Abstract
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.
Highlights
Malaria parasites infect over 300 million people around the world and the most virulent species, Plasmodium falciparum, kills 1–2 million individuals per year [1,2,3]
Traditional knowledge of gene regulation, learned largely from non-pathogenic model organisms such as E. coli, yeast, and mice, suggests that RNA for metabolic pathways are regulated in large part by DNA-binding transcriptional factors that are responsive to cellular metabolic needs
We demonstrate that the malaria-causing Plasmodium falciparum parasites, under lethal drug pressure from an antifolate with a known mechanism of action, are incapable of large reproducible changes in RNA levels for the target pathways, or for any other gene throughout the genome
Summary
Malaria parasites infect over 300 million people around the world and the most virulent species, Plasmodium falciparum, kills 1–2 million individuals per year [1,2,3]. It was expected that DNA microarrays would permit a quick, unbiased look at the mode of action of antimalarial drugs, simple antimetabolites [17]. In Saccharomyces cerevisiae, Mycobacterium tuberculosis, Candida albicans, mammalian cells and even plants, specific antimetabolites up-regulated dozens of target-related RNA by greater than 10-fold [18,19,20,21,22,23]. In many of these systems confidence in the power of DNA microarrays to reveal mechanisms of drug action come from perturbation of wellunderstood metabolic pathways
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