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A G-protein-coupled receptor modulates gametogenesis via PKG-mediated signaling cascade in Plasmodium berghei

ABSTRACT: Gametogenesis is essential for malaria parasite transmission, but the molecular mechanism of this process remains to be refined. Here, we identified a G-protein-coupled receptor 180 (GPR180) that plays a critical role in signal transduction during gametogenesis in Plasmodium. P. berghei GPR180 was predominantly expressed in gametocytes and ookinetes and associated with the plasma membrane in female gametes and ookinetes. Knockout of pbgpr180 (Δpbgpr180) had no noticeable effect on blood-stage development but impaired gamete formation and reduced transmission of the parasites to mosquitoes. Transcriptome analysis revealed that a large proportion of the dysregulated genes in the Δpbgpr180 gametocytes had assigned functions in cyclic nucleotide signaling transduction. In the Δpbgpr180 gametocytes, the intracellular cGMP level was significantly reduced, and the cytosolic Ca2+ mobilization showed a delay and a reduction in the magnitude during gametocyte activation. These results suggest that PbGPR180 functions upstream of the cGMP-protein kinase G-Ca2+ signaling pathway. In line with this functional prediction, the PbGPR180 protein was found to interact with several transmembrane transporter proteins and the small GTPase Rab6 in activated gametocytes. Allele replacement of pbgpr180 with the P. vivax ortholog pvgpr180 showed equal competence of the transgenic parasite in sexual development, suggesting functional conservation of this gene in Plasmodium spp. Furthermore, an anti-PbGPR180 monoclonal antibody and the anti-PvGPR180 serum possessed robust transmission-blocking activities. These results indicate that GPR180 is involved in signal transduction during gametogenesis in malaria parasites and is a promising target for blocking parasite transmission.

ORGANISM(S): Plasmodium berghei

PROVIDER: GSE198287 | GEO | 2022/04/06

REPOSITORIES: GEO

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Project description:For malaria transmission, the parasite must undergo sexual differentiation into mature gametocytes. However, the molecular basis for this critical transition in the parasites life cycle is unknown. Six previously uncharacterized genes, Pfg14.744, Pfg14.745, Pfg14.748, Pfg14.763, Pfg14.752 and Pfg6.6 that are members of a 36 gene Plasmodium falciparum-specific subtelomeric superfamily were found to be expressed in parasites that are committed to sexual development as suggested by co-expression of Pfs16 and Pfg27. Northern blots demonstrated that Pfg14.744 and Pfg14.748 were first expressed before the parasites differentiated into morphologically distinct gametocytes, transcription continued to increase until stage II gametocytes were formed and then rapidly decreased. Immunofluorescence assays indicated that both proteins were only produced in the subpopulation of ring stage parasites that are committed to gametocytogenesis and both localized to the parasitophorous vacuole (PV)b of the early ring stage parasites. As the parasites continued to develop Pfg14.748 remained within the parasitophorous vacuole, while Pfg14.744 was detected in the erythrocyte. The 5' flanking region of either gene alone was sufficient to drive early gametocyte specific expression of green fluorescent protein (GFP). In parasites transfected with a plasmid containing the Pfg14.748 5' flanking region immediately upstream of GFP, fluorescence was observed in a small number of schizonts the cycle before stage I gametocytes were observed. This expression pattern is consistent with commitment to sexual differentiation prior to merozoite release and erythrocyte invasion. Further investigation into the role of these genes in the transition from asexual to sexual differentiation could provide new strategies to block malaria transmission. Microarray analysis was used to compare two clones derived from Plasmodium falciparum strain 3D7 parasites that differ in their ability to undergo gametocytogenesis. Clone G+ produces gametocytes and clone G- produces very few if any gametocytes. RNA was harvested from the cultures when the asexual parasitemia was 0.9-1.48% (day 4) (n=4) after setting up the gametocyte cultures and 5.2-5.58% (day 6) (n=4) prior to the appearance of morphologically distinct gametocytes and used to generate cDNA that was labeled with Cy3 or Cy5 and hybridized to the Plasmodium falciparum 70 mer oligonucleotide microarray developed by DeRisi and co-workers.

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A G-protein-coupled receptor modulates gametogenesis via PKG-mediated signaling cascade in Plasmodium berghei

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