| Background and purposeMalaria is one of the three major infectious diseases in the world,which is a serious threat to human health.Among Plasmodium species that infect humans,Plasmodium falciparum(P.falciparum)causes the highest mortality rate.Although the malaria RTS,S vaccine is available,there are problems with its short protection and weak protection for children under 5 years of age.Besides,increased drug resistance in Plasmodium and Anopheles also pose a new challenge to the malaria prevention and control.Therefore,further screening of new malaria vaccine and drug targets is urgent.The blood stage is the main stage of the appearance of the clinical symptoms of malaria.In the blood stage,merozoite surface proteins(MSPs)play an important role in invading erythrocytes and regulating host immune response,which are potential targets for the malaria vaccine and drug design.MSP8 is an important member of the merozoite surface proteins.In vitro and in vivo experiments of P.falciparum and P.yoelii(Plasmodium yoelii)showed that anti-MSP8 antibody could effectively inhibit Plasmodium invasion of RBC(Red blood cell)and protect the host from infection.However,the specific mechanisms by which MSP8 binds to erythrocytes and regulates immune response remain unclear.This study aims to screen and identify the erythrocyte membrane receptor bound to MSP8,explore the role of MSP8 protein in Plasmodium invasion,and explore the influence of MSP8 protein on the host immune system,so as to provide more efficient strategies and ideas for malaria vaccine development.MethodsThe binding of MSP8 to the erythrocyte membrane was preliminarily verified by the rosette assay,and the erythrocyte membrane receptor bound to MSP8 was screened by the tandem affinity purification binding mass spectrometry.The recombinant proteins of Band 3extracellular ring regions were expressed by E.coli expression system,and the interaction between MSP8 and Band 3 extracellular ring regions was verified by pull down and ELISA experiments.The interaction of Band 3 with the natural Plasmodium MSP8 protein was verified using immunomagnetic beads.The effect of MSP8 on the invasion of P.yoelii 17 XL parasites was evaluated by immunizing mice with Py MSP8 protein.The inhibitory effect of anti-Pf MSP8 and Band 3-L5 antibodies on the invasion of P.falciparum parasites was evaluated by in vitro invasion inhibition assay.The msp8 gene of P.yoelii 17 XL was knocked out using CRISPR-Cas9 gene editing technology,and the deletion of MSP8 gene and protein were verified by PCR and Western blot.Mice were infected with the msp8 knockout strain of P.yoelii 17XL(ΔMSP8 P.yoelii 17XL),and the parasitemia,survival rate,ratio of spleen to body and HE staining results of spleen sections of them were observed.Transcriptomic analysis was performed on the spleen of P.yoelii 17 XL infected mice,and differential genes were screened.q RT-PCR and Western blot were used to verify the expression level of differential genes.It was found that the expression level of CCL19 in the spleen of the mice infected with ΔMSP8 P.yoelii 17 XL was higher than that of the mice infected with WT P.yoelii 17 XL.Therefore,adeno-associated virus was used to decrease the expression level of CCR7 in the mice,and then the mice were infected with ΔMSP8 P.yoelii 17 XL strain to observe the parasitemia and survival rate.The transcription levels of malaria-related cytokines in the spleen of P.yoelii 17XL-infected mice were detected by q RT-PCR,and the concentration of the cytokine in the serum were detected by ELISA.ResultsThe results of rosette assay showed that MSP8 could bind to the erythrocyte membrane.Further through tandem affinity purification binding mass spectrometry,the erythrocyte membrane receptor bound to MSP8 was screened to be Band 3.The interaction between MSP8 and Band 3-L5 and L6 recombinant proteins was demonstrated by pull down and ELISA.Using immunomagnetic beads,only Band 3-L5 was able to bind to the native Plasmodium MSP8 protein.After immunizing mice with Py MSP8 protein,the mice could resist the attack of P.yoelii 17 XL strain and the survival rate was increased.In addition,in vitro invasion inhibition experiments demonstrated that both anti-Pf MSP8 and anti-Band 3-L5 antibodies could inhibit the invasion of P.falciparum.ΔMSP8 P.yoelii 17 XL was obtained using CRISPR-Cas9 gene editing technology.The deletion of pymsp8 gene and Py MSP8 protein in ΔMSP8 P.yoelii 17 XL parasites was verified by PCR and Western blot.The maximum parasitemia of the mice infected with ΔMSP8 P.yoelii 17 XL reached 27.42% on the 6th day after infection,while the maximum parasitemia of mice infected with wild type(WT)P.yoelii 17 XL reached 94.13%.In addition,the mice infected with ΔMSP8 P.yoelii17 XL recovered spontaneously,while the mice infected with WT P.yoelii 17 XL died.And the damage degree of the spleen of the ΔMSP8 P.yoelii 17XL-infected mice was lower than that of the WT P.yoelii 17XL-infected mice.Transcriptomic analysis and q RT-PCR showed that the transcription level of CCL19 in the spleen of the mice infected with ΔMSP8 P.yoelii17 XL was significantly higher than that of the mice infected with WT P.yoelii 17 XL,and the difference was tissue-specific.The expression level of CCL19 protein in the spleen of the mice infected with ΔMSP8 P.yoelii 17 XL was significantly higher than that of mice infected with WT P.yoelii 17 XL by Western blot analysis.After reducing the expression level of CCL19 receptor-CCR7 in mice and infecting them with ΔMSP8 P.yoelii 17 XL strain,the mortality of mice was significantly increased,and the expression level of CCR7 protein in the spleen of dead mice was significantly lower than that of self-healing mice.In addition,the transcription level of IFN-γ in the spleen and the content of IFN-γ in the serum of the mice infected with ΔMSP8 P.yoelii 17 XL were higher than those of the mice infected with WT P.yoelii 17 XL.ConclusionsMSP8 could bind to Band 3-L5 to play an important role in the invasion of parasites.In addition,the absence of MSP8 led to the self-healing of the P.yoelii 17 XL infected mice by increasing the expression of CCL19 in the spleen,which then stimulated the recruitment of immune cells expressing CCR7 protein to release IFN-γ cytokines,thereby killing parasites.Therefore,MSP8 might have an inhibitory effect on the host immune response to anti-malarial infection. |
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