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.Phosphatidylinositol is a crucial phospholipid in eukaryotic cells. It is a structural membrane lipid, and phosphorylation of the hydroxyl groups of its inositol head group by specific lipid kinases leads to the production of seven different phosphoinositide species, which have been found to be enriched in distinct cellular compartments. They play key roles in a multitude of cellular processes, such as membrane traffic, cell motility, cytoskeletal reorganization, DNA synthesis, the cell cycle, adhesion, and signal transduction (9). Approximately 1% of total lipids in mammalian cells are phosphoinositides, mainly phosphatidylinositol 4-monophosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] (45). Derivatives phosphorylated at the 3 position are considerably less abundant in mammalian cells. Phosphatidylinositol 3-monophosphate (PI3P) is a ubiquitous lipid in eukaryotic cells and is present in small amounts in mammalian cells (classically Ͻ15% of PI4P), while PI3P is as abundant as PI4P in the yeast Saccharomyces cerevisiae (2). It has been suggested that one of the functions of these lipids is to establish membrane identity (46); PI4P predominates at the Gol...

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

“…The P. berghei proteins showing the highest sequence similarity to human mTOR are the single predicted phosphatidylinositol-3-kinase (PI3K) (PBANKA_111490) and the two predicted phosphatidylinositol-4 kinases (PI4Ks) (PBANKA_110940 and PBANKA_072200), which show conservation primarily in the kinase catalytic domain. Although little is known about the activity of the two predicted Plasmodium PI4Ks, PbPI3K is an essential gene (57), and treatment with the known PI3K inhibitors wortmannin and LY294002 reduced PI3P production by PfPI3K in vitro and inhibited blood stage parasite growth (58). LY294002 does not phenocopy Torin2 inhibition of P. berghei liver stages, however, and the inhibition of P. falciparum replication by wortmannin and LY294002 (58) is much more modest than what we observed with either Torin1 or Torin2.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…A similar phosphatase acting upon PtdIns(4,5)P 2 , called VPA0450, was identified in Vibrio parahaemolyticus (185), and a previously described acid phosphatase of Legionella that inhibits superoxide production of neutrophil cells is also capable of PtdIns(4,5)P 2 hydrolysis (1323). The intracellular parasite, Plasmodium falciparum that causes malaria also produces its own inositol lipid kinases, a class III PI3K ortholog PfPI3K (1533,1596), a PI4K ortholog PFE0485w (826), and a PIP 5-kinase (864) and also increases the levels of several inositol lipid isomers in the infected red blood cells (399,1533). A plasmodium PLC enzyme has also been described (1245), and activation of Ca 2ϩ signaling within the parasite has also been observed (37).…”

Isoprenoid Biosynthesis Inhibition Disrupts Rab5 Localization and Food Vacuolar Integrity in Plasmodium falciparum

“…Fosmidomycin inhibited protein prenylation and particularly reduced prenylation of proteins of around 25 kDa. The malaria parasite expresses at least 11 small GTPases with predicted molecular masses of between 23 and 27 kDa, each of which is predicted to be geranylgeranylated (34,35). These proteins likely comprise the dominant band that labels with [ 3 H]farnesol and are recognized by antifarnesyl antibodies at 25 kDa (16).…”