Antioxidant defences of Spironucleus vortens: Glutathione is the major non-protein thiol

The aerotolerant hydrogenosome-containing piscine diplomonad, Spironucleus vortens, is able to withstand high fluctuations in O₂ tensions during its life cycle. In the current study, we further investigated the O₂ scavenging and antioxidant defence mechanisms which facilitate the survival of S. vortens under such oxidizing conditions. Closed O₂ electrode measurements revealed that the S. vortens ATCC 50386 strain was more O₂ tolerant than a freshly isolated S. vortens intestinal strain (Sv1). In contrast to the related human diplomonad, Giardia intestinalis, RP-HPLC revealed the major non-protein thiols of S. vortens to be glutathione (GSH, 776 nmol/10⁷ cells) with cysteine and H2S as minor peaks. Furthermore, antioxidant proteins of S. vortens were assayed enzymatically and revealed that S. vortens possesses superoxide dismutase and NADH oxidase (883 and 37.5nmol/min/mg protein, respectively), but like G. intestinalis, lacks catalase and peroxidase activities. Autofluorescence of NAD(P)H and FAD alongside the fluorescence of the GSH-adduct in monochlorobimane-treated live organisms allowed the monitoring of redox balances before and after treatment with inhibitors, metronidazole and auranofin. H₂O₂ was emitted into the exterior of S. vortens at a rate of 2.85 pmol/min/10⁶ cells. Metronidazole and auranofin led to depletion of S. vortens intracellular NAD(P)H pools and an increase in H₂O₂ release with concomitant oxidation of GSH, respectively. Garlic-derived compounds completely inhibited O₂ consumption by S. vortens (ajoene oil), or significantly depleted the intracellular GSH pool of the organism (allyl alcohol and DADS). Hence, antioxidant defence mechanisms of S. vortens may provide novel targets for parasite chemotherapy.