratocytes was also examined by gelatin zymography and immunoblot analysis.
In the absence of keratocytes, the PAO1-conditioned medium increased the extent of collagen degradation. The conditioned medium also promoted keratocyte-mediated collagen degradation. Of the pseudomonal pathogenic factors examined, only elastase degraded collagen directly as well as stimulated keratocyte-mediated collagen degradation. Culture supernatant of elastase-deficient P. aeruginosa (lasR or lasB) mutants had no effect on collagen degradation in the absence or presence of keratocytes. Elastase also induced the conversion of the inactive precursors of MMP-1, -2, -3, and -9 produced by keratocytes to the active forms of the enzymes.
These results suggest that pseudomonal elastase both degrades type I collagen directly and promotes collagen degradation mediated by keratocytes, the latter effect being likely attributable, at least in part, to the activation of proMMPS:
通过对负责降解胶原基质的铜绿假单胞菌致病因子进行表征，研究铜绿假单胞菌诱导角膜溃疡的病理生物学。
将含有（或不含有）兔角膜细胞的I型胶原三维凝胶在铜绿假单胞菌PAO1菌株的培养上清液或假单胞菌致病因子（弹性蛋白酶、脂多糖或外毒素A）存在的情况下孵育，24小时后通过测量释放的羟脯氨酸来评估胶原降解程度。还通过明胶酶谱和免疫印迹分析检测角膜细胞产生的基质金属蛋白酶（MMPs）的激活情况。
在没有角膜细胞的情况下，PAO1条件培养基增加了胶原降解程度。该条件培养基还促进了角膜细胞介导的胶原降解。在所检测的假单胞菌致病因子中，只有弹性蛋白酶直接降解胶原并刺激角膜细胞介导的胶原降解。弹性蛋白酶缺陷型铜绿假单胞菌（lasR或lasB）突变体的培养上清液在有无角膜细胞的情况下对胶原降解均无影响。弹性蛋白酶还诱导角膜细胞产生的MMP-1、-2、-3和-9的无活性前体转化为酶的活性形式。
这些结果表明，假单胞菌弹性蛋白酶既能直接降解I型胶原，又能促进角膜细胞介导的胶原降解，后一种作用可能至少部分归因于前MMPs的激活。
