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

<div><p>(A) The three mutants isolated display increased resistance to artemisinin. YPGE plates with or without 4 μM artemisinin were used. <i>nde1Δ ndi1Δ</i> exhibited severe growth defect in nonfermentable media.</p><p>(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. <i>ADH1-NDE1</i> and <i>ADH1-SIP5</i> here denote constructs that express <i>NDE1</i> and <i>SIP5</i> under the control of <i>ADH1</i> promoter. The results of <i>ADH1-NDI1</i> are similar to that of <i>ADH1-NDE1</i> and are not shown on the two plates<i>.</i></p><p>(C) Expression of PfNDI1 in <i>ndi1</i>Δ restores yeast sensitivity to artemisinin. Plates used here are SG-Ura (with or without 8 μM artemisinin).</p><p>Art, artemisinin; SG, synthetic yeast media with glycerol as the carbon source; WT, wild type.</p></div

Artemisinin Inhibits Yeast Respiratory Growth by Depolarizing the Mitochondrial Membrane

<div><p>(A) Artemisinin (Art) inhibits yeast growth in nonfermentable media. In YPD the effect of artemisinin is minimal, whereas in YPG, artemisinin is highly effective.</p><p>(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.</p><p>(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.</p></div

Artemisinin Generates ROS in Yeast

<div><p>When applicable, 8 μM artemisinin was used.</p><p>(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 <i>NDE1</i> driven by <i>ADH1</i> promoter.</p><p>(B) Isolated artemisinin-resistant strains are not cross-resistant to paraquat or peroxide. Shown here are the wild-type (WT) parental strain (BY4742), <i>nde1</i>Δ and <i>ndi1</i>Δ on YPD plates without or with 0.02% paraquat.</p><p>(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.</p></div