title: Antibiotics induce redox-related physiological alterations as part of their lethality.
abstract: Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Dwyer
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: Peter A
authors.initials: PA
authors.lastname: Belenky
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: Jason H
authors.identifier: http://orcid.org/0000-0003-0921-4657
authors.initials: JH
authors.lastname: Yang
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: I Cody
authors.initials: IC
authors.lastname: MacDonald
authors.affiliation: Departments of Chemistry and.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Martell
authors.affiliation: Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Noriko
authors.initials: N
authors.lastname: Takahashi
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: Clement T Y
authors.initials: CT
authors.lastname: Chan
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115;Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114;
authors.forename: Michael A
authors.initials: MA
authors.lastname: Lobritz
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: Dana
authors.initials: D
authors.lastname: Braff
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: Eric G
authors.initials: EG
authors.lastname: Schwarz
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Ye
authors.affiliation: Chemistry, and.
authors.forename: Mekhala
authors.initials: M
authors.lastname: Pati
authors.affiliation: Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Maarten
authors.initials: M
authors.lastname: Vercruysse
authors.affiliation: Chemistry, and.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Ralifo
authors.affiliation: Department of Systems Biology, Columbia University, New York, NY 10032; and.
authors.forename: Kyle R
authors.initials: KR
authors.lastname: Allison
authors.affiliation: Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115;
authors.forename: Ahmad S
authors.initials: AS
authors.lastname: Khalil
authors.affiliation: Departments of Chemistry and.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
authors.affiliation: Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Graham C
authors.initials: GC
authors.lastname: Walker
authors.affiliation: Howard Hughes Medical Institute,Departments of Biomedical Engineering andCenter of Synthetic Biology, Boston University, Boston, MA 02215;Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115;Department of Medicine, Boston University School of Medicine, Boston, MA 02118 jcollins@bu.edu.
authors.forename: James J
authors.initials: JJ
authors.lastname: Collins
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000737:chemistry; D002374:Catalase / Q000378:metabolism*; D019069:Cell Respiration; D004260:DNA Repair; D004926:Escherichia coli / Q000187:drug effects; D029968:Escherichia coli Proteins / Q000378:metabolism; D005456:Fluorescent Dyes; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism*; D016296:Mutagenesis; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017382:Reactive Oxygen Species
chemical_list: D000900:Anti-Bacterial Agents; D000975:Antioxidants; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D002374:Catalase; C051253:katG protein, E coli; D051719:MutS DNA Mismatch-Binding Protein; D010100:Oxygen
keywords: DNA repair; mutagenesis; reactive oxygen species