Systematically Altering Bacterial SOS Activity under Stress Reveals Therapeutic Strategies for Potentiating Antibiotics

Abstract

The bacterial SOS response is a DNA damage repair network that is strongly implicated in both survival and acquired drug resistance under antimicrobial stress. The two SOS regulators, LexA and RecA, have therefore emerged as potential targets for adjuvant therapies aimed at combating resistance, although many open questions remain. For example, it is not well understood whether SOS hyperactivation is a viable therapeutic approach or whether LexA or RecA is a better target. Furthermore, it is important to determine which antimicrobials could serve as the best treatment partners with SOS-targeting adjuvants. Here we derived Escherichia coli strains that have mutations in either lexA or recA genes in order to cover the full spectrum of possible SOS activity levels. We then systematically analyzed a wide range of antimicrobials by comparing the mean inhibitory concentrations (MICs) and induced mutation rates for each drug-strain combination. We first show that significant changes in MICs are largely confined to DNA-damaging antibiotics, with strains containing a constitutively repressed SOS response impacted to a greater extent than hyperactivated strains. Second, antibiotic-induced mutation rates were suppressed when SOS activity was reduced, and this trend was observed across a wider spectrum of antibiotics. Finally, perturbing either LexA or RecA proved to be equally viable strategies for targeting the SOS response. Our work provides support for multiple adjuvant strategies, while also suggesting that the combination of an SOS inhibitor with a DNA-damaging antibiotic could offer the best potential for lowering MICs and decreasing acquired drug resistance. IMPORTANCE Our antibiotic arsenal is becoming depleted, in part, because bacteria have the ability to rapidly adapt and acquire resistance to our best agents. The SOS pathway, a widely conserved DNA damage stress response in bacteria, is activated by many antibiotics and has been shown to play central role in promoting survival and the evolution of resistance under antibiotic stress. As a result, targeting the SOS response has been proposed as an adjuvant strategy to revitalize our current antibiotic arsenal. However, the optimal molecular targets and partner antibiotics for such an approach remain unclear. In this study, focusing on the two key regulators of the SOS response, LexA and RecA, we provide the first comprehensive assessment of how to target the SOS response in order to increase bacterial susceptibility and reduce mutagenesis under antibiotic treatment.

Keywords: DNA damage; LexA; RecA; SOS pathway; adjuvant therapy; antibiotic resistance; mutagenesis.

FIG 1

Engineered lexA and recA variants in E. coli displaying a range of SOS activities. The LexA protein is represented by blue ovals, and the various LexA cleavage mutants are labeled in blue. RecA is shown as red ovals, and variants are labeled in red. Five LexA variants and two RecA variants allow for examination of the spectrum of SOS activation. Activated RecA filaments lead to cleavage of LexA and inducible expression of SOS genes in the WT strain. Inactivation of LexA self-cleavage (S119A) or deletion of recA (ΔrecA) inactivates the response. Mutations in the LexA protein can either decrease (G80A) or increase (E86P) the rate of self-cleavage relative to the WT strain and thus affect the level of SOS induction. Deletion of lexA (Delta) or hyperactivation of RecA (recA730) leads to constitutive expression of SOS genes.

FIG 2

Heat map of relative MICs of different SOS variants with various antibiotics. The MIC values of the WT strain, shown in white numbers, are listed in micrograms per milliliter. MIC values represent the average from at least four independent determinations performed on separate days. “ND” represents a condition and strain in which the MIC was not determined. The raw MIC values of all strains are shown in Table S1 in the supplemental material.

FIG 3

Impact of lexA variants on cell growth under sublethal doses of stress. (A) Growth curves of the strains exposed to increasing, yet sublethal, levels of ciprofloxacin stress. The data are represented as the mean of 3 independent measurements, and the error bars reflect the standard errors of the 3 replicates. Growth curves under mitomycin C and nitrofurantoin stress are shown in Fig. S4 in the supplemental material. (B) Estimated fitness of the variant strains relative to the WT strain under different types of antibiotic stress. The mean fitness of each strain relative to the WT strain was calculated from 3 independent competition experiments. Error bars represent the standard errors from the three trials. For strains in which no colonies were detected for the variant strain, the top of the error bar represents the limit of detection. Significant P values are noted (*, <0.05; **, <0.005).

FIG 4

Impact of the SOS activities on E. coli mutation rates. (A) Mutation rates of strains grown under no stress or after exposure to sublethal doses of ciprofloxacin. Circles and squares represent the maximum likelihood mutation rate calculated from 6 replicate cultures, and the error bars represent the 95% confidence intervals. (B) Heat map of relative mutation rates across strains and antibiotic stressors. The heat map captures the reduction or increase in the mutation rate relative to the WT strain under that antibiotic. Values listed for the WT are the mutation rate (×10⁻⁹) per generation under each condition. Black boxes represent conditions where no resistant colonies could be detected. (C) Mutation rates of strains across a wide panel of antimicrobial agents. Data are the same as in panel B, with values shown to offer a complementary perspective for comparison between antibiotics. The squares represent individual mutation rates, and the black bars represent the mean of mutation rates under the conditions tested; error bars were removed for clarity and included in Fig. S5 in the supplemental material, with raw values listed in Table S2 in the supplemental material. Strains that did not show resistant colonies under a particular antibiotic stress are marked with an asterisk in the color of that antibiotic. The attributed values of the asterisked strain denote the mutation rate detection limit under those conditions and were excluded from the calculation of the mean mutation rate.