Phosphatidylinositol 3-phosphate, an essential lipid in Plasmodium, localizes to the food vacuole membrane and the apicoplast

Abstract

Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasite Plasmodium falciparum establishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile of P. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. The Plasmodium genome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted in Plasmodium berghei, and in vitro growth of P. falciparum was sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential in Plasmodium blood stages. For intraparasitic PI3P localization, transgenic P. falciparum that expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function in Plasmodium appear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.

Fig. 1.

Analysis of phospholipids by thin-layer chromatography (TLC) and HPLC. Equal numbers of highly enriched P. falciparum-infected red blood cells (iRBC) and uninfected red blood cells (RBC) were labeled with H₃³²PO₄ for 1 h at 37°C, and phospholipids were extracted. (A) Separation by TLC. PhosphorImager acquisition data are shown. The positions of the major radiolabeled phospholipids phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), PI-monophosphates (PIP), and PI-bisphosphates (PIP₂), identified with respect to the retention factor value of unlabeled authentic standards, are indicated. (B to E) The zones corresponding to the PI-monophosphates and to the PI-bis/trisphosphates were recovered from the silica plate, and the lipids were deacylated and analyzed by HPLC. Shown are the radioactivity profiles of phosphatidylinositol monophosphates (B and D) and phosphatidylinositol bis- and trisphosphates (C and E) in uninfected red blood cells (B and C) and infected red blood cells (D and E) over time. (E) (Inset) Enlargement and overlay of the zone corresponding to PI(3,4,5)P₃ with the profile of infected RBC in gray and that of uninfected RBC in black.

Fig. 2.

Effect of wortmannin treatment on phosphoinositide synthesis in infected erythrocytes. Highly enriched infected red blood cell cultures were pretreated for 30 min with 100 nM wortmannin and were subsequently labeled for 1 h with H₃³²PO₄ in the presence of 100 nM wortmannin. The incorporation of radioactivity into individual phosphoinositides is expressed as a percentage of that for control cells incubated without wortmannin. Error bars correspond to SEMs (n = 5). P values were determined by unpaired two-tailed t tests and are indicated. The variations in the PI(3,4)P₂ and PI(3,4,5)P₃ levels were not statistically significant with respect to the values for PI4P and PI(4,5)P₂.

Fig. 3.

Generation of GFP-2×FYVE expressing P. falciparum parasites. (A) Schematic representation of the recombination event leading to the integration of GFP-2×FYVE into P. falciparum strain 3D7attB. The two plasmids used for cotransfection are represented at the top: plasmid pINT, carrying the integrase expression unit (int) and a neomycin resistance cassette (neo), and plasmid pLN-GFP-2×FYVE, carrying the GFP-2×FYVE fusion gene under the control of the calmodulin promoter and a blasticidin resistance cassette (bsd) flanking the attP site. The attB site is present in the genomic cg6 locus in strain 3D7attB (37). Recombination mediated by the integrase between the attP and attB sites yielded strain 3D7attB-GFP-2×FYVE. The primers used in PCR analysis of transgenic parasites are indicated by arrows. Large arrowheads indicate the transcriptional directions of genes and expression cassettes. The diagram is not drawn to scale. (B) PCR analysis of genomic DNA of the transgenic parasite population was performed to confirm the correct integration of GFP-2×FYVE into the P. falciparum genome. The relative positions of the primers used are indicated in panel A. Lanes 1 to 3 demonstrate the integration of pLN-GFP-2×FYVE at the attB locus (lane 1, primers 202/211; lane 2, primers 202/251; lane 3, primers 202/242. Lane 4 shows the presence of the blasticidin resistance gene (primers 207/211). Expected fragment sizes were 1,800 bp, 4,260 bp, 4,880 bp and 346 bp for lanes 1 to 4, respectively. M, molecular weight marker, with fragment sizes indicated on the left.

Fig. 4.

GFP fluorescence in parasites expressing GFP-2×FYVE or GFP-2×FYVE^C215S. (A) Cells were fixed and analyzed for GFP fluorescence. Nuclei were stained with Hoechst stain (blue stain in the merged images). (B) Colocalization of GFP-2×FYVE and the food vacuole membrane marker CRT using rabbit anti-CRT. Serial images of a Z-stack acquisition (0.38 μm step) are displayed. Arrowheads indicate protrusions from the food vacuole membrane that are colabeled with CRT and GFP-2×FYVE, while arrows indicate zones of intense GFP-2×FYVE staining in the absence of the food vacuole marker. A corresponding phase-contrast image is shown in the lower left corner.

Fig. 5.

Colocalization of GFP-2×FYVE with the apicoplast. GFP-2×FYVE expressing parasites at the indicated developmental stages were incubated with rabbit anti-ACP as an apicoplast marker. Nuclei were stained with Hoechst stain (Hoe).

Fig. 6.

Localization of GFP-2×FYVE by cryoimmunoelectron microscopy. (A) Section through a young schizont showing most of the gold label on or near the food vacuole membrane (arrows). Arrowheads point to what should be considered the background. Hz, hemozoin crystals; N, parasite nucleus; RBC, erythrocyte cytosol. (B and C) The membranes of electron-lucent (B) (arrow) and electron-dense (C) (asterisks) vesicles neighboring the food vacuole (Hz) are strongly labeled. Cytostomal vesicles (Cy) are not labeled. (D to E) General (D) and close-up (D′ and E) views of young schizonts showing the strong labeling of the apicoplast, mostly within the lumen. The four membranes of the apicoplast are clearly identified (E, arrow). A, apicoplast; P, parasite.

Fig. 7.

Schematic representation of the phosphoinositide metabolism in Plasmodium. Phosphoinositides and metabolites are represented by ovals. The corresponding enzymes together with their EC nomenclature are shown in unbordered shaded boxes, and the predicted or confirmed P. falciparum genes are shown in shaded boxes with dark borders. The putative synthesis of PI(3,4)P₂ and PI(3,4,5)P₃ through PI3-kinase or PI4P-5-kinase is indicated by dotted arrows. DAG, diacylglycerol; I(1,4,5)P₃ (IP₃), inositol trisphosphate; PIS, PI-synthase; PI-PLC, PI-specific phospholipase C.