Molecular characterization of the MRPA transporter and antimony uptake in four New World Leishmania spp. susceptible and resistant to antimony☆

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Highlights

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Level of expression of Pgp is increased in the SbIII-resistant L. guyanensis and L. amazonensis lines.
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Incorporation of antimony was reduced in the SbIII-resistant L. guyanensis, L. amazonensis and L. braziliensis lines.
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Down-regulation of AQP1 protein was observed in the SbIII-resistant L. guyanensis and L. amazonensis lines.
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Rates of SbIII efflux are higher in the SbIII-resistant lines of L. guyanensis and L. braziliensis.
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Mechanisms of antimony-resistance of the MRPA gene are different among species of Leishmania analyzed.

Abstract

ATP-binding cassette (ABC) transporters have been associated with drug resistance in various diseases. The MRPA gene, a transporter of ABCC subfamily, is involved in the resistance by sequestering metal-thiol conjugates in intracellular vesicles of Leishmania parasite. In this study, we performed the molecular characterization of the MRPA transporter, analysis of P-glycoprotein (Pgp) and aquaglyceroporin-1 (AQP1) expression, and determination of antimony level in antimony-susceptible and -resistant lines of L. (V.) guyanensis, L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum. PFGE analysis revealed an association of chromosomal amplification of MRPA gene with the drug resistance phenotype in all SbIII-resistant Leishmania lines analyzed. Levels of mRNA from MRPA gene determined by real-time quantitative RT-PCR showed an increased expression of two fold in SbIII-resistant lines of Leishmania guyanensis, Leishmania amazonensis and Leishmania braziliensis. Western blot analysis revealed that Pgp is increased in the SbIII-resistant L. guyanensis and L. amazonensis lines. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed a reduction in the accumulation of this element in SbIII-resistant L. guyanensis, L. amazonensis and L. braziliensis lines when compared to their susceptible counterparts. Interestingly, a down-regulation of AQP1 protein was observed in the SbIII-resistant L. guyanensis and L. amazonensis lines, contributing for decreasing of SbIII entry in these lines. In addition, efflux experiments revealed that the rates of SbIII efflux are higher in the SbIII-resistant lines of L. guyanensis and L. braziliensis, that may explain also the low SbIII concentration within of these parasites. The BSO, an inhibitor of γ-glutamylcysteine synthetase enzyme, reversed the SbIII-resistance phenotype of L. braziliensis and caused an increasing in the Sb intracellular level in the LbSbR line. Our data indicate that the mechanisms of antimony-resistance are different among species of Leishmania analyzed in this study.

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Author and article information

Contributors

Silvane M.F. Murta

Journal

Journal ID (nlm-ta): Int J Parasitol Drugs Drug Resist

Journal ID (iso-abbrev): Int J Parasitol Drugs Drug Resist

Title: International Journal for Parasitology, Drugs and Drug Resistance

Publisher: Elsevier

ISSN (Electronic): 2211-3207

Publication date PMC-release: 5 September 2013

Publication date (Electronic): 5 September 2013

Publication date Collection: December 2013

Volume: 3

Pages: 143-153

Affiliations

[a ]Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou – CPqRR/FIOCRUZ, Belo Horizonte 30190-002, Minas Gerais, Brazil

[b ]Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Minas Gerais, Brazil

[c ]Centre de Recherche en Infectiologie, CHUL, Québec G1V 4G2, QC, Canada

Author notes

[* ]Corresponding author. Address: Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas ‘René Rachou’, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002 Belo Horizonte, MG, Brazil. Tel.: +55 31 3349 7780; fax: +55 31 32953115. silvane@ 123456cpqrr.fiocruz.br

Article

Publisher ID: S2211-3207(13)00014-6

DOI: 10.1016/j.ijpddr.2013.08.001

PMC ID: 3862441

PubMed ID: 24533304

SO-VID: 44bfc39f-7b32-462d-9825-10eaa54409e9

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 29 September 2012

Date revision received : 20 August 2013

Date accepted : 21 August 2013

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