<div><p>(A) 5 μg purified anti-MSP1<sub>19</sub> human IgG1 (JS1) was subjected to SDS-PAGE under nonreducing (lane 1) or reducing (lane 2) conditions on 4%–15% polyacrylamide gradient gels and stained with Simply... more
<div><p>(A) 5 μg purified anti-MSP1<sub>19</sub> human IgG1 (JS1) was subjected to SDS-PAGE under nonreducing (lane 1) or reducing (lane 2) conditions on 4%–15% polyacrylamide gradient gels and stained with Simply Blue or immunoblotted with anti-human IgG-HRP.</p><p>(B) Under nonreducing conditions and after transfer to nitrocellulose, the human anti-MSP1<sub>19</sub> IgG1 (JS1) detects the recombinant GST-MSP1<sub>19</sub> fusion protein (lane 3) but not the GST alone control (lane 4). Localization of MSP1<sub>19</sub> by IFA.</p><p>(C) Schizont- and merozoite-stage parasites from the transgenic PbPbM19 and PbPfM19 lines were incubated with human Abs JS1 or JS2 (1:100), rabbit αPbM19 (1:1,000), or αPfM19 (1:1,000). After incubation with goat anti-rabbit Alexa-conjugated Ig (1:1,000) and FITC-conjugated anti-human IgG Fc (1:200), slides were washed and mounted in Vectrashield anti-fade. Parasites were visualized by fluorescence microscopy ×100 magnification, with the same fields photographed using filters to detect Alexa and FITC.</p><p>(D) JS1 reactive with MSP1<sub>19</sub> on methanol-acetone-fixed smears of merozoites and erythrocytes infected with P. falciparum (strain 3D7) ×40 magnification. JS2 gave similar results. No specific fluorescence was detected with an irrelevant human IgG1 (B10) recognizing MSP1<sub>19</sub> from P. yoelii [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030072#ppat-0030072-b024" target="_blank">24</a>].</p></div
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The detection of pathogens by polymerase chain reaction (PCR) in clinical samples, such as blood, urine, or feces, requires initial sample preparation to remove polymerase inhibitors and to concentrate the target DNA. Here we show for the... more
The detection of pathogens by polymerase chain reaction (PCR) in clinical samples, such as blood, urine, or feces, requires initial sample preparation to remove polymerase inhibitors and to concentrate the target DNA. Here we show for the first time that immunomagnetic separation can be used to recover pathogens from whole blood and then used for PCR analysis. With antibodies to the merozoite surface protein (MSP1), the malaria-causing parasite Plasmodium falciparum was purified and concentrated from clinical samples. The recovered parasites were used directly for in vitro DNA amplification. The PCR product was subsequently analyzed by a colorimetric 96-well microtiter plate assay. The results from examining 117 patients attending a clinic in the Borai district, Thailand, demonstrate that the combined method with immunomagnetic separation followed by PCR increases the group of positively diagnosed patients compared with microscopic examination of stained blood films. Analysis of 1 I...
Research Interests: Immunology, Malaria, Medicine, Plasma, Anemia, and 4 moreSma, Haptoglobin, Malarial Anemia, and Hemolysis
<p>(A) <i>Pf</i>ARF1/GFP was expressed using the CRT promoter (construct indicated above the images) and detected by epifluorescence microscopy. <i>Pf</i>ARF1/GFP was largely located in discrete dots; Figure... more
<p>(A) <i>Pf</i>ARF1/GFP was expressed using the CRT promoter (construct indicated above the images) and detected by epifluorescence microscopy. <i>Pf</i>ARF1/GFP was largely located in discrete dots; Figure details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1</a>. (B) The chimeric <i>Pf</i>ARF<sup>1-17/+4/-5</sup>AK2<sup>18-37</sup>/GFP was expressed using the CRT promoter (construct indicated above the images). Live cell imaging of the <i>Pf</i>ARF<sup>1-17/+4/-5</sup>AK2<sup>18-37</sup>/GFP parasite line at the late trophozoite stage showed that the pattern of GFP expression was similar to that of <i>Pf</i>AK2/GFP. Figure details are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1</a>. The <i>Pf</i>ARF<sup>1-17/+4/-5</sup>AK2<sup>18-37</sup>/GFP transgenic parasite was subjected to (C) hypotonic, or (D) SLO and saponin lysis and cell fractionation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">Fig 2</a>. No degradation of the fusion protein was detected after Proteinase K treatment of either the SLO or the saponin pellet fractions, indicating that the protein is protected from the protease and therefore within the PPM. The labelling is as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">2</a>.</p
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<p>Shows the location in the three-dimensional model of P. falciparum MSP1<sub>19</sub> of residues in the first epidermal growth factor domain, which on mutation affect binding by JS1 or JS2. Mutation of... more
