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.

Evidence of a cellulosic layer in Pandoravirus tegument and the mystery of the genetic support of its biosynthesis

“…These homologies were barely significant with the 10 best hits, identity percentages in amino acids varying between 24.7% and 23.9%, query coverages between 24% and 25%, and e-values ranging between 2e-15 and 3e-15 (Supplementary File 1). For this ORF594, no homology with a cellulose synthase was found by BLAST analyses neither against the nr database, nor against the conserved domain database (CDD) (Marchler-Bauer et al, 2017). Orthologs of this ORF594 were found by BLASTp analyses in all the 9 other pandoraviruses, with e-values varying from 3.41e-11 to 0.006; but none of them harbored the same bcsA domain when searched for by DELTA-BLAST analysis or in the Conserved Domain Database.…”

“…The cellulose is the most common biologic macromolecule, biosynthesized by vegetals, algae, some bacteria, and by some marine animals as Ascidiae (Chen et al, 2016; Helenius et al, 2006; Nakashima et al, 2004). It is also a major component of the cyst of Acanthamoeba, the unique host of pandoraviruses demonstrated to date (Magistrado-Coxen et al, 2019). It is composed of polymers of β (1,4) glucose subunits.…”

“…Phylogenetic reconstruction was performed using the Maximum Likelihood method with the MEGA6 software (Tamura et al, 2013). Conserved domains were also searched for by DELTA-BLAST analyses against the Conserved Domain Database (Marchler-Bauer et al, 2017).…”

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

“…Under their own promoters, GFP-tagged Luke, Jonah, and Leo lectins are not expressed by trophozoites but are strongly expressed in encysting protists (28) (also see below). In contrast, under a constitutive GAPDH promoter, Luke-GFP with two CBM49s and Jonah-GFP with a single CAA domain were each present in vesicles, but not on the surface, of the vast majority of transfected trophozoites (Figs.…”

“…To better understand its structure, we recently identified chitin and three sets of cellulose- and chitin-binding lectins in the cyst wall of A. castellanii Neff, which is the best studied strain of the protist (28). These lectins are present in 12 copies (Luke), 5 copies (Jonah), or 14 copies (Leo) in the cyst wall, which make them by far the most abundant proteins.…”