Gene expression analysis of antimony resistance in Leishmania tropica using quantitative real-time PCR focused on genes involved in trypanothione metabolism and drug transport
- 96 Downloads
Pentavalent antimonials remain the treatment of choice for all the clinical forms of leishmaniasis. The increasing rates of antimony resistance are becoming a serious health problem in treatment of anthroponotic cutaneous leishmaniasis (ACL). Accordingly, unraveling molecular markers is crucial for improving medication strategies and monitoring of drug-resistant parasites. Different studies have suggested the importance of genes involved in trypanothione metabolism and drug transport. In this regard, present study was designed to investigate the RNA expression level of five genes including γ-GCS, ODC, TRYR (involved in trypanothione metabolism), AQP1 (acts in drug uptake) and MRPA (involved in sequestration of drug) in sensitive and resistant Leishmania tropica isolates. Seven antimony-resistant and seven antimony-sensitive L. tropica clinical isolates were collected from ACL patients. Drug sensitivity test was performed on the samples as well as reference strains; afterwards, gene expression analysis was performed on clinical isolates by quantitative real-time PCR. The results revealed that the average expression level of AQP1 gene was decreased (0.47-fold) in resistant isolates compared to sensitive ones whereas MRPA (2.45), γ-GCS (2.1) and TRYR (1.97) was upregulated in resistant isolates. The average expression of ODC (1.24-fold) gene was not different significantly between sensitive and resistant isolates. Our findings suggest that AQP1, MRPA, GSH1 and TRYR can be considered as potential molecular markers for screening of antimony resistance in some L. tropica clinical isolates.
KeywordsNatural antimony resistance Leishmania tropica Potential molecular marker Gene expression Quantitative real-time PCR
The authors would like to express their gratitude to Dr. S.R. Naddaf, Mrs. S. Charedar, Dr. M. Saffari and Dr. B. Akhoondi for their kind cooperation.
This research has been supported by Tehran University of Medical Sciences and health Services Grant no. 93-02-27-25242.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The study was approved by the Ethics Research Committee of the School of Public Health, Tehran University of Medical Sciences.
Informed consent was obtained from all individual participants included in the study.
- 1.Adaui V, Schnorbusch K, Zimic M, Gutiérrez A, Decuypere S, Vanaerschot M, DE Doncker S, Maes I, Llanos-Cuentas A, Chappuis F, Arévalo J, Dujardin JC (2011) Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony. Parasitology 138:183–193. https://doi.org/10.1017/S0031182010001095 CrossRefGoogle Scholar
- 7.Decuypere S, Rijal S, Yardley V, DE Doncker S, Laurent T, Khanal B, Chappuis F, Dujardin JC (2005) Gene expression analysis of the mechanism of natural Sb(V) resistance in Leishmania donovani isolates from Nepal. Antimicrob Agents Chemother 49:4616–4621. https://doi.org/10.1128/AAC.49.11.4616-4621.2005 CrossRefGoogle Scholar
- 8.Monte-Neto D, Coelho RL, Raymond AC, Legare F, Corbeil D, Melo J, Frezard MN, Ouellette F, M (2011) Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis. PLoS Negl Trop Dis 5:e1167. https://doi.org/10.1371/journal.pntd.0001167 CrossRefGoogle Scholar
- 9.El Fadili K, Messier N, Leprohon P, Roy G, Guimond C, Trudel N, Saravia NG, Papadopoulou B, Legare D, Ouellette M (2005) Role of the ABC transporter MRPA (PGPA) in antimony resistance in Leishmania infantum axenic and intracellular amastigotes. Antimicrob Agents Chemother 49:1988–1993. https://doi.org/10.1128/AAC.49.5.1988-1993.2005 CrossRefGoogle Scholar
- 15.Jeddi F, Mary C, Aoun K, Harrat Z, Bouratbine A, Faraut F, Benikhlef R, Pomares C, Pratlong F, Marty P, Piarroux R (2014) Heterogeneity of molecular resistance patterns in antimony-resistant field isolates of Leishmania species from the western Mediterranean area. Antimicrob Agents Chemother 58(8):4866–4874. https://doi.org/10.1128/AAC.02521-13 CrossRefGoogle Scholar
- 16.Kazemi-Rad E, Mohebali M, Khadem-Erfan MB, Saffari M, Raoofian R, Hajjaran H, Hadighi R, Khamesipour A, Rezaie S, Abedkhojasteh H, Heidari M (2013) Identification of antimony resistance markers in Leishmania tropica field isolates through a cDNA-AFLP approach. Exp Parasitol 135(2):344–349. https://doi.org/10.1016/j.exppara.2013.07.018 CrossRefGoogle Scholar
- 17.Kumar D, Singh R, Bhandari V, Kulshrestha A, Negi NS, Salotra P (2012) Biomarkers of antimony resistance: need for expression analysis of multiple genes to distinguish resistance phenotype in clinical isolates of Leishmania donovani. Parasitol Res 111:223–230. https://doi.org/10.1007/s00436-012-2823-z CrossRefGoogle Scholar
- 20.Mandal S, Maharjan M, Singh S, Chatterjee M, Madhubala R (2010) Assessing aquaglyceroporin gene status and expression profile in antimony-susceptible and -resistant clinical isolates of Leishmania donovani from India. J Antimicrob Chemother 65:496–507. https://doi.org/10.1093/jac/dkp468 CrossRefGoogle Scholar
- 24.Mukherjee A, Padmanabhan PK, Singh S, Roy G, Girard I, Chatterjee M, Ouellette M, Madhubala R (2007) Role of ABC transporter MRPA, gamma-glutamylcysteine synthetase and ornithine decarboxylase in natural antimony-resistant isolates of Leishmania donovani. J Antimicrob Chemother 59:204–211. https://doi.org/10.1093/jac/dkl494 CrossRefGoogle Scholar
- 27.Plourde M, Coelho A, Keynan Y, Larios OE, Ndao M, Ruest A, Roy G, Rubinstein E, Ouellette M (2012) Genetic polymorphisms and drug susceptibility in four isolates of Leishmania tropica obtained from Canadian soldiers returning from Afghanistan. PLoS Negl Trop Dis 6(1):e1463. https://doi.org/10.1371/journal.pntd.0001463 CrossRefGoogle Scholar
- 33.Torres DC, Adaui V, Ribeiro-Alves M, Romero GA, Arévalo J, Cupolillo E, Dujardin JC (2010) Targeted gene expression profiling in Leishmania braziliensis and Leishmania guyanensis parasites isolated from Brazilian patients with different antimonial treatment outcomes. Infect Genet Evol 10(6):727–733. https://doi.org/10.1016/j.meegid.2010.05.006 CrossRefGoogle Scholar