A genetically hard-wired metabolic transcriptome in Plasmodium falciparum fails to mount protective responses to lethal antifolates.

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites.

Regulatory hotspots in the malaria parasite genome dictate transcriptional variation.

The determinants of transcriptional regulation in malaria parasites remain elusive. The presence of a well-characterized gene expression cascade shared by different Plasmodium falciparum strains could imply that transcriptional regulation and its natural variation do not contribute significantly to the evolution of parasite drug resistance. To clarify the role of transcriptional variation as a source of stain-specific diversity in the most deadly malaria species and to find genetic loci that dictate variations in gene expression, we examined genome-wide expression level polymorphisms (ELPs) in a genetic cross between phenotypically distinct parasite clones. Significant variation in gene expression is observed through direct co-hybridizations of RNA from different P. falciparum clones. Nearly 18% of genes were regulated by a significant expression quantitative trait locus. The genetic determinants of most of these ELPs resided in hotspots that are physically distant from their targets. The most prominent regulatory locus, influencing 269 transcripts, coincided with a Chromosome 5 amplification event carrying the drug resistance gene, pfmdr1, and 13 other genes. Drug selection pressure in the Dd2 parental clone lineage led not only to a copy number change in the pfmdr1 gene but also to an increased copy number of putative neighboring regulatory factors that, in turn, broadly influence the transcriptional network. Previously unrecognized transcriptional variation, controlled by polymorphic regulatory genes and possibly master regulators within large copy number variants, contributes to sweeping phenotypic evolution in drug-resistant malaria parasites.

Characterization of human malaria parasite Plasmodium falciparum eIF4E homologue and mRNA 5' cap status.

The mRNA 5' cap is an essential structural feature for translation of eukaryotic mRNA. Translation is initiated by recognition of the cap by the translation initiation factor eIF4E. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite eIF4E and its interaction with capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA cap binding activity. We used this protein to purify P. falciparum capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified capped mRNAs shows that 34 features were more than twofold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The putatively uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal.

Artemisinin effectiveness in erythrocytes is reduced by heme and heme-containing proteins.

Artemisinin loses its antimalarial activity on prolonged exposure to erythrocytes, especially alpha-thalassemic erythrocytes. In this report, we show that the major artemisinin-inactivating factor in cytosol of normal erythrocytes was heat-labile but a heat-stable factor from alpha-thalassemic cells also played a significant role in reducing artemisinin effectiveness, which was shown to be heme released from hemoglobin (Hb). Studies of fractionated lysate from genetically normal erythrocytes revealed that the protein fraction with molecular weight greater than 100 kDa was capable of reducing artemisinin effectiveness more readily than lower molecular weight fraction. Catalase and Hb A, but not selenoprotein glutathione peroxidase, were capable of reducing artemisinin effectiveness. Hemin (ferriprotoporphyrin IX) also reduced artemisinin effectiveness in a concentration- and time-dependent manner. It is concluded that heme and heme-containing proteins in erythrocyte are largely responsible for reducing artemisinin effectiveness and may contribute to resistance of Plasmodium falciparum infecting alpha-thalassemic erythrocytes observed in vitro.