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J W Barnwell

Publications and source records attributed to J W Barnwell.

At least 19 recordsLinked to original sources
PubMed2006

Characterization and application of multiple genetic markers for Plasmodium malariae.

Plasmodium malariae, a protozoan parasite that causes malaria in humans, has a global distribution in tropical and subtropical regions and is commonly found in sympatry with other Plasmodium species of humans. Little is known about the genetics or population structure of P. malariae. In the present study, we describe polymorphic genetic markers for P. malariae and present the first molecular epidemiological data for this parasite. Six microsatellite or minisatellite markers were validated using 76 P. malariae samples from a diverse geographical range. The repeat unit length varied from 2 to 17 bp, and up to 10 different alleles per locus were detected. Multiple genotypes of P. malariae were detected in 33 of 70 samples from humans with naturally acquired infection. Heterozygosity was calculated to be between 0.236 and 0.811. Allelic diversity was reduced for samples from South America and, at some loci, in samples from Thailand compared with those from Malawi. The number of unique multilocus genotypes defined using the 6 markers was significantly greater in Malawi than in Thailand, even when data from single genotype infections were used. There was a significant reduction in the multiplicity of infection in symptomatic infections compared with asymptomatic ones, suggesting that clinical episodes are usually caused by the expansion of a single genotype.

Alleles↗
PubMed2002

Extensive polymorphism in the plasmodium vivax merozoite surface coat protein MSP-3alpha is limited to specific domains.

Plasmodium merozoites are covered by a complex coat of surface proteins. Several of the Merozoite Surface Proteins (MSPs) that make up this coat have been proposed as vaccine candidates although some of the MSPs are known to be highly polymorphic. We present here the first survey and analysis of the polymorphism in the recently characterized P. vivax surface protein PvMSP-3alpha. Full length or partial sequences were obtained for the Pvmsp-3alpha gene from isolates originating in Central and South America, Asia and the Pacific. The Pvmsp-3alpha sequence is remarkably diverse, but this extensive diversity is largely restricted to certain domains of the encoded protein. An acidic C-terminal domain and a smaller hydrophilic N-terminus are relatively conserved, while a central domain containing coiled-coil heptad repeats is highly polymorphic and in some isolates of P. vivax is partially deleted. Unlike other MSPs, there is no evidence of allelic families of PvMSP-3alpha gene sequences, and no evidence that certain patterns of polymorphism group within isolates of similar geographical origin. The distribution and nature of polymorphism suggest that there are functional restrictions on mutations in this gene, and have implications for inclusion of PvMSP-3alpha as a candidate in a P. vivax vaccine.

Amino Acid Sequence↗
PubMed2001

A Plasmodium falciparum homologue of Plasmodium vivax reticulocyte binding protein (PvRBP1) defines a trypsin-resistant erythrocyte invasion pathway.

Invasion of erythrocytes by Plasmodium merozoites is an intricate process involving multiple receptor-ligand interactions. The glycophorins and an unknown trypsin sensitive factor are all erythrocyte receptors used during invasion by the major human pathogen Plasmodium falciparum. However, only one erythrocyte receptor, Glycophorin A, has a well-established cognate parasite ligand, the merozoite protein erythrocyte binding antigen-175 (EBA-175). The involvement of several other parasite proteins during invasion have been proposed, but no direct evidence links them with a specific invasion pathway. Here we report the identification and characterization of P. falciparum normocyte binding protein 1 (PfNBP1), an ortholog of Plasmodium vivax reticulocyte binding protein-1. PfNBP1 binds to a sialic acid dependent trypsin-resistant receptor on the erythrocyte surface that appears to be distinct from known invasion receptors. Antibodies against PfNBP1 can inhibit invasion of trypsinized erythrocytes and two P. falciparum strains that express truncated PfNBP1 are unable to invade trypsinized erythrocytes. One of these strain, 7G8, also does not invade Glycophorin B-negative erythrocytes. PfNBP1 therefore defines a novel trypsin-resistant invasion pathway and adds a level of complexity to current models for P. falciparum erythrocyte invasion.

Animals↗
PubMed2001

Plasmodium vivax merozoite surface proteins-3beta and-3gamma share structural similarities with P. vivax merozoite surface protein-3alpha and define a new gene family.

The genes encoding two merozoite surface proteins of Plasmodium vivax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. vivax lambdagt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. vivax lambdaDASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75677 Da, and 3' and 5' RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. vivax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. vivax genes have putative signal peptides, large central domains that contain >20% alanine residues bound by charged regions, are predicted to form alpha-helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low (<40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3alpha, Pvmsp-3beta, and Pvmsp-3gamma. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparum.

