Yeast model uncovers dual roles of mitochondria in action of artemisinin - PubMed (original) (raw)

Yeast model uncovers dual roles of mitochondria in action of artemisinin

Wei Li et al. PLoS Genet. 2005 Sep.

Abstract

Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Artemisinin Inhibits Yeast Respiratory Growth by Depolarizing the Mitochondrial Membrane

(A) Artemisinin (Art) inhibits yeast growth in nonfermentable media. In YPD the effect of artemisinin is minimal, whereas in YPG, artemisinin is highly effective. (B) Yeast growth is inhibited by artemisinin in YPG with an IC50 that is comparable to that required to kill cultured malaria parasites. Relative growth in the presence of artemisinin was measured against to that of the yeast grown in the absence of artemisinin. Experiments shown were performed three times in liquid YPG media. Error bars represent standard errors of the mean for each assay. (C) Artemisinin depolarizes mitochondrial membrane. The peak shift toward the left represents a decrease of fluorescence signal indicating the loss of membrane potential. Cells were grown in YPG with or without artemisinin (Art) for 2 h.

Figure 2

Figure 2. The Genetic Screen for Artemisinin-Resistant Mutations Identified Genes in the Electron Transport Chain or in the Pathway of Respiratory Control

(A) The three mutants isolated display increased resistance to artemisinin. YPGE plates with or without 4 μM artemisinin were used. nde1Δ ndi1Δ exhibited severe growth defect in nonfermentable media. (B) Increased activities of NADH dehydrogenases exacerbate artemisinin sensitivity, and Sip5 may be positioned upstream of NADH dehydrogenases. Plates are all SG-Ura (with or without 4 μM artemisinin) to prevent plasmid loss. ADH1-NDE1 and ADH1-SIP5 here denote constructs that express NDE1 and SIP5 under the control of ADH1 promoter. The results of ADH1-NDI1 are similar to that of ADH1-NDE1 and are not shown on the two plates_._ (C) Expression of PfNDI1 in _ndi1_Δ restores yeast sensitivity to artemisinin. Plates used here are SG-Ura (with or without 8 μM artemisinin). Art, artemisinin; SG, synthetic yeast media with glycerol as the carbon source; WT, wild type.

Figure 3

Figure 3. Artemisinin Generates ROS in Yeast

When applicable, 8 μM artemisinin was used. (A) Artemisinin-resistant strains generate fewer ROS. Yeast untreated with artemisinin was used as the control. The experiment was performed three times with similar results. NDE1 denotes the overexpressor strain of NDE1 driven by ADH1 promoter. (B) Isolated artemisinin-resistant strains are not cross-resistant to paraquat or peroxide. Shown here are the wild-type (WT) parental strain (BY4742), _nde1_Δ and _ndi1_Δ on YPD plates without or with 0.02% paraquat. (C) Iron is possibly involved in artemisinin (Art) activation. Addition of BPS to the medium reduces yeast's sensitivity to artemisinin, whereas BPS alone does not enhance general yeast survival on drug-free plates. We did not use a higher amount of BPS to further reduce the iron level because a severe reduction in iron dramatically affects yeast growth on YPG.

References

    1. Meshnick SR. Artemisinin: mechanisms of action, resistance and toxicity. Int J Parasitol. 2002;32:1655–1660. - PubMed
    1. Asawamahasakda W, Benakis A, Meshnick SR. The interaction of artemisinin with red cell membranes. J Lab Clin Med. 1994;123:757–762. - PubMed
    1. Bhisutthibhan J, Pan XQ, Hossler PA, Walker DJ, Yowell CA, et al. The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin. J Biol Chem. 1998;273:16192–16198. - PubMed
    1. Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, et al. Artemisinins target the SERCA of Plasmodium falciparum . Nature. 2003;424:957–961. - PubMed
    1. Mercereau-Puijalon O, Fandeur T. Antimalarial activity of artemisinins: identification of a novel target? Lancet. 2003;362:2035–2036. - PubMed

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