RESUMO
The cyst wall of the eye pathogen Acanthamoeba castellanii contains cellulose and has ectocyst and endocyst layers connected by conical ostioles. Cyst walls contain families of lectins that localize to the ectocyst layer (Jonah) or the endocyst layer and ostioles (Luke and Leo). How lectins and an abundant laccase bind cellulose and why proteins go to locations in the wall are not known and are the focus of the studies here. Structural predictions identified ß-jelly-roll folds (BJRFs) of Luke and sets of four disulfide knots (4DKs) of Leo, each of which contains linear arrays of aromatic amino acids, also present in carbohydrate-binding modules of bacterial and plant endocellulases. Ala mutations showed that these aromatics are necessary for cellulose binding and proper localization of Luke and Leo in the Acanthamoeba cyst wall. BJRFs of Luke, 4DKs of Leo, a single ß-helical fold (BHF) of Jonah, and a copper oxidase domain of the laccase each bind to glycopolymers in both layers of deproteinated cyst walls. Promoter swaps showed that ectocyst localization does not just correlate with but is caused by early encystation-specific expression, while localization in the endocyst layer and ostioles is caused by later expression. Evolutionary studies showed distinct modes of assembly of duplicated domains in Luke, Leo, and Jonah lectins and suggested Jonah BHFs originated from bacteria, Luke BJRFs share common ancestry with slime molds, while 4DKs of Leo are unique to Acanthamoeba.IMPORTANCEAcanthamoebae is the only human parasite with cellulose in its cyst wall and conical ostioles that connect its inner and outer layers. Cyst walls are important virulence factors because they make Acanthamoebae resistant to surface disinfectants, hand sanitizers, contact lens sterilizers, and antibiotics applied to the eye. The goal here was to understand better how proteins are targeted to specific locations in the cyst wall. To this end, we identified three new proteins in the outer layer of the cyst wall, which may be targets for diagnostic antibodies in corneal scrapings. We used structural predictions and mutated proteins to show linear arrays of aromatic amino acids of two unrelated wall proteins are necessary for binding cellulose and proper wall localization. We showed early expression during encystation causes proteins to localize to the outer layer, while later expression causes proteins to localize to the inner layer and the ostioles.
RESUMO
In this paper, we present the most relevant facts on multidrug resistance (MDR) in the protozoan parasite Entamoeba histolytica. MDR in E. histolytica presents characteristics similar to transformed mammalian cells. E. histolytica drug resistant mutants show cross-resistance to several drugs, and as in mammalian cells the resistance is reverted by verapamil. Six P-glycoprotein-like genes (EhPgp) have been cloned and characterized. Apparently, four of thses genes are transcribed in drug-resistance mutants (EhPgh1, EhPgp2, EhPgp5 and EhPgp6), although only Egpgp1, EhPgp5 and EhPgp6 transcripts were clearly detected. The open reading frame (ORF) of the four completely full length genes is about 1300 amino acids long. EhPgp1, EhPgp2 and EhPgp5 have between 64 and 67 percent of positional identity among them, while EhPgp6 shows 38 to 46 percent positional identity to the other ameba genes. Insterestingly, the phylogenetic tree suggested that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins. Differential gene expression in drug-resistant mutants was detected when specific probes for each EhPgp gene were used. To understand the differential expression of EhPgp genes we initiated the characterization of the upstream flanking regions of EhPgp1 and EhPgp5 genes. Upstream sequences showed between 53 and 66 percent of positional identity to Dictyostelium discoideum promoters