Displaying publications 1 - 20 of 38 in total

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  1. Fulltext Characterisation of a truncated Toxoplasma gondii surface antigen 2 (SAG2) secreted by the methylotrophic yeast Pichia pastoris
    Lau YL, Shamilah H, Fong MY
    Trop Biomed, 2006 Dec;23(2):186-93.
    PMID: 17322821 MyJurnal
    A truncated form of surface antigen 2 (SAG2) of the protozoan parasite Toxoplasma gondii was cloned and expressed in the methylotrophic yeast Pichia pastoris. This recombinant antigen, designated as recSAG2-N, contained only the N-terminal half of the native SAG2. The recSAG2-N was secreted by the Pichia pastoris into the culture supernatant, and it was harvested by using the trichloroacetic acid precipitation method. Specificity of recSAG2-N was evaluated in western blot assays. Fifty human serum samples, including 32 from confirmed cases of toxoplasmosis, were tested. Results from the assays showed that recSAG2-N reacted with sera from the toxoplasmosis cases only. In vivo experiments showed that serum from mice which received recSAG2-N reacted with the native SAG2 of T. gondii.
    Matched MeSH terms: Protozoan Proteins/metabolism*
  2. Experimental Study on Plasmodium knowlesi Normocyte Binding Protein Xa Region II (PkNBPXaII) for Erythrocyte Binding
    Wong KC, Lai MY, De Silva JR, Cheong FW, Fong MY, Lau YL
    Trop Biomed, 2021 Jun 01;38(2):143-148.
    PMID: 34172703 DOI: 10.47665/tb.38.2.051
    Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.
    Matched MeSH terms: Protozoan Proteins/metabolism*
  3. Polymorphism in the pvcrt-o and pvmdr-1 genes of Plasmodium vivax associated with a better prognosis for malaria
    Alves-Junior ER, Dombroski TCD, Nakazato L, Dutra V, Neves-Costa JD, Katsuragawa TH, et al.
    Trop Biomed, 2022 Sep 01;39(3):421-427.
    PMID: 36214439 DOI: 10.47665/tb.39.3.012
    The early molecular identification of strains of Plasmodium vivax that have a worse prognosis is important to stratify the risk of complications and choice of conduct made by medical teams. Thus, the aim of the present study was to associate the presence of polymorphisms in the pvmdr-1 and pvcrt-o resistance genes of P. vivax in patients with better or worse prognosis. This cross-sectional epidemiological study was conducted based on data obtained from the records of 120 patients diagnosed with malaria in the Brazilian Amazon. The T958M and F1076L mutations of the pvmdr-1 gene had a frequency of 3.3 and 4.2%, respectively, and primo-infected patients had a 17 times greater chance of being infected with protozoa with the T958M mutation compared to patients with previous episodes. Regarding pvcrt-o, the C393T and T786C polymorphisms had a frequency of 14.2 and 3.3%, respectively, and self-declared white patients had a 3.1 times greater chance of being infected with protozoa with the C393T polymorphism. In addition, patients with this pvcrt-o polymorphism had lower concentrations of C-reactive protein, indicating a better prognosis. These data present clues of genetic indicators useful for assessing the virulence of the parasite and the prognosis of patients with vivax malaria.
    Matched MeSH terms: Protozoan Proteins/metabolism
  4. Cloning and expression of Toxoplasma gondii dense granule antigen 2 (GRA2) gene by Pichia pastoris
    Lau YL, Fong MY, Idris MM, Ching XT
    Southeast Asian J. Trop. Med. Public Health, 2012 Jan;43(1):10-6.
    PMID: 23082548
    Detection of Toxoplasma gondii infection is essential in pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several Toxoplasma antigens, including dense granule antigens (GRAs) has high potential as diagnostic reagents. In the present study, we produced GRA2 using Pichia pastoris system. RNA of T. gondii RH strain tachyzoite was used as a template to produce cDNA clones of full-length GRA2 via reverse transcriptase PCR. Amplicons were inserted into pPICZalpha A and the recombinant plasmid transformed into P. pastoris, X-33 strain. The expressed recombinant protein was identified by SDS-PAGE and Western blotting. A recombinant protein of -28 kDa was produced, which could be detected by toxoplasmosis positive human sera indicating that the recombinant protein retained its antigenicity. The present study indicates that P. pastoris-expressed GRA2 should be useful for detection of Toxoplasma infection.
