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.
Highlights
The Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasito‐ phorous vacuole following the parasite’s invasion of red blood cells
P. knowlesi infection in each patient was confirmed by microscopic examination of Giemsa-stained thin and thick blood smears and polymerase chain reaction (PCR) amplification using diagnostic primers [12]
Unlike the findings on the old strains [π: 0.0082, 0.0123, 0.0076], the present study found relatively higher diversity among the P. knowlesi RAP-1 (PkRAP-1) of the recent isolates [π: 0.01298], and diversity was much higher in exon 2
Summary
The Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasito‐ phorous vacuole following the parasite’s invasion of red blood cells. Polymorphic proteins are often favoured by positive selection, in which selective forces, such as immune responses and drugs, drive the genes expressing these antigenic proteins to accumulate mutations and maintain them in the population [2] This strategy enables the parasite to manifest antigenically different alleles to thwart the host’s immune response. The Plasmodium merozoite invasion of red blood cells involves binding, apical orientation and secretion of apical organelle contents known as rhoptries, micronemes and dense granules [3,4,5]. Proteins in these organelles have been implicated in key aspects of invasion. RAP-1 forms a complex with smaller proteins, RAP-2 or RAP-3, and deletion of the RAP-1 gene results in mistargeting of RAP-2 to the rhoptries [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
