Genetic diversity, haplotypes and allele groups of Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia

Background: The monkey malaria parasite Plasmodium knowlesi is now recognized as the fifth species of Plasmodium that can cause human malaria. Like the region II of the Duffy binding protein of P. vivax (PvDBPII), the region II of the P. knowlesi Duffy binding protein (PkDBP alpha II) plays an essential role in the parasite's invasion into the host's erythrocyte. Numerous polymorphism studies have been carried out on PvDBPII, but none has been reported on PkDBP alpha II. In this study, the genetic diversity, haplotyes and allele groups of PkDBP alpha II of P. knowlesi clinical isolates from Peninsular Malaysia were investigated. Methods: Blood samples from 20 knowlesi malaria patients and 2 wild monkeys (Macaca fascicularis) were used. These samples were collected between 2010 and 2012. The PkDBP alpha II region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and haplotypes of PkDBP alpha II were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes. Results: Fifty-three PkDBP alpha II sequences from human infections and 6 from monkeys were obtained. Comparison at the nucleotide level against P. knowlesi strain H as reference sequence showed 52 synonymous and 76 nonsynonymous mutations. Analysis on the rate of these mutations indicated that PkDBP alpha II was under purifying (negative) selection. At the amino acid level, 36 different PkDBP alpha II haplotypes were identified. Twelve of the 20 human and 1 monkey blood samples had mixed haplotype infections. These haplotypes were clustered into 2 distinct allele groups. The majority of the haplotypes clustered into the large dominant group. Conclusions: Our present study is the first to report the genetic diversity and natural selection of PkDBP alpha II. Hence, the haplotypes described in this report can be considered as novel. Although a high level of genetic diversity was observed, the PkDBP alpha II appeared to be under purifying selection. The distribution of the haplotypes was skewed, with one dominant major and one minor group. Future study should investigate PkDBP alpha II of P. knowlesi from Borneo, which hitherto has recorded the highest number of human knowlesi malaria.