    Matched MeSH terms: Protozoan Proteins/metabolism
  5. Fulltext Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum
    Matsubayashi M, Teramoto-Kimata I, Uni S, Lillehoj HS, Matsuda H, Furuya M, et al.
    J Biol Chem, 2013 Nov 22;288(47):34111-34120.
    PMID: 24085304 DOI: 10.1074/jbc.M113.515544
    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.
    Matched MeSH terms: Protozoan Proteins/metabolism
  6. Fulltext Evaluation of Pichia pastoris-expressed recombinant rhoptry protein 2 of Toxoplasma gondii for its application in diagnosis of toxoplasmosis
    Chang PY, Fong MY, Nissapatorn V, Lau YL
    Am J Trop Med Hyg, 2011 Sep;85(3):485-9.
    PMID: 21896809 DOI: 10.4269/ajtmh.2011.11-0351
    Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZα A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).
    Matched MeSH terms: Protozoan Proteins/metabolism*
  7. Fulltext Two Genetically Distinct Plasmodium knowlesi Duffy Binding Protein Alpha Region II (PkDBPαII) Haplotypes Demonstrate Higher Binding Level to Fy(a+b+) Erythrocytes than Fy(a+b--) Erythrocytes
    Liew CC, Lau YL, Fong MY, Cheong FW
    Am J Trop Med Hyg, 2020 05;102(5):1068-1071.
    PMID: 32189613 DOI: 10.4269/ajtmh.19-0836
    Invasion of human erythrocytes by merozoites of Plasmodium knowlesi involves interaction between the P. knowlesi Duffy binding protein alpha region II (PkDBPαII) and Duffy antigen receptor for chemokines (DARCs) on the erythrocytes. Information is scarce on the binding level of PkDBPαII to different Duffy antigens, Fya and Fyb. This study aims to measure the binding level of two genetically distinct PkDBPαII haplotypes to Fy(a+b-) and Fy(a+b+) human erythrocytes using erythrocyte-binding assay. The binding level of PkDBPαII of Peninsular Malaysian and Malaysian Borneon haplotypes to erythrocytes was determined by counting the number of rosettes formed in the assay. Overall, the Peninsular Malaysian haplotype displayed higher binding activity than the Malaysian Borneon haplotype. Both haplotypes exhibit the same preference to Fy(a+b+) compared with Fy(a+b-), hence justifying the vital role of Fyb in the binding to PkDBPαII. Further studies are needed to investigate the P. knowlesi susceptibility on individuals with different Duffy blood groups.
    Matched MeSH terms: Protozoan Proteins/metabolism
  8. Fulltext Influence of the pfmdr1 Gene on In Vitro Sensitivities of Piperaquine in Thai Isolates of Plasmodium falciparum
    Mungthin M, Watanatanasup E, Sitthichot N, Suwandittakul N, Khositnithikul R, Ward SA
    Am J Trop Med Hyg, 2017 03;96(3):624-629.
    PMID: 28044042 DOI: 10.4269/ajtmh.16-0668
    Piperaquine combined with dihydroartemisinin is one of the artemisinin derivative combination therapies, which can replace artesunate-mefloquine in treating uncomplicated falciparum malaria in Thailand. The aim of this study was to determine the in vitro sensitivity of Thai Plasmodium falciparum isolates against piperaquine and the influence of the pfmdr1 gene on in vitro response. One hundred and thirty-seven standard laboratory and adapted Thai isolates of P. falciparum were assessed for in vitro piperaquine sensitivity. Polymorphisms of the pfmdr1 gene were determined by polymerase chain reaction methods. The mean and standard deviation of the piperaquine IC50 in Thai isolates of P. falciparum were 16.7 ± 6.3 nM. The parasites exhibiting chloroquine IC50 of ≥ 100 nM were significantly less sensitive to piperaquine compared with the parasite with chloroquine IC50 of < 100 nM. No significant association between the pfmdr1 copy number and piperaquine IC50 values was found. In contrast, the parasites containing the pfmdr1 86Y allele exhibited significantly reduced piperaquine sensitivity. Before nationwide implementation of dihydroartemisinin-piperaquine as the first-line treatment in Thailand, in vitro and in vivo evaluations of this combination should be performed especially in areas where parasites containing the pfmdr1 86Y allele are predominant such as the Thai-Malaysian border.
