Molecular & Biochemical Parasitology 196 (2014) 45–52 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Antioxidant defences of Spironucleus vortens: Glutathione is the major non-protein thiol C.F. Williams a,∗ , N. Yarlett b , M.A. Aon c , D. Lloyd a a Cardiff School of Biosciences, Main Building, Museum Avenue, Cardiff, CF10 3AT, Wales, UK b Departments of Chemistry and Physical Sciences, and Haskins Laboratories, Pace University, New York, NY 10038, USA c Johns Hopkins University, School of Medicine, Division of Cardiology, 720 Rutland Ave., 1059 Ross Building, Baltimore, MD 21205, USA a r t i c l e i n f o Article history: Received 2 May 2013 Received in revised form 21 July 2014 Accepted 28 July 2014 Available online 4 August 2014 Keywords: Protozoan Metronidazole Garlic Reactive oxygen species Superoxide dismutase a b s t r a c t The aerotolerant hydrogenosome-containing piscine diplomonad, Spironucleus vortens, is able to with- stand high fluctuations in O 2 tensions during its life cycle. In the current study, we further investigated the O 2 scavenging and antioxidant defence mechanisms which facilitate the survival of S. vortens under such oxidizing conditions. Closed O 2 electrode measurements revealed that the S. vortens ATCC 50386 strain was more O 2 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/10 7 cells) with cysteine and H 2 S as minor peaks. Furthermore, antioxidant proteins of S. vortens were assayed enzymatically and revealed that S. vortens possesses superoxide dis- mutase and NADH oxidase (883 and 37.5 nmol/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. H 2 O 2 was emitted into the exterior of S. vortens at a rate of 2.85 pmol/min/10 6 cells. Metronidazole and auranofin led to depletion of S. vortens intracellular NAD(P)H pools and an increase in H 2 O 2 release with concomitant oxidation of GSH, respectively. Garlic-derived compounds completely inhibited O 2 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. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Spironucleus vortens is a protozoan fish parasite capable of causing high mortalities in the ornamental aquaculture indus- try [1]. The organism primarily inhabits the middle to posterior intestinal tract of the host, a microaerobic environment contain- ing 33–66 M O 2 (JM Whittamore and RW Wilson, pers. com.). S. vortens is usually described as an anaerobic organism, utilizing O 2 - sensitive enzymes, e.g. pyruvate:ferredoxin oxidoreductase (PFOR) and hydrogenase, which play crucial roles in cellular metabolism and redox balance [2]. However, during its life cycle, S. vortens trophozoites are able to tolerate high fluctuations in O 2 ten- sions, especially during extra-intestinal systemic infection. Indeed, trophozoites have been isolated from head lesions of fish with hole-in-the-head disease, an environment which is likely to be ∗ Corresponding author. Tel.: +44 2920874048; fax: +44 2920874305. E-mail address: catrinwilliams@hotmail.com (C.F. Williams). quite aerobic in comparison to that of the intestinal tract [1]. Trophozoites are also expelled into the faeces of the host, where a small proportion of parasites are able to survive for more than 36 days outside the host [3]. Thus, in order to protect important intracellular anaerobic processes, S. vortens rapidly consumes O 2 from its immediate surroundings, thus creating its own optimal micro-environment [2]. Consumption of O 2 by S. vortens does not involve a phosphorylating respiratory chain, as the organism lacks cytochromes [4]. Instead, its hydrogenosomes are flavin-rich, have a trans-membrane electrochemical potential, and are labelled by an antibody raised against hydrogenase from Blastocystis hominis [5]. The mechanisms employed by S. vortens to detoxify O 2 have not been elucidated and may provide novel pathways for chemother- apy. Redox status plays a key role in cell survival. Indeed, ultra- dian oscillatory redox mechanisms are conserved in evolutionary diverse cells, from yeasts to cardiomyocytes, and are essential for cellular coherence and survival [6]. Redox cycling of intracellu- lar thiols forms the core of rhythmicity in yeast and heart cells http://dx.doi.org/10.1016/j.molbiopara.2014.07.010 0166-6851/© 2014 Elsevier B.V. All rights reserved.