Genetic Diversity and Population Genetic Structure Analysis of Plasmodium knowlesi Thrombospondin-Related Apical Merozoite Protein (TRAMP) in Clinical Samples.

The simian malaria parasite Plasmodium knowlesi causes a high number of zoonotic infections in Malaysia. The thrombospondin-related apical merozoite protein (TRAMP) is an essential ligand for binding to the erythrocyte cell surface, whereby it facilitates the invasion. This study is the first attempt to determine the genetic diversity, phylogeography, natural selection and population structure from 97 full-length PkTRAMP gene sequences originating from Malaysia. We found low levels of nucleotide diversity (π~0.0065) for the full-length gene despite samples originating from geographically separated regions (i.e., Peninsular Malaysia and Malaysian Borneo). The rate of synonymous substitutions was significantly higher than that of non-synonymous substitutions, indicating a purifying selection for the full-length gene within the clinical samples. The population genetic analysis revealed that the parasite population is undergoing a significant population expansion. The analysis of the amino acid sequence alignment of 97 PkTRAMP sequences identified 15 haplotypes, of which a major shared haplotype was noted Hap 1 (n = 68, Sarawak; n = 34, Sabah; n = 12, Peninsular Malaysia; n = 22). The phylogenetic analysis using DNA sequences identified two clusters that separated due to geographical distance and three mixed clusters with samples from both Peninsular Malaysia and Malaysian Borneo. Population structure analyses indicated two distinct sub-populations (K = 2). Our findings point to the potential for independent parasite evolution, which could make zoonotic malaria control and elimination even more challenging.

Genetic Diversity and Population Genetic Analysis of Plasmodium falciparum Thrombospondin Related Anonymous Protein (TRAP) in Clinical Samples from Saudi Arabia.

The thrombospondin related anonymous protein (TRAP) is considered one of the most important pre-erythrocytic vaccine targets. Earlier population genetic studies revealed the TRAP gene to be under strong balancing natural selection. This study is the first attempt to analyze genetic diversity, natural selection, phylogeography and population structure in 199 clinical samples from Saudi Arabia using the full-length PfTRAP gene. We found the rate of nonsynonymous substitutions to be significantly higher than that of synonymous substitutions in the clinical samples, indicating a strong positive or diversifying selection for the full-length gene and the Von Willebrand factor (VWF). The nucleotide diversity was found to be π~0.00789 for the full-length gene; however, higher nucleotide diversity was observed for the VWF compared to the thrombospondin repeat region (TSP). Deduction of the amino acid sequence alignment of the PNP repeat region in the Saudi samples revealed six genotypes characterized by tripeptide repeat motifs (PNP, ANP, ENP and SNP). Haplotype network, population structure and population differentiation analyses indicated four distinct sub-populations in spite of the low geographical distance between the sampling sites. Our results suggest the likeliness of independent parasite evolution, creating opportunities for further adaptation, including host transition, and making malaria control even more challenging.