Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genusAcanthamoeba. Although not allAcanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenicAcanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenicAcanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoebaisolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed withAcanthamoeba-conditioned medium as well as withAcanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenicAcanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.
TLR4-dependent lipopolysaccharide signalling in epithelial cells is independent of extracellular protease activity
