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Biomedical subjects

A F Cowman

Publications and source records attributed to A F Cowman.

At least 37 records · Page 2Linked to original sources
PubMed2000

Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway.

The plastid of Plasmodium falciparum (or 'apicoplast') is the evolutionary homolog of the plant chloroplast and represents a vestige of a photosynthetic past. Apicoplast indispensability indicates that it still provides essential functions to parasites. Similar to plant chloroplasts, the apicoplast is dependent on many nucleus-encoded genes to provide these functions. The apicoplast is surrounded by four membranes, two more than plant chloroplasts. Thus, protein targeting to the apicoplast must overcome additional membrane barriers. In P.falciparum we have analyzed apicoplast targeting using green fluorescent protein (GFP). We demonstrate that protein targeting is at least a two-step process mediated by bipartite N-terminal pre-sequences that consist of a signal peptide for entry into the secretory pathway and a plant-like transit peptide for subsequent import into the apicoplast. The P.falciparum transit peptide is exceptional compared with other known plastid transit peptides in not requiring serine or threonine residues. The pre-sequence components are removed stepwise during apicoplast targeting. Targeting GFP to the apicoplast has also provided the first opportunity to examine apicoplast morphology in live P. falciparum.

Amino Acid Sequence↗
PubMed2000

clag9: A cytoadherence gene in Plasmodium falciparum essential for binding of parasitized erythrocytes to CD36.

The propensity of isolates of the malaria parasite Plasmodium falciparum to delete a segment of chromosome 9 has provided positional information that has allowed us to identify a gene necessary for cytoadherence. It has been termed the cytoadherence-linked asexual gene (clag9). clag9 encodes at least nine exons and is expressed in blood stages. The hydrophobicity profile of the predicted CLAG9 protein identifies up to four transmembrane domains. We show here that targeted gene disruption of clag9 ablated cytoadherence to C32 melanoma cells and purified CD36. DNA-induced antibodies to the clag9 gene product reacted with a polypeptide of 220 kDa in the parental malaria clone but not in clones with a disrupted clag9 gene.

Animals↗
PubMed2000

Genomic distribution and functional characterisation of two distinct and conserved Plasmodium falciparum var gene 5' flanking sequences.

Approximately 50 highly diverse var genes distributed throughout the haploid genome of the malaria parasite Plasmodium falciparum code for PfEMP1 variants located on the surface of infected erythrocytes. PfEMP1 is involved in cytoadherence of parasitised red blood cells and undergoes antigenic variation through differential expression of var genes. Members of the var gene family are located in chromosome-internal positions on chromosomes 4, 7, 8 and 12, and in subtelomeric regions of all chromosomes. Here we show that there are two distinct and conserved types of 5' upstream regions (var17-type and 5B1-type) of var genes, and suggest that most subtelomeric var genes are flanked by a var17-type 5' upstream sequence. In contrast, 5B1-type 5' upstream are localised to chromosomes that have been shown to contain var genes within chromosome-internal regions. Transcriptional analysis using RT-PCR revealed that var genes flanked by either type of 5' upstream sequence are transcribed in in vitro cultured trophozoite stage parasites. In addition, we have shown that the 5' flanking sequences of four different var genes are able to drive transient expression of the cat reporter gene. Our results suggest that at least the minimal regulatory sequences required for transcription of var genes are conserved among both subgroups of the var gene family. Furthermore, these sequences provide new markers for the investigation of the chromosomal organisation of var genes.

5' Untranslated Regions↗
PubMed2000

Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum.

Throughout the latter half of this century, the development and spread of resistance to most front-line antimalarial compounds used in the prevention and treatment of the most severe form of human malaria has given cause for grave clinical concern. Polymorphisms in pfmdr1, the gene encoding the P-glycoprotein homologue 1 (Pgh1) protein of Plasmodium falciparum, have been linked to chloroquine resistance; Pgh1 has also been implicated in resistance to mefloquine and halofantrine. However, conclusive evidence of a direct causal association between pfmdr1 and resistance to these antimalarials has remained elusive, and a single genetic cross has suggested that Pgh1 is not involved in resistance to chloroquine and mefloquine. Here we provide direct proof that mutations in Pgh1 can confer resistance to mefloquine, quinine and halofantrine. The same mutations influence parasite resistance towards chloroquine in a strain-specific manner and the level of sensitivity to the structurally unrelated compound, artemisinin. This has important implications for the development and efficacy of future antimalarial agents.

ATP-Binding Cassette Transporters↗
PubMed2000

Functional conservation of the malaria vaccine antigen MSP-119across distantly related Plasmodium species.

