An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites

Abstract

Gametocytes differentiation to gametes (gametogenesis) within mosquitos is essential for malaria parasite transmission. Both reduction in temperature and mosquito-derived XA or elevated pH are required for triggering cGMP/PKG dependent gametogenesis. However, the parasite molecule for sensing or transducing these environmental signals to initiate gametogenesis remains unknown. Here we perform a CRISPR/Cas9-based functional screening of 59 membrane proteins expressed in the gametocytes of Plasmodium yoelii and identify that GEP1 is required for XA-stimulated gametogenesis. GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption. The identification of GEP1 being essential for gametogenesis provides a potential new target for intervention of parasite transmission.

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

“…It is likely that the GCα topology is orientated such that the twin GC catalytic domains face the cytosol in order to allow synthesis of cGMP to activate PKG, the majority of which is cytosolic (Hopp et al, 2012). 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.…”

“…The signal therefore likely represents a C-terminally truncated proteolytic fragment resulting from protein instability during detergent extraction. A similar phenomenon has also been observed in P. yoelii, where GCα migrates at ~175 kDa instead of the predicted full-length ~450 kDa (Jiang et al, 2020), as well as in T. gondii, where TgGC predominantly migrates at ~125 kDa and ~75 kDa, with only small amounts of the ~ 460 kDa species corresponding to full-length protein detected in some studies (Bisio et al, 2019, Brown and Sibley, 2018, Jia et al, 2017, Yang et al, 2019. Given the evidence suggesting that the Toxoplasma GC needs to be full length to be functional, it seems likely that full-length P. falciparum GCα protein is present but below detection limits in our experiments.…”

“…Synthesis of cGMP by GCα has been linked to the stimulation of gametogenesis by xanthurenic acid (XA) (Muhia et al, 2001), but it is not clear whether XA stimulates GCα activity directly or indirectly through other protein mediators. However, a study in P. yoelii has recently identified a protein called GEP1 that interacts with GCα, showing that both are required for XA-stimulated gametogenesis (Jiang et al, 2020). Although cGMP synthesis and PKG activation are required for merozoite egress, the nature of the upstream signal that activates GCα in Plasmodium blood stages is unknown.…”

Rapid kinetics of lipid second messengers controlled by a cGMP signalling network coordinates apical complex functions inToxoplasmatachyzoites

“…It is interesting that GC would serve to regulate secretion of micronemes in both Toxoplasma and Plasmodium, but that Plasmodium would require an addtitional variant compared to Toxoplasma. Some have suggested a stage specific function for PfGCa and PfGCb, but this is still not clear since there is no experimental evidence for Plasmodium GCa in mosquito stages and Plasmodium GCb KO mutants have not been fully characterized in the blood stage in which they are apparently not essential (Jiang et al 2020;Moon et al 2009). However, given the highly conserved nature of these genes, we argue that the difference in variant number probably reflects different cellular architectures (such as apical complex differences, modes of egress etc.)…”

“…Similarly, Plasmodium species possess two GCs, type a and type b (GCa + GCb), and the Plasmodium yoelli protein PyGCb has been shown to have an apical localization in sporozoites, critical for ookinete traversal of the mosquito midgut (Gao et al 2018). PyGCa however has a role in blood stage gametocytogenesis and its roles in intraerythrocytic blood stage and mosquito stage have yet to be determined (Jiang et al 2020).…”