The Plasmodium falciparum CCCH zinc finger protein ZNF4 plays an important role in gametocyte exflagellation through the regulation of male gametocyte enriched transcripts

Abstract

CCCH zinc finger proteins (ZFPs) function mainly as RNA-binding proteins (RBPs) where they play a central role in the mRNA metabolism. Over 27 CCCH-ZFPs are encoded in the genome of the human malaria parasite Plasmodium falciparum, the causative agent of malaria tropica. However, little is known about their functions. In this study, we characterize one member of the PfCCCH-ZFP named ZNF4. We show that ZNF4 is highly expressed in mature gametocytes where it predominantly localizes to the cytoplasm. Targeted gene disruption of ZNF4 showed no significant effect in asexual blood stage replication and gametocyte development while male gametocyte exflagellation was significantly impaired leading to reduced malaria transmission in the mosquito. Comparative transcriptomics between wildtype (WT) and the ZNF4-deficient line (ZNF4-KO) demonstrated the de-regulation of about 473 genes (274-upregulated and 199 down-regulated) in mature gametocytes. Most of the down-regulated genes show peak expression in mature gametocyte with male enriched genes associated to the axonemal dynein complex formation and cell projection organization highly affected, pointing to the phenotype in male gametocyte exflagellation. Up-regulated genes are associated to ATP synthesis. Our combine data therefore indicate that ZNF4 is a CCCH zinc finger protein which plays an important role in male gametocyte exflagellation through the regulation of male gametocyte enriched genes. Author SummaryCCCH ZFPs have gained significant interest due to their ability to interact with RNA and control different RNA metabolic processes. The role of CCCH ZFPs in the malaria parasite Plasmodium falciparum has not been well studied. In this study we report the functional characterization of a PfCCCH-ZFP named ZNF4. Using mouse anti-ZNF4 antisera and a ZNF4-HA tag parasite line we show that the protein is mainly expressed in the cytosol of mature gametocytes. To determine the function of ZNF4, we generated a ZNF4 knockout parasite line and we found that the asexual blood stages and gametocytes developed normally while the ability of the male gametocytes to exflagellate as well as the further development of the parasite in the mosquito was significantly impaired. Interestingly, when the transcriptome of mature gametocytes of the ZNF4-KO was compared to the WT, down-regulated genes were mainly male gametocyte enriched genes associated to processes involved in gametocyte exflagellation such axonemal dynein complex formation and cell projection organization indicating that ZNF4 plays an important role in male gametocyte exflagellation through the regulation of male gametocyte enriched transcripts.