Functional genomics investigation of PfAdoMetDC/ODC co-inhibition of Plasmodium falciparum

Polyamines are ubiquitous components of all living cells and their depletion usually causes cytostasis, a strategy employed for treatment of West-African trypanosomiasis. To evaluate polyamine-depletion as an antimalarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC) in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine-depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses: the increased abundance of the transcripts for lysine decarboxylase (LDC) and ornithine aminotransferase (OAT) as well as the decreased abundance of that for S-adenosylmethionine synthetase (AdoMet synthetase). Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparumrespond to alleviate the detrimental effects of polyamine-depletion via regulation of its transcriptome and subsequently the proteome and metabolome. Keywords: functional genomics, time course, reference design, drug exposure, stress response, polyamine depletion, cytostasis Transcriptional profiling of PfAdoMetDC/ODC co-inhibited P. falciparum was performed with in-house spotted, oligonucleotide microarray analysis as previously reported [Bozdech, Z., Zhu, J., Joachimiak, M., Cohen, F., Pulliam, B., and DeRisi, J. (2003) Genome Biol. 4(2), R9]. Parasites were simultaneously treated in the schizont stage with the inhibitors, DFMO and MDL73811, alongside untreated controls in duplicate (i.e. 2 biological replicates A, B). A time course study was performed and samples were harvested at three time points in the trophozoite stage [19, 27 and 34 hours post-invasion (hpi)], just before and after cytostasis. RNA was isolated and cDNA synthesis was performed. A reference design microarray experiment was conducted and a composite reference pool was compiled from all the samples included in the experiment. Samples were fluorescently labelled with Cy5 dye and the reference pool with Cy3 and co-hybridized overnight. Where possible, technical replication was performed.