Transcriptional Regulation Of PfHDAC1 And PfHDA1 During The Growth And Development Of Plasmodium Falciparum At Asexual Stage And Gametocyte Stage

Objective: Malaria is a disease transmitted by the female Anopheles mosquito and is caused by a single-celled parasite of the genus Plasmodium.Epidemiological studies have found that in low transmission areas of malaria,Plasmodium falciparum prefers to become gametocytes with up-regulated expression of gametocytogenesis determinants PfAP2-G and gametocyte-related genes,accompanied by down-regulated expression of PfHDAC1 and up-regulated expression of PfHDA1,suggesting that PfHDAC1 and PfHDA1 may be involved in the gametocytogenesis process of Plasmodium falciparum,but the function and mechanism of the deacetylases PfHDAC1 and PfHDA1 are unclear.In this study,we performed conditional knockout of PfHDAC1 and PfHDA1 based on CRISPR-Cas9 system,combined with RNA-seq and ChIP-seq to investigate the functions and regulatory mechanisms of PfHDAC1 and PfHDA1 in the asexual stage of Plasmodium falciparum infection invasion stage and gametocyte production,which can help us to understand the histone deacetylases of Plasmodium falciparum in depth.This will help us to understand the important role of histone deacetylase family in the growth and development of Plasmodium,and provide a theoretical basis for the target screening of future antimalarial drugs.Methods: 1.Bioinformatics analysis.The sequence of PfHDAC1(PF3D7＿0925700)and PfHDA1(PF3D7＿1472200)were obtained and the glm S-Ribozyme was predicted from Plasmo DB website.2.Gene editing.The acquisition of PfHDAC1 and PfHDA1 conditional knockout strain and fusion-tagged strain.3.Western blot.The PfHDAC1 and PfHDA1 conditional knockout strain and the fusion-tagged strain were tested by Western blot for normal expression of the genetically modified target protein and quantified using TY1,Flag and Actin antibodies,respectively.4.RT-qPCR to detect the transcript level of PfHDAC1 and PfHDA1 in each period.5.IFA identification of the localization of PfHDAC1 and PfHDA1.6.Flow cytometry analysis of growth rate of PfHDAC1 conditional knockout strain.The results were analyzed by continuous sampling during the first growth cycle,and erythrocytes were fixed immediately after sampling at 12 h,24 h,36 h and 48 h,respectively,and were uniformly stained with SYBR GREEN and then analysis on the machine.7.Gametocyte induction assay.The gametocyte production rate were investigated and counting of PfHDAC1 and PfHDA1 conditional knockout strains and controls,controlling the starting parasitemia rate and erythrocyte pressure.8.RT-qPCR was performed to detect the relative expression of infection invasion genes and gametocyte-related genes.9.RNA-seq.RNA samples from PfHDAC1 and PfHDA1 conditional knockout strains and controls were collected and whole genome sequencing was performed.10.ChIP-seq.ChIP-fixed samples of late schizont stage PfHDAC1 and PfHDA1 fusion-tagged strains were collected,incubated with ChIP-grade Flag antibody,purified to obtain DNA interacting with target proteins,and whole-genome sequencing was performed to observe the distribution and enrichment on Plasmodium chromosome.11.ChIP-qPCR.ChIP-fixed samples of PfHDAC1 and PfHDA1 conditional knockout strains and controls were collected,ChIP experiments were performed using H3K9-Ace antibody to detect enrichment in H3K9-Ace levels in the promoter region by ChIP-qPCR.Results: 1.Gene modified strains of PfHDAC1 and PfHDA1 were successfully obtained.The monoclonal strains were obtained by limited dilution method after WR and BSD drug screening.PCR assay and sequencing results demonstrated gene modification successfully.Western blot verified that the gene modified strains could normally express the target proteins.2.The expression level of PfHDAC1 and PfHDA1 were highest in the late schizont stage.3.PfHDAC1 and PfHDA1 were localized in the nucleus of Plasmodium falciparum.They were localized in a punctate manner during the ring stage,and diffused in the nucleus during the late trophozoite and late schizont stages.4.PfHDAC1 regulates infection invasion genes in the asexual stage and affects the growth rate.after the conditional knockout of PfHDAC1,the growth rate became faster.RNA-seq showed that the expression of most of the infection invasion genes was upregulated significantly,ChIPseq results showed that PfHDAC1 was significantly enriched in the promoter region of the infection invasion genes.The ChIP-qPCR results demonstrated that H3K9-Ace levels increased in the promoter regions of the most infection invasion genes.5.PfHDAC1 conditional knockout promoted gametocytogenesis while the expression of early gametocytogenesis genes was upregulated.RNA-seq showed an increase in early gametocytogenesis genes expression,and ChIP-seq showed that PfHDAC1 was highly enriched in the promoter region of early gametocytogenesis genes.ChIP-seq results showed that PfHDAC1 was highly enriched in the promoter region of the early gametocytogenesis genes.ChIP-qPCR results also verified that H3K9-Ace levels in the promoter region of early gametocytogenesis genes increased.6.PfHDA1 affects gametocyte production by regulating the expression of PfHDA2.Gametocyte induction experiment revealed that both gametocyte production rate and gametocyte formation rate decreased after PfHDA1 conditional knockout,early gametocytogenesis genes were downregulated and PfHDA2 expression was up-regulated.ChIP-seq results showed that PfHDA1 was significantly enriched in the PfHDA2 promoter region,and ChIP-qPCR results also verified that the level of H3K9-Ace in the PfHDA2 promoter region increased after PfHDA1 conditional knockout.Conclusion: 1.PfHDAC1 and PfHDA1 are highly expressed in the late schizont stage.2.PfHDAC1 and PfHDA1 are localized in the nucleus of Plasmodium falciparum.3.Conditional knockout of PfHDAC1 affects the growth rate of the asexual stage of Plasmodium falciparum and promotes the production of gametocytes;Conditional knockout of PfHDA1 reduces the production of gametocytes.4.PfHDAC1 mediates the growth and development of Plasmodium falciparum at different stages by regulating the expression of infection invasion genes and early gametocytogenesis genes;PfHDA1 mediates the production of gametocytes by regulating the expression of PfHDA2.