DNA Breaks-Mediated Fitness Cost Reveals RNase HI as a New Target for Selectively Eliminating Antibiotic-Resistant Bacteria

Abstract

Antibiotic resistance often generates defects in bacterial growth called fitness cost. Understanding the causes of this cost is of paramount importance, as it is one of the main determinants of the prevalence of resistances upon reducing antibiotics use. Here we show that the fitness costs of antibiotic resistance mutations that affect transcription and translation in Escherichia coli strongly correlate with DNA breaks, which are generated via transcription-translation uncoupling, increased formation of RNA-DNA hybrids (R-loops), and elevated replication-transcription conflicts. We also demonstrated that the mechanisms generating DNA breaks are repeatedly targeted by compensatory evolution, and that DNA breaks and the cost of resistance can be increased by targeting the RNase HI, which specifically degrades R-loops. We further show that the DNA damage and thus the fitness cost caused by lack of RNase HI function drive resistant clones to extinction in populations with high initial frequency of resistance, both in laboratory conditions and in a mouse model of gut colonization. Thus, RNase HI provides a target specific against resistant bacteria, which we validate using a repurposed drug. In summary, we revealed key mechanisms underlying the fitness cost of antibiotic resistance mutations that can be exploited to specifically eliminate resistant bacteria.

Keywords: DNA breaks; RNase HI targeting; antibiotic resistance; fitness cost; repurposed drug.

Fig. 1.

DNA breaks correlate with the fitness cost of resistance. (A) Fitness cost (red bars) and SOS induction (blue bars) of sensitive bacteria and Str^R and Rif^R mutants in LB at 4 h. The strains are ordered from lower to higher fitness cost (top to bottom). The dashed line indicates no SOS induction. Error bars represent mean ± SD of independent biological replicates (n ≥ 5). NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-sample two-tailed Student’s t-test). (B) Correlation between average fitness cost (y axis) and average SOS induction (x axis) per genotype, representing the data from (A). The blue line represents the logarithmic regression line, and the gray area represents the 95% CI. (C) SOS induction (blue bars) and fitness cost (red bars) of sensitive bacteria and Erm^R mutants in LB at 4 h. The dashed line indicates no SOS induction. Error bars represent mean ± SD of independent biological replicates (n = 6). NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-sample two-tailed Student’s t-test).

Fig. 2.

Compensatory evolution cause reduction of DNA breaks. (A) SOS induction (blue bars) and fitness cost (red bars), of sensitive (left) double-resistant (center) and double-resistant carrying a compensatory mutation (right) bacteria in LB at 4 h. The dashed line indicates no SOS induction. Error bars represent mean ± SD of independent biological replicates (n ≥ 3). NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-sample two-tailed Student’s t-test for evaluating individual genotypes, and two-tailed unpaired Student’s t-test for comparing different genotypes). (B) Fitness cost (red bars), and SOS induction (blue bars) of sensitive, ancestral RpsL^K43T RpoB^H526Y and nine compensated clones in LB at 4 h. The black dashed line indicates no SOS induction. The gray dashed lines mark the cost/SOS of the ancestral double mutant. Error bars represent mean ± SD of independent biological replicates (n ≥ 3). NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed unpaired Student’s t-test).

Fig. 3.

Replication speed affects the cost of resistance. (A) Left panel: correlation of the fitness cost between time 0 and 4 h (y axis) and between time 0 and 24 h (y axis), in LB. Right panel: correlation of the fitness cost between time 0 and 4 h (y axis) and between time 4 and 24 h (y axis), in LB. Black lines represent the linear regressions if the costs were identical. Blue lines represent the regression lines, and the gray areas represent the 95% CIs. Data from the experiments shown in figure 1A. (B) Schematic representation of DNA replication of Escherichia coli during fast (left) or slow (right) growth. Black dots represent the origin of replication, and red lines represent transcription forks; green arrows mark regions of potential replication–transcription conflicts. (C) Fitness cost (red bars), and SOS induction (blue bars) of sensitive bacteria and Str^R and/or Rif^R mutants in minimal medium at 8 h. The strains are ordered from lower to higher cost (top to bottom). The dashed line indicates no SOS induction. Error bars represent mean ± SD of independent biological replicates (n = 6). NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-sample two-tailed Student’s t-test). (D) Aliquots of serial dilutions (approximately from 5 × 10⁷ to 5 cells) of sensitive and RpsL^K43N RpoB^H526Y strains carrying the expression vector pCA24N -gfp, either empty (top, strains RB1317 and RB1321) or harboring the gene dnaA (bottom, strains RB1290 and RB1294) and either in the absence or the presence of the inducer IPTG. Each experiment included three biological replicates and three experiments were performed; representative data sets are shown.

Fig. 4.

RNAse HI strongly influences the fitness of resistant bacteria. (A) Correlation between the fitness cost of resistance mutations in wild-type (y axis) or ΔrnhA backgrounds (x axis). The values corresponding to the wild-type background (y axis) are those already shown in figure 1A. The values corresponding to the ΔrnhA background (x axis) are those shown in supplementary figure S6A, Supplementary Material online. (B)  Correlation between the fitness cost of resistance mutations in the absence of the RNase HI inhibitor (y axis) or in its presence (x axis). The values corresponding to the absence of the RNase HI inhibitor (y axis) are those already shown in figure 1A. (C) Correlation between the fitness cost of resistance mutations in the presence of the RHI001 (y axis) or in the ΔrnhA background (x axis). The values corresponding to the ΔrnhA background (x axis) and to the presence of RHI001 (y axis) are those already shown in panels (A) and (B), respectively. Error bars represent mean ± SD of independent biological replicates (n ≥ 3). The black line in panels (A–C) represents the linear regression if the costs were identical. (D) Fitness cost of sensitive bacteria and Str^R and/or Rif^R mutants in the presence of the RNase HI inhibitor RHI001 (red bars). For comparison, the corresponding values in the absence of the inhibitor (data from the experiments shown in fig. 1A) or in a ΔrnhA background (data from the experiments shown in panel A and supplementary fig. S6A, Supplementary Material online) are represented as white and black bars, respectively. Error bars represent mean ± SD of independent biological replicates (n = 3). NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed unpaired Student’s t-test) (see also supplementary figs. S6–S8, Supplementary Material online).

Fig. 5.

Lack of RNase HI favors outcompetition of resistant mutants by sensitive bacteria. (A and B) Frequency of single-resistant mutants during three independent long-term competitions in rich medium against sensitive bacteria either in a genetic background including RNase HI (blue lines) or in a ΔrnhA background (red lines), imposing a strong (1:1,500 dilutions, A) or a mild (1:50 dilutions, B) bottlenecks. (C and D) Frequency (C) and bacterial loads (log₁₀ CFUs/g feces, D) of double-resistant mutants during six independent long-term competitions against sensitive bacteria in mice, either in genetic backgrounds including RNase HI (blue lines) or in ΔrnhA backgrounds (red lines). Data are represented until extinction was observed in all six animals (13 days in the competitions in ΔrnhA backgrounds). The black dashed line represents the limit of detection. (E) Total (sensitive + double mutant) bacterial loads (log₁₀ CFUs/g feces) of the strains carrying RNase HI (black lines) or in the ΔrnhA background (gray lines) during the competitions in the mouse gut. (F) Bacterial loads (log₁₀ CFUs/g feces) at day 13 after gavage and after the week-long streptomycin treatment of sensitive (diamonds) and double-resistant bacteria (circles) either in a genetic background carrying RNase HI (blue) or in a ΔrnhA background (red). Error bars represent mean ± SD of the results in different mice (n = 6).