A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

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.

Nucleosome landscape and control of transcription in the human malaria parasite

“…The predominant enrichment with FAIRE-seq that we observed during the erythrocytic cycle is consistent with a relatively permissive transcriptional status of P. falciparum's genes in general (Salcedo-Amaya et al 2009). Such a global model to control gene expression is supported by the growing evidence that the parasite lacks transcriptional plasticity, as suggested by the absence of positive or negative feedbacks observed at the transcriptional level in response to metabolic stresses (Ganesan et al 2008;Le Roch et al 2008). Furthermore, it has been shown that the parasite preinitiation complex interacts with stage specific ''active'' and ''inactive'' promoters (Gopalakrishnan et al 2009).…”

“…Although genome-wide gene expression analyses have shown remarkable variations of steadystate mRNA levels with a tightly regulated cascade of transcripts throughout the parasite life cycle (Bozdech et al 2003;Le Roch et al 2003), only a handful of transcription factors have been identified to date (Coulson et al 2004;Balaji et al 2005;Llinas et al 2006). Moreover, recent studies have demonstrated that the parasite has a limited capacity to regulate the expression of its genes in response to metabolic stresses (Ganesan et al 2008;Le Roch et al 2008). Altogether, these findings present an enigma as to how gene expression is regulated with regards to the paucity of transcription factors and the apparent absence of gene feedback circuits.…”

Transcriptional variation in the malaria parasitePlasmodium falciparum

“…The specific transcription factors identified seem insufficient to mount a wide range of transcriptional responses. (2) Analysis of transcriptional responses immediately or very soon after environmental perturbations failed to detect large alterations in the levels of transcripts related with the challenge, in contrast to similar studies in other eukaryotes (Gunasekera et al 2007;Ganesan et al 2008;Le Roch et al 2008;Young et al 2008). This led some investigators to conclude that P. falciparum has an unresponsive, hard-wired transcriptome (Ganesan et al 2008;Le Roch et al 2008).…”

“…(2) Analysis of transcriptional responses immediately or very soon after environmental perturbations failed to detect large alterations in the levels of transcripts related with the challenge, in contrast to similar studies in other eukaryotes (Gunasekera et al 2007;Ganesan et al 2008;Le Roch et al 2008;Young et al 2008). This led some investigators to conclude that P. falciparum has an unresponsive, hard-wired transcriptome (Ganesan et al 2008;Le Roch et al 2008). This is controversial because other studies detected changes in transcription upon various challenges (Functional Genomics Workshop Group et al 2007;Oakley et al 2007;Natalang et al 2008;Tamez et al 2008;Hu et al 2010), but the changes observed were of low amplitude and could be explained by cell cycle arrest or sexual differentiation, or may represent pathways leading to parasite death, rather than being authentic protective transcriptional responses (Functional Genomics Workshop Group et al 2007;Natalang et al 2008).…”

Epigenetics in Plasmodium: What Do We Really Know?

“…Overall, the in vitro experiments described above offer some valuable leads on how Plasmodium might respond epigenetically to changing host conditions, although the real situation will, of course, be more multifactorial. Just how flexible the intraerythrocytic transcriptional "cascade" actually is remains controversial, since some studies have reported a very "hardwired" transcriptional pattern: there is almost no transcriptional response when cultured parasites are treated with lethal antifolate drugs, for example (48). However, P. falciparum may well have evolved to modify its transcriptional program only in response to specific in vivo stimuli (not including the evolutionarily recent phenomenon of drug treatment).…”