    Matched MeSH terms: Protozoan Proteins/metabolism
  9. Fulltext Entamoeba histolytica: Quantitative Proteomics Analysis Reveals Putative Virulence-Associated Differentially Abundant Membrane Proteins
    Ng YL, Olivos-García A, Lim TK, Noordin R, Lin Q, Othman N
    Am J Trop Med Hyg, 2018 12;99(6):1518-1529.
    PMID: 30298805 DOI: 10.4269/ajtmh.18-0415
    Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.
    Matched MeSH terms: Protozoan Proteins/metabolism
  10. Fulltext The Plasmodium falciparum male gametocyte protein P230p, a paralog of P230, is vital for ookinete formation and mosquito transmission
    Marin-Mogollon C, van de Vegte-Bolmer M, van Gemert GJ, van Pul FJA, Ramesar J, Othman AS, et al.
    Sci Rep, 2018 10 08;8(1):14902.
    PMID: 30297725 DOI: 10.1038/s41598-018-33236-x
    Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.
    Matched MeSH terms: Protozoan Proteins/metabolism*
  11. Fulltext Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials
    Tindall SM, Vallières C, Lakhani DH, Islahudin F, Ting KN, Avery SV
    Sci Rep, 2018 02 06;8(1):2464.
    PMID: 29410428 DOI: 10.1038/s41598-018-20816-0
    Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.
    Matched MeSH terms: Protozoan Proteins/metabolism
  12. Fulltext Coordination of apicoplast transcription in a malaria parasite by internal and host cues
    Kobayashi Y, Komatsuya K, Imamura S, Nozaki T, Watanabe YI, Sato S, et al.
    Proc Natl Acad Sci U S A, 2023 Jul 11;120(28):e2214765120.
    PMID: 37406097 DOI: 10.1073/pnas.2214765120
    The malaria parasite Plasmodium falciparum has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain poorly understood, despite this organelle being crucial for the parasite life cycle. Here, we identify a nuclear-encoded apicoplast RNA polymerase σ subunit (sigma factor) which, along with the α subunit, appears to mediate apicoplast transcript accumulation. This has a periodicity reminiscent of parasite circadian or developmental control. Expression of the apicoplast subunit gene, apSig, together with apicoplast transcripts, increased in the presence of the blood circadian signaling hormone melatonin. Our data suggest that the host circadian rhythm is integrated with intrinsic parasite cues to coordinate apicoplast genome transcription. This evolutionarily conserved regulatory system might be a future target for malaria treatment.
    Matched MeSH terms: Protozoan Proteins/metabolism
  13. Fulltext Plasmodium knowlesi genome sequences from clinical isolates reveal extensive genomic dimorphism
    Pinheiro MM, Ahmed MA, Millar SB, Sanderson T, Otto TD, Lu WC, et al.
    PLoS One, 2015;10(4):e0121303.
    PMID: 25830531 DOI: 10.1371/journal.pone.0121303
    Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology.
    Matched MeSH terms: Protozoan Proteins/metabolism
  14. Fulltext Identification and characterisation of heat shock protein 70 in thermal stressed Blastocystis sp
    Gaythri T, Suresh K, Subha B, Kalyani R
    PLoS One, 2014;9(9):e95608.
    PMID: 25180903 DOI: 10.1371/journal.pone.0095608
    Protistan parasites in order to ensure their viability and demonstrate successful progression in their life cycle need to respond towards various environmental stressors. Blastocystis sp. is known to be the most commonly found intestinal protistan parasite in any human stool surveys and has been incriminated to be responsible for diarrhea and bloating stomach. The present study demonstrates for the first time the presence of HSP70 in subtypes of Blastocystis sp. when the cultures were subjected to temperature of 39 and 41 °C where the growth of parasites was reduced to a minimum to majority being granular forms. The growth of parasites exposed to higher temperatures however doubled compared to the controls when the parasites were re-cultured back at 37 °C. Upon thermal stress at 41 °C, subtype 3 and subtype 5 isolates' growth reached up to 2.97 × 10(6) and 3.05 × 10(6) cells/ml compared to their respective controlled culture tubes at 37 °C which peaked only at 1.34 × 10(6) and 1.70 × 10(6) cells/ml respectively. The designed primer set that amplified Blastocystis sp. subtype 7 HSP70 gene in subtypes 1, 3 and 5 was against a conserved region. The gene was amplified at 318 bp. The multiple sequence alignment showed that the targeted sequence length ranges from 291-295 bp. The pair wise alignment result showed that the sequence identity among the four sequence ranges from 88% to 96%. These findings were further evidenced by the up regulation of HSP70 gene in thermal stressed isolates of subtype 3 and 5 at 41 °C. Higher number of granular forms was significantly found in thermal stressed isolates of subtype 3 and 5 which implicates that this life cycle stage has a role in responding to thermal stress.
