Study On The Mechanism Of NIF Protein Family Mediating Artemisininresistance In Plasmodium Falciparum

Objective:Malaria is an infectious disease of parasitic protozoa transmitted by the vector Anopheles mosquitoes.In 2006,WHO recommended artemisinin-based combination therapy（ACT）as the first-line treatment for non-severe malaria in endemic areas,resulting in a significant reduction in the number of malaria infections worldwide.However,due to the genetic mutation of malaria parasites and drug abuse,malaria parasites have developed resistance to artemisinin.Genome-wide association study（GWAS）revealed that the mutations of multiple gene loci in the genome of P.falciparum were closely related to artemisinin resistance.Among them,Y1133N and V1157L loci mutations of P.falciparum NIF4 protein（Plasmo DB ID:PF3D7＿1012700）were associated with artesunate and dihydroartemisinin resistance.NIF4 belongs to the NIF protein family,which is homologous to the yeast FCP/SCP protein family,and has homologs in all apicomplexa.The NIF protein family in P.falciparum contains four members:NIF2,NIF3,NIF4 and TIM50.Previous findings found that:（1）The CPDc domain of Pf NIF4 contains the core motif"Dx Dx（T/V）"with FCP/SCP family members.（2）Pf NIF4 was expressed in the ring,trophozoite,schizont and gametocyte stages,and located in the nucleus of Plasmodium.（3）Conditional knockdown of Pf NIF4 inhibited merozoite invasion and decreased gametocyte rate.（4）The Pf NIF4 knock-down strain showed decreased sensitivity to artemether（AM）and dihydroartemisinin（DHA）.In view of the above results,the present study aimed to determine the effect of Pf NIF4 knockdown on the susceptibility of artemisinin and its derivatives and drug combinations.To explore the interaction proteins of NIF4 in the erythrocyte,analyze the correlation between Pf NIF2 protein and artemisinin resistance,so as to clarify the molecular mechanism of NIF protein family mediating artemisinin resistance,and to provide theoretical basis for the development of new antimalarial drug targets.Methods:1.The drug IC₅₀and ring-stage survival assay(RSA_0-3h)were used to detect t he sensitivity of Pf NIF4 knockdown strain(Pf NIF4^{i KD})to artemisinin and its derivatives and compatibility drugs.2.RNA-seq was used to analyze the effect of Pf NIF4^{i KD}on th e transcription levels of drug resistance-related genes.3.Selection-linked integration（SLI）technology was used to construct Pf NIF4^{Turbo ID}worm strain with Turbo ID fusion at the C-terminus of endogenous NIF4 protein.The interacting proteins of Pf NIF4 in the eryt hrocyte were analyzed by Co-IP and LC-MS/MS experiments.4.The domin,core motif and sequence conservation of Pf NIF2 protein in various genera of Plasmodium were pre dicted by bioinformatics analysis.5.Based on the SLI technique,the Pf NIF2^GFPstrain wi th a GFP tag at the C-terminus of Pf NIF2 protein and Pf NIF2^KOstrain with Pf NIF2 pr otein knockout were constructed.Meanwhile,to study the role of Ser²⁹⁷and Ser³⁰¹phos phorylation sites of Pf NIF2 protein,The Pf NIF2 full-length（WT）vector,two serine to alanine（S-to-A）and two serine to aspartic acid（S-to-D）vectors were constructed based on p Lyn-FRB-m Cherry-nmd3-BSD plasmid vector.Pf NIF2^WT,Pf NIF2^S-to-Aand Pf NIF2^S-to-Dstrains were obtained by using BSD.6.Pf NIF2^GFPstrain was used to detect the locali zation and expression of Pf NIF2 in the erythrocyte by indirect immunofluorescence assa y（IFA）and Western blot.The Pf NIF2^KOstrain was used to examine the role of Pf NIF2 protein in the growth and development of erythrocytic stage.7.IC₅₀and RSA_0-3hassay s were used to investigate the effect of NIF2 knockout on the sensitivity of P.falciparu m to artemisinin derivatives and their compatibility drugs.8.Phenotypic analysis of Pf NI F2^WT,Pf NIF2^S-to-Aand Pf NIF2^S-to-Dstrains were performed to elucidate the effect of Ser²⁹⁷and Ser³⁰¹phosphorylation site mutations on Pf NIF2 protein function.9.RNA-seq anal ysis of WT and NIF2^KOstrains at the schizont stage were performed to evaluate the eff ect of Pf NIF2 protein on the transcriptional level of schizont stage,and q RT-PCR exper iments were performed to verify the experimental results of RNA-seq analysis.Results:1.Pf NIF4 protein knockdown significantly reduced the ring survival rate of Pf NIF4^{i KD}strains at 700,350,175 and 87.5 n M DHA concentrations,indicating that Pf NIF4^{i KD}increased the susceptibility of the