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Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines

BACKGROUND: Tryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules. Reports suggest that drug-resistant parasites may increase the levels of TXNPx and other enzymes, thereby protecting them against oxi...

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Autores principales: Andrade, Juvana M, Murta, Silvane M F
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261743/
https://www.ncbi.nlm.nih.gov/pubmed/25174795
http://dx.doi.org/10.1186/1756-3305-7-406
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author Andrade, Juvana M
Murta, Silvane M F
author_facet Andrade, Juvana M
Murta, Silvane M F
author_sort Andrade, Juvana M
collection PubMed
description BACKGROUND: Tryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules. Reports suggest that drug-resistant parasites may increase the levels of TXNPx and other enzymes, thereby protecting them against oxidative stress. METHODS: In this study, the gene encoding cytosolic TXNPx (cTXNPx) was characterized in lines of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum that are susceptible and resistant to potassium antimony tartrate (Sb(III)). We investigated the levels of mRNA and genomic organization of the cTXNPx gene. In addition, we transfected the Leishmania lines with the cTXNPx gene and analysed the susceptibility of transfected parasites to Sb(III) and to hydrogen peroxide (H(2)O(2)). RESULTS: Northern blot and real-time reverse transcriptase polymerase chain reaction analyses revealed that the level of TXNPx mRNA was approximately 2.5-fold higher in the Sb(III)-resistant L. braziliensis line than in the parental line. In contrast, no significant difference in cTXNPx mRNA levels between the L. infantum lines was observed. Southern blot analyses revealed that the cTXNPx gene is not amplified in the genome of the Sb(III)-resistant Leishmania lines analysed. Functional analysis of cTXNPx was performed to determine whether overexpression of the enzyme in L. braziliensis and L. infantum lines would change their susceptibility to Sb(III). Western blotting analysis showed that the level of cTXNPx was 2 to 4-fold higher in transfected clones compared to non-transfected cells. Antimony susceptibility test (EC(50) assay) revealed that L. braziliensis lines overexpressing cTXNPx had a 2-fold increase in resistance to Sb(III) when compared to the untransfected parental line. In addition, these clones are more tolerant to exogenous H(2)O(2) than the untransfected parental line. In contrast, no difference in Sb(III) susceptibility and a moderate index of resistance to H(2)O(2) was observed in L. infantum clones overexpressing cTXNPx. CONCLUSION: Our functional analysis revealed that cTXNPx is involved in the antimony-resistance phenotype in L. braziliensis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1756-3305-7-406) contains supplementary material, which is available to authorized users.
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spelling pubmed-42617432014-12-10 Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines Andrade, Juvana M Murta, Silvane M F Parasit Vectors Research BACKGROUND: Tryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules. Reports suggest that drug-resistant parasites may increase the levels of TXNPx and other enzymes, thereby protecting them against oxidative stress. METHODS: In this study, the gene encoding cytosolic TXNPx (cTXNPx) was characterized in lines of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum that are susceptible and resistant to potassium antimony tartrate (Sb(III)). We investigated the levels of mRNA and genomic organization of the cTXNPx gene. In addition, we transfected the Leishmania lines with the cTXNPx gene and analysed the susceptibility of transfected parasites to Sb(III) and to hydrogen peroxide (H(2)O(2)). RESULTS: Northern blot and real-time reverse transcriptase polymerase chain reaction analyses revealed that the level of TXNPx mRNA was approximately 2.5-fold higher in the Sb(III)-resistant L. braziliensis line than in the parental line. In contrast, no significant difference in cTXNPx mRNA levels between the L. infantum lines was observed. Southern blot analyses revealed that the cTXNPx gene is not amplified in the genome of the Sb(III)-resistant Leishmania lines analysed. Functional analysis of cTXNPx was performed to determine whether overexpression of the enzyme in L. braziliensis and L. infantum lines would change their susceptibility to Sb(III). Western blotting analysis showed that the level of cTXNPx was 2 to 4-fold higher in transfected clones compared to non-transfected cells. Antimony susceptibility test (EC(50) assay) revealed that L. braziliensis lines overexpressing cTXNPx had a 2-fold increase in resistance to Sb(III) when compared to the untransfected parental line. In addition, these clones are more tolerant to exogenous H(2)O(2) than the untransfected parental line. In contrast, no difference in Sb(III) susceptibility and a moderate index of resistance to H(2)O(2) was observed in L. infantum clones overexpressing cTXNPx. CONCLUSION: Our functional analysis revealed that cTXNPx is involved in the antimony-resistance phenotype in L. braziliensis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1756-3305-7-406) contains supplementary material, which is available to authorized users. BioMed Central 2014-08-29 /pmc/articles/PMC4261743/ /pubmed/25174795 http://dx.doi.org/10.1186/1756-3305-7-406 Text en © Andrade and Murta; licensee BioMed Central Ltd. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Andrade, Juvana M
Murta, Silvane M F
Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines
title Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines
title_full Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines
title_fullStr Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines
title_full_unstemmed Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines
title_short Functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible Leishmania braziliensis and Leishmania infantum lines
title_sort functional analysis of cytosolic tryparedoxin peroxidase in antimony-resistant and –susceptible leishmania braziliensis and leishmania infantum lines
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261743/
https://www.ncbi.nlm.nih.gov/pubmed/25174795
http://dx.doi.org/10.1186/1756-3305-7-406
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