Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba

Acanthamoeba keratitis is a vision-threatening infection caused by pathogen ic species of the genus Acanthamoeba. Although not all Acanthamoeba spp, ca n cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role i n ocular pathology, we used colorimetric, cytopathic, and zymographic assay s to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using ate-linked protein as a substrate , showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers o f immortalized corneal epithelial cells in four-well plates were used for c ytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited m arked CPE on immortalized corneal epithelial cells, while nonpathogenic iso lates did not exhibit CPE. Protease zymography was performed with Acanthamo eba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-c onditioned medium. The zymographic protease assays showed various banding p atterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba i solates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains o nly partial inhibition was observed by using PMSF. The pathogenic Acanthamo eba strains grown under typical laboratory conditions without epithelial ce lls exhibited one overexpressed protease band of 107 kDa in common; this pr otease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.