Three stages in the development of the cyst wall of the eye pathogen Acanthamoeba castellanii

Abstract

9When deprived of nutrients, trophozoites of the eye pathogen Acanthamoeba castellanii make 10 a cyst wall, which contains cellulose and has two layers connected by cone-shaped ostioles. 11 We recently showed chitin is also present and identified three sets of lectins, which localize to 12 the ectocyst layer (Jonah lectin) or the endocyst layer and ostioles (Luke and Leo lectins). To 13 Acanthamoeba was used to discover and characterize roles of actin and associated proteins in 54 the cytoskeleton, phagocytosis, and cell motility (16). Whole genome sequences of A. 55 castellanii identified >500 genes derived from bacteria by HGT, which is the greatest number 56 of any eukaryote described to date (17). A mannose-binding protein on the surface of 57 trophozoites, secreted proteases, and pore-forming peptides each appear to contribute to 58 contact-mediated cytolysis of corneal epithelial cells by Acanthamoebae (18)(19)(20). 59 The Acanthamoeba cyst wall, which forms when trophozoites are starved, is an 60 important virulence factor, because it makes cysts resistant to surface disinfectants, sterilizing 61 agents in contact lens solutions, and antibiotics applied to the eye (21, 22). Fifty years ago, 62 the cyst wall was shown to contain cellulose and have two microfibril-dense layers (outer 63 ectocyst and an inner endocyst), as well as conical structures (ostioles) that connect the layers 64 (23, 24). During excystation, the trophozoites escape through one of the ostioles, which were 65 not counted (25). Although the electron micrographs were beautiful, none of the cyst wall 66 proteins were identified, and there was minimal information as to how the two layers and 67 ostioles are formed. Further, monoclonal antibodies, potentially useful as diagnostic reagents, 68 were made to trophozoites but not to cysts (26, 27). 69To better understand its structure, we recently identified chitin and three sets of 70 cellulose-and chitin-binding lectins in the cyst wall of A. castellanii Neff, which is the best 71 studied strain of the protist (28). These lectins are present in 12 copies (Luke), 5 copies 72 (Jonah), or 14 copies (Leo) in the cyst wall, which make them by far the most abundant 73 proteins. These lectins contain carbohydrate-binding modules that were shown to bind 74 cellulose in Dictyostelium and plants (CBM49s of Luke lectins), were previously identified in 75 bacteria but were uncharacterized (choice of anchor A (CAA) domains of Jonah lectins), or are 76 unique to Acanthamoebae (8-Cys domains of Leo lectins) (Fig. 1) (29-31). A representative 77 Jonah lectin is present in the ectocyst layer, while representative Luke and Leo lectins are 78 present in the endocyst layer and ostioles. 79 Here we used structured illumination microscopy (SIM), probes for glycopolymers, and 80 green fluorescent protein (GFP) tags for cyst wall lectins to determine the sequence of events 81 (stages) involved in the formation of the A. castellanii cyst wall during encystation (32). To 82 characterize their carbohydrate-bi...