PfAP2-G2 Is Associated To Production And Maturation Of Gametocytes In Plasmodium Falciparum Via Regulating The Expression Of PfMDV-1

Objective:Malaria only spread by female Anopheles and caused by protozoan parasites of Plasmodium.Plasmodium falciparum is the most lethal in the species that infect humans.The entire lifecycle of P.falciparum includes stages that develop in the human liver and blood as well as a mosquito vector.Though the symptoms of the disease are associated with the asexual growth of the parasites in the human blood,the parasites must undergo sexual differentiation to form female and male gametocytes,so that the mature gametocytes in the human peripheral blood can be taken up by the mosquito and transmitted.Therefore,gametocyte is an attractive target for eliminating malaria.Therefore,in order to achieve the grand plan of eradicating malaria,gametophytic cells are undoubtedly one of the most important targets in the research process.Different from other eukaryotes,a class of highly conserved and AP2 domain-containing protein family(ApiAP2)has been found in Plasmodium.So far,the ApiAP2 family is the largest transcriptional factor family of Plasmodium and the members are expressed in the entire life cycle of Plasmodium.Previous studies of AP2-G2 in P.berghei demonstrated that PbAP2-G2 disruptants almost completely lacked mature gametocytes,suggesting that this transcriptional factor played a critical role in the maturation of gametocytes.However,the role and molecular mechanisms of AP2-G2 in P.falciparum remain unclear.In this study,PfAP2-G2 knockout strains and tagged strains were obtained through the CRISPR-Cas9 system,and we analyzed the phenotype of PfAP2-G2 knockout strains in the gametocyte stage.We further explored the mechanisms of these phenotypes at the transcriptome level and proteome level,and clarified the regulatory pathway of the PfAP2-G2,which provide a theory for further research on the important role of the ApiAP2 family in Plasmodium falciparum.Methods:1.Bioinformatics analysis.The sequence of Pf3D7₁408200(PfAP2-G2)gene was obtained and the domain was predicted from PlasmoDB website.2.The acquisition of PfAP2-G2 knockout parasite strain and PfAP2-G2-3TY1-3Flag tag parasite strain.After expanded culture,PfAP2-G2 knockout plasmid and PfAP2-G2-3TY1-3Flag tag plasmid and Cas9 plasmid were extracted by plasmid extraction kit and then identified by PCR and by DNA sequencing.PfAP2-G2 knockout plasmids and PfAP2-G2-3TY1-3Flag tag plasmid were electrotransfected with Cas9 plasmid into the purified schizont,respectively.The high purity of PfAP2-G2 knockout parasite strain and PfAP2-G2-3TY1-3Flag tag parasite strain were obtained by monoclone after screening with WR99210 and BSD,the successful acquisition of PfAP2-G2 knockout parasite strain and PfAP2-G2-3TY1-3Flag tag parasite strain were confirmed by PCR and DNA sequencing.3.Western blot.The expression of tag of PfAP2-G2 were detected by Western blot.NF54 parasites and mixed stages of PfAP2-G2-3TY1-3Flag tag parasites protein were separated by SDS-PAGE.The expression of PfAP2-G2-3TY1-3Flag tag was determined by the reaction of anti-Flag tag mouse antibody with PVDF membrane printed with antigen.4.Transcription level of PfAP2-G2 mRNA was detected by RT-qPCR.Total RNA were extracted from ring stage,trophozoite stage and schizont stage parasites according to the instructions of RNA extraction kit.cDNA were obtained by using Kit with gDNAEraser.Primers were designed to detect the transcriptional level of PfAP2-G2 mRNA in these stages of Plasmodium falciparum by real-time PCR.5.Indirect immunofluorescence assay.To detect the location of PfAP2-G2,the parasites of each asexual stage and NF54 strain were treated with 0.25%Triton X-100,then incubated with anti-Flag antibody.After DAPI staining,the parasites were treated with anti-quenching tablet and then observed by fluorescence microscope.6.Gametocyte induction of PfAP2-G2 knockout parasite strain and NF54 strain.Gametocyte induction started with the same initial parasitemia and blood pressure.The asexual parasites were eliminated with N-Acetyl-D-glucosamine.We determined the effect of knocking out PfAP2-G2 on gametocytes development via daily parasitemia of PfAP2-G2 knockout parasite strain and NF54 strain.7.Detecting the effect of knocking out PfAP2-G2 on female/male gametocyte ratio by IFA.The ring stage parasites of PfAP2-G2 knockout parasite strain and NF54 strain before N-Acetyl-D-glucosamine treated were treated with 0.25%Triton X-100,and then incubated with anti-pfs16 antibody and anti-?