Genetic polymorphism of the thrombospondin-related apical merozoite protein (TRAMP) of Plasmodium knowlesi in Malaysia.
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ABSTRACT:
SUBMITTER: Ng YL
PROVIDER: S-EPMC9664425 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Genetic polymorphism of the thrombospondin-related apical merozoite protein (TRAMP) of Plasmodium knowlesi in Malaysia.
Ng Yee Ling YL Lau Yee Ling YL Hamid Mohd Hafizi Abdul MHA Jelip Jenarun J Ooi Choo Huck CH Mudin Rose Nani RN Jaimin Joel Judson JJ Fong Mun Yik MY
Parasitology research 20221115 1
Plasmodium knowlesi is a simian malaria parasite that causes significant zoonotic infections in Southeast Asia, particularly in Malaysia. The Plasmodium thrombospondin-related apical merozoite protein (TRAMP) plays an essential role in the invasion of the parasite into its host erythrocyte. The present study investigated the genetic polymorphism and natural selection of the full length PkTRAMP from P. knowlesi clinical isolates from Malaysia. Blood samples (n = 40) were collected from P. knowles ...[more]
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Project description:The simian malaria parasite Plasmodium knowlesi causes a high number of zoonotic infections in Malaysia. The thrombospondin-related apical merozoite protein (TRAMP) is an essential ligand for binding to the erythrocyte cell surface, whereby it facilitates the invasion. This study is the first attempt to determine the genetic diversity, phylogeography, natural selection and population structure from 97 full-length PkTRAMP gene sequences originating from Malaysia. We found low levels of nucleotide diversity (π~0.0065) for the full-length gene despite samples originating from geographically separated regions (i.e., Peninsular Malaysia and Malaysian Borneo). The rate of synonymous substitutions was significantly higher than that of non-synonymous substitutions, indicating a purifying selection for the full-length gene within the clinical samples. The population genetic analysis revealed that the parasite population is undergoing a significant population expansion. The analysis of the amino acid sequence alignment of 97 PkTRAMP sequences identified 15 haplotypes, of which a major shared haplotype was noted Hap 1 (n = 68, Sarawak; n = 34, Sabah; n = 12, Peninsular Malaysia; n = 22). The phylogenetic analysis using DNA sequences identified two clusters that separated due to geographical distance and three mixed clusters with samples from both Peninsular Malaysia and Malaysian Borneo. Population structure analyses indicated two distinct sub-populations (K = 2). Our findings point to the potential for independent parasite evolution, which could make zoonotic malaria control and elimination even more challenging.
| S-EPMC9689803 | biostudies-literature
Project description:BackgroundThe simian malaria parasite Plasmodium knowlesi has been reported to cause significant numbers of human infection in South East Asia. Its merozoite surface protein-3 (MSP3) is a protein that belongs to a multi-gene family of proteins first found in Plasmodium falciparum. Several studies have evaluated the potential of P. falciparum MSP3 as a potential vaccine candidate. However, to date no detailed studies have been carried out on P. knowlesi MSP3 gene (pkmsp3). The present study investigates the genetic diversity, and haplotypes groups of pkmsp3 in P. knowlesi clinical samples from Peninsular Malaysia.MethodsBlood samples were collected from P. knowlesi malaria patients within a period of 4 years (2008-2012). The pkmsp3 gene of the isolates was amplified via PCR, and subsequently cloned and sequenced. The full length pkmsp3 sequence was divided into Domain A and Domain B. Natural selection, genetic diversity, and haplotypes of pkmsp3 were analysed using MEGA6 and DnaSP ver. 5.10.00 programmes.ResultsFrom 23 samples, 48 pkmsp3 sequences were successfully obtained. At the nucleotide level, 101 synonymous and 238 non-synonymous mutations were observed. Tests of neutrality were not significant for the full length, Domain A or Domain B sequences. However, the dN/dS ratio of Domain B indicates purifying selection for this domain. Analysis of the deduced amino acid sequences revealed 42 different haplotypes. Neighbour Joining phylogenetic tree and haplotype network analyses revealed that the haplotypes clustered into two distinct groups.ConclusionsA moderate level of genetic diversity was observed in the pkmsp3 and only the C-terminal region (Domain B) appeared to be under purifying selection. The separation of the pkmsp3 into two haplotype groups provides further evidence of the existence of two distinct P. knowlesi types or lineages. Future studies should investigate the diversity of pkmsp3 among P. knowlesi isolates in North Borneo, where large numbers of human knowlesi malaria infection still occur.