<p>Shows the location in the three-dimensional model of P. falciparum MSP1<sub>19</sub> of residues in the first epidermal growth factor domain, which on mutation affect binding by JS1 or JS2. Mutation of Cys<sup>28</sup> shown in red completely ablated binding of both mAbs (12.10 and 12.8) and JS1 or JS2. Mutation of the partnering Cys<sup>12</sup>, also shown in red, while ablating binding by the murine mAbs, had no effect on the binding by JS1 or JS2. Arg<sup>20</sup> and Asn<sup>33</sup> in salmon had intermediate effects on binding as determined by SPR analysis when mutated to more neutral or negatively charged side-chains (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030072#ppat-0030072-t001" target="_blank">Table 1</a>). Three further substitutions at Lys<sup>40</sup>, Lys<sup>29</sup>, and Asn<sup>39</sup> seen in brown had minor effects on binding when the interaction was studied by ELISA. The model of P. falciparum MSP1<sub>19</sub> was generated by PyMol using atomic coordinates available from NCBI under accession number PDB: 1CEJ.</p
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Malaria in pregnancy is characterised by the sequestration of Plasmodium falciparum-infected erythrocytes in placental intervillous spaces. Placental parasites express a specific phenotype, which allows them to cytoadhere to chondroitin... more
Malaria in pregnancy is characterised by the sequestration of Plasmodium falciparum-infected erythrocytes in placental intervillous spaces. Placental parasites express a specific phenotype, which allows them to cytoadhere to chondroitin sulfate A expressed by syncytiotrophoblasts. Malaria infection during pregnancy allows the acquisition of antibodies against placental parasites, these antibodies are thought to be involved in protection during subsequent pregnancies. To investigate the development of a cellular response to placental parasites during pregnancy, peripheral blood mononuclear cells were collected from women at the time of their confinement. The study was performed in Cameroon where malaria transmission is perennial. In vitro cell proliferation and cytokine production were measured in response to non-malarial activators (concanavalin A and PPD), a recombinant protein from P. falciparum MSP-1, and erythrocytes infected by two P. falciparum lines, RP5 and W2. Like placenta...
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Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacu-ole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of... more
Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacu-ole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-ter-minal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subse-quent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-ter-minal 37 amino ...
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Cellular immune response to Plasmodium falciparum after pregnancy is related to previous placental infection and parity
A promising area of research on novel anti-malarial drugs has been the disruption of the acylation of key proteins during the parasitic life cycle. Inhibition of an enzyme responsible for one type of acylation, N-myristoylation, has been... more
A promising area of research on novel anti-malarial drugs has been the disruption of the acylation of key proteins during the parasitic life cycle. Inhibition of an enzyme responsible for one type of acylation, N-myristoylation, has been observed to have pleiotropic effects with severe impacts on parasite development, growth, and multiplication. In Plasmodium falciparum, N-myristoyltransferase (NMT) co-translationally catalyzes the transfer of a 14carbon moiety from myristoyl coenzyme A to a glycine residue at the amino terminus of a wide range of proteins. This acyl chain is important for the localization of NMT-substrates to specific membranes, allowing them to play roles in host-cell invasion, parasite motility, and protein trafficking and degradation. As a result, NMT has been validated as a therapeutic target for diseases such as malaria.
The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either... more
The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.
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We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were... more
We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, a...
Research Interests: Immune response, Malaria, Biological Sciences, Infection and immunity, Mice, and 13 moreFemale, Animals, Infection, Monoclonal Antibodies, Plasmodium falciparum, Protozoan Proteins, Monoclonal Antibody, Plasmodium yoelii, Growth Factor, Enzyme Linked Immunosorbent Assay, Disulfide bond, Merozoite surface protein 1, and Medical and Health Sciences
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In this issue of Cell Host & Microbe, Zhang et al. (2017) show that translational repression through eIF2α phosphorylation mediated by PK4 kinase activity plays a key role in artemisinin resistance in recrudescent malaria infections.... more
In this issue of Cell Host & Microbe, Zhang et al. (2017) show that translational repression through eIF2α phosphorylation mediated by PK4 kinase activity plays a key role in artemisinin resistance in recrudescent malaria infections. Targeting this druggable process could extend the lifespan of current frontline treatments.
