De novosynthesis of phosphatidylcholine is essential for the promastigote but not amastigote stage inLeishmania major

Abstract

Phosphatidylcholine (PC) is the most abundant type of phospholipids in eukaryotes constituting ~30% of total lipids in Leishmania. PC synthesis mainly occurs via the choline branch of the Kennedy pathway (choline ⇒ choline-phosphate ⇒ CDP-choline ⇒ PC) and the N-methylation of phosphatidylethanolamine (PE). In addition, Leishmania parasites can acquire PC and other lipids from the host or culture medium. In this study, we assessed the function and essentiality of choline ethanolamine phosphotransferase (CEPT) in Leishmania major which is responsible for the final step of the de novo synthesis of PC and PE. Our data indicate that CEPT is localized in the endoplasmic reticulum and possesses the activity to generate PC from CDP-choline and diacylglycerol. Targeted deletion of CEPT is only possible in the presence of an episomal CEPT gene in the promastigote stage of L. major. These chromosomal null parasites require the episomal expression of CEPT to survive in culture, confirming its essentiality during the promastigote stage. In contrast, during in vivo infection of BALB/c mice, these chromosomal null parasites appeared to lose the episomal copy of CEPT while maintaining normal levels of virulence, replication and cellular PC. Therefore, while the de novo synthesis of PC/PE is indispensable for the proliferation of promastigotes, intracellular amastigotes appear to acquire most of their lipids through salvage and remodeling.

Ethanolaminephosphate cytidyltransferase is essential for survival, lipid homeostasis and stress tolerance inLeishmania major

“…Loss of CPCT has no impact on promastigote growth in culture or their virulence in mice [45]. A similar study on CEPT which is directly responsible for the generation of diacyl-PE and PC revealed that this enzyme is only required for the promastigote but not amastigote stage [18]. These findings indicate that intracellular amastigotes can survive and proliferate without de novo PC synthesis by replying on the uptake/remodeling of host lipids.…”

“…4). The fact that EPCT is indispensable for amastigotes is intriguing, as amastigotes can acquire enough PC, which is far more abundant than PE [18]. One possibility is that the PE scavenging pathway, either direct uptake or remodeling of host lipids, is insufficient and amastigotes need certain level of de novo synthesis to meet the demand for PE.…”

“…S7). Such defects were not found with the episomal overexpression EPT, CPCT, CEPT, or FPPS (an essential enzyme required for sterol synthesis) [14, 18,44,45]. As a cytosolic protein, EPCT generates CDP-EtN which is a substrate for EPT and CEPT to synthesize PME and diacyl-PE, respectively (Fig.…”

“…Genes conferring resistance to blasticidin (BSD) and puromycin (PAC) were then cloned between the upstream and downstream flanking sequences to generate pUC18-KO-EPCT:BSD and pUC18-KO-EPCT:PAC, respectively. To generate the EPCT+/-heterozygotes (∆EPCT::BSD/EPCT), wild type (WT) L. major promastigotes were transfected with linearized BSD knockout fragment (derived from pUC18-KO-EPCT:BSD) by electroporation and transfectants showing resistance to blasticidin were selected and later confirmed to be EPCT+/-by Southern blot as previously described [18]. To delete the second chromosomal allele of EPCT, we used an episome assisted approach as previously described [42].…”

Sphingolipids protect ergosterol in theLeishmania majormembrane from sterol-specific toxins

“…In mammalian cells, phospholipids regulate cholesterol access [33]. We did not examine amastigotes because they salvage lipids from the host macrophage [34][35][36], the membranes are substantially similar in spt2 -, ipcs -, and wild type amastigotes [20,37], and contaminating macrophage membranes could obscure results.…”