Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii

Abstract

Lipid flipping in the membrane bilayers is a widespread eukaryotic phenomenon that is catalyzed by assorted P4-ATPases. Its occurrence, mechanism, and importance in apicomplexan parasites have remained elusive, however. Here we show that Toxoplasma gondii , an obligate intracellular parasite with high clinical relevance, can salvage phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) but not phosphatidylcholine (PtdCho) probes from its milieu. Consistently, the drug analogs of PtdCho are broadly ineffective in the parasite culture. NBD-PtdSer imported to the parasite interior is decarboxylated to NBD-PtdEtn, while the latter is not methylated to yield PtdCho, which confirms the expression of PtdSer decarboxylase but a lack of PtdEtn methyltransferase activity and suggests a role of exogenous lipids in membrane biogenesis of T. gondii . Flow cytometric quantitation of NBD-probes endorsed the selectivity of phospholipid transport and revealed a dependence of the process on energy and protein. Accordingly, our further work identified five P4-ATPases ( Tg P4-ATPase1-5), all of which harbor the signature residues and motifs required for phospholipid flipping. Of the four proteins expressed during the lytic cycle, Tg P4-ATPase1 is present in the apical plasmalemma; Tg P4-ATPase3 resides in the Golgi network along with its noncatalytic partner Ligand Effector Module 3 ( Tg Lem3), whereas Tg P4-ATPase2 and Tg P4-ATPase5 localize in the plasmalemma as well as endo/cytomembranes. Last but not least, auxin-induced degradation of Tg P4-ATPase1-3 impaired the parasite growth in human host cells, disclosing their crucial roles during acute infection. In conclusion, we show selective translocation of PtdEtn and PtdSer at the parasite surface and provide the underlying mechanistic and physiological insights in a model eukaryotic pathogen.

Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis

“…Here we demonstrate that T. gondii encodes for 6 type IV ATPases and 4 CDC50 cofactors with different functions and fitness associated to their deletion. Importantly, some of the data presented here are supported by an overlapping study (30).…”

“…During this process, the transmembrane region remainsstructurally rigid based on its interaction with the CDC50 partner (27). Among the repertoire of P4-ATPases in apicomplexan parasites, some are essential for parasite survival (28)(29)(30) yet little is known about their biological roles and enzymatic functions. Beside the lipid composition, membrane fusion is a universal process that also involves a machinery composed of SNAREs (for "soluble N-Ethylmaleimide-sensitive factor (NSF)-attachment protein receptor") (31).…”

The flexibility of Apicomplexa parasites in lipid metabolism

“…These polar headgroups are used by the parasite for the production of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) [ 55 , 74 ]. Chen and colleagues have shown that PE and PS are able to be taken up directly from the host environment, unlike PC that thus has to be generated directly by the parasite from host-scavenged and or de novo–made precursors [ 75 ]. A very recent study suggests that parasite lysophopholipase could be used to generate the phosphocholine for parasite PC synthesis from existing lysolipids from the host during P .…”

“…Both proteins under starvation showed no deleterious effect on tachyzoite growth, indicating it is unlikely they are involved in nutrient adaptation. ATP-dependent PL transporters for PS and PE are present in the plasma membrane of tachyzoites [ 75 ].…”

“…gondii strain, the early fixation of a P4 flippase gene SNP supported a critical role of lipid homeostasis [ 35 ]. Other flippases have been identified in Toxoplasma , but their roles have not been fully elucidated [ 75 ].…”