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Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis

Audrey Gerstel ,Jordi Zamarreño Beas ,Yohann Duverger,Emmanuelle Bouveret,Frédéric Barras ,Béatrice Py

Abstract

The level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli.

Introduction

Drug combination is a potent strategy against the worrying rise of multi-drug resistant bacteria as it reduces the chance of resistance acquisition. However, several instances of antagonisms between drugs have been reported. In fact, a thorough investigation of growth phenotypes caused by pair-wise combination of over 250 compounds, including neglected antibiotics, FDA approved human drugs and food additives, revealed antagonism to be more prevalent than synergy. For instance, drugs causing oxidative stress, such as paraquat or plumbagin, were found to antagonize antibiotics of different families including quinolones. This was consistent with the previous observation that redox-cycling compounds (RCC) such as paraquat or plumbagin enhanced both survival and persister formation in the presence of the oxolinic acid fluoroquinolone.

Methods

Bacterial strains and growth conditions

The transcriptional PsoxS::lacZ fusion was constructed as described in Ezraty et al. [33]. The PsoxS promoter region fused to lacZ encompassed a region from the 111 nucleotides upstream the transcriptional soxS start site to the 21 first nucleotides of the soxS-coding region. The E. coli K-12 strain MG1655 and its derivatives used in this study are listed in S1 Table. Deletion mutations were introduced by P1 transduction. Transductants were verified by PCR, using primer pairs hybridizing upstream and downstream of the deleted gene. E. coli strains were grown at 37°C in Luria-Bertani (LB) rich medium. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.1 mM), arabinose (0.2%), glucose (0.2%) and anhydrotetracycline (aTc) (2 μM) were added when required. Solid media contained 1.5% agar. Antibiotics were used at the following concentrations: chloramphenicol 25 μg/mL, kanamycin 30 μg/mL, and ampicillin 50 μg/mL.

Discussion

Understanding the influence of environmental conditions on level of antibiotic resistance is a prerequisite to monitor and control bacterial antibiotic resistance. Previously, we showed that iron limitation enhanced level of resistance of E. coli to aminoglycosides, and that Fe-S cluster biogenesis regulation played a key role in this unexpected link [8,41]. Here we show that Fe-S homeostasis connects ROS producing compound, RCC, and resistance level to fluoroquinolones.

Acknowledgments

We thank all members of the Py group (Marseille), the Ezraty group (Marseille) and the Barras unit (Paris) for fruitful discussions. We thank Y. Dennis and R. Barré (IMM transcriptomic platform) for qRT-PCR experiments.

Citation: Gerstel A, Zamarreño Beas J, Duverger Y, Bouveret E, Barras F, Py B (2020) Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis. PLoS Genet 16(11): e1009198. https://doi.org/10.1371/journal.pgen.1009198.

Editor: Melanie Blokesch, Swiss Federal Institute of Technology Lausanne (EPFL), SWITZERLAND.

Received: May 26, 2020; Accepted: October 15, 2020; Published: November 2, 2020.

Copyright: © 2020 Gerstel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by grants from the CNRS, the JPIAMR “Combinatorial” grant, Aix‐Marseille Université, Institut Pasteur and the ANR-10-LABX-62-IBEID. A.G. and J.Z. were supported by fellowships from Ministère de l’Enseignement et de la Recherche. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: no authors have competing interests.

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