The C-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP-119) is at present a leading malaria vaccine candidate. Antibodies against the epidermal growth factor-like domains of MSP-1 19are associated with immunity to P. falciparum and active immunization with recombinant forms of the molecule protect against malaria challenge in various experimental systems. These findings, with the knowledge that epidermal growth factor-like domains in other molecules have essential binding functions, indicate the importance of this protein in merozoite invasion of red blood cells. Despite extensive molecular epidemiological investigations, only limited sequence polymorphism has been identified in P. falciparum MSP-119 (refs. 9-11). This indicates its sequence is functionally constrained, and is used in support of the use of MSP-119 as a vaccine. Here, we have successfully complemented the function of most of P. falciparum MSP-119 with the corresponding but highly divergent sequence from the rodent parasite P. chabaudi. The results indicate that the role of MSP-119 in red blood cell invasion is conserved across distantly related Plasmodium species and show that the sequence of P. falciparum MSP-119 is not constrained by function.

Amino Acid Sequence↗
PubMed2000

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species.

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.

Animals↗
PubMed1999

The adhesion of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A is mediated by P. falciparum erythrocyte membrane protein 1.

Chondroitin sulfate A (CSA) is an important receptor for the sequestration of Plasmodium falciparum in the placenta, but the parasite ligand involved in adhesion has not previously been identified. Here we report the identification of a var gene transcribed in association with binding to CSA and present evidence that the P. falciparum erythrocyte membrane protein 1 product of the gene is the parasite ligand mediating CSA binding. Description of this gene and the implication of P. falciparum erythrocyte membrane protein 1 as the parasite ligand paves the way to a more detailed understanding of the pathogenesis of placental infection and potential therapeutic strategies targeting the interaction.

Animals↗
PubMed1999

Transfection of the human malaria parasite Plasmodium falciparum.

In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.

Animals↗
PubMed1999

Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand.

Resistance to quinoline-containing compound has been associated with the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene. We analyzed wild P. falciparum isolates with high levels of chloroquine and mefloquine resistance for their macrorestriction maps of chromosome 5 and sequence of pfmdr1. Two types of chromosome 5 amplification were found. Eleven of 62 resistant isolates displayed Bgl 1 fragments larger than 100 kb. Twenty-nine isolates possessed multiple copies of the fragments. We failed to detect any amplification of this region on chromosome 5 in 22 mefloquine-resistant isolates, suggesting that other mechanisms can mediate the mefloquine-resistant phenotype. There was no direct association between pfmdr1 mutations and chloroquine sensitivity. Resistant lines could have Asn-86 and Tyr-184 or Phe-184, the predicted sequence of those chloroquine-sensitive isolates. No mutation at Asn-1042 and Asp-1246 was detected among these chloroquine-resistant isolates. Therefore, a few base substitutions in the pfmdr1 gene may not be sufficient to account for all chloroquine-resistant phenotypes.

Amodiaquine↗
PubMed1998

Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum.

A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or "apicoplast," is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), beta-ketoacyl-ACP synthase III (FabH), and beta-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.

Animals↗
PubMed1998

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria.

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

Alleles↗
PubMed1998

Effect of altered serum lipid concentrations on the IC50 of halofantrine against Plasmodium falciparum.

Halofantrine (Hf) is a highly lipophilic antimalarial which significantly associates with triglyceride (TG) rich plasma lipoproteins, and this is likely manifest as a decrease in the free fraction of drug. This study assessed the effect of using growth media containing 10% serum containing different concentrations of TG (i.e. TG-rich plasma lipoproteins) on the IC50 of Hf determined using continuous in vitro culture of Plasmodium falciparum. Serum was collected from a human subject in either a fasted state or at various times after ingestion of a fatty meal. There was a linear and statistically significant 2.5-fold increase in the IC50 of Hf across a 6-fold range of increasing TG concentrations, with the increased IC50 values being ascribed to a decreased free fraction of Hf in the growth media due to sequestration by TG-rich lipoproteins. Chloroquine diphosphate, which is hydrophilic and not significantly bound by TG-rich lipoproteins, was used as a control and its IC50 values were independent of TG concentrations. These data indicate that consideration should be given to the adoption of standard conditions for the collection of serum with respect to pre- or postprandial states, and that subject- and disease-related factors which alter plasma lipoprotein profiles should be considered when interpreting the IC50 profile of Hf (and possibly other lipophilic antimalarials). Furthermore, although food is known to affect the pharmacokinetics of Hf, these data suggest that altered plasma lipoprotein profiles could also influence its pharmacodynamic profile.

Animals↗
PubMed1997

Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria in humans. An important class of drugs in malaria treatment is the sulfone/sulfonamide group, of which sulfadoxine is the most commonly used. The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resistance to this drug. In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference. We have expressed eight alleles of P. falciparum PPPK-DHPS in Escherichia coli and purified the functional enzymes to homogeneity. Strikingly, the Ki for sulfadoxine varies by almost three orders of magnitude from 0.14 microM for the DHPS allele from sensitive isolates to 112 microM for an enzyme expressed in a highly resistant isolate. Comparison of the Ki of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds. These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of P. falciparum are central to the mechanism of resistance to sulfones and sulfonamides.

Animals↗