    Matched MeSH terms: Protozoan Proteins/metabolism*
  15. In silico identification and validation of a novel hypothetical protein in Cryptosporidium hominis and virtual screening of inhibitors as therapeutics
    Shrivastava AK, Kumar S, Sahu PS, Mahapatra RK
    Parasitol Res, 2017 May;116(5):1533-1544.
    PMID: 28389892 DOI: 10.1007/s00436-017-5430-1
    Computational approaches to predict structure/function and other biological characteristics of proteins are becoming more common in comparison to the traditional methods in drug discovery. Cryptosporidiosis is a major zoonotic diarrheal disease particularly in children, which is caused primarily by Cryptosporidium hominis and Cryptosporidium parvum. Currently, there are no vaccines for cryptosporidiosis and recommended drugs are ineffective. With the availability of complete genome sequence of C. hominis, new targets have been recognized for the development of effective and better drugs and/or vaccines. We identified a unique hypothetical protein (TU502HP) in the C. hominis genome from the CryptoDB database. A three-dimensional model of the protein was generated using the Iterative Threading ASSEmbly Refinement server through an iterative threading method. Functional annotation and phylogenetic study of TU502HP protein revealed similarity with human transportin 3. The model is further subjected to a virtual screening study form the ZINC database compound library using the Dock Blaster server. A docking study through AutoDock software reported N-(3-chlorobenzyl)ethane-1,2-diamine as the best inhibitor in terms of docking score and binding energy. The reliability of the binding mode of the inhibitor is confirmed by a complex molecular dynamics simulation study using GROMACS software for 10 ns in the water environment. Furthermore, antigenic determinants of the protein were determined with the help of DNASTAR software. Our findings report a great potential in order to provide insights in the development of new drug(s) or vaccine(s) for treatment and prophylaxis of cryptosporidiosis among humans and animals.
    Matched MeSH terms: Protozoan Proteins/metabolism*
  16. Fulltext Genetic polymorphism of the thrombospondin-related apical merozoite protein (TRAMP) of Plasmodium knowlesi in Malaysia
    Ng YL, Lau YL, Hamid MHA, Jelip J, Ooi CH, Mudin RN, et al.
    Parasitol Res, 2023 Jan;122(1):195-200.
    PMID: 36378331 DOI: 10.1007/s00436-022-07716-z
    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. knowlesi malaria patients from Peninsular Malaysia and Malaysian Borneo. The PkTRAMP gene was amplified using PCR, followed by cloning into a plasmid vector and sequenced. Results showed that the nucleotide diversity of PkTRAMP was low (π: 0.009). Z-test results indicated negative (purifying) selection of PkTRAMP. The alignment of the deduced amino acid sequences of PkTRAMP of Peninsular Malaysia and Malaysian Borneo revealed 38 dimorphic sites. A total of 27 haplotypes were identified from the amino acid sequence alignment. Haplotype analysis revealed that there was no clustering of PkTRAMP from Peninsular Malaysia and Malaysian Borneo.
    Matched MeSH terms: Protozoan Proteins/metabolism
  17. Genetic polymorphism and clustering of the Plasmodium cynomolgi Duffy binding protein 1 region II of recent macaque isolates from Peninsular Malaysia
    Latif ENM, Noordin NR, Shahari S, Amir A, Lau YL, Cheong FW, et al.
    Parasitol Res, 2024 Jan 19;123(1):105.