| S-EPMC5209848 | biostudies-literature
Project description:BACKGROUND: Malaria caused by Plasmodium vivax is a major public health problem worldwide that affects 70-80 million people in the Middle East, Asia, Western Pacific, South America and the Caribbean. Despite its epidemiological importance, few antigens from this parasite species have been characterized to date compared to Plasmodium falciparum, due in part to the difficulties of maintaining an in vitro culture of P. vivax. This study describes the identification of the P. falciparum thrombospondin-related apical merozoite protein homologue in P. vivax (PvTRAMP) and examines its potential to be further evaluated as vaccine candidate. METHODS: The gene encoding PvTRAMP was identified through an extensive search of the databases hosting the genome sequence of P. vivax. Genes adjacent to pvtramp were identified in silico to determine the degree of similarity between the protein sequences encoded by equivalent chromosomic fragments in P. falciparum and Plasmodium knowlesi. The pvtramp gene was amplified from cDNA of P. vivax schizont stages, cloned and expressed in Escherichia coli. Anti-PvTRAMP antisera was obtained by inoculating rabbits with PvTRAMP B cell epitopes produced as synthetic peptides in order to assess its recognition in parasite lysates by Western blot and in intact parasites by indirect immunofluorescence. The recognition of recombinant PvTRAMP by sera from P. vivax-infected individuals living in endemic areas was also assessed by ELISA. RESULTS: The PfTRAMP homologue in P. vivax, here denoted as PvTRAMP, is a 340-amino-acid long antigen encoded by a single exon that could have a potential role in cytoadherence, as indicated by the presence of a thrombospondin structural homology repeat (TSR) domain. According to its transcription and expression profile, PvTRAMP is initially located at the parasite's apical end and later on the parasite surface. Recombinant PvTRAMP is recognized by sera from infected patients, therefore, indicating that it is targeted by the immune system during a natural infection with P. vivax. CONCLUSIONS: The results of this work support conducting further studies with PvTRAMP to evaluate its immunogenicity and protection-inducing ability in the Aotus animal model.
| S-EPMC3020679 | biostudies-literature
Project description:BACKGROUND:Human infections due to the monkey malaria parasite Plasmodium knowlesi is on the rise in most Southeast Asian countries specifically Malaysia. The C-terminal 19 kDa domain of PvMSP1P is a potential vaccine candidate, however, no study has been conducted in the orthologous gene of P. knowlesi. This study investigates level of polymorphisms, haplotypes and natural selection of full-length pkmsp1p in clinical samples from Malaysia. METHODS:A total of 36 full-length pkmsp1p sequences along with the reference H-strain and 40 C-terminal pkmsp1p sequences from clinical isolates of Malaysia were downloaded from published genomes. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 and MEGA 5.0 software. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (F ST ) and population structure of parasite was determined using Arlequin v3.5 and STRUCTURE v2.3.4 software. RESULTS:Comparison of 36 full-length pkmsp1p sequences along with the H-strain identified 339 SNPs (175 non-synonymous and 164 synonymous substitutions). The nucleotide diversity across the full-length gene was low compared to its ortholog pvmsp1p. The nucleotide diversity was higher toward the N-terminal domains (pkmsp1p-83 and 30) compared to the C-terminal domains (pkmsp1p-38, 33 and 19). Phylogenetic analysis of full-length genes identified 2 distinct clusters of P. knowlesi from Malaysian Borneo. The 40 pkmsp1p-19 sequences showed low polymorphisms with 16 polymorphisms leading to 18 haplotypes. In total there were 10 synonymous and 6 non-synonymous substitutions and 12 cysteine residues were intact within the two EGF domains. Evidence of strong purifying selection was observed within the full-length sequences as well in all the domains. Shared haplotypes of 40 pkmsp1p-19 were identified within Malaysian Borneo haplotypes. CONCLUSIONS:This study is the first to report on the genetic diversity and natural selection of pkmsp1p. A low level of genetic diversity and strong evidence of negative selection was detected and observed in all the domains of pkmsp1p of P. knowlesi indicating functional constrains. Shared haplotypes were identified within pkmsp1p-19 highlighting further evaluation using larger number of clinical samples from Malaysia.
