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LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici

Increased drug efflux compromises the efficacy of a large panel of treatments in the clinic against cancer or bacterial, fungal, and viral diseases, and in agriculture due to the emergence of multidrug-resistant pathogenic fungi. Until recently, to demonstrate increased drug efflux, the use of label...

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Autores principales: Fouché, Guillaume, Rosati, Dominique, Venet, Catherine, Josserand, Hervé, Latorse, Marie-Pascale, Debieu, Danièle, Fillinger, Sabine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9331226/
https://www.ncbi.nlm.nih.gov/pubmed/35893552
http://dx.doi.org/10.3390/microorganisms10081494
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author Fouché, Guillaume
Rosati, Dominique
Venet, Catherine
Josserand, Hervé
Latorse, Marie-Pascale
Debieu, Danièle
Fillinger, Sabine
author_facet Fouché, Guillaume
Rosati, Dominique
Venet, Catherine
Josserand, Hervé
Latorse, Marie-Pascale
Debieu, Danièle
Fillinger, Sabine
author_sort Fouché, Guillaume
collection PubMed
description Increased drug efflux compromises the efficacy of a large panel of treatments in the clinic against cancer or bacterial, fungal, and viral diseases, and in agriculture due to the emergence of multidrug-resistant pathogenic fungi. Until recently, to demonstrate increased drug efflux, the use of labeled drugs or fluorescent dyes was necessary. With the increasing sensitivity of detection devices, direct assessment of drug efflux has become realistic. Here, we describe a medium-throughput method to assess the intracellular drug concentration in the plant pathogenic fungus Zymoseptoria tritici cultivated in the presence of a sublethal fungicide concentration. As a model fungicide, we used the succinate-dehydrogenase inhibitor boscalid. The boscalid concentration was assessed in the different culture fractions using mass spectrometry linked to liquid chromatography (LC-MS/MS). The ratio between the intracellular and total boscalid amount was used as an inversed proxy for the efflux activity. Using isogenic mutant strains known for their differential efflux capacities, we validated the negative correlation between the intracellular boscalid concentration and efflux activity. In addition, intra-cellular fungicide accumulation explains the susceptibility of the tested strains to boscalid. This assay may be useful in lead development when a new molecule displays good inhibitory activity against its isolated target protein but fails to control the target organism.
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spelling pubmed-93312262022-07-29 LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici Fouché, Guillaume Rosati, Dominique Venet, Catherine Josserand, Hervé Latorse, Marie-Pascale Debieu, Danièle Fillinger, Sabine Microorganisms Article Increased drug efflux compromises the efficacy of a large panel of treatments in the clinic against cancer or bacterial, fungal, and viral diseases, and in agriculture due to the emergence of multidrug-resistant pathogenic fungi. Until recently, to demonstrate increased drug efflux, the use of labeled drugs or fluorescent dyes was necessary. With the increasing sensitivity of detection devices, direct assessment of drug efflux has become realistic. Here, we describe a medium-throughput method to assess the intracellular drug concentration in the plant pathogenic fungus Zymoseptoria tritici cultivated in the presence of a sublethal fungicide concentration. As a model fungicide, we used the succinate-dehydrogenase inhibitor boscalid. The boscalid concentration was assessed in the different culture fractions using mass spectrometry linked to liquid chromatography (LC-MS/MS). The ratio between the intracellular and total boscalid amount was used as an inversed proxy for the efflux activity. Using isogenic mutant strains known for their differential efflux capacities, we validated the negative correlation between the intracellular boscalid concentration and efflux activity. In addition, intra-cellular fungicide accumulation explains the susceptibility of the tested strains to boscalid. This assay may be useful in lead development when a new molecule displays good inhibitory activity against its isolated target protein but fails to control the target organism. MDPI 2022-07-25 /pmc/articles/PMC9331226/ /pubmed/35893552 http://dx.doi.org/10.3390/microorganisms10081494 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Fouché, Guillaume
Rosati, Dominique
Venet, Catherine
Josserand, Hervé
Latorse, Marie-Pascale
Debieu, Danièle
Fillinger, Sabine
LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici
title LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici
title_full LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici
title_fullStr LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici
title_full_unstemmed LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici
title_short LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici
title_sort lc-ms/ms-based fungicide accumulation assay to demonstrate efflux activity in the wheat pathogen zymoseptoria tritici
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9331226/
https://www.ncbi.nlm.nih.gov/pubmed/35893552
http://dx.doi.org/10.3390/microorganisms10081494
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