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The Duffy binding protein (PkDBP?II) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

ABSTRACT: The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBP?II) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBP?II haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated.The PkDBP?II of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes.Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBP?II. Interestingly, the PkDBP?II of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBP?II haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes.This study is the first report of phenotypic difference between PkDBP?II haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

SUBMITTER: Lim KL

PROVIDER: S-EPMC5553923 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

Lim Khai Lone KL Amir Amirah A Lau Yee Ling YL Fong Mun Yik MY

Malaria journal 20170811 1

<h4>Background</h4>The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaqu ...[more]

PMID: 28800732

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Project description:Plasmodium knowlesi is one of the monkey malaria parasites that can cause human malaria. The Duffy binding protein of P. knowlesi (PkDBP?II) is essential for the parasite's invasion into human and monkey erythrocytes. A previous study on P. knowlesi clinical isolates from Peninsular Malaysia reported high level of genetic diversity in the PkDBP?II. Furthermore, 36 amino acid haplotypes were identified and these haplotypes could be separated into allele group I and allele group II. In the present study, the PkDBP?II of clinical isolates from the Malaysian states of Sarawak and Sabah in North Borneo was investigated, and compared with the PkDBP?II of Peninsular Malaysia isolates.Blood samples from 28 knowlesi malaria patients were used. These samples were collected between 2011 and 2013 from hospitals in North Borneo. The PkDBP?II region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and phylogenetics of PkDBP?II haplotypes were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes.Forty-nine PkDBP?II sequences were obtained. Comparison at the nucleotide level against P. knowlesi strain H as reference sequence revealed 58 synonymous and 102 non-synonymous mutations. Analysis on these mutations showed that PkDBP?II was under purifying (negative) selection. At the amino acid level, 38 different PkDBP?II haplotypes were identified. Twelve of the 28 blood samples had mixed haplotype infections. Phylogenetic analysis revealed that all the haplotypes were in allele group I, but they formed a sub-group that was distinct from those of Peninsular Malaysia. Wright's FST fixation index indicated high genetic differentiation between the North Borneo and Peninsular Malaysia haplotypes.This study is the first to report the genetic diversity and natural selection of PkDBP?II of P. knowlesi from Borneo Island. The PkDBP?II haplotypes found in this study were distinct from those from Peninsular Malaysia. This difference may not be attributed to geographical separation because other genetic markers studied thus far such as the P. knowlesi circumsporozoite protein gene and small subunit ribosomal RNA do not display such differentiation. Immune evasion may possibly be the reason for the differentiation.

Project description:The zoonotic malaria parasite Plasmodium knowlesi has become an emerging threat to South East Asian countries particular in Malaysia. A recent study from Sarawak (Malaysian Borneo) discovered two distinct normocyte binding protein xa (Pknbpxa) types of P. knowlesi. In the present study, the Pknbpxa of clinical isolates from Peninsular Malaysia and Sabah (Malaysian Borneo) were investigated for the presence of Pknbpxa types and natural selection force acting on the gene.Blood samples were collected from 47 clinical samples from Peninsular Malaysia (n = 35) and Sabah (Malaysian Borneo, n = 12) were used in the study. The Pknbpxa gene was successfully amplified and directly sequenced from 38 of the samples (n = 31, Peninsular Malaysia and n = 7, Sabah, Malaysian Borneo). The Pknbpxa sequences of P. knowlesi isolates from Sarawak (Malaysian Borneo) were retrieved from GenBank and included in the analysis. Polymorphism, genetic diversity and natural selection of Pknbpxa sequences were analysed using DNAsp v 5.10, MEGA5. Phylogentics of Pknbpxa sequences was analysed using MrBayes v3.2 and Splits Tree v4.13.1. The pairwise F ST indices were used to determine the genetic differentiation between the Pknbpxa types and was calculated using Arlequin 3.5.1.3.Analyses of the sequences revealed Pknbpxa dimorphism throughout Malaysia indicating co-existence of the two types (Type-1 and Type-2) of Pknbpxa. More importantly, a third type (Type 3) closely related to Type 2 Pknbpxa was also detected. This third type was found only in the isolates originating from Peninsular Malaysia. Negative natural selection was observed, suggesting functional constrains within the Pknbpxa types.This study revealed the existence of three Pknbpxa types in Malaysia. Types 1 and 2 were found not only in Malaysian Borneo (Sarawak and Sabah) but also in Peninsular Malaysia. A third type which was specific only to samples originating from Peninsular Malaysia was discovered. Further genetic studies with a larger sample size will be necessary to determine whether natural selection is driving this genetic differentiation and geographical separation.

Project description: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?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?II. In this study, the genetic diversity, haplotyes and allele groups of PkDBP?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?II region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and haplotypes of PkDBP?II were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes. RESULTS: Fifty-three PkDBP?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?II was under purifying (negative) selection. At the amino acid level, 36 different PkDBP?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?II. Hence, the haplotypes described in this report can be considered as novel. Although a high level of genetic diversity was observed, the PkDBP?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?II of P. knowlesi from Borneo, which hitherto has recorded the highest number of human knowlesi malaria.

Dataset Information

The Duffy binding protein (PkDBP?II) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

Publications

The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

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