A detailed dissection of the expression, localization, structure, and diagnostic potential of cyst wall proteins of the eye pathogen Acanthamoeba

Abstract

The cyst wall of Acanthamoeba castellanii (Ac) contains cellulose and has outer ectocyst and inner endocyst layers connected by conical ostioles. Previously, we used mass spectrometry of purified Ac walls to identify families of abundant lectins (Jonah, Luke, and Leo). With the goal of identifying and characterizing novel target proteins for the diagnosis of cysts in patients with Acanthamoeba keratitis (AK), we localized three additional GFP-tagged proteins (a multicopper oxidase (AKA laccase) and additional Jonah and Leo lectins) to the ectocyst layer of the Ac wall. Although all walls proteins are expressed only during encystation, the precise timing of expression plays a decisive factor in their subcellular localization. Promoter swaps experiments showed early expression causes proteins to localize to the ectocyst layer, while later expression causes proteins to localize to the endocyst layer and ostioles. Ac wall proteins originate from bacteria by horizontal gene transfer (beta-helical folds of Jonah and three cupredoxin-like domains of the laccase), share a common ancestry with wall proteins of slime molds (beta-jelly-roll folds of Luke), or are unique to Acanthamoebae (four disulfide knots of Leo). β-jelly-roll folds of Luke and disulfide knots of Leo both share a linear array of aromatic amino acids, which are necessary for binding cellulose and proper localization of these proteins in the cyst wall, a feature consistent with convergent evolution. Purified rabbit antibodies to recombinant proteins bound to the ectocyst layer (Jonah and laccase) and/or to the ostioles (Luke and Leo) of cysts of 10 of 11 Acanthamoeba isolates tested, including five T4 genotypes that cause most cases of AK. In summary, we combined basic science experiments to understand better the localization, origin, and structure of Ac cyst wall proteins with translational studies to test target proteins for diagnosis of cysts in eye infections.