title: Artemisinin induces calcium-dependent protein secretion in the protozoan parasite Toxoplasma gondii.
abstract: Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca2+-ATPases are potential drug targets in parasites.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
authors.forename: Kisaburo
authors.initials: K
authors.lastname: Nagamune
authors.forename: Wandy L
authors.initials: WL
authors.lastname: Beatty
authors.forename: L David
authors.initials: LD
authors.lastname: Sibley
mesh_terms: D000818:Animals; D037621:Artemisinins / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D005816:Genetic Complementation Test; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D010271:Parasites / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014122:Toxoplasma / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D037621:Artemisinins; C408878:MIC2 protein, Toxoplasma gondii; D008565:Membrane Proteins; D015800:Protozoan Proteins; D019284:Thapsigargin; C031327:artemisinin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium