Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation

Abstract text

Introduction: The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to "activation" of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. Amino-artemisinins exhibit better activity against Toxoplasma gondii than artemisinin, however, the role of ROS has not been investigated.

Materials and methods: T. gondii tachyzoites were grown in human foreskin fibroblasts. The T. gondii reference strain ME49 was treated with stepwise increasing amounts of artemisone and artemiside, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. The formation of reactive oxygen species (ROS) was assayed using dichlorofluorescein-diacetate (DCF-DA).

Results: The amino-artemisinins are effective in vitro against the non-heme-degrading T. gondii tachyzoites with IC 50 values of 50–70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 μM) of these two compounds within few days. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and – to a lesser extent – in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites.

Conclusions: Amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium, possibly linked to TGME49_236950, and the rhoptry neck protein RON2.