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.

Identifying how small molecules act to kill malaria parasites can lead to new “chemically validated” targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

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...