Co-inhibition of Plasmodium falciparum S-Adenosylmethionine Decarboxylase/Ornithine Decarboxylase Reveals Perturbation-specific Compensatory Mechanisms by Transcriptome, Proteome, and Metabolome Analyses

Abstract

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 anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ ornithine decarboxylase 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 as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine 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. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.Polyamines such as putrescine, spermidine, and spermine are small organic compounds containing two or more amino groups. At physiological pH, these polycations interact electrostatically with numerous anionic macromolecules, thereby stabilizing DNA, RNA, nucleoside triphosphates (e.g. ATP), phospholipids, and proteins (1, 2). These interactions with polyamines can alter DNA conformation, regulate replication and transcription, strengthen membranes, regulate ion channels, and protect DNA and phospholipids from oxidative stress (1-5). Yet polyamines are also implicated in apoptosis (5). Polyamine depletion generally causes cytostasis or growth arrest, which implies that these molecules are involved in cell cycle progression and regulation, and it is speculated that polyamines regulate cyclin degradation (1, 6, 7). Therefore, polyamines are essential for cellular growth, differentiation, and macromolecular synthesis and are ubiquitous components of all living cells, except two orders of Archaea (1). Polyamine metabolism is particularly important in rapidly proliferating cells and has been exploited in the treatment of cancer (1) and parasitic diseases (8). Polyamine metabolism of the malaria parasite Plasmodium falciparum is also a potential target for therapeutic intervention (9, 10).Polyamine and methionine metabolism are closely connected. This is particularly evident in Plasmodium where the two rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) 2 and S-adenosylme...

Metabolomic-Based Strategies for Anti-Parasite Drug Discovery

“…36 Global transcript analysis further revealed growth arrest after 19 h posttreatment and an increase in expression of lysine decarboxylase and ornithine aminotransferase as well as a decrease in expression of S-adenosylmethionine synthetase, supporting the metabolite data. 36 A second study aimed to validate glutamate dehydrogenase as a drug target using an untargeted LC-MS approach. 32 Heavy atom-labeled glutamine tracking was used to show that there is no difference in the labeling of the tricarboxylic acid cycle intermediates after knock out of this gene, nor is there an increased sensitivity to oxidative stresses.…”

“…88 A combination of the ODC inhibitor, eflornithine and 5′{[(Z)-4-amino-2-butenyl]methylamino}5′deoxyadenosine, an inhibitor of AdoMetDC, was added to late schizonts of P. falciparum, and changes to the metabolome were recorded using MRM of 172 metabolites. 36 Increases in putrescine and spermidine were recorded, but ornithine levels were unchanged due to an increase in ornithine aminotransferase expression, as determined by a global transcript analysis. 36 Global transcript analysis further revealed growth arrest after 19 h posttreatment and an increase in expression of lysine decarboxylase and ornithine aminotransferase as well as a decrease in expression of S-adenosylmethionine synthetase, supporting the metabolite data.…”

“…36 Increases in putrescine and spermidine were recorded, but ornithine levels were unchanged due to an increase in ornithine aminotransferase expression, as determined by a global transcript analysis. 36 Global transcript analysis further revealed growth arrest after 19 h posttreatment and an increase in expression of lysine decarboxylase and ornithine aminotransferase as well as a decrease in expression of S-adenosylmethionine synthetase, supporting the metabolite data. 36 A second study aimed to validate glutamate dehydrogenase as a drug target using an untargeted LC-MS approach.…”

Binding and inhibition of human spermidine synthase by decarboxylated S‐adenosylhomocysteine

“…[1][2][3][4][5][6][7] Spermidine is a major polyamine that serves multiple functions in growth including its role as a precursor of hypusine, which is essential for the activity of the eukaryotic initiation factor elF-5A. [8][9][10][11] Polyamines are formed in eukaryotes and in many, but not all, prokaryotes by the action of aminopropyltransferases.…”

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum

“…The finding that the initial rate of uptake of [ 3 H]spermidine uptake was significantly higher in the DFMO-treated cells than in untreated cells raises the possibility that the parasite might respond to polyamine depletion by the up-regulation of one or more transport pathways. However, a previous analysis of the transcriptional profile of intra-erythrocytic P. falciparum following DFMOinduced polyamine depletion did not identify altered expression of genes encoding proteins with similarity to known polyamine transporters (van Brummelen et al, 2009). …”