Genetic diversity and natural selection in the rhoptry-associated protein 1 (RAP-1) of recent Plasmodium knowlesi clinical isolates from Malaysia

Abstract

BackgroundThe Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasitophorous vacuole following the parasite’s invasion of red blood cells. Although there is some evidence that the protein is recognized by the host’s immune system, study of Plasmodium falciparum RAP-1 (PfRAP-1) suggests that it is not under immune pressure. A previous study on five old (1953–1962) P. knowlesi strains suggested that RAP-1 has limited genetic polymorphism and might be under negative selection. In the present study, 30 recent P. knowlesi isolates were studied to obtain a better insight into the polymorphism and natural selection of PkRAP-1.MethodsBlood samples from 30 knowlesi malaria patients were used. These samples were collected between 2010 and 2014. The PkRAP-1 gene, which contains two exons, was amplified by PCR, cloned into Escherichia coli and sequenced. Genetic diversity and phylogenetic analyses were performed using MEGA6 and DnaSP ver. 5.10.00 programs.ResultsThirty PkRAP-1 sequences were obtained. The nucleotide diversity (π) of exons 1, 2 and the total coding region (0.00915, 0.01353 and 0.01298, respectively) were higher than those of the old strains. Further analysis revealed a lower rate of non-synonymous (dN) than synonymous (dS) mutations, suggesting negative (purifying) selection of PkRAP-1. Tajima’s D test and Fu and Li’s D test values were not significant. At the amino acid level, 22 haplotypes were established with haplotype H7 having the highest frequency (7/34, 20.5 %). In the phylogenetic analysis, two distinct haplotype groups were observed. The first group contained the majority of the haplotypes, whereas the second had fewer haplotypes.ConclusionsThe present study found higher genetic polymorphism in the PkRAP-1 gene than the polymorphism level reported in a previous study. This observation may stem from the difference in sample size between the present (n = 30) and the previous (n = 5) study. Synonymous and non-synonymous mutation analysis indicated purifying (negative) selection of the gene. The separation of PkRAP-1haplotypes into two groups provides further evidence to the postulation of two distinct P. knowlesi types or lineages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1127-7) contains supplementary material, which is available to authorized users.

Genetic diversity in the C-terminus of merozoite surface protein 1 among Plasmodium knowlesi isolates from Selangor and Sabah Borneo, Malaysia

“…This was similar to the results for Pk-1-msp-42 reported by Pacheco et al, (2012) whereby the haplotype diversity of their P. knowlesi-infected orangutan samples was 0.0103 ± 0.0031. In addition, our result was also found to be highly diverse and similar to other P. knowlesi functional genes, such as Pk-DBPαII (Hd: 0.986 ± 0.008; π: 0.013 ± 0.002) (Fong et al, 2014), Pk-RAP-1 (Hd: 0.995 ± 0.009; π: 0.01298 ± 0.00091) (Rawa et al, 2016). However, this diversity was less than previously reported previously for both P. falciparum (see Mehrizi et al, 2008;Zamani et al, 2009;Pan et al, 2010;Viputtigul et al, 2013) and P. vivax (see Dias et al, 2011;Kang et al, 2012), which means that P. knowlesi MSP-142 appears to be less diverse than MSP-142 of P. falciparum and P. vivax.…”

Genetic clustering and polymorphism of the merozoite surface protein-3 of Plasmodium knowlesi clinical isolates from Peninsular Malaysia

“…The nucleotide diversity (π: 0.046 ± 0.011) was found to be high when compared to other P. knowlesi functional genes [39–41], considering that most of the haplotypes discovered in this study were unique. A similar observation has also been reported for other merozoite surface antigens such as eba175 , and this suggests that even where functional constraints exist, a range of haplotypes can still occur [45].…”

“…Nucleotide diversity (π: 0.046 ± 0.011) for the full length sequence was found to be several times higher compared to other P. knowlesi functional genes such as PkDBPαII (π: 0.012) [39], PkAMA-1 (π: 0.00501) [40] and PkRAP-1 (π: 0.01298) [41]. Diversity for Domain B (π: 0.067 ± 0.025) was found to be higher than that for Domain A (π: 0.039 ± 0.002).…”

Clustering and genetic differentiation of the normocyte binding protein (nbpxa) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia and Malaysia Borneo

“…The average pairwise nucleotide diversity per site (π) for Pknbpxa was 0.02186. This nucleotide diversity level is higher than the diversity of some other P. knowlesi functional genes such as PkDBPαII (π = 0.012) [ 23 ], PkAMA-1 (π = 0.00501) [ 24 ] and PkRAP-1 (π = 0.01298) [ 25 ]. Phylogenetic trees constructed using Bayesian method showed that the sequences were divided according to Pknbpxa cluster types (Fig.…”