Abstract
Human infections due to the monkey malaria parasite Plasmodium knowlesi are increasingly being reported from Malaysia. The parasite causes high parasitaemia, severe and fatal malaria in humans thus there is a need for urgent measures for its control. The MSP4 is a potential vaccine candidate, which is well studied in Plasmodium falciparum and Plasmodium vivax; however, no study has been conducted in the orthologous gene of P. knowlesi. In this study, we investigated the level of polymorphisms, haplotypes, natural selection and population structure of full-length pkmsp4 in 32 clinical samples from Malaysian Borneo along with 4 lab-adapted strains. We found low levels of polymorphism across the gene with exon I showing higher diversity than the exon II. The C- terminal epidermal growth factor (EGF) domains and GPI-anchored region within exon II were mostly conserved with only 2 non-synonymous substitutions. Although 21 amino acid haplotypes were found, the frequency of mutation at the majority of the polymorphic positions was low. We found evidence of negative selection at the exon II of the gene indicating existence of functional constraints. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. High population differentiation values were observed within parasite populations originating from Malaysian Borneo (Kapit, Sarikei and Betong) and laboratory-adapted strains obtained from Peninsular Malaysia and Philippines indicating distinct population structure. This is the first study to genetically characterize the full-length msp4 gene from clinical isolates of P. knowlesi from Malaysia and thus would be very useful for future rational vaccine studies. Further studies with higher number of samples and functional characterization of the protein will be necessary.
Highlights
Plasmodium knowlesi, a zoonotic malaria parasite of long-tailed and pig-tailed macaques is considered as the fifth Plasmodium species infecting humans and the most common cause of malaria in Malaysia
The signal peptide of the PkMSP4 protein was detected between amino acid positions 22 to 26 by the SignalP server S1 Fig. Alignment and comparison of deduced amino acid sequences between the lab-adapted strains and the clinical isolates from Sarawak showed that there was an amino acid substitution from arginine to cysteine (R33C) in all the clinical isolates (n = 34) from Sarawak Fig 1
All six cysteine residues within the epidermal growth factor (EGF)-domain of exon II were conserved in the clinical isolates as well in the lab-adapted strains from Peninsular Malaysia and Philippines
Summary
Plasmodium knowlesi, a zoonotic malaria parasite of long-tailed and pig-tailed macaques is considered as the fifth Plasmodium species infecting humans and the most common cause of malaria in Malaysia. Death due to P. knowlesi malaria has been reported in Sarawak and Sabah of Malaysian Borneo and rapid increase in parasitaemia has been shown to be associated with severe malaria and in some cases fatal in Malaysian Borneo [8,9]. Genetic studies and genomic studies on P. knowlesi clinical isolates from Malaysia have identified at least 3 sub-populations with their overall diversity even higher than P. falciparum and P. vivax, 2 of the populations were associated with primary primate hosts and one with geographical location [1,10,11,12]. Knowlesi from clinical isolates and macaques identified two distinct clusters which clustered geographically to Peninsular Malaysia and Malaysian Borneo [13]
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