Functional Analysis Of ApiAP2 Transcription Factors Of Plasmodium Yoelii During The Early-stage Development In Mosquito Host

Malaria is a mosquito-borne infectious disease caused by protozoan parasites of the genus Plasmodium.In 2017,there are 91 countries and regions all over the world which are still at risk of malaria,leading to an around 400,000 death.Plasmodium parasites are single-cell organisms that have a complex life cycle involving mosquitoes and vertebrate hosts.The development of malaria parasites in these two hosts are accompanied by various distinct morphological changes that are dependent on correspondent models for gene regulation.Currently,the transcription factors and transcriptional mechanisms for gene regulation are still remained to be elucidated.ApiAP2 family which contains 26 members in Plasmodium parasite genome is the most important transcription factor family.In our previous study,we used the mouse malaria parasite P.yoelii and applied the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9(CRISPR-Cas9)-based methods we described previously to research the expression and function of ApiAP2 protein family.In this paper,we introduce the three transcription factors,ap2-o2,ap2-o3 and ap2-o4,that are all having significant roles in the development of early mosquito stage of malaria parasites.Phenotypic analyses of ? ap2-o3 parasite display that AP2-03 has no role in asexual stages,gametocyte formation and male gametocyte exflagellation,but bears essential functions in the development from gametocyte to ookinete.To validate the expression model of AP2-03 in the parasites' whole life cycle,the transcription factor was tagged with 6×HA peptide.Interestingly,protein expression of this transcription factor cannot be detected in asexual blood stages and ookinete stage;it displays a female-specific expression in gametocyte stage.We also find out that AP2-03 is onlyfunctional in female gametocyte and obviously affects the translational level of P28 protein at activated state of female gametocyte.Further analyze the expression of AP2-03 all over the developmental stages ofmalaria parastie,we find out that the expression of this protein maybe have reverse relation with DNA replication process in various developmental stages.Through transcriptome analysis,almost all genes participate in DNA replication and repair process are up-regulated after ap2-o3 knockout.qRT-PCR validate the results from transcriptome next generation sequencing.In the zygote cells activated from female gametocyte,the nuclear ploidy of AP2-O3 positive zygotes is lower than AP2-O3 negative zygotes.Overexpression of AP2-O3 in zygote,the process from 2N to 4N is stoped and the development of mature ookinete is significantly defected.Above all,we suggest that AP2-O3 can transcriptionally repress expression of genes take part in DNA replication and repair.Transcription level of gene lc6 and zk11 which have no predict information are down-regulate after ap2-o3 knock out.Their protein level in female gametocyte is regulated by AP2-O3.Further analysis show that lc6 and zk11 are play significant roles in forming ookinete.We think that lc6 and zk11 maybe as downstream genes of AP2-O3 are account for the process form gametocyte to ookinete.AP2-O3 can transcriptionally activate expression of lc6 and zk11 to affect ookinete development.To validate the ap2-o4's function,phenotypic analyses conducted to show that PyAP2-O4 have no significant role in development of asexual blood stage,gametocyte stage and ookinete stage.But no one oocyst have been detected in ap2-o4 KO strain infected mosquitos at 4 dpi(day post infection).Furthermore,ap2-o4KO from P28::mCherry strain repeat the phenotype and the number of parasites in mosquito are decreased gradually from 1 to 3 dpi.The 6HA tagged AP2-O4 strain tell us that this transcription factor is nuclear localization and express in whole life cycle except the asexual blood stage.It shows that AP2-O4 may affect expression of some genes which are functional from ookinete to oocyst development.To find out the functions of ap2-o2,we conduct the phenotypic analyses of ap2-o2 knock-out(KO)strain parental from P.yoelii 17XNL.It is showing that ap2-o2 has redundant roles in asexual blood stage,gametocyte stage(gametocyte formation),ookinete stages(both retort and mature ookinete).But when the Pyap2-o2 mutant strain entering into the mosquito midgut,its ability of production of oocysts and sporozoites are decreased remarkably.To know the expression model of AP2-02,we tagged AP2-02 with sextuple hemagglutinin(6K×HA)tags.The immunofluorescence of tagged strain shows that AP2-02 is expressing in all developmental stages in two hosts except mature ookinete stage.AP2-02 is localizing at cell nuclear as a representative transcription factor.The expression of oocyst stage in mosquito midgut give us a hint that AP2-02 influence the expression of genes have roles in escaping immune attack from mosquito host.In this paper,we studied three significant transcription factors which are functional in malaria parasites development in early mosquito stage.We first propse and prove the relationship between the transcription factor AP2-03 and DNA replication and repair process of parasite.We also firstly identify two genes which are positively regulated by PyAP2-03 and essential for development from gametocyte to ookinete stage.The functional and expression research about Pyap2-o2 and Pyap2-o4 tell us that malaria parasites have complex regulational mechanism to fit its environment.This paper not only provides the new model for gene regulation in early mosquito stage,but also explain how these parasites manage to control their complex life cycle using a small number of sequence-specific ApiAP2 transcription factors.The original few days of malaria parasites entering into mosquito are mostly important for malaria transmission.The Plasmoidum parasites in this stage is potential ideal target for antimalaria drugs.It also inspires us to seek for new methods for malaria elimination.