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

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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Bibliographic Details
Published in:Microorganisms (Basel) 2022-07, Vol.10 (8)
Main Authors: Fouché, Guillaume, Rosati, Dominique, Venet, Catherine, Josserand, Hervé, Latorse, Marie-Pascale, Debieu, Danièle, Fillinger, Sabine
Format: Article
Language:English
Subjects:
Biochemical assays
Biological transport
Diseases and pests
Drug resistance in microorganisms
Fungi, Phytopathogenic
Fungicides
Liquid chromatography
Mass spectrometry
Microbiological research
Physiological aspects
Wheat
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Summary: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.
ISSN:2076-2607
2076-2607
DOI:10.3390/microorganisms10081494