Timing of DNA damage responses impacts persistence PNAS PLUS to fluoroquinolones Wendy W. K. Moka and Mark P. Brynildsena,1 aDepartment of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544 Edited by Bruce R. Levin, Emory University, Atlanta, GA, and approved May 23, 2018 (received for review March 13, 2018) Bacterial persisters are subpopulations of phenotypic variants in cultures, persisters to ofloxacin (OFL) (a FQ) and cells that died isogenic cultures that can survive lethal doses of antibiotics. Their from treatment exhibited indistinguishable DNA damage re- tolerances are often attributed to reduced activities of antibiotic sponses both during and after treatment (15). Further, those targets, which limit corruption and damage in persisters compared persisters required DNA repair machinery for survival, which with bacteria that die from treatment. However, that model does suggested that they suffered OFL-induced DNA damage (15). not hold for nongrowing populations treated with ofloxacin, a These and other studies have established that growth arrest and fluoroquinolone, where antibiotic-induced damage is comparable persistence are not equivalent (16–19), which suggests that addi- between cells that live and those that die. To understand how tional aspects of persister physiology must be considered to un- those persisters achieve this feat, we employed a genetic system derstand their impressive abilities to survive antibiotic exposure. that uses orthogonal control of MazF and MazE, a toxin and its In a recent study, we used orthogonal expression of the MazF- cognate antitoxin, to generate model persisters that are uniformly MazE toxin–antitoxin (TA) system to generate model persisters tolerant to ofloxacin. Despite this complete tolerance, MazF model for analysis (20). This model was constructed based on the im- persisters required the same DNA repair machinery (RecA, RecB, portance of TA systems to persistence (21–24), and, notably, and SOS induction) to survive ofloxacin treatment as their non- MazF accumulation has been employed by various groups to growing, WT counterparts and exhibited similar indicators of DNA study antibiotic tolerance (25, 26). An important feature of our damage from treatment. Further investigation revealed that, MazF persisters was that they were uniformly tolerant to OFL following treatment, the timing of DNA repair was critical to MazF treatments (20) whereas ∼90% of stationary-phase populations persister survival because, when repair was delayed until after were killed when treated with the same OFL dose (15). Further- growth and DNA synthesis resumed, survival was compromised. In more, our model system recapitulated features of WT persisters, addition, we found that, with nongrowing, WT planktonic and such as sensitivity toward aminoglycosides despite being tolerant biofilm populations, stalling the resumption of growth and DNA to FQs and β-lactams (8, 10, 20, 27, 28). Based on these results, we synthesis after the conclusion of fluoroquinolone treatment with a hypothesized that MazF persisters could be used to understand prevalent type of stress at infection sites (nutrient limitation) led to how growth-arrested cells could tolerate OFL treatments and give near complete survival. These findings illustrate that the timing of rise to persisters. events, such as DNA repair, following fluoroquinolone treatment is In this study, we examined how MazF model persisters were important to persister survival and provide further evidence that able to achieve complete tolerance to OFL whereas growth- knowledge of the postantibiotic recovery period is critical to inhibited WT populations could not. We confirmed that MazF understanding persistence phenotypes. persisters required a similar repertoire of DNA repair enzymes as WT OFL persisters for survival and that, following treatment, they persistence | SOS response | DNA damage | MazF | starvation can experience DNA fragmentation and metabolic perturbations ithin clonal bacterial cultures, a subpopulation of cells Significance Wknown as persisters can transiently survive doses of anti- – biotics that kill their isogenic kin (1 3). Unlike resistant mutants, Bacterial persisters are able to survive high concentrations of persisters are phenotypic variants, and their tolerances are not antibiotics that kill their genetically identical kin. Their tolerances attributed to heritable genetic changes that allow them to grow are thought to arise from decreased activity of cellular processes, in the presence of antibiotics. After the antibiotic is removed, which limits damage from antibiotics. However, persistence to persisters resume growth and replication and give rise to pop- fluoroquinolones in growth-inhibited populations