Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli

Abstract

Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.

Figure 1. Survival of the tisAB/istR mutants after ciprofloxacin exposure and complementation of the phenotype.

(A) Knockout strains of the toxin locus tisAB and its antitoxin istR-1 were exposed to 1 μg/ml ciprofloxacin in exponential growth phase and survival determined by spot plating for colony forming units. The graph is a representative of at least five independent experiments with similar results, error bars indicate the standard error. (B) MG1655 ΔtisAB carrying the tisAB region as a single-copy insertion in the lambda attachment site was treated as described in (A). wt, wild type.

Figure 2. Schematic of the tisAB/istR locus.

Only the LexA-controlled toxin tisB is translated in vivo; tisA contains the binding site for the constitutively expressed antitoxin RNA IstR-1 . The IstR-2 RNA is under LexA control and contains the entire IstR-1 RNA. Its role in tisAB regulation is currently unclear.

Figure 3. Induction of LexA-controlled promoters by ciprofloxacin.

Cells carrying plasmid-borne promoter-gfp fusions were exposed to 0.1 µg/ml ciprofloxacin in exponential phase. Fold induction is GFP fluorescence after 3 h (open bars) and 6 h (green bars) of exposure normalized to initial fluorescence. This graph is a representative of three independent experiments with similar results; error bars indicate the standard error.

Figure 4. Adaptive ciprofloxacin tolerance in E. coli.

Wild-type MG1655 and its ΔtisAB derivative were grown to exponential phase and exposed to 0.1 µg/ml ciprofloxacin (cipro/cip) for 3 h, after which 1 µg/ml ciprofloxacin was added (ciprofloxacin MIC is 0.016 µg/ml). As a control, a parallel culture was immediately exposed to 1 µg/ml. Viable cell number was determined by serial dilution and plating for colony forming units (CFU/ml). The data points are averages of three independent experiments; error bars indicate the standard error. wt, wild type.

Figure 5. TisB overproduction and antibiotic tolerance.

tisB was overexpressed in (A) MG1655 and (B) MG1655 ΔrecA in exponential phase from a low copy number vector and exposed to ciprofloxacin (1 µg/ml), ampicillin (50 µg/ml), streptomycin (25 µg/ml), or colistin (10 µg/ml). Survival after 3 h was compared to a control strain carrying vector without tisB. The graph shows averages of three independent experiments; error bars indicate the standard error.

Figure 6. TisB-dependent persister formation in SOS response mutants.

E. coli MG1655 and its derivatives ΔrecA, ΔrecA ΔtisAB, ΔrecA lexA300(Def), and ΔrecA lexA300(Def) ΔtisAB were grown to exponential phase and exposed to (A) ciprofloxacin at 1 µg/ml or (B) tobramycin at 20 µg/ml. Data are averages of at least three independent experiments; error bars indicate the standard error. wt, wild type.

Figure 7. Model of ciprofloxacin-induced persister formation.

Ciprofloxacin induces the SOS response, which up-regulates DNA repair functions. In a subpopulation of cells, the SOS response also induces the TisB toxin to a high level, which causes a decrease in proton motive force and ATP level, leading to multidrug tolerance.