Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes

The uptake and expression of extracellular DNA has been established as a mechanism for horizontal transfer of genes between bacterial species. Such transfer can support acquisition of advantageous elements, including determinants that affect the interactions between infectious organisms and their hosts. Here we show that erythrocyte-stage Plasmodium falciparum malaria parasites spontaneously take up DNA from the host cell cytoplasm into their nuclei. We have exploited this finding to produce levels of reporter expression in P.falciparum that are substantially improved over those obtained by electroporation protocols currently used to transfect malaria parasites. Parasites were transformed to a drug-resistant state when placed into cell culture with erythrocytes containing a plasmid encoding the human dihydrofolate reductase sequence. The findings reported here suggest that the malaria genome may be continually exposed to exogenous DNA from residual nuclear material in host erythrocytes.

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.

Evidence for clonal propagation in natural isolates of Plasmodium falciparum from Venezuela

We have analyzed 75 isolates of Plasmodium falciparum, collected in Venezuela during both the dry (November) and rainy (May–July) seasons, with a range of genetic markers including antigen genes and 14 random amplified polymorphic DNA (RAPD) primers. Thirteen P. falciparum stocks from Kenya and four other Plasmodium species are included in the analysis for comparison. Cross-hybridization shows that the 14 RAPD primers reveal 14 separate regions of the parasite's genome. The P. falciparum isolates are a monophyletic clade, significantly different from the other Plasmodium species. We identify three RAPD characters that could be useful as “tags” for rapid species identification. The Venezuelan genotypes fall into two discrete genetic subdivisions associated with either the dry or the rainy season; the isolates collected in the rainy season exhibit greater genetic diversity. There is significant linkage disequilibrium in each seasonal subsample and in the full sample. In contrast, no linkage disequilibrium is detected in the African sample. These results support the hypothesis that the population structure of P. falciparum in Venezuela, but not in Africa, is predominantly clonal. However, the impact of genetic recombination on Venezuelan P. falciparum seems higher than in parasitic species with long-term clonal evolution like Trypanosoma cruzi, the agent of Chagas' disease. The genetic structure of the Venezuelan samples is similar to that of Escherichia coli, a bacterium that propagates clonally, with occasional genetic recombination.

The pfmdr1 gene of Plasmodium falciparum confers cellular resistance to antimalarial drugs in yeast cells.

The exact role of the pfmdr1 gene in the emergence of drug resistance in the malarial parasite Plasmodium falciparum remains controversial. pfmdr1 is a member of the ATP binding cassette (ABC) superfamily of transporters that includes the mammalian P-glycoprotein family. We have introduced wild-type and mutant variants of the pfmdr1 gene in the yeast Saccharomyces cerevisiae and have analyzed the effect of pfmdr1 expression on cellular resistance to quinoline-containing antimalarial drugs. Yeast transformants expressing either wild-type or a mutant variant of mouse P-glycoprotein were also analyzed. Dose-response studies showed that expression of wild-type pfmdr1 causes cellular resistance to quinine, quinacrine, mefloquine, and halofantrine in yeast cells. Using quinacrine as substrate, we observed that increased resistance to this drug in pfmdr1 transformants was associated with decreased cellular accumulation and a concomitant increase in drug release from preloaded cells. The introduction of amino acid polymorphisms in TM11 of Pgh-1 (pfmdr1 product) associated with drug resistance in certain field isolates of P. falciparum abolished the capacity of this protein to confer drug resistance. Thus, these findings suggest that Pgh-1 may act as a drug transporter in a manner similar to mammalian P-glycoprotein and that sequence variants associated with drug-resistance pfmdr1 alleles behave as loss of function mutations.

Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

Variant antigens and endothelial receptor adhesion in Plasmodium falciparum.

Parasite-derived proteins expressed on the surface of erythrocytes infected with Plasmodium falciparum are important virulence factors, since they mediate binding of infected cells to diverse receptors on vascular endothelium and are targets of a protective immune response. They are difficult to study because they undergo rapid clonal antigenic variation in vitro, which precludes the derivation of phenotypically homogeneous cultures. Here we have utilized sequence-specific proteases to dissect the role of defined antigenic variants in binding to particular receptors. By selection of protease-resistant subpopulations of parasites on defined receptors we (i) confirm the high rate of antigenic variation in vitro; (ii) demonstrate that a single infected erythrocyte can bind to intercellular adhesion molecule 1, CD36, and thrombospondin; (iii) show that binding to intercellular adhesion molecule 1 and CD36 are functions of the variant antigen; and (iv) suggest that binding to thrombospondin may be mediated by other components of the infected erythrocyte surface.

Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum.

