旭硝子財団助成研究成果報告2018

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86Chaturong PutaporntipSequence diversity and evolution of the apical membrane antigen 1 gene (AMA1) of Plasmodium vivax-related primate malaria (Plasmodium knowlesi, P. cynomolgi and P. inui)(Project 2016)64旭硝子財団　助成研究成果報告（2018）TLR3-activated IDO expression, suggesting the multiple pathways in the inductive mechanism. Our finding indicated that activated TLR3 could encourage the stem cell and immunosuppressive properties of PDLSCs. Apical membrane antigen-1 (AMA1) of malaria parasites plays an important role in erythrocyte entry; thereby, it has been considered to be a vaccine candidate. The AMA1 genes of both Plasmodium falciparum and P. vivax (PfAMA1 and PvAMA1) contain 3 extracellular domains (DI, DII and DIII) and display sequence diversity among isolates. Contrasting selective pressures have been observed between these genes, in which the former seems to have evolved under positive selection while the latter exhibited population-specific differential selection profiles. This study aims to gain further insights into the extent of sequence diversity and evolution of this locus among P. knowlesi (PkAMA1, n=24), P. cynomolgi (PcyAMA1, n=9) and P. inui (PiAMA1, n=70), the simian malaria parasites known to be transmissible to humans under natural or experimental conditions. Analysis of the complete coding sequences of PkAMA1, PcyAMA1 and PiAMA1 has revealed 90, 144 and 176 nucleotide substitutions, resulting in 30, 84 and 47 amino acid changes, respectively. Meanwhile, analysis of PvAMA1 from 281 isolates (Thailand, n=231; Brazil, n=39; Indonesia, n=11) has shown 63 nucleotide substitutions causing 58 amino acid changes. On the other hand, 71 nucleotide substitutions were detected among 39 PfAMA1 sequences, leading to 61 amino acid changes. Nucleotide diversity of PcyAMA1 (¶±S.E.=0.0176±0.0077) significantly exceeded that of PvAMA1 (0.0077±0.0001) whereas PkAMA1 and PiAMA1 exhibited similar levels of nucleotide diversity (0.0047±0.0002 and 0.0050±0.0009, respectively). The levels of haplotype diversity of AMA1 of these simian malaria parasites and P. vivax were greater than 0.9, indicating that most isolates bore different sequences without notable skew towards particular ones. Significantly greater values of the rate of synonymous nucleotide substitutions per synonymous site (dS) in comparison with that of nonsynonymous substitutions per nonsynonymous site (dN) were observed in domain I and C-terminal portion spanning transmembrane and cytoplasmic tail of PkAMA1, and in all domains of PcyAMA1. However, no deviation from selective neutrality occurred in all domains in PiAMA1 and PvAMA1. Consistently, codon-specific analyses revealed that a number of codons have evolved under purifying selection and predominantly occurred in extracellular domains of PvAMA1 and its related simian malaria parasites. However, pervasive and episodic positively selected codons have been detected predominantly in domain 3 and the C-terminal part of these proteins, albeit relatively rare. By contrast, positive selection as evidenced by significantly greater dN than dS was observed in all 3 extracellular domains of PfAMA1. Evidence of intragenic recombination was observed across the AMA1 loci of these simian malaria parasites. The evolution of malarial AMA1 seems to have co-diverged with speciation of the genus Plasmodium in which congruent clustering of P. vivax and P. vivax-related species were obtained when phylogenetic trees were inferred from the AMA1 sequences, 18S rRNA and mitochondrial cytochrome b genes. Meanwhile, P. knowlesi populations from different geographic areas exhibited significant genetic differentiation as viewed from the fixation indices. Network analysis inferred from the PkAMA1 sequences has suggested two potential modes of transmission, i.e. a previously perceived cross-transmission from macaque natural hosts to humans and the other anthroponotic transmission. Taken together, besides being a strong candidate for human malaria vaccines, the AMA1 sequences of simian malaria parasites provide important information on the evolution and mode of transmission of simian malaria from macaque natural hosts to humans.