The most abundant cyst wall proteins of Acanthamoeba castellanii are lectins that bind cellulose and localize to distinct structures in developing and mature cyst walls

Abstract

Background

Acanthamoeba castellanii, which causes keratitis and blindness in under-resourced countries, is an emerging pathogen worldwide, because of its association with contact lens use. The wall makes cysts resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye.

Methodology/Principal findings

Transmission electron microscopy and structured illumination microscopy (SIM) showed purified cyst walls of A. castellanii retained an outer ectocyst layer, an inner endocyst layer, and conical ostioles that connect them. Mass spectrometry showed candidate cyst wall proteins were dominated by three families of lectins (named here Jonah, Luke, and Leo), which bound well to cellulose and less well to chitin. An abundant Jonah lectin, which has one choice-of-anchor A (CAA) domain, was made early during encystation and localized to the ectocyst layer of cyst walls. An abundant Luke lectin, which has two carbohydrate-binding modules (CBM49), outlined small, flat ostioles in a single-layered primordial wall and localized to the endocyst layer and ostioles of mature walls. An abundant Leo lectin, which has two unique domains with eight Cys residues each (8-Cys), localized to the endocyst layer and ostioles. The Jonah lectin and glycopolymers, to which it binds, were accessible in the ectocyst layer. In contrast, Luke and Leo lectins and the glycopolymers, to which they bind, were mostly inaccessible in the endocyst layer and ostioles.

Conclusions/Significance

The most abundant A. castellanii cyst wall proteins are three sets of lectins, which have carbohydrate-binding modules that are conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique to Acanthamoebae (8-Cys of Leo). Cyst wall formation is a tightly choreographed event, in which lectins and glycopolymers combine to form a mature wall with a protected endocyst layer. Because of its accessibility in the ectocyst layer, an abundant Jonah lectin is an excellent diagnostic target.

Author summary

A half century ago, investigators identified cellulose in the Acanthamoeba cyst wall, which has two layers and conical ostioles that connect them. Here we showed cyst walls contain three large sets of cellulose-binding lectins, which localize to the ectocyst layer (a Jonah lectin) or to the endocyst layer and ostioles (Luke and Leo lectins). We used the lectins to establish a sequence for cyst wall assembly when trophozoites are starved and encyst. In the first stage, a Jonah lectin and glycopolymers were present in dozens of distinct vesicles. In the second stage, a primordial wall contained small, flat ostioles outlined by a Luke lectin. In the third stage, a Jonah lectin remained in the ectocyst layer, while Luke and Leo lectins moved to the endocyst layer and ostioles. A description of the major events during cyst wall development is a starting point for mechanistic studies of its assembly.