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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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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. |
format | Online Article Text |
id | pubmed-9331226 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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