Essential Roles for GPI-anchored Proteins in African Trypanosomes Revealed Using Mutants Deficient in GPI8

Abstract

The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion of GPI8 (to give ⌬gpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non-protein-linked GPI molecules, some of which are surface located. Procyclic ⌬gpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression. INTRODUCTIONTrypanosoma brucei is the heteroxenous, hemoflagellate protozoan parasite responsible for Sleeping Sickness in humans and Nagana in domestic animals in the tsetse belt of subSaharan Africa. All life cycle stages of the parasite utilize glycosylphosphatidylinositol (GPI) anchors as the predominant method for attaching proteins to their plasma membrane. In the mammalian host, these proteins include one subunit of the heterodimeric transferrin receptor (Schell et al., 1991), an alanine-rich protein of unknown function (Nolan et al., 2000), and the variant surface glycoprotein (VSG) (Ferguson et al., 1988) essential for evasion of the host's immune system.The bloodstream forms of T. brucei differentiate into procyclic forms once ingested by the tsetse fly vector. This differentiation involves remodeling of the surface by shedding the VSG coat and replacing it with an invariant coat of GPI-anchored proteins known as procyclins (Roditi et al., 1989). There are four types of procyclin, three bearing between 18 and 30 internal -Glu-Pro-repeats (EP1, EP2, and EP3; see Acosta-Serrano et al., 2000 for alignment), and one with a -Gly-Pro-Glu-Glu-Thr-(GPEET) repeat region . Although EP1 and EP3 both undergo N-glycosylation, EP2 and GPEET do not, although the latter is phosphorylated on the threonine residues of the repeat region (Butikofer et al., 1999). All procyclin isoforms are anchored by a GPI modified with a heterogeneous poly-Nacetyllactosamine side chain (Treumann et al., 1997), widely believed to form a glycocalyx over the cell surface. Displayed above this, both EP and GPEET procyclins are thought to adopt a rod-like conformation (Roditi et al., 1989;Treumann et al., 1997). Although the N-termini of the...

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

“…In procyclic form T. brucei, these large poly-N-acetyl-lactosamine glycans are absent, and the N-linked oligosaccharide repertoire is limited to oligomannose (Man 9-5 GlcNAc 2 ) structures (6, 47, 51, 52). Thus, in this life cycle stage, Gal appears to be largely restricted to the poly-N-acetyl-lactosamine-containing side chains of the procyclin GPI anchors (6,47) and of the free GIPLs (7)(8)(9). An obvious hypothesis is that these GPI side chain poly-N-acetyl-lactosamine structures play an important (possibly anti-adhesive) protective role on the cell surface.…”

“…Following ingestion in a blood meal, the stumpy trypomastigote form differentiates into the dividing procyclic form that colonizes the tsetse midgut. The procyclic trypanosomes express a radically different cell surface coat made up of about 3 ϫ 10 6 procyclin glycoproteins (3)(4)(5)(6) and a smaller number of free glycoinositol phospholipids (GIPLs) 1 (7)(8)(9). The procyclins are polyanionic, rod-like (6, 10) proteins encoded by four procyclin genes: GPEET, which encodes a protein with five or six Gly-Pro-Glu-Glu-Thr repeats, and EP1, EP2, and EP3, which encode proteins with 18 -30 Glu-Pro repeats (11).…”

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

“…Among three common components, a structural difference is seen in the catalytic component GPI8. Human GPI8 is a type I membrane protein having a transmembrane domain near the C terminus (13), whereas TbGPI8 lacks a transmembrane domain (3,19). The transmembrane domain of human GPI8 is not essential for the enzyme activity (13).…”

“…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).…”

“…TbGPI8, T. brucei homologue of the GPI8 gene, has been cloned (19) and its essential role in GPI anchoring has been elucidated by gene disruption (3). We speculated that trypanosome GPI transamidase might be significantly different from its human counterpart for three reasons.…”