Amino Acid Sequence↗
PubMed2001

Profiling the malaria genome: a gene survey of three species of malaria parasite with comparison to other apicomplexan species.

We have undertaken the first comparative pilot gene discovery analysis of approximately 25,000 random genomic and expressed sequence tags (ESTs) from three species of Plasmodium, the infectious agent that causes malaria. A total of 5482 genome survey sequences (GSSs) and 5582 ESTs were generated from mung bean nuclease (MBN) and cDNA libraries, respectively, of the ANKA line of the rodent malaria parasite Plasmodium berghei, and 10,874 GSSs generated from MBN libraries of the Salvador I and Belem lines of Plasmodium vivax, the most geographically wide-spread human malaria pathogen. These tags, together with 2438 Plasmodium falciparum sequences present in GenBank, were used to perform first-pass assembly and transcript reconstruction, and non-redundant consensus sequence datasets created. The datasets were compared against public protein databases and more than 1000 putative new Plasmodium proteins identified based on sequence similarity. Homologs of previously characterized Plasmodium genes were also identified, increasing the number of P. vivax and P. berghei sequences in public databases at least 10-fold. Comparative studies with other species of Apicomplexa identified interesting homologs of possible therapeutic or diagnostic value. A gene prediction program, Phat, was used to predict probable open reading frames for proteins in all three datasets. Predicted and non-redundant BLAST-matched proteins were submitted to InterPro, an integrated database of protein domains, signatures and families, for functional classification. Thus a partial predicted proteome was created for each species. This first comparative analysis of Plasmodium protein coding sequences represents a valuable resource for further studies on the biology of this important pathogen.

Animals↗
PubMed2000

Two Plasmodium falciparum genes express merozoite proteins that are related to Plasmodium vivax and Plasmodium yoelii adhesive proteins involved in host cell selection and invasion.

Two related Plasmodium falciparum genes and their encoded proteins have been identified by comparative analyses with Plasmodium vivax reticulocyte binding protein 2 (PvRBP-2). The P. falciparum genes have a structure which suggests that they may be the result of an evolutionary duplication event, as they share more than 8 kb of closely related nucleotide sequence but then have quite divergent unique 3' ends. Between these shared and unique regions is a complex set of repeats, the nature and number of which differs between the two genes, as well as between different P. falciparum strains. Both genes encode large hydrophilic proteins, which are concentrated at the invasive apical end of the merozoite and are predicted to be more than 350 kDa, with an N-terminal signal sequence and a single transmembrane domain near their C termini. Importantly, they also share gene structure and amino acid homology with the Plasmodium yoelii 235-kDa rhoptry protein family, which is also related to PvRBP-2. Together these Plasmodium proteins define an extended family of proteins that appear to function in erythrocyte selection and invasion. As such, they may prove to be essential components of malaria vaccine preparations.

Amino Acid Motifs↗
PubMed2000

Cross-species interactions between malaria parasites in humans.

The dynamics of multiple Plasmodium infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.

Adolescent↗
PubMed2000

Genetic diversity and dynamics of plasmodium falciparum and P. vivax populations in multiply infected children with asymptomatic malaria infections in Papua New Guinea.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3alpha, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6.6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co-infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5-14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

Adolescent↗
PubMed1999

Stage-specific expression of 14-3-3 in asexual blood-stage Plasmodium.

This paper reports the identification of 14-3-3 in Plasmodium. 14-3-3 is an evolutionarily conserved protein that is most noted as a mediator in signal transduction events and cell cycle regulation. The complete cDNA (approximately 2.6 kb) and gDNA (approximately 3.4 kb) of a Plasmodium knowlesi 14-3-3 (Pk14-3-3) is reported. The gene has three introns; two near the beginning and one close to the end of the coding sequence. Also reported, is the gDNA of the Plasmodium falciparum homologue (Pf14-3-3). Unlike in many other organisms, where multiple gene copies and different functional isoforms exist, Plasmodium 14-3-3 is encoded as a single-copy gene. Northern blot analyses show that the Pk14-3-3 transcript in asexual blood stages begins to be expressed in the ring-stage, predominates in young trophozoites, and thereafter declines. An antiserum produced against recombinant Pk14-3-3 reacts via immunoblot and immunoprecipitation with the approximately 30 kDa and the approximately 32 kDa Pk14-3-3 and Pf14-3-3 proteins, respectively. Protein expression in P. knowlesi closely mimics the pattern of the transcript.

14-3-3 Proteins↗
PubMed1999

Karyotype and synteny among the chromosomes of all four species of human malaria parasite.

