Inhibition of mutation and combating the evolution of antibiotic resistance

Abstract

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.

Figure 1. Survival and Mutation of E. coli Mutants In Vivo after Starting Antibiotic Therapy

Survival and mutation of ΔlacZ and lexA(S119A) mutants of E. coli ATCC 25922 in thighs of neutropenic mice at 24-h intervals after starting therapy with (A) ciprofoxacin or (B) rifampicin. Open circles and triangles correspond to the total cfu/thigh of the ΔlacZ and lexA(S119A) strains, respectively. Solid circles and triangles represent the number of drug-resistant ΔlacZ and lexA(S119A) cfu/thigh, respectively.

Figure 2. Survival of E. coli Mutants In Vitro

Survival on solid media containing 40 ng/ml ciprofloxacin of ΔlacZ control and (A) NER and recombination mutants with wild-type sensitivity, (B) recombination mutants that were hypersensitive to ciprofloxacin, and (C) lexA(S119A) and inducible polymerase mutants.

Figure 3. Proposed Response to Ciprofloxacin

In the absence of homologous sequences, free DSBs are repaired by nuclease and polymerase-dependent IR (pathway A). In the presence of a suitable homologous sequence, free DSBs may be repaired by RDR (pathway B). This involves resectioning of the DNA ends by RecBC and loading of RecA onto the ssDNA produced. These RecA-ssDNA filaments catalyze D-loop formation and repair of the DSB. This pathway may also contribute to the repair of replication forks when they encounter the free DSB. Finally, replication forks that encounter topoisomerases that are covalently-bound to the DNA are repaired by recombination-dependent fork repair (pathway C). This involves RecG-mediated fork regression and RuvC cleavage to produce DSEs where RecBC mediates RecA-ssDNA filament formation. These filaments catalyze strand invasion of a homologous sequence where PriA, and possibly Pol II, help to reestablish a processive replication fork. With sufficient accumulation of DSBs and collapsed forks, persistent RecA-ssDNA filaments induce levels of LexA cleavage sufficient to de-repress the error prone polymerases, Pol IV and Pol V, which cooperate to induce mutations (pathway D). Once resistance-conferring mutations are made, DSBs and collapsed forks cease to accumulate and RecA-filaments no longer persist. Subsequently, the cellular concentration of LexA increases, shutting down expression of the pro-mutagenic polymerases. See text for details.