Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi

Mejía-Jaramillo et al. Parasites & Vectors 2011, 4:169 http://www.parasitesandvectors.com/content/4/1/169 RESEARCH Open Access Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi Ana M Mejía-Jaramillo, Geysson J Fernández, Lina Palacio and Omar Triana-Chávez* Abstract Background: Chagas disease is a neglected illness, with limited treatments, caused by the parasite Trypanosoma cruzi. Two drugs are prescribed to treat the disease, nifurtimox and benznidazole, which have been previously reported to have limited efficacy and the appearance of resistance by T. cruzi. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of the genes involved, probably in its mechanisms of action. Results: The differential genes expression of a sensitive Trypanosoma cruzi strain and its induced in vitro benznidazole-resistant phenotypes was studied. The stepwise increasing concentration of BZ in the parental strain generated five different resistant populations assessed by the IC50 ranging from 10.49 to 93.7 μM. The resistant populations maintained their phenotype when the BZ was depleted from the culture for many passages. Additionally, the benznidazole-resistant phenotypes presented a cross-resistance to nifurtimox but not to G418 sulfate. On the other hand, four of the five phenotypes resistant to different concentrations of drugs had different expression levels for the 12 genes evaluated by real-time PCR. However, in the most resistant phenotype (TcR5x), the levels of mRNA from these 12 genes and seven more were similar to the parental strain but not for NTR and OYE genes, which were down-regulated and over-expressed, respectively. The number of copies for these two genes was evaluated for the parental strain and the TcR5x phenotype, revealing that the NTR gene had lost a copy in this last phenotype. No changes were found in the enzyme activity of CPR and SOD in the most resistant population. Finally, there was no variability of genetic profiles among all the parasite populations evaluated by performing low-stringency single-specific primer PCR (LSSP-PCR) and random amplified polymorphic DNA RAPD techniques, indicating that no clonal selection or drastic genetic changes had occurred for the exposure to BZ. Conclusion: Here, we propose NTR as the major marker of the appearance of resistance to BZ. Background American trypanosomiasis, or Chagas disease, is a neglected parasitic illness widely spread throughout the Americas, from the Southern United States to Argentina and Chile. Trypanosoma cruzi currently infects at least 7,694,500 individuals and between 60 and 80 million remain at risk of T. cruzi infection in endemic countries [1,2]. There is no vaccine to prevent the infection and chemotherapy is restricted to two nitroheterocyclic compounds: nifurtimox (NFX (4[(5-nitrofurfurylidene) amino]-3-methylthiomorpholine-1,1-dioxide) and * Correspondence: Grupo Biología y Control de Enfermedades Infecciosas-BCEI-SIU, Instituto de Biología, Universidad de Antioquia, Medellín, Colombia benznidazole (BZ (N-benzyl-2-nitroimidazole-1-acetamide) [3,4]. BZ is a nitroheterocyclic compound that contains a nitro group linked to an imidazole ring. As a pro-drug, BZ undergoes activation by enzymatic activity to have cytotoxic effects within the parasite, which is catalyzed by nitroreductases (NTRs) [5]. Because there are two possible enzymes acting on it, there are two proposed hypotheses for its toxic action. The first one postulates the generation of reactive oxygen species (ROS) following a one-electron reduction caused by NTR type II enzymatic activity. Under aerobic conditions, this induces the production of superoxide anions and causes re-cycling of the drug [6]. However, it is now known © 2011 Mejía-Jaramillo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mejía-Jaramillo et al. Parasites & Vectors 2011, 4:169 http://www.parasitesandvectors.com/content/4/1/169 that T. cruzi possesses both enzymatic and non-enzymatic antioxidant defenses, making it unlikely that ROS production affects the viability of the parasite, at least at the doses used to treat the disease [7-11]. The second hypothesis proposes a two-electron reduction of the drug by NTR type I. This reaction goes through a nitroso species, to a hydroxylamine derivative using NADPH as a source of electron donors. Hydroxylamine can react to produce a nitrenium cation, which induces DNA strand breaks. Moreover, the high electrophilic intermediaries may affect other molecules within the cell [12]. There are two trypanosomal enzymes with this type of activity: prostaglandin F2a synthase or old yellow enzyme (OYE), which mediates two-electron reduction in NFX under anaerobic conditions [13] and nitroreductase I (NTR). This second enzyme has already shown strong experimental evidence associated with cross-resistance to NFX and BZ [5,12,14,15]. Despite the great efforts made to understand BZ’s mode of action, it is not yet completely clear; even less thoroughly studied are the initially acquired mechanisms of resistance. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of genes involved in its mechanisms of action [16-18]. Many studies have been based on differential gene expression analysis in high concentrations of BZ in resistant phenotypes [19-26], but the commencement of this condition remains to be understood. For this reason, the identification of genes that are differentially expressed during progression through the susceptible drug population to a resistant phenotype in T. cruzi populations may help to further our grasp of acquired stable resistance mechanisms, including the basis of the drug’s mode of action. Additionally, it is also important to identify gene expression and/or genetic alterations as markers of either sensitivity or resistance responses, which could be useful for treatment prognosis and as potential new therapeutic targets. Therefore, the aim of this study was to examine the initial expression changes of parasites submitted to a stepwise concentration of BZ. For this reason, we chose 19 genes suggested to be involved in escaping BZ cytotoxic effects. Their RNA expression was quantified by real-time PCR (RT-qPCR). We also investigated other issues concerning drug resistance, such as the stability of BZ-resistant phenotypes without drug pressure over a long period of time as well as the cross-resistance with other nitroheterocyclic and non-nitroheterocyclic drugs. Materials and methods Reagents BZ and NFX were purified by organic extraction from Rochagan™ (...truncated)