Contents lists available at ScienceDirect IJP: Drugs and Drug Resistance journal homepage: www.elsevier.com/locate/ijpddr Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani Aditya Verma a,b , Vasundhra Bhandari a,1 , Deepak Kumar Deep a , Shyam Sundar c , Jean Claude Dujardin d , Ruchi Singh a , Poonam Salotra a,* a ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, India b Symbiosis School of Biomedical Sciences, Symbiosis International University, Pune, India c Institute of Medical Sciences, Banaras Hindu University, Varanasi, India d Unit of Molecular Parasitology, Department of Parasitology, Institute of Tropical Medicine, Antwerp, Belgium ARTICLE INFO Keywords: Leishmania donovani Drug resistance Paromomycin Transcriptome ABC transporters Nitric oxide Visceral leishmaniasis ABSTRACT Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL) in the Indian subcontinent. Paromomycin (PMM), an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R) and the corresponding PMM sensitive (PMM-S) parasites revealed modulated expression of 500 genes (1.5 fold cut off) in PMM-R parasites. Selected genes were validated for their modulated expression by quan- titative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a) reduced oxidative phosphorylation; b) increased glyco- somal succinate fermentation and substrate level phosphorylation; c) dependency on lipids and amino acids for energy generation; d) reduced DNA synthesis and increased DNA damage repair and e) decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca 2+ channel that are also modulators of ABC transpor- ters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO) levels while reactive oxygen species (ROS) level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania. 1. Introduction Visceral leishmaniasis (VL) or kala-azar is the most severe form of leishmaniasis and may be fatal, if left untreated. In the absence of any vaccine, control of VL relies mainly on chemotherapy. Existing drugs for VL have serious drawbacks in terms of safety, efficacy, cost, and development of resistance. Miltefosine (MIL) was considered as one of the main pillars of VL elimination program in India; however, reports of relapses following MIL treatment and decline in its efficacy raised concerns regarding its utility in VL control (Bhandari et al., 2012; Sundar et al., 2012; Rijal et al., 2013). In a recent study the declining efficacy of MIL for the treatment of post kala-azar dermal leishmaniasis (PKDL) patients, considered to be a major reservoir for transmission of the disease, has been reported (Ramesh et al., 2015). Another drug, amphotericin B, is associated with toxicity (Srivastava et al., 2011) and its liposomal formulation (AmBisome) has been recommended as a first line treatment of VL in the Indian subcontinent (World Health Organisation, 2010). Paromomycin (PMM) is another treatment option for VL control in the Indian subcontinent and has been found to be effective as mono- therapy as well as in combination with other drugs (Sinha et al., 2011; Sundar et al., 2011; Rahman et al., 2017). Development of resistance towards aminoglycosides such as PMM has been frequently en- countered in a variety of bacteria and PMM resistance in Leishmania can readily be induced in vitro (El-On et al., 1991; Maarouf et al., 1998; Hendrickx et al., 2012). A report of increased ED 90 towards PMM in http://dx.doi.org/10.1016/j.ijpddr.2017.10.004 Received 7 September 2017; Received in revised form 4 October 2017; Accepted 9 October 2017 * Corresponding author. National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi, 110029, India. 1 Present Address: School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India. E-mail address: salotra@vsnl.com (P. Salotra). IJP: Drugs and Drug Resistance 7 (2017) 370–377 Available online 10 October 2017 2211-3207/ © 2017 Indian Council of Medical Research-National Institute of Pathology. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T