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.

Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.

18Acanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because 19 of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are 20 resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. We used 21 transmission electron microscopy, as well as structured illumination microscopy and probes for 22 cellulose and chitin, to show that purified cyst walls of A. castellanii retain an outer ectocyst 23 layer, an inner endocyst layer, and conical ostioles that connect the layers. Mass spectrometry 24 showed candidate cyst wall proteins are dominated by three families of lectins (named here 25 Luke, Leo, and Jonah), each of which binds to microcrystalline cellulose and to a lesser degree 26 chitin. A Jonah lectin, which has one choice-of-anchor A (CAA) domain, localizes to the 27 ectocyst layer of mature cyst walls. Luke lectins, which have two or three carbohydrate-binding 28 modules (CBM49), localize to the endocyst layer and ostioles. A Leo lectin, which has two 29 domains with eight Cys residues each (8-Cys), also localizes to the endocyst layer and 30 ostioles. In summary, the most abundant A. castellanii cyst wall proteins are three sets of 31 lectins, which have carbohydrate-binding modules that are conserved (CBM49s of Luke), 32 newly characterized (CAA of Jonah), or unique to Acanthamoebae (8-Cys of Leo). Despite 33 their lack of common ancestry, Luke and Leo lectins both localize to the endocyst layer and 34 ostioles, while the Jonah lectin localizes to the ectocyst layer. 35IMPORTANCE 36 Fifty years ago, investigators identified cellulose in the Acanthamoeba cyst wall, which has an 37 outer ectocyst layer, an inner endocyst layer, and conical ostioles that connect the layers. 38Here we show cyst walls also contain chitin and three large sets of cellulose-and chitin-binding 39 lectins, which have distinct localizations. The Acanthamoeba cyst wall, therefore, is more 40 complicated than cyst walls of Entamoeba and Giardia (causes of dysentery and diarrhea, 41 respectively), which have a single layer, a single glycopolymer, and small sets of one or two 42 lectins. In contrast, the Acanthamoeba cyst wall is far simpler than the walls of fungi and 43 plants, which have multiple layers, numerous glycopolymers, and hundreds of proteins. In 44 addition to providing a better understanding of the cell biology and biochemistry of the 45 Acanthamoeba cyst wall, these studies may lead to diagnostic antibodies that bind to cyst wall 46 proteins and/or therapeutics that target chitin. 47 100 file S1) (41). The GST-AcCBM49 expression construct was designed to replicate that used to 101 determine the carbohydrate binding properties of SlCBM49, which is a C-terminal 102 carbohydrate-binding module of the Solanum lycopersicum (tomato) cellulase SlGH9C (42). In 103 both intact cysts and purified cyst walls, GST-AcCBM49 predominantly labeled the ectocyst 104 layer, WGA highlighted the ostioles, and CFW stained the endocyst...

9When deprived of nutrients, trophozoites of the eye pathogen Acanthamoeba castellanii make 10 a cyst wall, which contains cellulose and has two layers connected by cone-shaped ostioles. 11 We recently showed chitin is also present and identified three sets of lectins, which localize to 12 the ectocyst layer (Jonah lectin) or the endocyst layer and ostioles (Luke and Leo lectins). To 13 Acanthamoeba was used to discover and characterize roles of actin and associated proteins in 54 the cytoskeleton, phagocytosis, and cell motility (16). Whole genome sequences of A. 55 castellanii identified >500 genes derived from bacteria by HGT, which is the greatest number 56 of any eukaryote described to date (17). A mannose-binding protein on the surface of 57 trophozoites, secreted proteases, and pore-forming peptides each appear to contribute to 58 contact-mediated cytolysis of corneal epithelial cells by Acanthamoebae (18)(19)(20). 59 The Acanthamoeba cyst wall, which forms when trophozoites are starved, is an 60 important virulence factor, because it makes cysts resistant to surface disinfectants, sterilizing 61 agents in contact lens solutions, and antibiotics applied to the eye (21, 22). Fifty years ago, 62 the cyst wall was shown to contain cellulose and have two microfibril-dense layers (outer 63 ectocyst and an inner endocyst), as well as conical structures (ostioles) that connect the layers 64 (23, 24). During excystation, the trophozoites escape through one of the ostioles, which were 65 not counted (25). Although the electron micrographs were beautiful, none of the cyst wall 66 proteins were identified, and there was minimal information as to how the two layers and 67 ostioles are formed. Further, monoclonal antibodies, potentially useful as diagnostic reagents, 68 were made to trophozoites but not to cysts (26, 27). 69To better understand its structure, we recently identified chitin and three sets of 70 cellulose-and chitin-binding lectins in the cyst wall of A. castellanii Neff, which is the best 71 studied strain of the protist (28). These lectins are present in 12 copies (Luke), 5 copies 72 (Jonah), or 14 copies (Leo) in the cyst wall, which make them by far the most abundant 73 proteins. These lectins contain carbohydrate-binding modules that were shown to bind 74 cellulose in Dictyostelium and plants (CBM49s of Luke lectins), were previously identified in 75 bacteria but were uncharacterized (choice of anchor A (CAA) domains of Jonah lectins), or are 76 unique to Acanthamoebae (8-Cys domains of Leo lectins) (Fig. 1) (29-31). A representative 77 Jonah lectin is present in the ectocyst layer, while representative Luke and Leo lectins are 78 present in the endocyst layer and ostioles. 79 Here we used structured illumination microscopy (SIM), probes for glycopolymers, and 80 green fluorescent protein (GFP) tags for cyst wall lectins to determine the sequence of events 81 (stages) involved in the formation of the A. castellanii cyst wall during encystation (32). To 82 characterize their carbohydrate-bi...