Mutations in the Staphylococcus aureus Global Regulator CodY confer tolerance to an interspecies redox-active antimicrobial
Anthony M. Martini, Sara A. Alexander, Anupama Khare
Abstract
Bacteria often exist in multispecies communities where interactions among different species can modify individual fitness and behavior. Although many competitive interactions have been described, molecular adaptations that can counter this antagonism and preserve or increase fitness remain underexplored. Here, we characterize the adaptation of Staphylococcus aureus to pyocyanin, a redox-active interspecies antimicrobial produced by Pseudomonas aeruginosa, a co-infecting pathogen frequently isolated from wound and chronic lung infections with S. aureus. Using experimental evolution, we identified mutations in a conserved global transcriptional regulator, CodY, that confer tolerance to pyocyanin and thereby enhance survival of S. aureus.
Introduction
Microorganisms commonly live in the presence of other microbial species, whether in diverse environmental niches or in association with a host [1–3]. These polymicrobial communities can be structurally and functionally dynamic in part through the balance of cooperative and competitive interactions among members [4,5]. Through these interactions, microbial species can impact the fitness, behaviors, and adaptation of other constituent members of the community [6–8]. Notably, antimicrobial effects of several secreted compounds have been shown to mediate interbacterial antagonism in vitro, enhancing the relative fitness of the producing species [9,10]. How community members may adapt to these antagonistic interactions is, however, less well-characterized.
Materials and method
Bacterial strains and growth conditions
All strains and plasmids used in this study are described in S2 Table. Bacteria were cultured at 37 °C with shaking at 300 rpm in modified M63 medium [16] (13.6 g/L KH2PO4, 2 g/L (NH4)2SO4, 0.4 µM ferric citrate, 1 mM MgSO4; pH adjusted to 7.0 with KOH) supplemented with 0.3% glucose, 1x ACGU solution (Teknova), 1x Supplement EZ (Teknova), 0.1 ng/L biotin, and 2 ng/L nicotinamide for all experiments. For the co-culture experiments (shown in Fig 3 and S15 Fig), and experiments testing the effect of other P.
Results
Experimental evolution selects for PYO tolerance in S. aureus
We first determined the bactericidal effect of PYO on the S. aureus strain JE2 by quantifying survival of exponential phase cells upon treatment with a range of PYO concentrations. PYO production by P. aeruginosa can range from 0 to ~30 µg/mL (~150 µM) depending on medium composition and infection site [31,44] although higher concentrations have been observed [30,45] and are likely to exist in local microenvironments. We tested PYO concentrations that covered this range and observed a concentration-dependent effect of PYO on S. aureus cell density, including moderate growth reduction at 12.5 and 25 µM, growth inhibition at 50 and 100 µM, and killing at 200 and 400 µM (Fig 1A).
Discussion
Constituents of polymicrobial communities can exhibit competitive behaviors that affect other community members [9]. Such selective pressures within these communities can promote adaptations that maintain or shift the balance of community form and function [4], but the breadth of these mechanisms is not well-characterized. In this study, we use experimental evolution to investigate the adaptive response of S. aureus, a widespread pathogen frequently identified in antibiotic-resistant and polymicrobial infections, to the redox-active antimicrobial PYO, produced by the co-infecting pathogen P. aeruginosa. We show that recurrent treatment with a bactericidal concentration of PYO selects for increased S.
Acknowledgments
We would like to acknowledge the Center for Cancer Research (CCR) Genomics Core for RNA-sequencing and whole-genome sequencing, and the Brinsmade lab (Georgetown University) for providing the pKM16 plasmid. We thank Susan Gottesman, Gisela Storz, Tiffany Zarrella, Kalinga Pavan Thushara Silva, and Stefan Katharios-Lanwermeyer for comments on the manuscript, and members of the Gottesman, Ramamurthi, and Khare labs for discussion and feedback throughout the study. This work used the computational resources of the NIH High Performance Computing Biowulf Cluster (http://hpc.nih.gov).
Citation: Martini AM, Alexander SA, Khare A (2025) Mutations in the Staphylococcus aureus Global Regulator CodY confer tolerance to an interspecies redox-active antimicrobial. PLoS Genet 21(3): e1011610. https://doi.org/10.1371/journal.pgen.1011610
Editor: Jennifer Herman, Texas A&M University, UNITED STATES OF AMERICA
Received: December 5, 2024; Accepted: February 6, 2025; Published: March 7, 2025
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: The whole genome and RNA sequencing data have both been deposited at NCBI Short Read Archive (SRA) associated with BioProject PRJNA1122578.
Funding: This work was supported by funding from the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research to AK, AMM, and SAA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
