Abstract
Phosphatidylcholine (PC) is a major cell membrane constituent and precursor of important second messengers. In Leishmania parasites, PC synthesis can occur via the choline branch of the Kennedy pathway, the N-methylation of phosphatidylethanolamine (PE), or the remodeling of exogenous phospholipids. To investigate the role of de novo PC synthesis in Leishmania major, we focused on the cholinephosphate cytidylyltransferase (CPCT) which catalyzes the formation of CDP-choline, a key intermediate in the choline branch of the Kennedy pathway. Without CPCT, L. major parasites cannot incorporate choline into PC, yet the CPCT-null mutants contain similar levels of PC and PE as wild type parasites. Loss of CPCT does not affect the growth of parasites in complete medium or their virulence in mice. These results suggest that other mechanisms of PC synthesis can compensate the loss of CPCT. Importantly, CPCT-null parasites exhibited severe growth defects when ethanolamine and exogenous lipids became limited or when they were co-cultured with certain bacteria that are known to be members of sandfly midgut microbiota. These findings suggest that Leishmania employ multiple PC synthesis pathways to utilize a diverse pool of nutrients, which may be crucial for their survival and development in the sandfly.
Highlights
Leishmaniases are a group of neglected tropical diseases transmitted through the bite of female phlebotomine sandflies[1]
PC synthesis from choline in L. major, we focused on a cholinephosphate cytidylyltransferase (CPCT) ortholog (Tritrypdb ID: LmjF.18.1330, 592 amino acids) which is expected to catalyze the production of CDP-choline from CTP and choline-P (Fig. 1)
The endogenous CPCT alleles were deleted from L. major wild type (WT) parasites and the resulting cpct− mutants were confirmed by Southern blot (Fig. 2 and Fig. S1)
Summary
Leishmaniases are a group of neglected tropical diseases transmitted through the bite of female phlebotomine sandflies[1]. In addition to de novo synthesis, Leishmania parasites acquire lipids from the media (for promastigotes) or host (for amastigotes)[12,13,14,15,16]. In Leishmania, CDP-EtN is utilized to synthesize either plasmenylethanolamine (PME)[23] or 1,2-diacyl-PE (Fig. 1) These two biosynthetic routes, collectively known as the Kennedy pathway[24], is responsible for the production of the majority of PC and PE in many mammalian cell types and Trypanosoma brucei, a kinetoplastid parasite closely related to Leishmania species[22,25,26,27,28]. Why would Leishmania parasites retain multiple, seemingly redundant PC synthesis pathways (Fig. 1)? What is the relative contribution of each pathway to the overall PC production during the promastigote and amastigote stages of Leishmania? And, does each mechanism favor the synthesis of particular PC species (variants in fatty acyl chain length and saturation)? Addressing these questions will provide novel insights into the physiological role of PC synthesis in Leishmania and may facilitate the development of new treatments
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