An unusual and vital protein with guanylate cyclase and P4-ATPase domains in a pathogenic protist

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.

Plasmodium falciparumguanylyl cyclase-alpha and the activity of its appended P4-ATPase domain are essential for cGMP synthesis and blood stage egress

“…Our observed localisation for P. falciparum GCα is reminiscent of that recently described for GCα in P. yoelii gametocytes (Jiang et al, 2020). In contrast, in Toxoplasma (Bisio et al, 2019, Brown and Sibley, 2018, Gunay-Esiyok et al, 2019, Yang et al, 2019 the single GC has been localised to the plasma membrane, primarily at the apical pole, in extracellular parasites.…”

“…Although the single GC in the related apicomplexan parasite T. gondii (Bisio et al, 2019, Brown and Sibley, 2018, Gunay-Esiyok et al, 2019, Yang et al, 2019 and GCβ in P.…”

“…The physical linkage of a guanylyl cyclase with a P4-ATPase domain is unique to Plasmodium and its apicomplexan and ciliate relatives (Baker et al, 2017, Bisio et al, 2019, Brown and Sibley, 2018, Gao et al, 2018, Gunay-Esiyok et al, 2019, Yang et al, 2019, but the functional significance of this unusual coupled architecture is unknown.…”

Co-option of Plasmodium falciparum PP1 for egress from host erythrocytes

“…4a) 31,45,46 . Termed GCα, the protein is present across apicomplexan parasites, and in Toxoplasma gondii is also essential for egress [47][48][49][50] . Given the function we identified for PfPP1 in egress and phosphoregulation of PfGCα, we measured the effect of PfPP1-DD knockdown on cellular cGMP levels, finding that destabilization of the phosphatase enzyme reduces the second messenger in schizont-stage parasites by~2-fold ( Fig.…”

Phosphatidylinositol synthesis, its selective salvage, and inter-regulation of anionic phospholipids in Toxoplasma gondii

“…TgPIS-mAID-3xHA mutant also exhibited egress defect in normal cultures (-IAA), which was radically accentuated upon inclusion of auxin ( Supplementary Figure 3a). To test whether curtailed egress may be caused by slow replication of the mutant, we examined induced egress in response to activation of cGMP signaling by zaprinast, a phosphodiesterase inhibitor 51,52 . Indeed, we recorded a marked recovery in egress of the auxin-treated mutant but the impairment was still evident ( Supplementary Figure 3b).…”