ring（0-3h）parasite to DHA.Treatment with 2.5 m M Glc N significantly reduced the IC₅₀of the Pf NIF4^{i KD}strain to artemether（AM）and dihydroartemisinin（DHA）while the sensitivity of Pf NIF4^{i KD}strain to other drugs,such as quinolones（chloroquine,piperaquine,amodiaquine）and aminolols（mefloquine,lumefantrine,quinine）did not change significantly.2.Pf NIF4 knockdown RNA-seq results showed that 90,422 and 587 up-regulated genes and 416,312 and 778 down-regulated genes were detected in the ring,trophozoite and schizont stages,respectively,indicating that Pf NIF4^{i KD}had a greater influence in the schizont stage.According to GO analysis,genes related to RNA metabolism and proteins related to drug targets were enriched in up-regulated genes,and 52%of genes enriched in down-regulated genes were associated with invasion in the schizont stage.3.The construction of the Pf NIF4^{Turbo ID}strain was verified by PCR identification,and the Pf NIF4-FKBP-Turbo ID fusion protein was mainly located in the nucleus using IFA with anti-FKBP antibody.4.Co-IP and LC-MS/MS results showed that there were 24 Pf NIF4-interacting proteins,including ribosomal subunits,RNA splicing factors（RRP43 and SF3A1）,chromatin related proteins(heterochromatin protein 1[HP1]and GCN5 and AP2 domain transcription factors.5.Bioinformatics analysis showed that the core motif of the NIF2 protein domain in various genera of Plasmodium is DLDE/DT,while the core motif of Pf NIF2 protein in P.falciparum is DLDET.6.Pf NIF2 tagged strains(Pf NIF2^GFP),Pf NIF2 knockout strains(Pf NIF2^KO)and Pf NIF2 phosphorylation site mutant strains(Pf NIF2^WT,Pf NIF2^S-to-A,Pf NIF2^S-to-D)were verified by PCR and Western blot.7.Pf NIF2 was highly expressed at the schizont stage and was distributed as punctate and scattered in the cytoplasm,where it colocalized with the microsomal protein AMA1.8.There was a difference in the infection rate between Pf NIF2^KOstrain and wild type（WT）strain in the third cycle of growth monitoring,but there was no difference in the morphology.There was no significant difference in the number of merozoites and the percentage of schizont rupture between Pf NIF2^KOand wild type（WT）strains.However,the Pf NIF2^KOstrain formed a significantly reduced number of rings.9.Knockout of Pf NIF2 protein increased the susceptibility of P.falciparum to AS but did not enhance the susceptibility to CQ.10.Phenotypic reversion experiments showed that the infection rates of Pf NIF2^S-to-Aand Pf NIF2^S-to-Dmutants were higher than those of Pf NIF2^KObut lower than those of WT and Pf NIF2^WT.11.RNA-seq results of the Pf NIF2 knockout showed that 40 genes were identified to be up-regulated and 182 genes to be down-regulated in the Pf NIF2^KOstrain at the schizont stage compared with the wild-type strain 3D7.12.GO analysis showed that Pf NIF2 protein involved cell components including rhoptry,neck of rhoptry,myosin complex,microneme,apical complex,and host cell membrane.The molecular functions involved include:host cell surface receptor binding,actin filament binding and sulfur compound binding.The biological processes involved include:host environment movement,entry into host,evasion of host immune response,and cell adhesion;therefore,the Pf NIF2 protein affects cell adhesion and evades the host immune response.The results of q RT-PCR showed that the 14 genes analyzed included（AMA1）,RAP2,merozoite surface protein（MSP2/4/7,MSA180）,RON2/3/4/5/6/12,rhoptry associated protein（RAP2）,and rhoptry protein（RON2/3/4/5/6/12）.Among the high molecular weight rhoptry structural proteins（Rho PH2/3）,8 genes（AMA1,MSP7,MSA180,RON2,RON3,RON4,RHOPH2,RHOPH3）were down-regulated,suggesting that Pf NIF2protein may affect the invasion of host erythrocytes by down-regulating the transcription level of invasion related genes.Conclusion:1.Pf NIF4 protein knockdown increased susceptibility to DHA in ring stage（0-3h）.2.In the schizont stage,down-regulated genes in RNA-seq analysis were associated with invasion.3.The Pf NIF4^{Turbo ID}strain was correctly constructed and located in the nucleus and there were 24 interacting proteins identified by Pf NIF4^{Turbo ID}parasite affinity purification.4.The core motif of the Pf NIF2 protein is DLDET,Knockout of Pf NIF2 protein increased the susceptibility of P.falciparum to AS,affect the invasion of erythrocytes by merozoites and mutations at phosphorylation sites 297 and 301 of Pf NIF2 inhibited parasite growth.