Transcription And Expression Analysis Of The Var3Genes Of Plasmodium Falciparum

Malaria is a major threat to human life and health, especially in sub-SaharanAfrica. An estimated300-500million clinical cases occur each year, and660,000deaths. It is a disease that mainly affects immune naive individuals, especially thechildren under5years, and women who under first or second pregnancy. Malariavaccine development has been quite slow due to the complicated life cycle andfrequent antigenic variation of the Plasmodium falciparum （P. falciparum） parasites.According to the previously reported, the pathogenicity of P. falciparum is closelyrelated to its antigenic variation, and P. falciparum erythrocyte membrane protein1（PfEMP1） is the best characterized variant surface antigen. Members of thePfEMP1family mediate the cyto-adherence of infected erythrocytes to host receptors,allowing parasites to avoid splenic clearance and an immense sequence variationwithin the protein family has evolved to escape host antibody responses.The PfEMP1family is encoded by var genes, including fifty-six var genes plusthree small var-like genes. It has previously been shown that var genes can besub-grouped into five groups （UpsA, UpsB, UpsC, UpsD and UpsE） based on theconserved upstream sequence and genomic locations. The var3genes were alsoclassified to group A, however, they are very different from the other var genes interms of sequence length and secondary structures of the encoded proteins. The threevar3genes （PF3D7₀100300, PF3D7₀600400and PF3D7₀937600） are located insubtelomeric regions of chromosome1,6and9, respectively. Var3genes are around4kb, and the secondary structures of the extracellular regions of the encodedproteins contained only one DBLα and one DBLε domain, whereas the otherPfEMP1proteins are composed of multi-DBL-domains and a cystine-richinterdomain region （CIDR）. Thus, var3genes encoded “mini-PfEMP1s” contain avery compressed architecture and an atypical PfEMP1structure. Further, theorthologs of the three var3genes are detected in nearly all parasite isolates, which are as conserved as the var2csa gene that encodes VAR2CSA associated withpregnancy associated malaria （PAM）. However, the function of the “mini-PfEMP1s”encoded by the var3genes is still not known. In this study, the transcription andexpression of the individual var3genes were analyzed by quantitative RT-PCR,Western-blot and immunofluorescence.The transcription of the three var-like genes in the P. falciparum3D7clone wasperformed by quantitative PCR, with the gene encoding seryl-tRNA synthetaseas theinternal control. Results showed that the transcription of the three genes started early aftererythrocyte invasion, which was in a similar way as other var genes, but with a relativelyshort time window. The transcription peaked at16h post invasion and terminatedimmediately afterwards, which was obviously different from the other var genes.To further characterize the expression of the encoded proteins, recombinantproteins and specific antibodies were generated for western-blot andimmunofluorescence.First, the P. falciparum （3D7clone） parasites were cultured in humanerythrocytes according to standard methods. Then the infected erythrocytes at threedevelopmental stages, including ring stage, late trophozoite stage and schizont stage,were further purified by gradient Percoll centrifugation. Western-blot was performedwith anti-ATS （acidic terminal segment）, anti-PF3D7₀937600, anti-PF3D7₀100300and anti-PF3D7₀600400IgG as primary antibody respectively. Results showed thatthe encoded protein of PF3D7₀600400could be detected in both late trophozoitestage and schizont stage; while the encoded protein of PF3D7₀937600andPF3D7₀100300could only by detected in late trophozoite stage and schizent stage,respectively.Surface immunofluorescence assays showed that the distribution of proteinsencoded by the three var-like genes on the infected red blood cells was in a typicalpunctuate pattern of PfEMP1.