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-B/GPI10 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.

Figure 1

TbGPI10 is a functional homologue of PIG-B/GPI10. (A) Restoration of the surface expression of GPI-anchored protein Thy-1 on mouse PIG-B-deficient S1A-b cells with TbGPI10. S1A-b cells transiently transfected with an empty vector; human PIG-B and TbGPI10 plasmids were stained for Thy-1 and analyzed in a flow cytometer. (B) Restoration of biosynthesis of the mature GPI-anchor precursors with TbGPI10 in S1A-b cells. Wild-type S1A cells (lane 1), PIG-B-deficient S1A-b cells (lane 2), and stable transfectants of S1A-b with TbGPI10 (lane 3) or human PIG-B (lane 4) were labeled with d-[³H]mannose in the presence of tunicamycin. Radiolabeled lipids were analyzed by TLC. DPM, dolichol phosphate mannose; M2 and M3/H6, intermediates with two and three mannoses, respectively; H7 and C/H8, complete GPI anchors. (C) Rescue of lethality of GPI10 knockout S. cerevisiae with TbGPI10. (Upper) Yeast multicopy vector (p425) carrying S. cerevisiae GPI10 (pScGPI10), TbGPI10 (pTbGPI10), and human PIG-B (pHsPIG-B) was introduced into wild-type (GPI10) and GPI10 knockout (gpi10) S. cerevisiae. The transformants were inoculated on plates as indicated. In the presence of geneticin, the rescued gpi10 strains, but not the GPI10 strains, can grow, because disruption of GPI10 was by replacement with kanamycin resistance gene. (Lower) Single colonies of GPI10 and gpi10 S. cerevisiae bearing pTbGPI10 were inoculated in 50 ml of SD medium in the presence of leucine (the selection marker for pTbGPI10), cultured to stationary phase, and then plated onto SD plates in the presence of leucine. The colonies were then replica plated onto SD plates with or without leucine. More than 50% of wild-type cells lost pTbGPI10 (no growth without leucine), whereas none of GPI10 knockout cells were able to lose the plasmid, confirming that GPI10 is essential for growth of S. cerevisiae. Colonies that grew on the nonselective plates but not on the selective plates were circled.

Figure 2

Essentiality of TbGPI10 for bloodstream form of T. brucei. (A) Knockout strategy. A restriction map of TbGPI10 and its flanking regions and two targeting constructs in which TbGPI10 was replaced with HYG or NEO are shown. A probe for Southern blotting (Sal–Sph probe) and predicted fragments detected with this probe are indicated above the restriction map. (B) Southern blot analysis of drug-resistant clones. Expected positions of wild-type and homologous recombinant fragments are indicated on the left, and size markers are on the right. WT, wild-type; kbp, kilobase pair. (C) Stability of episomal plasmids in the presence or absence of chromosomal TbGPI10. TbGPI10 double knockout cells bearing episomal TbGPI10 plasmids with BLE were cultured in the absence of drug selection. As a positive control for a loss of episomal plasmids without drug selection, a neomycin-resistant, heterozygous TbGPI10 knockout procyclic clone was transfected with an episomal plasmid bearing BLE. At passages, DNA was prepared, digested with BamHI, and analyzed by Southern blot hybridization with BLE probe to detect episomal DNA and Sal–Sph probe to detect chromosomal TbGPI10. Size markers are on the right. Bands shown in each panel had the same mobility, although they are not well aligned in the figure because of uneven running.

Figure 3

Disruption of TbGPI10 in the procyclic form of T. brucei. (A) Southern blot analysis of homologous recombination. Samples of SalI and XhoI cut DNA of wild-type strain 427 (WT, lane 1), single TbGPI10 knockout clone (H-R, lane 2), and five double knockout clones derived from H-R (HN-R, lanes 3–7) were hybridized with a Sal–Sph probe. Expected positions of wild-type and homologous recombinant fragments are indicated on the left, and size markers are on the right. (B) Microscopic observation of the TbGPI10 knockout procyclics in culture. Wild-type and the knockout mutant procyclics were inoculated into flasks treated for nonadherent culture (Sumilon, Tokyo, MS-2005R; Upper; nonadherent) and regular nontreated flasks for adherent culture (Iwaki, Chiba, Japan, 3100-025; Lower; adherent) at a concentration of 10⁵ per ml. On days 3 and 6 of culture at 27°C, procyclics were observed under a phase contrast microscope.

Figure 4

Defective GPI biosynthesis, GPI anchoring, and surface expression of procyclins in TbGPI10 knockout procyclics. (A) GPI biosynthesis. Wild-type (WT) and doubly disrupted mutant (−/−), which were transformed with an empty vector (Mock) or TbGPI10 plasmid (TbGPI10), were used. The membranes were incubated with GDP-[³H]mannose to label GPI, and aliquots were subjected to TLC directly (−) or after digestion with α-mannosidase (M) or GPI-PLD (D). Identities of mannolipids are shown on the left of chromatograms. Designations of mannolipids from TbGPI10-disruptant are tentative. M1 and M2, intermediates containing one and two mannoses; M2(acyl) and M2(lyso), M2 species with acylation on inositol and with a lack of sn-2 fatty acid; M3(acyl), an intermediate bearing three mannoses with acylation on inositol; A′-like, an intermediate bearing three mannoses with ethanolamine phosphate on the third mannose; PP3, A′-like intermediate with acylation on inositol; PP1, complete GPI precursor (a lyso form of PP3). The spots that appeared after GPI-PLD-treatments (lanes 3, 6, and 9) are inositol-acylated GPI glycans. (B) Incorporation of myristic acid into procyclins. Lane 1, wild-type; lane 2, single TbGPI10-disruptant; lane 3, double TbGPI10-disruptant; lane 4, double TbGPI10-disruptant bearing an empty plasmid; lane 5, double TbGPI10-disruptant bearing TbGPI10 plasmid. Size markers are on the right. (C) Surface expression of EP procyclins. Single and double TbGPI10-disruptant clones were stained with anti-EP procyclins (shaded lines) or control (dotted line) monoclonal antibodies and analyzed in a FACScan. (D) Pulse-chase analysis of EP procyclins. Double TbGPI10-disrupted mutant bearing TbGPI10 (Left) or empty (Right) plasmid was pulse-labeled with [¹⁴C]proline for 30 min and chased for indicated time periods. At each time point, aliquots of samples were separated into supernatants and cell pellets, solubilized by detergent, and immunoprecipitated with anti-EP procyclins antibody. Immunoprecipitates were analyzed by SDS/PAGE and autoradiography.

Figure 5

Effect of defective GPI anchor biosynthesis on the infection of procyclic T. brucei in tsetse fly midgut. Flies were infected with procyclic forms, thereafter fed three times per week in vitro with defibrinated horse blood, and dissected on days 14 and 24. Midguts were scored for degrees of infection as heavy (100–300 trypanosomes per field in 10 fields with the ×20 objective, black section), intermediate (between “heavy” and “weak”, dark gray section), weak (less than three trypanosomes, light gray section), and negative (no trypanosome detectable, white section).