Gopinathan U, Ramakrishna T, Willcox M, Rao C M, Balasubramanian D, Kulkarni A, Vemuganti G K, Rao G N
Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.
Exp Eye Res. 2001 Apr;72(4):433-42. doi: 10.1006/exer.2000.0971.
Mycotic keratitis, being frequently refractive to most of the currently available antifungal therapy, continues to pose a therapeutic challenge to the clinician. In keratitis of infectious etiology stromal dissolution may be brought about by a combination of agent and host factors. An understanding of the source and nature of corneal tissue damage is essential for evolving more effective therapeutic modalities in the treatment of fungal keratitis. In the present study, we have characterized the extracellular proteases produced in vitro by corneal fungal pathogens namely the Aspergillus flavus and Fusarium solani when collagen was provided as the sole nitrogen source. In addition, fungal infected rabbit corneas were investigated for proteolytic activities and nature of inflammatory reaction. Gelatin zymography detected protease bands with molecular mass ranging from 100 to 200 kDa in the culture extracts of A. flavus, and a single major band of molecular mass approximately 200 kDa in the culture extracts of F. solani. A basal proteolytic activity of mass 65 kDa was visualized in all uninfected and infected rabbit corneal extracts. Infected corneas in addition revealed the presence of additional proteolytic species of mass 92 and 200 kDa. The enzyme inhibitory profile suggested that fungal cultures in vitro contained predominantly serine protease activity and to a lesser extent metalloprotease activity. However, fungal infected corneal homogenates showed the presence of metalloproteinase activity alone, the enzymatic activities entirely being sensitive to ethylene diamine tetra acetate (EDTA), a metalloprotease inhibitor. Interestingly, the serine proteolytic activity detected in fungal cultures in vitro was not present in the fungal infected corneas in vivo. However, the possible role of fungal serine proteases in the activation of corneal matrix metalloproteinases (MMPs) cannot be ruled out. Based on the criteria of molecular mass, proteolytic activity in the presence of calcium at neutral pH, and sensitivity to inhibition by a metalloprotease inhibitor, the 65 and 92 kDa gelatinases were identified as MMP 2 and MMP 9, respectively. The expression of 92 and 200 kDa gelatinases correlated positively with the amount of polymorphonuclear cells present in the infected tissues. Activated resident corneal cells or inflammatory cells may largely contribute to the increased proteolytic activities in fungal infected corneas resulting in tissue matrix degradation in fungal keratitis.
真菌性角膜炎对目前大多数可用的抗真菌治疗常常具有耐药性，这继续给临床医生带来治疗挑战。在感染性病因的角膜炎中，基质溶解可能由病原体和宿主因素共同导致。了解角膜组织损伤的来源和性质对于开发更有效的真菌性角膜炎治疗方法至关重要。在本研究中，我们对角膜真菌病原体黄曲霉和茄病镰刀菌在以胶原蛋白作为唯一氮源时体外产生的细胞外蛋白酶进行了表征。此外，我们还研究了真菌感染的兔角膜的蛋白水解活性和炎症反应的性质。明胶酶谱分析在黄曲霉的培养提取物中检测到分子量在100至200 kDa之间的蛋白酶条带，在茄病镰刀菌的培养提取物中检测到一条分子量约为200 kDa的主要条带。在所有未感染和感染的兔角膜提取物中均可见到分子量为65 kDa的基础蛋白水解活性。感染的角膜还显示存在分子量为92和200 kDa的其他蛋白水解物种。酶抑制谱表明，真菌体外培养物主要含有丝氨酸蛋白酶活性，金属蛋白酶活性较低。然而，真菌感染的角膜匀浆仅显示存在金属蛋白酶活性，其酶活性完全对金属蛋白酶抑制剂乙二胺四乙酸（EDTA）敏感。有趣的是，在体外真菌培养物中检测到的丝氨酸蛋白水解活性在体内真菌感染的角膜中并不存在。然而，不能排除真菌丝氨酸蛋白酶在激活角膜基质金属蛋白酶（MMP）中的可能作用。根据分子量标准、中性pH下钙存在时的蛋白水解活性以及对金属蛋白酶抑制剂抑制的敏感性，分别将65 kDa和92 kDa的明胶酶鉴定为MMP 2和MMP 9。92 kDa和200 kDa明胶酶的表达与感染组织中多形核细胞的数量呈正相关。活化的角膜驻留细胞或炎症细胞可能在很大程度上导致真菌感染角膜中蛋白水解活性增加，从而导致真菌性角膜炎中的组织基质降解。
