Acanthamoeba castellanii genome reannotation and multi-omic encystation profiling reveals cyst wall proteins and carbohydrate-active enzymes

Acanthamoeba castellanii is both a key evolutionary outgroup to humans and a free-living amoeba that causes eye infections. During infection, trophozoites damage the cornea and convert to drug-resistant cysts. Systematic characterization of encystation pathways has been hindered by poor genome annotation. We used long-read RNA sequencing across an encystation time course to improve annotation accuracy from 52% to 98%. Orthogroup analyses showed one-third of Acanthamoeba genes are absent in humans and so may be drug targets. We identified 20 cyst wall protein families. Multiple carbohydrate-active enzymes and carbohydrate-binding modules also localize to the cyst wall, including homologs of cellulose synthase, chitin synthase, expansin, and loosenin. A plant-like endocellulase fused to a pollen antigen domain localizes to pockets embedded in the cyst wall. We also used antibodies to endogenous cyst wall proteins to observe encystation of Acanthamoeba in a human corneal epithelium tissue model, demonstrating the potential translational impact of these resources.