Acanthamoeba castellanii: Characterization of an adhesin molecule

Acanthamoeba castellanii is a free-living protozoan that causes keratitis i n humans and has been associated with pneumonia and granulomatous amebic en cephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In th is study, several mouse monoclonal antibodies (MAb) generated to whole Acan thamoeba trophozoites identified surface membrane epitopes by ELISA and IFA . Nine antibodies inhibited adherence of [S-35]-methionine-labeled Acantham oeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium per iodate treatment, but not proteinase K digestion, of whole Acanthamoeba des troyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7 H6, suggesting that the adherence epitopes are carbohydrates. Other antibod ies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2 A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the pep tide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa ban d on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirm ing that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adher ence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in so luble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibite d from binding to whole trophozoites nor membrane extracts by mannose or ma nnan in competitive binding assays. When our Acanthamoeba membrane preparat ions were electrophoresed and immunoblotted with alpha-D-mannosylated-bioti n albumin, no bands were recognized in the >207 kDa range by our adherence- associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein. (C) 1999 Academic Press.