is not as cut- ulations with similar susceptibility to the antibiotic used for treat- and-dried, with survivors of treatment exhibiting similar DNA ment as cells in the original population (4). Given their potential to damage as cells that die. In this article, we use a model system of repopulate infections, persisters are considered to be important persistence to reveal that the timing of events, such as DNA contributors to chronic and relapsing infections (5–7). To devise repair, following fluoroquinolone treatment is critical to survival treatments against this threat, better understanding of persister and show that the same is true for WT populations. These data physiology will be enabling (8–10). highlight the importance of processes following antibiotic Persisters are generally thought to originate from growth- treatments to persister phenotypes and establish that timing arrested cells because such cells harbor less active cellular pro- matters for genetically susceptible bacteria struggling to sur- cesses than their growing counterparts and thus suffer limited vive fluoroquinolone treatments. damage from antibiotic treatment. However, this paradigm is not MICROBIOLOGY generalizable to all types of persisters (2, 11). For example, Author contributions: W.W.K.M. and M.P.B. designed research; W.W.K.M. performed re- Wakamoto et al. (12) demonstrated that persistence to isoniazid search; W.W.K.M. and M.P.B. analyzed data; and W.W.K.M. and M.P.B. wrote the paper. in Mycobacterium smegmatis depends on a dynamic balance be- The authors declare no conflict of interest. tween cell growth and stochastic expression of the prodrug ac- This article is a PNAS Direct Submission. tivating enzyme, KatG. In addition, some classes of antibiotics, Published under the PNAS license. such as fluoroquinolones (FQs), which target topoisomerases 1To whom correspondence should be addressed. Email: [email protected]. and are on the World Health Organization’s essential medicine This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. list (13), are capable of killing both growing and nongrowing 1073/pnas.1804218115/-/DCSupplemental. bacteria (14). It was recently reported that, in stationary-phase Published online June 18, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1804218115 PNAS | vol. 115 | no. 27 | E6301–E6309 Downloaded by guest on September 27, 2021 reflective of DNA damage, such as accumulation of dNTPs (29). cleavable LexA were the most important DNA damage and repair We further provide evidence that resumption of growth and DNA systems tested (15). These data provided strong genetic evidence synthesis was slower in MazF model persisters compared with a for the use of this model system to interrogate the survival strat- growth-arrested control that had lower persister levels, and we egies of persisters to OFL in growth-inhibited WT populations. demonstrate that delaying DNA repair compromised MazF per- sister survival. Collectively, these observations suggested that the MazF Persisters Experience DNA Breaks Following OFL Treatment. timing of events, such as DNA repair and reinitiation of growth, Given that the whole population of MazF persisters was tolerant was important for survival from FQ damage in nongrowing cells. to OFL, we could use population-wide measurements to evaluate To examine the relevance of these findings to WT persister levels, whether they suffered DNA damage as a result of treatment. To we delayed growth resumption and DNA synthesis with nutrient demonstrate that drug-stabilized DNA–gyrase complexes, which deprivation, which can occur at infection sites (30–32), following are the cause of lethal DNA double-stranded breaks (DSBs) (36), FQ treatment, and discovered that such scenarios can increase were formed in MazF model persisters following OFL treatment, survival of WT populations to near 100%. These results demon- we measured cleavage sites in an 11-kb stretch of genomic DNA in strate that the timing of molecular events following FQ treatment OFL-treated cells. We used Southern blotting to examine the is critical to the persistence phenotype and suggest that strategies integrity of the region encompassing dnaA-dnaN-recF-gyrB as this to reduce FQ persistence in growth-arrested populations, which locus was previously shown to be fragmented as a result of are notoriously difficult to treat, would be to stimulate DNA treatment with another quinolone antibiotic, oxolinic acid (37). synthesis and/or slow DNA repair. We observed DNA fragmentation in MazF persisters after expo- sure to OFL although we note that damage was reduced com- Results pared with the toxin-free, growing control, which bears only an MazF Persisters Require DNA Repair Systems to Survive OFL Treatment.