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A F Cowman

Publications and source records attributed to A F Cowman.

At least 19 recordsLinked to original sources
PubMed2005

A comparison of match-only algorithms for the analysis of Plasmodium falciparum oligonucleotide arrays.

This study is motivated by two data sets which employ a custom Plasmodium falciparum version of the Affymetrix GeneChip, containing only perfect match (PM) oligonucleotides. A PM-only chip cannot be analysed using the standard Affymetrix-supplied software. We compared the performance of three match-only algorithms on these data: the Match Only Integral Distribution (MOID) algorithm, Robust Multichip Analysis (RMA), and the Model Based Expression Index (MBEI). We validated the differential expression of several genes using quantitative reverse transcriptase-PCR. We also performed a comparison using two publicly available 'benchmarking' data sets: the Latin Square spike-in data set generated by Affymetrix, and the Gene Logic dilution series. Since we know what the true fold changes are in these special data sets, they are helpful for assessment of expression algorithms.

Algorithms↗
PubMed2001

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes.

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.

Amino Acid Sequence↗
PubMed2001

Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria.

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-1(19) from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-1(19), we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum-immune humans and P. chabaudi-immune mice. In some individual human sera, MSP-1(19) antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-1(19)-based vaccines.

Adult↗
PubMed2001

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites.

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and gamma irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

Animals↗
PubMed2001

Functional analysis of drug resistance in Plasmodium falciparum in the post-genomic era.

Malaria has plagued humans throughout recorded history and results in the death of over 2 million people per year. The protozoan parasite Plasmodium falciparum causes the most severe form of malaria in humans. Chemotherapy has become one of the major control strategies for this parasite; however, the development of drug resistance to virtually all of the currently available drugs is causing a crisis in the use and deployment of these compounds for prophylaxis and treatment of this disease. The genome sequence of P. falciparum is providing the informational base for the use of whole-genome strategies such as bioinformatics, microarrays and genetic mapping. These approaches, together with the availability of a high-resolution genome linkage map consisting of hundreds of microsatellite markers and the advanced technologies of transfection and proteomics, will facilitate an integrated approach to address important biological questions. In this review we will discuss strategies to identify novel genes involved in the molecular mechanisms used by the parasite to circumvent the lethal effect of current chemotherapeutic agents.

Animals↗
PubMed2001

Multiple var gene transcripts are expressed in Plasmodium falciparum infected erythrocytes selected for adhesion.

Adherence of Plasmodium falciparum-infected erythrocytes to the post-capillary endothelium is an important characteristic of malaria infection. The adhesion is mediated predominantly by P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), a clonally variant protein expressed on the surface of infected red blood cells that appears to be a target of protective immunity. A multi-membered var gene family encodes PfEMP1 and switching expression of different var genes conveys different antigenic and adhesive properties to infected red blood cells. Knowledge about transcriptional control of phenotypic expression, or the mechanisms that allow multiple binding specificities, is very limited. Here, we describe a series of phenotypic selection experiments, which resulted in the expression of different PfEMP1 and the detection of multiple full-length var gene transcripts in the mature trophozoite stage. However, a dominant form of PfEMP1 appeared to be expressed, which suggested that most var transcripts do not lead to a surface expressed PfEMP1 molecule. Parasites bound to specific receptors still expressed multiple full-length var genes and mature trophozoites selected for increased adhesion to a specific receptor retained the ability to bind to multiple receptors. Our findings suggest that a defined adhesive phenotype can be associated with expression of multiple var genes.

Alternative Splicing↗
PubMed2001

An EBA175 homologue which is transcribed but not translated in erythrocytic stages of Plasmodium falciparum.

Plasmodia species can bind to the Duffy blood group antigen (Plasmodium vivax and P. knowlesi) or glycophorin A (P. falciparum) on human erythrocytes as receptors for the invasion of merozoites in the asexual life cycle. A number of proteins have been identified in P. vivax, P. knowlesi and P. falciparum that serve as parasite ligands for these interactions and this group of proteins form the erythrocyte binding protein (EBP) family. The availability of sequence data generated as part of the P. falciparum Genome Project has allowed the identification of other genes related to the known EBP family members. We describe the Psi EBA165 gene and show that it has four exons, a structure identical to that described for EBA175. Analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) has shown that all introns are spliced and that this gene is transcribed. The predicted protein would have the same structure as EBA175 containing the F1/F2 domains, a cysteine-rich region followed by a predicted transmembrane region and a short cytoplasmic tail, but the coding region of Psi EBA165 contains frameshifts. It was possible that the frameshifts may be corrected in the transcript, or alternatively, a mechanism could operate that allowed the translation machinery to read through the frameshifts. Antibodies that recognise EBA165 fusion proteins could not detect this protein in the P. falciparum parasites tested. Additionally, it was possible to disrupt the Psi EBA165 gene without affecting the parasite's ability to invade and grow in erythrocytes. These results suggest that the Psi EBA165 gene is a transcribed pseudogene.