| S-EPMC5853062 | biostudies-literature
Project description:BackgroundPlasmodium knowlesi a parasite of the macaques is currently the most common cause of human malaria in Malaysia. The thrombospondin-related adhesive protein (TRAP) gene is pre-erythrocytic stage antigen. It is a well-characterized vaccine candidate in Plasmodium vivax and Plasmodium falciparum, however, no study has been done in the orthologous gene of P. knowlesi. This study investigates nucleotide diversity, haplotypes, natural selection and population differentiation of full-length pktrap genes in clinical samples from Malaysia.MethodsForty full-length pktrap sequences from clinical isolates of Malaysia along with the reference H-strain were downloaded from published databases. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. McDonald-Kreitman test was conducted using P. vivax and Plasmodium coatneyi as ortholog sequence in DnaSP 5.10 software. Population genetic differentiation index (FST) of parasite populations was determined using Arlequin v3.5. Phylogenetic relationships between trap ortholog genes were determined using MEGA 5.0 software.ResultsComparison of 40 full-length pktrap sequences along with the H-strain identified 74 SNPs (53 non-synonymous and 21 synonymous substitutions) resulting in 29 haplotypes. Analysis of the full-length gene showed that the nucleotide diversity was lower compared to its nearest ortholog pvtrap. Domain-wise analysis indicated that the proline/asparagine rich region had higher nucleotide diversity compared to the von Willebrand factor domain and the thrombospondin-type-1 domain. McDonald-Kreitman test identified that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. knowlesi was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. knowlesi and P. vivax. The von Willebrand factor domain also indicated balancing selection using MK test, however, it did not give significant results when tested with P. coatneyi as an outgroup. Phylogenetic analysis of full-length genes identified three distinct sub-clusters of P. knowlesi, one originating from Peninsular Malaysia and two originating from Malaysian Borneo. High population differentiation values was observed within samples from Peninsular Malaysia and Malaysian Borneo.ConclusionsThis study is the first to report on the genetic diversity and natural selection of full-length pktrap. Low level of genetic diversity was found across the full-length gene of pktrap. Balancing selection of the von Willebrand factor domain indicated that TRAP could be a target in inducing immune response against P. knowlesi infections. However, higher number of samples would be necessary to further confirm the findings.
| S-EPMC6062916 | biostudies-literature
Project description:BackgroundThe merozoite surface protein-1 (MSP-1) gene encodes for a leading malaria vaccine candidate antigen. However, its extensive polymorphic nature represents a major obstacle to the development of a protective vaccine. Previously, a pilot study was carried out to explore the sequence variation of the C-terminal 42 kDa fragment within P. knowlesi MSP-1 gene (PkMSP-142) based on 12 clinical samples; however, further study on an adequate sample size is vital in estimating the genetic diversity of the parasite population.MethodsIn the present study, we included a larger sample size of P. knowlesi (83 samples) covering eight states of Malaysia to determine the genetic polymorphism, natural selection and haplotype groups of the gene fragment coding PkMSP-142. The region flanking PkMSP-142 was amplified by PCR and directly sequenced. Genetic diversity, haplotype diversity, population genetic differentiation and natural selection were determined in order to study the polymorphic characteristic of PkMSP-142.ResultsA high level of genetic diversity (Hd = 0.970 ± 0.007; л = 0.01079 ± 0.00033) was observed among the 83 P. knowlesi samples, confirming the extensive genetic polymorphism exhibited among the P. knowlesi population found in Malaysia. A total of 18 distinct haplotypes with 17 amino acid changes were identified, whereby 15 were new haplotypes. High population differentiation values were observed within samples from Peninsular Malaysia and Malaysian Borneo. The 42 kDa fragments of P. knowlesi from Malaysian Borneo were found to be acting on balancing selection whilst purifying selection was suggested to act on isolates from Peninsular Malaysia. The separation of PkMSP-142 haplotypes into two main groups based on geographical separation has further supported the existence of two distinct P. knowlesi lineages.ConclusionsA high level of genetic diversity was observed among PkMSP-142 in Malaysia, whereby most of the polymorphisms were found within the 33 kDa region. Taken together, these data will be useful in order to understand the nature of P. knowlesi population in Malaysia as well as the design and development of a MSP-142 based knowlesi malaria vaccine.
| S-EPMC6282282 | biostudies-literature
Project description:Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.
| S-EPMC10920329 | biostudies-literature
Project description: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.
