Plasmodium knowlesi clinical isolates from Malaysia show extensive diversity and strong differential selection pressure at the merozoite surface protein 7D (MSP7D)

Background High proportion of human cases due to the simian malaria parasite Plasmodium knowlesi in Malaysia has been a cause of concern, as it can be severe and fatal. Merozoite surface protein 7 (MSP7) is a multigene family which forms a non-covalent complex with MSP-1 prior to receptor-ligand recognition in Plasmodium falciparum and thus an important antigen for vaccine development. However, no study has been done in any of the ortholog family members in Plasmodium knowlesi from clinical samples. Thus in this study we investigated the level of polymorphism, haplotypes, and natural selection acting at the pkmsp-7D gene in clinical samples from Malaysia. Methods We analyzed 36 full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) which were orthologous to pvmsp7H (PVX_082680) from clinical isolates of Malaysia available from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection. Results Analysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in P. vivax ( msp7H ). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5’ and 3’ regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5’ and 3’ regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population. Conclusions High genetic diversity with hyper-variable SNPs and strong evidence of positive natural selection at the central region of MSP7D indicated exposure of the region to host immune pressure. Negative selection at the 5’ and the 3’ regions of MSP7D might be because of functional constraints at the unexposed regions during the merozoite invasion process of P. knowlesi . No evidence of geographical clustering among the clinical isolates from Malaysia indicated uniform selection pressure in all populations. These findings highlight the further evaluation of the regions and functional characterization of the protein as a potential blood stage vaccine candidate for P. knowlesi .