title: Carbohydrate analysis of Acanthamoeba castellanii.
abstract: We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting "cyst wall" material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.
authors.affiliation: School of Biological and Chemical Sciences, Birkbeck College, University of London, London, UK.
authors.forename: Ricky
authors.initials: R
authors.lastname: Dudley
authors.forename: Edward L
authors.initials: EL
authors.lastname: Jarroll
authors.forename: Naveed Ahmed
authors.initials: NA
authors.lastname: Khan
mesh_terms: D015823:Acanthamoeba Keratitis / Q000469:parasitology; D048248:Acanthamoeba castellanii / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D046529:Microscopy, Electron, Transmission; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D063065:Organophosphonates / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: C436083:2-(2-oxo-2-((3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy)aminoethyl)phosphonic acid, (2,2-dimethyl-1-oxopropoxy)methyl ester sodium; D000577:Amides; D002241:Carbohydrates; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D063065:Organophosphonates; D011725:Pyridines; C108830:Y 27632; D015638:Cytochalasin D; D014638:Vanadates; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D054460:rho-Associated Kinases; D017027:Protein Tyrosine Phosphatases