    PMID: 38240877 DOI: 10.1007/s00436-024-08125-0
    Plasmodium cynomolgi is a simian malaria parasite that has been increasingly infecting humans. It is naturally present in the long-tailed and pig-tailed macaques in Southeast Asia. The P. cynomolgi Duffy binding protein 1 region II [PcDBP1(II)] plays an essential role in the invasion of the parasite into host erythrocytes. This study investigated the genetic polymorphism, natural selection and haplotype clustering of PcDBP1(II) from wild macaque isolates in Peninsular Malaysia. The genomic DNA of 50 P. cynomolgi isolates was extracted from the macaque blood samples. Their PcDBP1(II) gene was amplified using a semi-nested PCR, cloned into a plasmid vector and subsequently sequenced. The polymorphism, natural selection and haplotypes of PcDBP1(II) were analysed using MEGA X and DnaSP ver.6.12.03 programmes. The analyses revealed high genetic polymorphism of PcDBP1(II) (π = 0.026 ± 0.004; Hd = 0.996 ± 0.001), and it was under purifying (negative) selection. A total of 106 haplotypes of PcDBP1(II) were identified. Phylogenetic and haplotype analyses revealed two groups of PcDBP1(II). Amino acid length polymorphism was observed between the groups, which may lead to possible phenotypic difference between them.
    Matched MeSH terms: Protozoan Proteins/metabolism
  18. Fulltext Sero-diagnostic evaluation of Toxoplasma gondii recombinant Rhoptry antigen 8 expressed in E. coli
    Sonaimuthu P, Fong MY, Kalyanasundaram R, Mahmud R, Lau YL
    Parasit Vectors, 2014;7:297.
    PMID: 24986686 DOI: 10.1186/1756-3305-7-297
    Toxoplasma gondii infects all warm-blooded animals, including humans. Early diagnosis and determining the infective stage are critical for effectively treating immunosuppressed individuals and pregnant women with toxoplasmosis. Among the rhoptry proteins of the parasite, Rhoptry protein 8 (ROP8), is known to be expressed during the early stages of T. gondii infection and is involved in parasitophorous vacuole formation. In this study, we have investigated the diagnostic efficacy of recombinant ROP8 (rROP8).
    Matched MeSH terms: Protozoan Proteins/metabolism*
  19. Fulltext Genetic diversity, haplotypes and allele groups of Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia
    Fong MY, Lau YL, Chang PY, Anthony CN
    Parasit Vectors, 2014;7:161.
    PMID: 24693997 DOI: 10.1186/1756-3305-7-161
    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 (PkDBPαII) 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 PkDBPαII. In this study, the genetic diversity, haplotyes and allele groups of PkDBPαII of P. knowlesi clinical isolates from Peninsular Malaysia were investigated.
    Matched MeSH terms: Protozoan Proteins/metabolism*
  20. Fulltext Erythrocyte-binding assays reveal higher binding of Plasmodium knowlesi Duffy binding protein to human Fya+/b+ erythrocytes than to Fya+/b- erythrocytes
    Fong MY, Cheong FW, Lau YL
    Parasit Vectors, 2018 Sep 26;11(1):527.
    PMID: 30257710 DOI: 10.1186/s13071-018-3118-8
    BACKGROUND: The merozoite of the zoonotic Plasmodium knowlesi invades human erythrocytes via the binding of its Duffy binding protein (PkDBPαII) to the Duffy antigen on the eythrocytes. The Duffy antigen has two immunologically distinct forms, Fya and Fyb. In this study, the erythrocyte-binding assay was used to quantitatively determine and compare the binding level of PkDBPαII to Fya+/b+ and Fya+/b- human erythrocytes.

    RESULTS: In the erythrocyte-binding assay, binding level was determined by scoring the number of rosettes that were formed by erythrocytes surrounding transfected mammalian COS-7 cells which expressed PkDBPαII. The assay result revealed a significant difference in the binding level. The number of rosettes scored for Fya+/b+ was 1.64-fold higher than that of Fya+/b- (155.50 ± 34.32 and 94.75 ± 23.16 rosettes, respectively; t(6) = -2.935, P = 0.026).

    CONCLUSIONS: The erythrocyte-binding assay provided a simple approach to quantitatively determine the binding level of PkDBPαII to the erythrocyte Duffy antigen. Using this assay, PkDBPαII was found to display higher binding to Fya+/b+ erythrocytes than to Fya+/b- erythrocytes.

    Matched MeSH terms: Protozoan Proteins/metabolism*
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