Identification of new targets for the diagnosis of cysts (four) and trophozoites (one) of the eye pathogen Acanthamoeba

Acanthamoebae, which are free-living amoebae, cause corneal inflammation (keratitis) and blindness, if not diagnosed and effectively treated. While trophozoites adhere to and damage the cornea, Acanthamoeba cysts, the walls of which contain cellulose and have two layers connected by conical ostioles, are the diagnostic form by microscopy of the eye or of corneal scrapings. We recently used structural and experimental methods to characterize cellulose-binding domains of Luke and Leo lectins, which are abundant in the inner layer and ostioles. However, no antibodies have been made to these lectins or to a Jonah lectin and a laccase, which are abundant in the outer layer. Here we used confocal microscopy to show that rabbit antibodies to recombinant Luke, Leo, Jonah, and laccase generally support localizations of GFP-tagged proteins in walls of transfected Acanthamoebae. Rabbit antibodies to all four wall proteins efficiently detected calcofluor white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC, including five T4 genotypes that cause most cases of keratitis. Laccase shed into the medium during encystation was detected by an enzyme-linked immunoassay. We also used structural and experimental methods to characterize the mannose-binding domain of an Acanthamoeba mannose-binding protein and showed that rabbit antibodies to the mannose-binding domain efficiently detected trophozoites of all 11 Acanthamoeba isolates. We conclude that four wall proteins are all excellent targets for diagnosing Acanthamoeba cysts in the eye or corneal scrapings, while the mannose-binding domain is an excellent target for identifying trophozoites in cultures of corneal scrapings. ImportanceFree-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of cysts by microscopy of the eye or of corneal scrapings, using calcofluor-white that unfortunately cross-reacts with fungi and plants. Alternatively, Acanthamoeba infections are diagnosed by identification of trophozoites in cultures of scrapings. Here we showed that rabbit antibodies to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently detect calcofluor-white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rabbit antibodies to the mannose-binding domain of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identifies trophozoites of all 11 ATCC isolates. In summary, four wall proteins and the ManBD appear to be excellent targets for diagnosis of Acanthamoeba cysts and trophozoites, respectively.