| S-EPMC6872184 | biostudies-literature
Project description:BackgroundRecent reports from Thailand reveal a substantial surge in Plasmodium knowlesi cases over the past decades, with a more than eightfold increase in incidence by 2023 compared to 2018. This study investigates temporal changes in genetic polymorphism associated with the escalating transmission of P. knowlesi malaria in Thailand over time using the prominent vaccine candidate, pkmsp1 as a marker.MethodsTwenty-five P. knowlesi samples collected in 2018-2023 were sequenced for the 42-kDa region of pkmsp1 and compared with 24 retrieved sequences in 2000-2009, focusing on nucleotide diversity, natural selection, recombination rate, and population differentiation.ResultsSeven unique haplotypes were identified in recent samples, compared to 15 in earlier samples. Nucleotide and haplotype diversity were lower in recent samples (π = 0.016, Hd = 0.817) than in earlier samples (π = 0.018, Hd = 0.942). Significantly higher synonymous substitution rates were observed in both sample sets (dS-dN = 2.77 and 2.43, p < 0.05), indicating purifying selection and reduced genetic diversity over time. Additionally, 8 out of 17 mutation points were located on predicted B-cell epitopes, suggesting an adaptive response by the parasites to evade immune recognition. Population differentiation analysis using the fixation index (Fst) revealed high genetic differentiation between parasite populations in central and southern Thailand or Malaysia. Conversely, the relatively lower Fst value between southern Thailand and Malaysia suggests a closer genetic relationship, possibly reflecting historical gene flow.ConclusionThis study highlights a decline in genetic diversity and evidence of purifying selection associated with the recently increased incidence of P. knowlesi malaria in Thailand. The minor genetic differentiation between P. knowlesi populations from southern Thailand and Malaysia suggests a shared recent ancestry of these parasites and underscores the need for coordinated efforts between the two countries for the elimination of P. knowlesi.
| S-EPMC11562464 | biostudies-literature
Project description:BackgroundThe C-terminal 42 kDa domain of Plasmodium knowlesi merozoite surface protein 1 (PkMSP1) is a potential asexual blood-stage vaccine candidate, however, only a limited number of clinical isolates have been analysed from Malaysia and no inter-country comparative diversity study has been conducted. In the present study, nucleotide diversity, haplotypes and natural selection levels of pkmsp1 in clinical samples from geographically distinct regions of Malaysia and Thailand were investigated. The overall population structure of the parasite from the region was determined.MethodsEleven full-length pkmsp1 sequences obtained from clinical isolates of Malaysia along with the H-strain were downloaded from the database for domain wise characterization of pkmsp1 gene. Additionally, 76 pkmsp-142 sequences from Thailand and Malaysia were downloaded from the database for intra and inter-population analysis. DnaSP 5.10 and MEGA 5.0 software were used to determine genetic diversity, polymorphism, haplotypes and natural selection. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (FST) of parasites were analysed using Arlequin v3.5.ResultsSequence analysis of 11 full-length pkmsp1 sequences along with the H-strain identified 477 (8.4%) polymorphic sites, of which 107 were singleton sites. The overall diversity observed in the full-length genes were high in comparison to its ortholog pvmsp1 and the 4 variable domains showed extensive size variations. The nucleotide diversity was low towards the pkmsp1-42 compared to the conserved domains. The 19 kDa domain was less diverse and completely conserved among isolates from Malaysian Borneo. The nucleotide diversity of isolates from Peninsular Malaysia and Thailand were higher than Malaysian Borneo. Network analysis of pkmsp1-42 haplotypes showed geographical clustering of the isolates from Malaysian Borneo and grouping of isolates from Peninsular Malaysia and Thailand. Population differentiation analysis indicated high FST values between parasite populations originating from Malaysian Borneo, Peninsular Malaysia and Thailand attributing to geographical distance. Moderate genetic differentiation was observed for parasite populations from Thailand and Peninsular Malaysia. Evidence of population expansion and purifying selection were observed in all conserved domains with strongest selection within the pkmsp1-42 domain.ConclusionsThis study is the first to report on inter country genetic diversity and population structure of P. knowlesi based on msp1. Strong evidence of negative selection was observed in the 42 kDa domain, indicating functional constrains. Geographical clustering of P. knowlesi and moderate to high genetic differentiation values between populations identified in this study highlights the importance of further evaluation using larger number of clinical samples from Southeast Asian countries.
| S-EPMC6267868 | biostudies-literature