PURIFICATION OF PSEUDOMONAS-AERUGINOSA PROTEASES AND MICROSCOPIC CHARACTERIZATION OF PSEUDOMONAL PROTEASE-INDUCED RABBIT CORNEAL DAMAGE
PURIFICATION OF PSEUDOMONAS-AERUGINOSA PROTEASES AND MICROSCOPIC CHARACTERIZATION OF PSEUDOMONAL PROTEASE-INDUCED RABBIT CORNEAL DAMAGE
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DOI：
10.1128/iai.19.2.630-648.1978
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发表时间：
1978-01-01
影响因子：
3.1
通讯作者：
GRAY, LD
中科院分区：
文献类型：
--
作者：
KREGER, AS;GRAY, LD
采用硫酸铵沉淀、UltrogelAcA 54凝胶过滤、平板等电聚焦等方法分离了3株铜绿假单胞菌角膜强毒株的胞外蛋白酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、聚丙烯酰胺凝胶薄层等电聚焦、免疫扩散和免疫电泳程序以及其他已知假单胞菌产物的存在测试来确定制剂的纯度。角膜内注射亚微克量的蛋白酶后4-6小时，对兔角膜病变进行光镜和电镜检查，发现上皮、内皮和角膜细胞变性和坏死，多形核白细胞浸润、变性和坏死，基质蛋白多糖基质的特征性网状结构、胶体铁染色和钌红染色消失，基质蛋白多糖基质的变性和坏死程度减轻。结构正常的胶原纤维的分散;以及坏死角膜中血浆蛋白和纤维蛋白的积累。这些结构改变与先前在实验性铜绿假单胞菌角膜炎期间观察到的结构改变非常相似，并且这种相似性支持假单胞菌蛋白酶至少部分地负责假单胞菌诱导的角膜炎的快速和广泛的液化坏死特征的想法。假单胞菌蛋白酶可通过引起角膜蛋白多糖基质的损失引起严重的角膜损伤，从而导致未损伤的胶原纤维的分散、角膜基质的弱化以及随后的后弹力层膨出形成和前房压力引起的角膜穿孔。
Extracellular proteases of 3 cornea-virulent strains of P. aeruginosa were isolated by sequential ammonium sulfate precipitation, Ultrogel AcA 54 gel filtration and flat-bed isoelectric focusing. The purity of the preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer isoelectric focusing in polyacrylamide gel, immunodiffusion and immunoelectrophoretic procedures and tests for the presence of other known pseudomonal products. Light microscopic and EM examination of rabbit corneal lesions observed 4-6 h after the intracorneal injection of submicrogram amounts of the proteases revealed: degeneration and necrosis of epithelium, endothelium and keratocytes; infiltration, degeneration and necrosis of polymorphonuclear leukocytes; loss of the characteristic weblike pattern, colloidal Fe staining and ruthenium red staining of the stromal proteoglycan ground substance; dispersal of structurally normal appearing collagen fibrils; and accumulation of plasma proteins and fibrin in the necrotic corneas. These structural alterations are very similar to those observed previously during experimental P. aeruginosa keratitis, and this similarity supports the idea that pseudomonal proteases are responsible, at least in part, for the rapid and extensive liquefaction necrosis characteristic of pseudomonal-induced keratitis. Pseudomonal proteases may elicit severe corneal damage by causing the loss of the corneal proteoglycan ground substance, thus resulting in dispersal of undamaged collagen fibrils, weakening of the corneal stroma and subsequent descemetocele formation and corneal perforation by the anterior chamber pressure.