Identification and characterization of Acanthamoeba cyst wall proteins

ABSTRACT: Acanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for sugar polymers to show that purified cyst walls of A. castellanii retain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight cysteines each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for sugar polymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and sugar polymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and sugar polymers to which they bind are mostly inaccessible in the ectocyst layer and ostioles. In summary, these results show the most abundant A. castellanii CWPs are three sets of lectins, which localize to either the ectocyst layer (Jonah) or endocyst layer and ostioles (Luke and Leo).

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Acanthamoeba Castellanii Str. Neff

SUBMITTER: Angelo Lopez  

LAB HEAD: John Crawford Samuelson

PROVIDER: PXD011826 | Pride | 2019-05-21

REPOSITORIES: Pride