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Plasmodium knowlesi, a common parasite of macaques, is recognised as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in... more
Plasmodium knowlesi, a common parasite of macaques, is recognised as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P. knowlesi infection in humans.
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The anaphase promoting complex/cyclosome (APC/C) is a highly conserved multi-subunit E3 ubiquitin ligase that controls mitotic division in eukaryotic cells by tagging cell cycle regulators for proteolysis. APC3 is a key component that... more
The anaphase promoting complex/cyclosome (APC/C) is a highly conserved multi-subunit E3 ubiquitin ligase that controls mitotic division in eukaryotic cells by tagging cell cycle regulators for proteolysis. APC3 is a key component that contributes to APC/C function. Plasmodium, the causative agent of malaria, undergoes atypical mitotic division during its life cycle. Only a small subset of APC/C components has been identified in Plasmodium and their involvement in atypical cell division is not well understood. Here, using reverse genetics we examined the localisation and function of APC3 in Plasmodium berghei. APC3 was observed as a single focus that co-localised with the centriolar plaque during asexual cell division in schizonts, whereas it appeared as multiple foci in male gametocytes. Functional studies using gene disruption and conditional knockdown revealed essential roles of APC3 during these mitotic stages with loss resulting in a lack of chromosome condensation, abnormal cyt...
Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role... more
Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and function of PhIL1, an IMC-associated protein on the motile and invasive stages of both human and rodent parasites. We show that PhIL1 is located in the IMC in all three invasive (merozoite, ookinete-, and sporozoite) stages of development, as well as in the male gametocyte and locates both at the apical and basal ends of ookinete and sporozoite stages. Proteins interacting with PhIL1 were identified, showing that PhIL1 was bound to only some proteins present in the glideosome motor complex (GAP50, GAPM1-3) of both P. falciparum and P. berghei. Analysis of PhIL1 function using gene targeting approaches indicated that the protein is required...
Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC... more
Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. ...
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Research Interests: Biological Sciences, Molecular, Processing, Parasite, Animals, and 9 moreMonoclonal Antibodies, Peptides, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Plasmodium falciparum, Protozoan Proteins, Amino Acid Sequence, Merozoite surface protein 1, Molecular Sequence Data, and Medical and Health Sciences
The antibody response to two different epitopes located in the C-terminal 19kDa fragment of the Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) has been studied using a competitive ELISA based on the inhibition of monoclonal... more
The antibody response to two different epitopes located in the C-terminal 19kDa fragment of the Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) has been studied using a competitive ELISA based on the inhibition of monoclonal antibody (MoAb) binding by serum samples. Sera from children aged three to eight years who suffered clinical symptoms of malaria, or were partially immune with an asymptomatic infection, and from adults all living in The Gambia, West Africa were tested. The results suggest that the antibody response to MSP-1(19) has a role in naturally-acquired immunity in Gambian individuals.
Research Interests: Microbiology, Aging, Medical Microbiology, Humans, Child, and 15 moreImmunity, Animals, Parasite Immunology, Monoclonal Antibodies, Gambia, Adult, Plasmodium falciparum, Veterinary Sciences, Molecular weight, Protozoan Proteins, Epitopes, Enzyme Linked Immunosorbent Assay, Merozoite surface protein 1, Child preschool, and Malaria Falciparum
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Optical tweezers have been used extensively to measure the mechanical properties of individual biological molecules. Over the past 10-15 years optical trapping studies have revealed important information about the way in which motor... more
Optical tweezers have been used extensively to measure the mechanical properties of individual biological molecules. Over the past 10-15 years optical trapping studies have revealed important information about the way in which motor proteins convert chemical energy to mechanical ...
