Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei

Abstract

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B͞GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.T rypanosoma brucei is a protozoan parasite invading humans and other mammals by transmission via tsetse flies. It causes sleeping sickness in humans and nagana disease in domestic animals living in the ''tsetse belt'' in central Africa. These are serious medical and agricultural problems for which safe and effective therapeutic and protective measures are highly desirable (1, 2).T. brucei has two distinct proliferative stages, a bloodstream stage living free in mammalian blood and an insect stage (or procyclic form) living in the midguts of tsetse flies. The cell surface of both stages of this unicellular parasite is covered by a large amount of glycosylphosphatidylinositol (GPI)-anchored proteins (3, 4): 10 7 variant surface glycoproteins per cell for the bloodstream form of the parasite and 3 ϫ 10 6 to 6 ϫ 10 6 procyclins (or procyclic acidic repetitive proteins) per cell of the procyclic form of the parasite (4-6), corresponding to 10% and 1-3%, respectively, of total proteins in these parasite stages (7,8). T. brucei evades the host's immune response by expressing structurally different forms of variant surface glycoproteins (4). Procyclins are thought to protect procyclic cells from digestion by the digestive enzymes in the fly (4, 6). In addition, T. brucei expresses a number of other GPI-anchored proteins, such as transferrin receptors in the bloodstream form (3, 4). Thus, the importance of GPI anchors for the survival and infection of T. brucei has been suggested, leading to the notion that the GPI biosynthesis pathway may be a good target for chemo...

Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

“…The transposon donor cassette has been cloned into pT11-bs, a version of the pT13-11 plasmid that can be propagated in the bloodstream stage, to create pSgl35. Although the original description of these plasmids noted that pT13-11 propagated poorly in bloodstream T. brucei, in contrast to pT13-11 in procyclic forms (26), other investigators did not find this to be a problem (11), although an initial lag in growth of G418-resistant plasmid-containing cells has been observed by us and by others.…”

GPI transamidase of Trypanosoma brucei has two previously uncharacterized (trypanosomatid transamidase 1 and 2) and three common subunits

“…Culture of the trypanosome, gene disruption, complementation with episomal plasmids, Southern blotting, flow-cytometric analysis, and GPI biosynthesis analysis were carried out as described (2). Drug-resistant clones were selected with 50 g͞ml geneticin (Invitrogen) or 10 g͞ml blasticidin S (Funakoshi, Tokyo).…”

“…The cell surfaces of these forms are covered by a large amount of glycosylphosphatidylinositol (GPI)-anchored proteins, variant surface glycoproteins in the bloodstream form and procyclins in the procyclic form, corresponding to 10% and 1-3%, respectively, of total cellular proteins (1). The GPI biosynthesis pathway is a candidate target for development of chemotherapeutic agents because GPI anchoring is essential for the life of the bloodstream form (2,3).…”

Probing Enzymes Late in the Trypanosomal Glycosylphosphatidylinositol Biosynthetic Pathway with Synthetic Glycosylphosphatidylinositol Analogues

“…served between eukaryotes, consists of NH 2 CH 2 CH 2 -PO 4 H-6Man␣1-2Man␣1-6Man␣1-4GlcN␣1-6-D-myoinositol-1-HPO 4 -lipid, where the lipid can be diacylglycerol, lyso-acylglycerol, alkylacylglycerol, or ceramide, and is often further decorated with additional ethanolamine phosphate and/or carbohydrate groups and/or fatty acid attached to the inositol residue (inositol acylation) in a species-and tissue-specific manner. Biosynthesis of GPI in T. brucei ( Figure 1, panel a), which occurs in the endoplasmic reticulum, is initiated by the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI) to generate N-acetyl-glucosaminylphosphatidylinositol (1, GlcNAc-PI), which is de-N-acetylated to produce glucosaminylphosphatidylinositol (2, GlcN-PI) (17).…”

“…De-N-acetylation is a prerequisite for the subsequent mannosylation of GlcN-PI (18), which requires the sequential action of three distinct mannosyltransferases (MTI, MTII, and MTIII) to form trimannosyl-glucosaminylphosphatidylinositol (Man 3 GlcN-PI). MTIII, a Dol-P-Man: Man 2 GlcN-PI ␣(1-2) mannosyltransferase, has been shown genetically to be essential in bloodstream form T. brucei (4). From GlcN-PI onward there are significant differences in the GPI biosynthetic pathways of T. brucei and mammalian cells.…”