Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

Abstract

Background: Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.

Results: Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.

Conclusions: This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.

Figure 1

Morphological analysis of Plasmodium falciparum following inhibition of PfSpdSyn with cyclohexylamine. (A) Parasites treated with cyclohexylamine at invasion were arrested in the early trophozoite stage, correlating close to the time of maximal expression of this transcript in the intaerythrocytic developmental cycle (IDC). Untreated parasites matured through mid- into late trophozoites at comparable time points. Arrows indicate the Pearson correlation (r) between samples. In its 48 h lifecycle, the expression of PfSpdSyn is maximally expressed at 18 hours post-invasion (hpi) in a window of expression from 10-40 hpi. (B) Pearson correlations of untreated and treated samples, showing poor correlations of untreated samples to their treated counterparts at later time points (25 and 30 hpi). The approximate time of arrest (between 10 and 18 hpi) is indicated by the highest correlations to the untreated samples at 18 hpi.

Figure 2

Levels of selected polyamines following PfSpdSyn inhibition. Values represent the concentration of polyamine per 10¹⁰ cells, measured from two independent experiments performed in duplicate. Error bars are given as the standard error of the mean. Statistical significance is indicated by *** equivalent to 99% (p < 0.01) and * to 90% (p < 0.10) determined through a heteroscedastic Student t-test analysis.

Figure 3

Regulation of transcripts, proteins and metabolites of the polyamine biosynthetic pathway following cyclohexylamine inhibition. The relative expression (to untreated control samples) of each transcript and its encoding protein is indicated at each time point sampled. Increased/decreased levels of selected metabolites are indicated. Additionally, regulation of transcripts in purine metabolism and various methyltransferases are indicated.