Abstract
Purpose: :
This study was conducted to investigate the role of alkaline serine protease(ALP) and phospholipase B (PLB) of Aspergillus fumigatus in the regulation of matrix metalloproteinase (MMP) 2 and 9 in a mouse model of experimental keratitis.
Methods: :
RNA interference of ALP and PLB in Aspergillus fumigatus was transformed by a specific vector of plasmid pBC-hygro containing a dsRNA coding sequence and a GFP sequence. Mice were then challenged with 106 CFU ALP and PLB gene-silenced Aspergillus fumigatus(ΔALP and ΔPLB). The severity of keratomycosis in the animals was scored visually with the aid of a dissecting microscope and slit lamp. Eyes were enucleated at 1, 2, 4, and 8 days after fungal challenge and homogenized to evaluate the production of MMP-2, MMP-9 mRNA and protein by real-time PCR and western blot,respectively.
Results: :
After RNA interference, the inhibition of ALP and PLB gene expression in the transformant were 65.35% and 40.94%. The scores ofΔALP and ΔPLB challenged eyes was obviously lower than the controls from 2 day to 8 day after fungal challenge (p<0.05). In the eyes challenged by ΔALP, it was the expression of MMP-9 protein (p<0.05) but not MMP-9 mRNA (p>0.05) that statistically downregulationed, and the MMP-2 mRNA and protein were slightly upregulationed from 4 day to 8 day after challenge. However, in the eyes challenged by ΔPLB, MMP-9, MMP-2 mRNA and protein were both significantly downregulated.
Conclusions: :
The results imply that ALP and PLB have important roles in corneal destruction during fungal keratitis by regulating the expression of MMP-9 and MMP-2 in cornea, and suggests that targeting ALP and PLB of the pathogenic Aspergillus fumigatus may be a novel therapeutic strategy for fungal keratitis.
Keywords: cornea: basic science • enzymes/enzyme inhibitors