Insights into the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in Leishmania parasites and characterization of a choline kinase from Leishmania infantum.

The protozoan parasite Leishmania infantum is a causative agent of the disease visceral leishmaniasis, which can be fatal if not properly treated. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis pathways are attractive targets for new antileishmanial compounds since these Leishmania cell membrane phospholipids are important for parasite morphology and physiology. In this work we observed Leishmania synthesize PC and PE from extracellular choline and ethanolamine, respectively, suggesting the presence of CDP-choline and CDP-ethanolamine pathways. In addition, Leishmania converted PE to PC, indicating the parasite possesses phosphatidylethanolamine N-methyltransferase (PEMT) activity. The first step in the biosynthesis of PC or PE requires the phosphorylation of choline or ethanolamine by a kinase. We cloned the gene encoding a putative choline/ethanolamine kinase from Leishmania infantum and expressed and purified the encoded recombinant protein. The enzyme possesses choline kinase activity with a Vmax of 3.52µmol/min/mg and an apparent Km value of 0.089mM with respect to choline. The enzyme can also phosphorylate ethanolamine in vitro, but the apparent Km for ethanolamine is 850-fold greater than for choline. In an effort to probe requirements for small molecule inhibition of Leishmania choline kinase, the recombinant enzyme was evaluated for the ability to be inhibited by novel quaternary ammonium salts. The most effective inhibitor was N-iodomethyl-N,N,-dimethyl-N-(6,6-diphenyl hex-5-en-1-yle) ammonium iodide, denoted compound C6. In the presence of 4mM compound C6, the Vmax/Km decreased to approximately 1% of the wild-type catalytic efficiency. In addition, in Leishmania cells treated with compound C6 choline transport was inhibited.

Phosphatidylethanolamine synthesis is required for optimal virulence of Brucella abortus.

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.

Fatty acid incorporation by Rhodnius prolixus midgut.

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.

Inhibition of phosphatidylcholine biosynthesis and cell proliferation in Trypanosoma cruzi by ajoene, an antiplatelet compound isolated from garlic.

Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 microM ajoene, while 100 microM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 microM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 microM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.

Lipid metabolism in electroplax.

The in vivo labeling of electrocyte lipids is followed after injection of radioactive glycerol and two fatty acids, oleate and arachidonate, into the electric organ of an elasmobranch (Discopyge tschudii). De novo synthesis of lipids and acyl-exchange reactions are operative in the electrocyte. The three precursors are preferentially incorporated into phosphatidylcholine, phosphatidylinositol, and triacylglycerols. The highest specific activities are attained by triacylglycerols and polyphosphoinositides. Electrocyte stacks from electric organ show an efficient and continuous esterification of oleate and arachidonate into lipids after several hours of incubation. Except for an apparently more active labeling of triacylglycerols, which is attributed to the larger availability of free fatty acid precursors under the in vitro experimental conditions, the pattern of lipid labeling is similar to that attained in vivo. 32P-labeled lipids are also steadily produced in electrocyte stacks (24 h of incubation with [32P]phosphate) using glucose as the sole exogenous source of energy. Polyphosphoinositides are the lipids preferentially labeled. The ability to sustain the labeling of lipids under in vitro conditions renders isolated electrocyte stacks an interesting model for future research on lipid involvement in cholinergic function.

Phospholipid composition and [14C]glycerol incorporation into glycerolipids of toad retina and brain.

The phospholipid composition as well as the in vivo [14C]glycerol uptake in lipids was found to be similar in the toad brain and retina. The choroid lipid labeling was markedly different. An in vitro time-course study of [14C]glycerol incorporation in toad retina lipids disclosed that under the conditions of these experiments: (1) retina is able to rapidly synthesize phosphatidic acid from the radioactive precursor; (2) the sequence phosphatidic acid-diacylglycerol-triacylglycerol operates; (3) a high rate of phosphatidylinositol de novo biosynthesis takes place; (4) phosphoglycerides of choline and of ethanolamine are also heavily labeled after a lag period; (5) in vivo labeling profiles resembled those obtained in vitro mainly regarding phosphatidylinositol biosynthesis; and (6) the presence of glycerol kinase in the CNS is suggested.