Glycosylation of the pathogen Acanthamoeba castellaniee

Cell surface glycoconjugates and their ligands play important roles in the interaction between the different cells of an organism as well as between symbionts/pathogens/parasites and their hosts. The protozoans, single-cellular eukaryotes, including amoebae and trypanosomatids express a range of novel glycosylated macromolecules such as lipophosphoglycans (LPG) and glycosylinositolphospholipids (GIPLs) as well as unique O-glycans. Acanthamoeba species are common free-living amoeba found in soil and water; however, they can also prove to be serious pathogens causing amoebic keratitis, particularly in contact lens wearers, and granulomatous amoebic encephalitis in immunocomprised patients. In the former case, a mannose-binding protein is involved in binding to the cornea; the end result of the infection can be the requirement for a corneal transplant. The encephalitis, though, in almost all cases proves lethal. The genome of an Acanthamoeba strain (Neff strain of A. castellanii, genotype T4) is in the process of being sequenced; however, little is known about its glycome. Over thirty years ago, a lipophosphonoglycan was described in terms of its fatty acid, inositol, phosphoethanolamine and monosaccharide content, although no full structure was determined; in retrospect, this glycoconjugate may well display similarities to lipophosphoglycans of trypanosomatids and of Entamoeba histolytica. Thus, a major goal of the proposed work is to characterise this lipophosphonoglycan using modern analytical techniques such as MALDI-TOF, ESI and GC mass spectrometry. Similar methods will be used to examine the N-glycans of this species to explore similarities and differences with other unicellular organisms such as trypanosomatids and the non-pathogenic amoeba Dictyostelium discoideum. The outcome will be a fuller understanding of the glycome of Acanthamoeba species; if glycomic differences between strains are observed, a new method for distinguishing Acanthamoeba isolates may result. Furthermore, glycobiosynthetic studies will also be facilitated, which may culminate in the identification of novel chemotherapeutic targets.

Cell surface glycoconjugates and their ligands play important roles in the interaction between the different cells of an organism as well as between symbionts/pathogens/parasites and their hosts. The protozoans, single-cellular eukaryotes, including amoebae and trypanosomatids express a range of novel glycosylated macromolecules such as lipophosphoglycans (LPG) and glycosylinositolphospholipids (GIPLs) as well as unique O-glycans. Acanthamoeba species are common free-living amoeba found in soil and water; however, they can also prove to be serious pathogens causing amoebic keratitis, particularly in contact lens wearers, and granulomatous amoebic encephalitis in immunocomprised patients. In the former case, a mannose-binding protein is involved in binding to the cornea; the end result of the infection can be the requirement for a corneal transplant. The encephalitis, though, in almost all cases proves lethal. At the beginning of this project, little was known about the glycome of these organisms. Over thirty years ago, a lipophosphonoglycan was described in terms of its fatty acid, inositol, phosphoethanolamine and monosaccharide content, although no full structure was determined; we obtained some further compositional data on this macromolecule. A major focus was on the N-glycans (i.e., carbohydrate chains N-linked to asparagine residues of proteins) of different Acanthamoeba strains in order to explore similarities and differences with other unicellular organisms such as trypanosomatids and the non-pathogenic amoeba Dictyostelium discoideum. Indeed, the N-glycans of this species are highly unusual and unique; glycomic differences between strains were observed. Furthermore, the N-glycans of another protozoan, the widespread sexually-transmitted parasite Trichomonas vaginalis, were also analysed and found to also present unusual strain- specific features.