-tubulin antibody,respectively.After DAPI staining,the parasites were treated with anti-quenching tablet and then counted the number of gametocytes and male gametocytes.8.RT-qPCR was used to identify the expression level of gametocyte specific genes in PfAP2-G2 knockout parasite strain and NF54 strain.Total RNA were extracted from ring stage and schizont stage parasites according to the instructions of RNA extraction kit.cDNA were obtained by using Kit with gDNA Eraser.The specific primers were designed to detect the expression level of gametocyte specific genes.9.RNA-seq.Total RNA were sequenced by Novogene company which were extracted from ring stage and schizont stage parasites of PfAP2-G2 knockout parasite strain and NF54 strain.The effect of PfAP2-G2 deletion to transcription level of other genes was identified by bioformatics analysis.10.CHIP-qPCR was used to identified the target genes of PfAP2-G2.DNA was extracted from ring stage and schizont stage parasites of PfAP2-G2 knockout parasite strain and NF54 strain which was interacted with PfAP2-G2.The specific primers were designed to detect the quantity of DNA,and CHIP-qPCR was used to identified the target genes of PfAP2-G2.Results:1.PfAP2-G2-3TY1-3Flag tag parasite and PfAP2-G2 knockout parasite were successfully obtained.We obtained the drug-resistant PfAP2-G2-3TY1-3Flag tag parasite and PfAP2-G2 knockout parasite after selection of WR99210 and BSD.The results of PCR and sequences and Western blot showed that the Plasmodium falciparum with 3TY1-3Flag tag was successfully obtained at C' terminal of PfAP2-G2,as well as showed that the domain was deleted from PfAP2-G2.2.In the asexual stages,PfAP2-G2 expressed the highest in ring stage,lowest in schizont stage.3.Indirect immunofluorescence assay showed nuclear localization of PfAP2-G2 in asexual stages.IFA of fixed parasites detected that at the ring stage PfAP2-G2 was shown as a single punctum at the nuclear periphery demarcated by DAPI staining,while in the trophozoite stage,it diffused and overlapped more extensively with the nucleus.However,the anti-Flag staining became much fainter at the schizont stage and the more prominent foci did not overlap with the DAPI staining.4.Phenotypic analysis of PfAP2-G2(-)parasites was compared with the NF54 strain.Although the PfAP2-G2(-)parasites and the NF54 control showed no significant difference in daily asexual parasitemia,there was?95%reduction in gametocytemia in the PfAP2-G2(-)parasites at day 9 after post gametocyte induction.However,gametocytes produced by PfAP2-G2(-)parasites retained normal morphology observed under a light microscope on day 9 post gametocyte induction.However,on day 12 post gametocyte induction when mature gametocytes were visible in the NF54 parasites,gametocytes could not be observed in the PfAP2-G2(-)parasites.5.On day 6 post gametocyte induction,we found that deletion of PfAP2-G2 had a substantial impact on the sex ratio as determined by differential staining with anti-Pfs16 and anti-?-tubulin-? antibodies,with the PfAP2-G2(-)parasites having significantly higher female/male gametocyte ratio(?9.1:1)than the NF54 parasites(?3.7:1).6.Compared with the NF54 parasites,PfAP2-G2 deletion resulted in significantly more genes upregulated(113 genes)than downregulated(12 genes)in the ring stage.In the schizont stage,however,41 and 102 genes were upregulated and downregulated,respectively in PfAP2-G2(-)parasites.we found that the expression level of PfMDV-1,which is essential to the development of male gametocytes,was significantly decreased in both the ring and schizont stages.The expression level of PfAP2-G,the master regulator of sexual commitment,was also decreased in the schizont stage,but not in the ring stage.7.qPCR analysis using primer pairs targeting to different upstream regions of the PfMDV-1 gene showed significant enrichment of the PfAP2-G2 protein at around 500 bp upstream of the start codon,which corresponds approximately to the transcriptional start site(TSS)of PfMDV-1 at both ring and schizont stages.This result suggests that PfMDV-1 is one of the targets of PfAP2-G2.Conclusion:1.PfAP2-G2 was highly expressed in the ring stage.2.PfAP2-G2 located in nuclear in asexual stage.3.PfAP2-G2 is essential for production and maturation of gametocytes in Plasmodium falciparum,especially the male gametocytes.4.PfMDV-1 is one of the target genes of PfAP2-G2,and PfAP2-G2 regulates the expression of PfMDV-1 to affect the development of gametocytes.