Enzymatic, clinical and histologic evaluation of corneal tissues in experimental fungal keratitis in rabbits

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.