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. 2022 Jul 25;10(8):1494.
doi: 10.3390/microorganisms10081494.

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

Guillaume Fouché  1 , Dominique Rosati  2 , Catherine Venet  2 , Hervé Josserand  2 , Marie-Pascale Latorse  2 , Danièle Debieu  1 , Sabine Fillinger  1
Affiliations

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

Guillaume Fouché et al. Microorganisms. .

Abstract

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.

Keywords: intracellular drug accumulation; medium-throughput assay; multidrug resistance; non-radioactive assay.

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Conflict of interest statement

Dominique Rosati, Catherine Venet and Marie-Pascale Latorse are employees of Bayer SAS, France. Guillaume Fouché’s salary for this work was funded by Bayer SAS. The present work did not yield any commercial or patent exploitations by Bayer SAS. Therefore, the authors disclose any conflict of interest.

Figures

Figure 1
Figure 1
Flowchart of the fraction preparation. F: fungicide fraction; C: intracellular fraction; T: total fraction; M: matrix fraction. YSS: YSS liquid medium; ACN: acetonitrile; WAT: water; P: pellet; SN: supernatant.
Figure 2
Figure 2
Intracellular accumulation of boscalid in Z. tritici mfs1 mutants during in vitro growth. Indicated values are the means of n = 3 technical replicates of IPO323, and of two independent mutants per MFS1 genotype.
Figure 3
Figure 3
Exponential correlation curve between the intracellular accumulation of boscalid (day 7) and EC50 values (day 7) of Z. tritici strains with different MFS1 genotypes. The randomly analyzed fourth strain SE31 is highlighted in red. The EC50 values are the means of 2–4 biological replicates. The intracellular boscalid percentages are the means of three independent measures, including two biological replicates, and the means of two independent mutants per MFS1 genotype. Error bars = standard errors.
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