Metabolomics profiling reveals new aspects of dolichol biosynthesis inPlasmodium 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. Polyprenols have been hardly detectable in eukaryotic cells under normal conditions with the exception of plants and sporulating yeast. Our metabolomics studies revealed that cis-polyisoprenoids are more prevalent and diverse in the 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 also observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. In addition, we confirmed the presence of an active cisprenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite. Isotopic labeling and metabolomic analyses of a conditional mutant of PfCPT or PfPPRD suggest that polyprenols may be able to substitute dolichols in their biological functions when dolichol synthesis is impaired in Plasmodium.Malaria is caused by protozoan parasites of the genus Plasmodium and most cases of lifethreatening 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 2 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 i...

Apicoplast-derived isoprenoids are essential for biosynthesis of GPI protein anchors, and consequently for egress and invasion inPlasmodium falciparum

“…Given that protein prenylation is adversely affected by fosmidomycin treatment, we tested whether GPI-AP localisation is also sensitive to fosmidomycin. Consistent with previous reports, GGOH supplementation allowed fosmidomycin treated parasites to mature to schizont stages (Supp anchoring following fosmidomycin treatment suggests there is an initial, residual pool of dolichols that supports GPI biosynthesis, consistent with previous reports that low levels of dolichols are sufficient to maintain glycosylation (23). As dolichols are not consumed through their participation in GPI biosynthesis, they appear to be more robust in the short term to fluctuations in IPP (S3 Fig) . In contrast, prenyl groups are covalently linked to the Cterminus of proteins with prenylation motifs and thereby consumed, so are dependent on the de novo synthesis of IPP for continuing prenylation.…”

“…Conversely, rapid inhibition of IPP synthesis by fosmidomycin left GPI-APs initially unaffected. This persistence of GPI-APs suggests that a low level of dolichols is sufficient for adequate GPI synthesis, as has been observed in other studies (23). Plasmodium parasites likely contribute to the maintenance of a surface glycocalyx on the merozoite plasma membrane, as well as acting as a large reservoir of GPI precursors (52).…”

“…In addition to their roles in GPI biosynthesis and protein C-mannosylation, neutral polyisoprenol/dolichol lipids can also contribute to the biophysical properties of cellular 25 membranes (55,56). Intriguingly, P. falciparum asexual parasite stages produce unusually high levels of cis-polyisoprenols, which are present at the same concentration as mature dolichols (23). In most eukaryotes, levels of cis-polyisoprenoids are very low due to their conversion to dolichol by a polyprenol reductase.…”

“…The factors and proteins that govern membrane invagination remain largely unknown, though sporozoite budding in the mosquito is at least in part regulated by the presence of the GPI anchor of circumsporozoite protein, possibly through the organisation of lipid-rich microdomains (57). GPI-APs in parasite membranes may play a role in these processes but free polyisoprenoids may also influence these membranes (23,55,56).…”

“…Apicoplast IPP is also expected to be used to synthesise dolichols, very long polyisoprenoid alcohols that are synthesised in the endoplasmic reticulum (ER) via the reduction of polyprenols (Fig 1B) (21)(22)(23). Dolichol can be phosphorylated to form dolichol-phosphate (dolichol-P) and subsequently dolichol-phospho-mannose (DPM), which acts as a sugar donor for a number of ER mannosyltransferases involved in glycophosphatidylinositol (GPI) biosynthesis and protein tryptophan C-mannosylation (24,25).…”