Graphical abstract

cGMP signaling is one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. Herein, we report an exclusive guanylate cyclase coupled with N-terminal P4-ATPase in a common parasitic protist, Toxoplasma gondii. This bulky protein (477-kD), termed TgATPaseP-GC to fairly reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite, whereas the corresponding cGMP-dependent protein kinase (TgPKG) is distributed in the cytomembranes. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9–assisted disruption aborts the lytic cycle of T. gondii. Besides, Cre/loxP–mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP and inhibited the parasite growth due to impairments in the motility-dependent egress and invasion events. Equally, repression of TgPKG by a similar strategy recapitulated phenotypes of the TgATPaseP-GC–depleted mutant. Notably, despite a temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans, and offers an excellent drug target against the parasitic protists.

is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in theiochondrion (PEPCK), whereas the other protein is otxpressed in achyzoites (PEPCK). Parasites with an intact glycolysis can tolerate genetic deletions ofPEPCK as well as of PEPCK, indicating their nonessential roles for tachyzoite survival.PEPCK can also be ablated in a glycolysis-deficient mutant, while PEPCK is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of PEPCK in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCK in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function ofPEPCK in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase (PyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.

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.

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