Amino Acid Sequence↗
PubMed2001

A gene family expressing a host-protective antigen of Echinococcus granulosus.

Echinococcus granulosus causes cystic hydatidosis in humans. A recombinant antigen vaccine has been developed, for use in the parasite's natural animal intermediate hosts, that may provide a new tool for control of hydatid disease transmission. The antigen, designated EG95, is encoded by a cDNA the features of which indicate it to be an incomplete copy of the associated mRNA. Characterisation of the gene(s) encoding the antigen was undertaken in order to enable subsequent study of genetic variability in the gene and associated protein in different parasite isolates. Southern hybridisation studies of E. granulosus genomic DNA probed with the eg95 cDNA revealed that the gene belonged to a gene family. DNA sequence analysis of cloned genomic fragments indicated that the gene family consists of at least seven members, one of which is a pseudogene. The gene having identity with the eg95 cDNA was cloned and sequenced, and the full length mRNA characterised. Genomic sequence and structure of the eg95 gene family members are highly conserved with respect to the gene encoding EG95. Four eg95-related genes are predicted to express an identical EG95 protein and all four were shown to be expressed in the oncosphere life-cycle stage. The full length EG95 protein has a predicted molecular mass of 16.9 kDa, secretory signal sequence, carboxy-terminal glycosylphosphatidylinositol hydrophobic anchor motif and a fibronectin type III domain. PCR amplification conditions were established which allow gene-specific characterisation of the eg95 gene in E. granulosus isolates from different host species and geographical locations.

5' Untranslated Regions↗
PubMed2001

Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the world's burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

Africa South of the Sahara↗
PubMed2001

A novel ligand from Plasmodium falciparum that binds to a sialic acid-containing receptor on the surface of human erythrocytes.

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor-ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid-dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte-binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid-dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease-resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid-dependent receptor on human erythrocytes.

Amino Acid Sequence↗
PubMed2001

Identification of proteins from Plasmodium falciparum that are homologous to reticulocyte binding proteins in Plasmodium vivax.

Plasmodium falciparum infections can be fatal, while P. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereas P. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5' end but are highly divergent at the 3' end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.

Amino Acid Sequence↗
PubMed2001

Plasmodium falciparum homologue of the genes for Plasmodium vivax and Plasmodium yoelii adhesive proteins, which is transcribed but not translated.

The 235-kDa family of rhoptry proteins in Plasmodium yoelii and the two reticulocyte binding proteins of P. vivax comprise a family of proteins involved in host cell selection and erythrocyte invasion. Here we described a member of the gene family found in P. falciparum (PfRH3) that is transcribed in its entirety, under stage-specific control, with correct splicing of the intron, but appears not to be translated, probably due to two reading frameshifts at the 5' end of the gene.

Amino Acid Sequence↗
PubMed2000

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells.

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.

Animals↗
PubMed2000

Targeted disruption of an erythrocyte binding antigen in Plasmodium falciparum is associated with a switch toward a sialic acid-independent pathway of invasion.

Erythrocyte invasion by Plasmodium requires molecules present both on the merozoite surface and within the specialized organelles of the apical complex. The Plasmodium erythrocyte binding protein family includes the Plasmodium falciparum sialic acid-binding protein, EBA-175 (erythrocyte binding antigen-175), which binds sialic acid present on glycophorin A of human erythrocytes. We address the role of the conserved 3'-cysteine rich region, the transmembrane, and cytoplasmic domains through targeted gene disruption. Truncation of EBA-175 had no measurable effect on either the level of EBA-175 protein expression or its subcellular localization. Similarly, there appears to be no impairment in the ability of soluble EBA-175 to be released into the culture supernatant after schizont rupture. Additionally, the 3'-cys rich region, transmembrane, and cytoplasmic domains of EBA-175 are apparently non-essential for merozoite invasion. In contrast, erythrocyte invasion via the EBA-175/glycophorin A route appears to have been disrupted to such a degree that the mutant lines have undergone a stable switch in invasion phenotype. As such, EBA-175 appears to have been functionally inactivated within the truncation mutants. The sialic acid-independent invasion pathway within the mutant parasites accounts for approximately 85% of invasion into normal erythrocytes. These data demonstrate the ability of P. falciparum to utilize alternate pathways for invasion of red blood cells, a property that most likely provides a substantial survival advantage in terms of overcoming host receptor heterogeneity and/or immune pressure.

Animals↗
PubMed2000

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells.

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.

Animals↗
PubMed2000

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum.

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.

Animals↗