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Research Interests: Membrane Proteins, Structural Analysis, Biological Sciences, Animals, Proteins, and 12 moreMolecular cloning, Protein Secondary Structure Prediction, Molecular Mass, Molecular weight, Protozoan Proteins, Amino Acid Sequence, Base Sequence, Plasmodium yoelii, DNA probes, Plasmodium Vivax, Molecular Sequence Data, and Medical and Health Sciences
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Research Interests: Microbiology, Calcium, Experimental parasitology, Mice, Parasite, and 15 moreFemale, Animals, Monoclonal Antibodies, Experimental, Antibody, Molecular weight, Monoclonal Antibody, Plasmodium yoelii, Polyacrylamide Gel Electrophoresis, Erythrocytes, Protein Binding, Chelating Agents, Ligands, Ethylene Glycol, and Chymotrypsin
Research Interests: Biological Chemistry, Biological Sciences, Circular Dichroism, Peptides, CHEMICAL SCIENCES, and 11 moreProtein Secondary Structure Prediction, Calmodulin, Plasmodium falciparum, Protozoan Proteins, Amino Acid Sequence, Protein Binding, Protein Denaturation, Plasmodium Knowlesi, Plasmodium berghei, Molecular Sequence Data, and Medical and Health Sciences
The salivarian trypanosomes have a unique capacity for antigenic variation at the cell surface. This phenomenon is their primary mechanism for evasion of the host's immune response. Variation is mediated through alternate expression... more
The salivarian trypanosomes have a unique capacity for antigenic variation at the cell surface. This phenomenon is their primary mechanism for evasion of the host's immune response. Variation is mediated through alternate expression of an extensive repertoire of variant surface glycoproteins (VSGs). Extensive amino acid sequence diversity is responsible for the antigenic diversity of VSGs. All the isolated VSGs of Trypanosoma brucei studied also contain an immunologically cross-reacting glycosyl side chain at the C-terminus, which probably represents a recognition site for proteolytic processing of the hydrophobic putative membrane-binding tail present on the synthesized molecule but not so far found on purified VSGs.
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A major protein found on the surface of the invasive stage of the malaria parasite Plasmodium falciparum, merozoite surface protein-1 (MSP1), has been proposed as a vaccine candidate. Antibodies which recognise a single fragment of this... more
A major protein found on the surface of the invasive stage of the malaria parasite Plasmodium falciparum, merozoite surface protein-1 (MSP1), has been proposed as a vaccine candidate. Antibodies which recognise a single fragment of this molecule (MSP1(19)), composed of 2 regions related to epidermal growth factor (EGF), also inhibit parasite growth in vitro. It is shown by direct expression of the individual EGF-like domains in Escherichia coli, that the first domain is the target of growth-inhibitory antibodies. A single amino acid difference influences the binding of some antibodies to this domain.
Research Interests: Gene expression, Biological Sciences, PCR, Escherichia coli, Animals, and 14 moreMonoclonal Antibodies, Epidermal Growth Factor, Plasmodium falciparum, Protozoan Proteins, Monoclonal Antibody, Amino Acid Sequence, Base Sequence, Growth Factor, Epitopes, Malaria Vaccines, Merozoite surface protein 1, Molecular Sequence Data, Malaria Falciparum, and Medical and Health Sciences
The merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum undergoes an extracellular proteolytic cleavage (secondary processing) intrinsic to successful erythrocyte invasion. In the T9/96 clone of P. falciparum... more
The merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum undergoes an extracellular proteolytic cleavage (secondary processing) intrinsic to successful erythrocyte invasion. In the T9/96 clone of P. falciparum the protease responsible has been characterised as a membrane-associated, calcium-dependent activity, sensitive to irreversible inhibitors of serine proteases. Here we extend these studies and show that secondary processing activity in intact merozoites of P. falciparum strains expressing the alternative dimorphic type of merozoite surface protein-1 has identical characteristics, and that the cleavage site is close to or identical to that in the protein from T9/96. The protease responsible is shown to be parasite-derived, and able to catalyse processing of native substrate only when present in the same membrane. Cleavage of the substrate follows apparent first order kinetics for at least 2 half-lives. It is concluded that secondary processing of both dimorphic forms of the P. falciparum merozoite surface protein-1 is a conserved event, mediated by a mechanistically conserved protease located on the merozoite surface. These observations provide clues to the identity of the protease and show that, irrespective of the dimorphic type, secondary processing results in the same, highly conserved region of the merozoite surface protein-1 remaining on the surface of the invading merozoite.