Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum

Abstract

the cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite. Malaria is caused by protozoan parasites of the genus Plasmodium and most cases of life-threatening malaria are attributable to infection with Plasmodium falciparum. The parasite has a complex life cycle that involves the human host and its vector, the Anopheles mosquitoes. All clinical features are caused during the asexual intraerythrocytic life cycle due to the repeated invasion of human red blood cells (RBCs). The asexual intraerythrocytic developmental cycle of P. falciparum lasts around 48 h, during which the parasite progresses through four morphologically different stages: ring, trophozoite, and schizont stages, ending with rupture of the erythrocyte and release of merozoites that will invade new erythrocytes. Transmission of the malaria parasite requires development of male and female gametocytes (gametocytogenesis), which are ingested by female mosquitoes during a blood meal and undergo sexual reproduction in the mosquito's midgut. Nondividing P. falciparum gametocytes take between 10 and 12 days to fully mature and progress through five morphologically distinct forms (stages I to V), which are different from other Plasmodium species. During their complex life cycle malaria parasites encounter different nutritional environments within and between hosts. The presence of de novo and salvage pathways gives parasites a great metabolic flexibility to cope with those changes 1,2. For example, mature RBCs are capable of only a few metabolic functions since transcription and translation is not present in these cells. However, a wide variety of metabolites are available to the parasite in the human plasma 3. Among the metabolic pathways that become inactive in mature RBCs is the mevalonate pathway which synthesizes the isoprenoid building blocks isopentenyl diphosphate (IPP) and d...

N-Glycosylation in Piroplasmids: Diversity within Simplicity

“…In apicomplexan parasites, as well as in eubacteria and plant chloroplasts, isoprenoid precursors are synthesized through the methylerythritol phosphate (MEP) pathway, while in other eukaryotes, synthesis takes place through the mevalonate pathway. The MEP pathway is a seven-step process that starts with pyruvate and ends with the synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), and, in apicomplexans, takes place in the apicoplast [ 25 ]. All MEP pathway enzymes were shown to be encoded in the genomes of B. bovis , B. microti , and B. orientalis , and expression of two of them (BoDXS and BoDXR) was experimentally demonstrated in merozoites (mz) of the latter [ 26 , 27 ].…”

“…It has been proposed for P. falciparum that the end products of the MEP pathway, IPP and DMAPP, are converted outside the apicoplast into farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), and that the final steps of dol-P synthesis take place at the cytoplasmic leaflet of the RER membrane [ 25 ]. Thus, some kind of transport system needs to carry these hydrophobic molecules through the hydrophilic cytoplasmic milieu.…”

“…There, an isoprenoid elongation step, catalyzed by cis-prenyltransferase (CPT) takes place, resulting in a polyprenyl diphosphate bearing a varying number of isoprene units which, in P. falciparum , has been determined to be 15 to 19. Dephosphorylation by polyprenyl diphosphate phosphatase yields polyprenol, which is then reduced by polyprenol reductase (PPRD) to dol [ 25 ]. Dol needs then to undergo phosphorylation to be utilized in the N -glycosylation and GPI synthesis pathways.…”

“…Homologous genes to those encoding the enzymes that participate in the N -glycosylation pathway, as well as in sugar nucleotide synthesis and the final steps of dol-P synthesis of Saccharomyces cerevisiae S288C and/or P. falciparum strain 3D7 [ 3 , 23 , 25 , 46 ] were searched for in the available genomes of B. bovis strain T2Bo [ 47 ], B. bigemina strain BOND [ 48 ], B. divergens strain 1802A [ 49 ], B. ovata strain Miyake [ 50 ], Babesia sp. Xinjang [ 51 ], B. microti strain R1 [ 52 ], T. parva strain Muguga [ 53 ], T. annulata strain Ankara [ 54 ], T. orientalis strain Shintoku [ 55 ], T. equi strain WA [ 17 ], and C. felis strain Winnie [ 56 ].…”

Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites

“…Longer-chain polyprenyl-PPs and related dolichols serve as membrane-bound glycan carriers for protein glycosylation, but these activities in Plasmodium appear to occur in the endoplasmic reticulum as they do in other organisms. 20,72,73 Linear polyprenyl alcohols have been found to be important components of plant membranes, especially chloroplast membranes, where they are proposed to modulate membrane structure, fluidity, and dynamics. 66,68,74,75 In the absence of other known roles for longer-chain polyprenyl-PPs in the apicoplast, we hypothesize that linear polyprenols or polyprenyl phosphates may serve as critical components of the apicoplast membranes and be required for maintaining membrane fluidity during organelle biogenesis (Figure 9).…”