GlmS mediated knock-down of a phospholipase expedite alternate pathway to generate phosphocholine required for phosphatidylcholine synthesis in Plasmodium falciparum.

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from the catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with up-regulation of phosphocholine and SAM levels; these results show up-regulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights the presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite.

Neutral lipid metabolism influences phospholipid synthesis and deacylation in Saccharomyces cerevisiae.

Establishment and maintenance of equilibrium in the fatty acid (FA) composition of phospholipids (PL) requires both regulation of the substrate available for PL synthesis (the acyl-CoA pool) and extensive PL turnover and acyl editing. In the present study, we utilize acyl-CoA synthetase (ACS) deficient cells, unable to recycle FA derived from lipid deacylation, to evaluate the role of several enzymatic activities in FA trafficking and PL homeostasis in Saccharomyces cerevisiae. The data presented show that phospholipases B are not contributing to constitutive PL deacylation and are therefore unlikely to be involved in PL remodeling. In contrast, the enzymes of neutral lipid (NL) synthesis and mobilization are central mediators of FA trafficking. The phospholipid:DAG acyltransferase (PDAT) Lro1p has a substantial effect on FA release and on PL equilibrium, emerging as an important mediator in PL remodeling. The acyl-CoA dependent biosynthetic activities of NL metabolism are also involved in PL homeostasis through active modulation of the substrate available for PL synthesis. In addition TAG mobilization makes an important contribution, especially in cells from stationary phase, to FA availability. Beyond its well-established role in the formation of a storage pool, NL metabolism could play a crucial role as a mechanism to uncouple the pools of PL and acyl-CoAs from each other and thereby to allow independent regulation of each one.

Degradation of lysophosphatidylcholine by lysosomes. Stimulation of lysophospholipase C by taurocholate and deficiency in Niemann-Pick fibroblasts.

Hydrolysis of 2-[1-14C]oleoyl phosphatidylcholine and of 1-[1-14C]oleoyl lysophosphatidylcholine by lysosomes prepared from rat liver using Triton WR-1339 has been studied. At pH 5.0 sodium taurocholate stimulated the release by the soluble lysosomal fraction of labelled lysophosphatidylcholine, diacyl- and monoacylglycerol and fatty acids from [14C]phosphatidylcholine. The time course of appearance of labelled products suggested that monoacylglycerol could be released as a result of the action of phospholipase A1 followed by lysophospholipase C or by the initial action of phospholipase C followed by monoacylglycerol lipase. The hydrolysis of 1-[14C]acyl lysophosphatidylcholine was also stimulated by sodium taurocholate under similar conditions; however, only release of monoacylglycerol was increased, whereas release of fatty acid was inhibited. Mg2+ inhibited the release of labelled monoacylglycerol and of fatty acid from lysophosphatidylcholine. The detergents deoxycholate and Triton X-100 and phospholipids were strongly inhibitory. 5'-AMP almost completely suppressed release of monoacylglycerol but increased release of fatty acid. Chloroquine strongly suppressed release of monoacylglycerol and only at high concentration (1.25 mM) diminished fatty acid release. In the presence of sodium taurocholate the predominant mechanism for degradation of phosphatidylcholine by the soluble fraction of lysosomes involves phospholipase A followed by phospholipase C. Assay of release of monoacylglycerol from [14C]lysophosphatidylcholine catalyzed by extracts of fibroblasts from patients with Niemann-Pick disease and controls in the presence of taurocholate revealed that lysophospholipase C activity was lacking in those cell lines that were deficient in sphingomyelinase. This suggests that lysophospholipase C and sphingomyelinase activities may be catalyzed by one enzyme.