The karyotype and chromosomes of the human malaria parasite Plasmodium falciparum have been well characterized in recent years. Here we present karyotype maps of the three other human malaria species, P. vivax, P. malariae and P. ovale. Chromosomes of these species were found to be of significantly higher molecular weight than those of P. falciparum. Some 14 P. vivax chromosomes were distinguishable, and 12-14 P. malariae and P. ovale chromosomes. The chromosome location of 15 genes, known to be present within five synteny groups between P. falciparum and the rodent malarias, were analyzed, and four of these synteny groups were found to be conserved between all of the human malaria species. In addition, a more detailed genome map of P. vivax was made using ten housekeeping and antigen genes. These data represent the first karyotype maps of all species of malaria which infect man.

Animals↗
PubMed1999

Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central domain.

Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.

Alanine↗
PubMed1999

Plasmodium vivax, P. cynomolgi, and P. knowlesi: identification of homologue proteins associated with the surface of merozoites.

We have identified a Plasmodium vivax merozoite surface protein (MSP) that migrates on SDS-polyacrylamide gels at a Mr of about 185 kDa. This protein was recognized by a P. vivax monoclonal antibody (mAb) that localizes the protein by immunofluorescence to the surface of merozoites and also immunoprecipitates this protein from NP-40 detergent extracts of [35S]methionine metabolically radiolabeled P. vivax schizonts. The P. vivax MSP does not become biosynthetically radiolabeled with [3H]glucoamine, [3H]myristate, [3H]palmitate, or [3H]mannose, indicating that this P. vivax MSP is not posttranslationally modified and bound to the merozoite membrane by a glycosylphosphatidylinositol (GPI) lipid anchor. Thus, in this respect, this protein is different from members of the MSP-1 protein family and from MSP-2 and MSP-4 of P. falciparum. The mAb cross-reacts with and outlines the surface of P. cynomolgi merozoites and immunoprecipitates a 150-kDa P. cynomolgi homologue. The mAb was used as an affinity reagent to purify the native homologous MSP from NP-40 extracts of P. cynomolgi mature schizonts in order to develop a specific polyclonal antiserum. The resulting anti-PcyMSP rabbit antiserum cross-reacts strongly with the P. vivax 185-kDa MSP and also recognizes an analogous 110-kDa protein from P. knowlesi. We have determined via an immunodepletion experiment that the 110-kDa P. knowlesi MSP corresponds to the PK 110 protein partially characterized earlier (Perler et al. 1987). The potential of P. vivax MSP as a vaccine candidate was addressed by conducting in vitro inhibition of erythrocyte invasion assays, and the IgG fraction of both the P. vivax MSP mAb and the P. cynomolgi MSP rabbit antiserum significantly inhibited entry of P. vivax merozoites. We denote, on a preliminary basis, these antigenically related merozite surface proteins PvMSP-185, PcyMSP-150, and PkMSP-110.

Animals↗
PubMed1999

Antigenic variation in malaria: a 3' genomic alteration associated with the expression of a P. knowlesi variant antigen.

Antigenic variation of malaria parasites was discovered in P. knowlesi, using a schizont-infected cell agglutination (SICA) assay to detect variant antigens expressed at the surface of infected erythrocytes. Later studies utilizing stable clones, Pk1(A+) and its direct derivative, Pk1(B+)1+, showed that SICA[+] clones express distinct parasite-encoded antigens of approximately 200 kDa. Here we identify a P. knowlesi variant antigen gene and cDNA and demonstrate that it encodes the 205 kDa variant antigen expressed by B+ parasites. This gene belongs to a multigene family, which we term SICAvar. Its ten-exon structure with seven cysteine-rich coding modules is unique compared to P. falciparum var genes. Further, we highlight a 3' genomic alteration that we predict is related to SICAvar gene switching.

Amino Acid Sequence↗
PubMed1999

Polymorphism at the merozoite surface protein-3alpha locus of Plasmodium vivax: global and local diversity.

Allelic diversity at the Plasmodium vivax merozoite surface protein-3alpha (PvMsp-3alpha) locus was investigated using a combined polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) protocol. Symptomatic patient isolates from global geographic origins showed a high level of polymorphism at the nucleotide level. These samples were used to validate the sensitivity, specificity, and reproducibility of the PCR/RFLP method. It was then used to investigate PvMsp3alpha diversity in field samples from children living in a single village in a malaria-endemic region of Papua New Guinea, with the aim of assessing the usefulness of this locus as an epidemiologic marker of P. vivax infections. Eleven PvMsp-3alpha alleles were distinguishable in 16 samples with single infections, revealing extensive parasite polymorphism within this restricted area. Multiple infections were easily detected and accounted for 5 (23%) of 22 positive samples. Pairs of samples from individual children provided preliminary evidence for high turnover of P. vivax populations.

Adolescent↗
PubMed1999

A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies.

BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite. MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far. RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein. CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.

Alleles↗