Original Contribution Reactive oxygen species regulates expression of iron–sulfur cluster assembly protein IscS of Leishmania donovani Krishn Pratap Singh a , Amir Zaidi a , Shadab Anwar a , Sanjeev Bimal b , Pradeep Das c , Vahab Ali a,n a Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, India 800007 b Department of Immunology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, India 800007 c Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, India 800007 article info Article history: Received 8 April 2014 Received in revised form 10 July 2014 Accepted 14 July 2014 Available online 22 July 2014 Keywords: Iron–sulfur protein Leishmania Reactive oxygen species (ROS) Drug resistance Amphotericin B abstract The cysteine desulfurase, IscS, is a highly conserved and essential component of the mitochondrial iron– sulfur cluster (ISC) system that serves as a sulfur donor for Fe–S clusters biogenesis. Fe–S clusters are versatile and labile cofactors of proteins that orchestrate a wide array of essential metabolic processes, such as energy generation and ribosome biogenesis. However, no information regarding the role of IscS or its regulation is available in Leishmania, an evolving pathogen model with rapidly developing drug resistance. In this study, we characterized LdIscS to investigate the ISC system in AmpB-sensitive vs resistant isolates of L. donovani and to understand its regulation. We observed an upregulated Fe–S protein activity in AmpB-resistant isolates but, in contrast to our expectations, LdIscS expression was upregulated in the sensitive strain. However, further investigations showed that LdIscS expression is positively correlated with ROS level and negatively correlated with Fe–S protein activity, independent of strain sensitivity. Thus, our results suggested that LdIscS expression is regulated by ROS level with Fe–S clusters/ proteins acting as ROS sensors. Moreover, the direct evidence of a mechanism, in support of our results, is provided by dose-dependent induction of LdIscS-GFP as well as endogenous LdIscS in L. donovani promastigotes by three different ROS inducers: H 2 O 2 , menadione, and Amphotericin B. We postulate that LdIscS is upregulated for de novo synthesis or repair of ROS damaged Fe–S clusters. Our results reveal a novel mechanism for regulation of IscS expression that may help parasite survival under oxidative stress conditions encountered during infection of macrophages and suggest a cross talk between two seemingly unrelated metabolic pathways, the ISC system and redox metabolism in L. donovani. & 2014 Elsevier Inc. All rights reserved. Introduction Leishmania donovani is a unicellular parasitic protozoon that causes visceral leishmaniasis (VL), a neglected tropical disease with severe systemic manifestations. If left untreated, it has a high fatality rate, ranked next to malaria as the most deadly protozoan disease [1]. The parasite exists in two host-specific forms: (i) extracellular promastigotes form in the midgut of sandfly (Phlebotomus spp.) vector and (ii) intracellular amastigotes form inside the host macrophages [2]. During its life cycle, Leishmania encounters and readily adapts to survive various hostile conditions such as oxidative stress due to heme digestion in the blood meal and midgut proteases in the sandfly, complement lysis in the blood upon transmission, and reactive oxygen and nitrogen species (ROS and RNS) generated during phagocytosis by host macrophages [3]. The survival of parasite relies mainly on the unique thiol-redox metabolism [4] and skills to suppress oxidative outbursts of the host defense mechanisms [5]. In recent decades, the parasites have encountered an additional stress imposed by antileishmanial drugs, the majority of which exert ROS-mediated cytotoxic effects [6], but emerging resistance to these drugs [7,8] proves the high adaptive capacity of this dynamic parasitic protozoa. The first line drug sodium stibogluconate is no more preferred for the treatment of VL patients in India because more than 65% cases show resistance or relapse in Bihar which con- tributes about 90% of the VL cases of India [9]. Thus, the second line drug liposomal preparation of Amphotericin B (AmpB) and miltefosine (first oral drug), alone or in combination, is presently Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine http://dx.doi.org/10.1016/j.freeradbiomed.2014.07.017 0891-5849/& 2014 Elsevier Inc. All rights reserved. Abbreviations: ISC, iron sulfur cluster; LdIscS, L. donovani IscS; Fe–S, iron–sulfur; PLP, pyridoxal phosphate; ROS, reactive oxygen species; AmpB, amphotericin B; GFP, Green fluorescent protein; H 2 O 2 , hydrogen peroxide n Corresponding author. Fax: þ91 612 2634379. E-mail address: vahab_ali@yahoo.com (V. Ali). Free Radical Biology and Medicine 75 (2014) 195–209