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. Bacterial populations contain a small number of dormant cells (persisters) that are tolerant to antibiotics. Persisters are not mutants, but rather phenotypic variants of regular cells. Persisters play a major role in resistance of bacterial biofilms to death, and are likely to be responsible for recalcitrance of chronic infections to antibiotics. A lead into the mechanism by which these specialized survivor cells arise comes from the fact that DNA damage induces the SOS response in bacteria, a signaling pathway that up-regulates DNA repair functions. SOS response induction also leads to expression in E. coli of a tisB “toxin” gene encoding a small membrane-acting peptide that leads to a decrease in ATP and can kill cells if artificially overexpressed. We reasoned that tisB may actually be a persister gene and its product induces reversible dormancy by shutting down cell metabolism. We show that a knockout of tisB resulted in a sharply decreased frequency of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage, whereas mild overproduction of the peptide induced persister formation. TisB-dependent persisters also were highly tolerant to unrelated antibiotics. It appears that production of persisters tolerant to all antimicrobials is a “side-effect” of fluoroquinolone antibiotics. Our results suggest that induction of TisB by the SOS response controls production of multidrug-tolerant cells and represents, to our knowledge, the first mechanism of persister formation.