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

When deprived of nutrients, trophozoites of the eye pathogen Acanthamoeba castellanii make a cyst wall, which contains cellulose and has two layers connected by cone-shaped ostioles. We recently showed chitin is also present and identified three sets of lectins, which localize to the ectocyst layer (Jonah lectin) or the endocyst layer and ostioles (Luke and Leo lectins). To determine how the cyst wall is made, we examined encysting protists using structured illumination microscopy, probes for glycopolymers, and tags for lectins. In the first stage (3 to 9 hr), cellulose, chitin, and a Jonah lectin were each made in dozens of encystation-specific vesicles. In the second stage (12 to 18 hr), a primordial wall contained both glycopolymers and Jonah lectin, while small, flat ostioles were outlined by a Luke lectin. In the third stage (24 to 36 hr), an ectocyst layer enriched in Jonah lectin was connected to an endocyst layer enriched in Luke and Leo lectins by large, conical ostioles. Jonah and Luke lectins localized to the same places in mature cyst walls (72 hr) independent of the timing of expression. The Jonah lectin and the glycopolymer bound by the lectin were accessible in the ectocyst layer of mature walls. In contrast, Luke and Leo lectins and glycopolymers bound by the lectins were mostly inaccessible in the endocyst layer and ostioles. These results show that cyst wall formation is a tightly choreographed event, in which glycopolymers and lectins combine to form a mature wall with a protected endocyst layer.