The pfmdr1 gene has been associated with a drug-resistant phenotype in Plasmodium falciparum, and overexpression of pfmdr1 has been associated with mefloquine- and halofantrine-resistant parasites, but little is known about the functional role of pfmdr1 in this process. Here, we demonstrate that the pfmdr1 gene expressed in a heterologous yeast system functions as a transport molecule and complements a mutation in ste6, a gene which encodes a mating pheromone a-factor export molecule. In addition, the pfmdr1 gene containing two mutations which are associated with naturally occurring chloroquine resistance abolishes this mating phenotype, suggesting that these genetic polymorphisms alter this transport function. Our results support the functional role of pfmdr1 as a transport molecule in the mediation of drug resistance and provide an assay system to address the nature of this transport function.

Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins.

Open reading frames in the Plasmodium falciparum genome encode domains homologous to the adhesive domains of the P. falciparum EBA-175 erythrocyte-binding protein (eba-175 gene product) and those of the Plasmodium vivax and Plasmodium knowlesi Duffy antigen-binding proteins. These domains are referred to as Duffy binding-like (DBL), after the receptor that determines P. vivax invasion of Duffy blood group-positive human erythrocytes. Using oligonucleotide primers derived from short regions of conserved sequence, we have developed a reverse transcription-PCR method that amplifies sequences encoding the DBL domains of expressed genes. Products of these reverse transcription-PCR amplifications include sequences of single-copy genes (including eba-175) and variably transcribed genes that cross-hybridize to multiple regions of the genome. Restriction patterns of the multicopy genes show a high degree of polymorphism among different parasite lines, whereas single-copy genes are generally conserved. Characterization of the single-copy genes has identified a gene (ebl-1) that is related to eba-175 and is likely to be involved in erythrocyte invasion.

CYP2D6: a global analysis of phenotypic and genotypic variation in search of radical cure of Plasmodium vivax malaria

Thesis (Master's)--University of Washington, 2016-06

Treatment of falciparum malaria in the age of drug-resistance

The growing problem of drug resistance has greatly complicated the treatment for falciparum malaria. Whereaschloroquine and sulfadoxine/ pyrimethamine could once cure most infections, this is no longer true and requiresexamination of alternative regimens. Not all treatment failures are drug resistant and other issues such asexpired antimalarials and patient compliance need to be considered. Continuation of a failing treatment policyafter drug resistance is established suppresses infections rather than curing them, leading to increasedtransmission of malaria, promotion of epidemics and loss of public confidence in malaria control programs.Antifolate drug resistance (i.e. pyrimethamine) means that new combinations are urgently needed particularlybecause addition of a single drug to an already failing regimen is rarely effective for very long. Atovaquone/proguanil and mefloquine have been used against multiple drug resistant falciparum malaria with resistance toeach having been documented soon after drug introduction. Drug combinations delay further transmission ofresistant parasites by increasing cure rates and inhibiting formation of gametocytes. Most currentlyrecommended drug combinations for falciparum malaria are variants of artemisinin combination therapy wherea rapidly acting artemisinin compound is combined with a longer half-life drug of a different class. Artemisininsused include dihydroartemisinin, artesunate, artemether and companion drugs include mefloquine, amodiaquine,sulfadoxine/ pyrimethamine, lumefantrine, piperaquine, pyronaridine, chlorproguanil/dapsone. The standard ofcare must be to cure malaria by killing the last parasite. Combination antimalarial treatment is vital not only tothe successful treatment of individual patients but also for public health control of malaria.

Population pharmacokinetics and pharmacodynamics of lumefantrine in young Ugandan children treated with artemether-lumefantrine for uncomplicated malaria

Background. The pharmacokinetics and pharmacodynamics of lumefantrine, a component of the most widely used treatment for malaria, artemether-lumefantrine, has not been adequately characterized in young children. Methods. Capillary whole-blood lumefantrine concentration and treatment outcomes were determined in 105 Ugandan children, ages 6 months to 2 years, who were treated for 249 episodes of Plasmodium falciparum malaria with artemether-lumefantrine. Results. Population pharmacokinetics for lumefantrine used a 2-compartment open model with first-order absorption. Age had a significant positive correlation with bioavailability in a model that included allometric scaling. Children not receiving trimethoprim-sulfamethoxazole with capillary whole blood concentrations <200 ng/mL had a 3-fold higher hazard of 28-day recurrent parasitemia, compared with those with concentrations >200 ng/mL (P =. 0007). However, for children receiving trimethoprim-sulfamethoxazole, the risk of recurrent parasitemia did not differ significantly on the basis of this threshold. Day 3 concentrations were a stronger predictor of 28-day recurrence than day 7 concentrations. Conclusions. We demonstrate that age, in addition to weight, is a determinant of lumefantrine exposure, and in the absence of trimethoprim-sulfamethoxazole, lumefantrine exposure is a determinant of recurrent parasitemia. Exposure levels in children aged 6 months to 2 years was generally lower than levels published for older children and adults. Further refinement of artemether-lumefantrine dosing to improve exposure in infants and very young children may be warranted. © 2016 The Author.

Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

This work reports the in vitro activity against Plasmodium falciparum blood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile.