Generation of reactive oxygen species coincides with membrane depolarization
To test whether depolarizing type I toxins trigger ROS formation, pBAD plasmids with respective toxin genes under control of the PBAD promoter were used for overexpression in E. coli K-12 wild type strain MG1655. Toxins TisB, HokB, and two DinQ variants with varying toxicity (less toxic DinQ-III and fully toxic DinQ-V)14 were selected. All four toxins contain a transmembrane helix (Fig. 1a) and are targeted towards the inner membrane12,13,14. Addition of the inducer L-arabinose caused specific transcription of toxin mRNAs (Fig. 1b), and resulted in the expected growth inhibition due to toxin production (Fig. 1c). HokB overexpression resulted in a drop in optical density, maybe due to leakage of cellular material through larger HokB pores13. The final optical density (300 min) was significantly lower than for all other toxins (P < 0.01, one-way ANOVA with post-hoc Tukey HSD). The potential-sensitive probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] was applied to monitor depolarization. Since DiBAC4(3) only enters depolarized cells, increasing cellular fluorescence is a direct measure for depolarization. All toxins, except DinQ-III, caused a significant increase in DiBAC4(3) fluorescence after 60 minutes of overexpression when compared to the empty vector control (Fig. 1d). The fluorescence value of ~4,300 arbitrary units (AU) in the empty vector control represented background fluorescence as revealed by fluorescence microscopy (Supplementary Fig. S1). DiBAC4(3) fluorescence was consistent with the proposed toxicity of the two DinQ variants, with DinQ-V causing higher fluorescence values than DinQ-III (~10,500 vs. ~6,700 AU, respectively). While TisB was comparable to DinQ-V, HokB caused the highest fluorescence values (~18,000 AU). We tentatively conclude that the degree of depolarization depends on the potential of the respective toxin, but cannot exclude that differences in toxin expression levels contributed to the observed differences in depolarization. Furthermore, HokB supposedly forms larger pores (~0.59 to 0.64 nm)13 than TisB (~0.15 nm)17. The larger pore size of HokB might support increased uptake of DiBAC4(3), which is congruent with strong depolarization of the inner membrane and ATP leakage13.
Formation of ROS was measured after 60 minutes of toxin overexpression using the fluorogenic dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). H2DCFDA is oxidized to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) by various ROS, including hydrogen peroxide, peroxyl radicals, and peroxynitrite24. Importantly, H2DCFDA is cell-permeable and expected to enter cells irrespective of pore formation or size. All toxins, except DinQ-III, caused a significant increase in DCF fluorescence compared to the empty vector control, indicating enhanced ROS formation (Fig. 1e). TisB and DinQ-V were again comparable, causing a DCF fluorescence increase of ~2.5-fold. As expected, HokB caused the strongest increase of ~3.5-fold. The toxin-dependent increase in ROS formation, therefore, matched the degree of depolarization (compare Fig. 1d,e). The depolarizing agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied at a final concentration of 50 µM to inhibit the growth of wild type MG1655 cells (Supplementary Fig. S2). CCCP caused a significant increase in DCF fluorescence of ~3.8-fold already after 30 minutes (Fig. 1e), but did not affect DCF fluorescence in cell-free reactions (Supplementary Fig. S3). ROS measurements using fluorescein dyes can be affected by an increase in the intrinsic fluorescence of cells28. However, in our experiments neither toxin expression nor CCCP treatment increased the intrinsic fluorescence (fold changes of 0.85 to 0.96). Collectively, our results indicate that depolarizing toxins cause a disturbance of metabolic functions with the potential to trigger ROS formation. Whether depolarization and ROS formation causally depend on each other, or are independent outcomes of toxin expression, remains speculative (see Discussion).
Depolarization by toxin TisB specifically induces the SoxRS regulon
To further investigate toxin-dependent ROS formation, TisB was selected as an established model toxin18,21. TisB (29 amino acids long) forms an alpha-helix with a hydrophilic side containing five charged amino acids (Supplementary Fig. S4). In a recent screen for TisB variants with altered toxicity, we identified the positively charged amino acid lysine at position 12 to be important for toxicity (unpublished results). The exchange of lysine with leucine (K12L) generated a TisB variant with attenuated toxicity. Upon addition of L-arabinose, a wild-type strain featuring TisB-K12L had a delay in growth inhibition of ~30 minutes in comparison to non-mutated TisB (Fig. 2a), resulting in a significantly higher optical density at 300 min (P < 0.01, one-way ANOVA with post-hoc Tukey HSD). As expected, depolarization by TisB-K12L was delayed as well, and DiBAC4(3) fluorescence values did not reach the levels of non-mutated TisB after four hours of induction (~7,900 vs. ~10,300 AU, respectively; Fig. 2b). Since tisB mRNA levels (Fig. 1b) and protein levels (Supplementary Fig. S5) were largely unaffected by the K12L mutation, we hypothesize that the attenuated toxicity of TisB-K12L is due to impaired pore formation or less effective passage of protons across the inner membrane. Upon overexpression of non-mutated TisB, progressive degradation of 16 S and 23 S rRNAs was observed12, and was also confirmed here (Fig. 2c). Even though 5 S rRNA and tisB mRNA itself were not affected to the same extent (Supplementary Fig. S6), other transcripts might be subject to degradation in the overexpression strain, which would clearly distort their quantification. Overexpression of the TisB-K12L variant, on the other hand, did not cause obvious rRNA degradation until 180 minutes post induction (Fig. 2c). RNA samples from TisB-K12L overexpression experiments at 60 minutes post induction were compared to pre-treatment samples to assess changes in transcript levels for genes from the oxidative stress response using quantitative RT-PCR. The pspA gene, encoding a bifunctional protein of the envelope stress response, was chosen as a positive control, since pspA is known to be induced by pore-forming proteins33. As expected, the transcript level of pspA was increased ~13-fold upon overexpression of TisB-K12L (Fig. 2d). Genes from the SoxRS regulon (response to superoxide and nitric oxide) showed a similar (~13-fold for sodA) or even higher induction (~20-fold for marB and ~145-fold for soxS). By contrast, genes from the OxyR regulon (response to hydrogen peroxide) were only slightly affected (~6-fold for dps, ~3-fold for grxA, ~2-fold for ahpF, and ~2-fold for trxC) or not affected at all (katG) (Fig. 2d). Considering that dps is the gene with the strongest induction within the OxyR regulon upon hydrogen peroxide stress (~180-fold)34, the increase observed here upon TisB-K12L overexpression appears negligible. Since treatment with CCCP for 30 minutes gave the strongest increase in ROS formation (Fig. 1e), it was tested whether CCCP activates the oxidative stress response. As expected, the SoxRS regulon genes were strongly induced (~164-fold for soxS and ~172-fold for marB). Furthermore, and in contrast to TisB-K12L overexpression experiments, genes from the OxyR regulon were induced as well, as observed for grxA (~103-fold) (Fig. 2e). These results indicated that CCCP caused enhanced formation of both superoxide and hydrogen peroxide, while TisB-dependent depolarization failed to produce enough hydrogen peroxide to fully induce the OxyR regulon.
To further confirm our findings, TisB and TisB-K12L were overexpressed in mutants lacking ROS-detoxifying enzymes. The Hpx− mutant lacks all three enzymes involved in hydrogen peroxide detoxification (Ahp, KatG, and KatE), and shows strongly enhanced DCF fluorescence upon addition of hydrogen peroxide (Supplementary Fig. S7). The SodAB− mutant lacks both cytoplasmic superoxide dismutases (SodA and SodB). In the Hpx− mutant and in the wild type, DCF fluorescence was increased to the same extent (2.5 to 3-fold) upon overexpression of non-mutated TisB (Fig. 2f). Surprisingly, overexpression of TisB-K12L did not increase fluorescence, neither in the wild type nor in the Hpx− mutant strain (Fig. 2f). In SodAB− cells, however, both non-mutated TisB and TisB-K12L provoked elevated DCF fluorescence values (Fig. 2f). These results confirmed that TisB overexpression resulted in formation of superoxide, but not hydrogen peroxide.
TisB contributes to ROS formation upon ciprofloxacin treatment
While plasmid-borne overexpression experiments are useful to evaluate effects of strong toxin production, chromosomal deletions are preferable to assess toxin functions under more physiological conditions. The fluoroquinolone antibiotic ciprofloxacin (CF) can be used to activate the SOS response and, consequently, TisB synthesis18,21. It was shown that a tisB deletion strain does not undergo depolarization upon CF treatment during exponential phase18. We therefore exposed wild-type and ΔtisB cultures to CF and measured DCF fluorescence over time (Fig. 3a). An increase in DCF fluorescence was only observed at very high CF concentrations (1,000x MIC). As supposed by our findings with TisB overexpression strains, the ΔtisB strain scored lower fluorescence values (e.g., ~4450 AU in ΔtisB vs. ~6500 AU in wild type after six hours of treatment). However, at lower CF concentrations (100x MIC), differences were not significant. These data indicate that, at very high ciprofloxacin concentrations, TisB contributes to ROS formation in a wild-type background. We performed the same experiment with double deletion strain Δ1-41 ΔistR, which lacks both the antitoxin gene istR-1 and an inhibitory structure in the 5′ untranslated region of the tisB mRNA. Due to deletion of both inhibitory RNA elements, TisB production is easily excited by addition of CF, resulting in a highly persistent phenotype18,35. In Δ1-41 ΔistR cultures, DCF fluorescence increased over time and was significantly higher than in wild-type cultures irrespective of the CF concentration (Fig. 3a). Intrinsic fluorescence did not account for the changes in DCF fluorescence: strain Δ1-41 ΔistR did not show an increase in intrinsic fluorescence at all (fold changes of 0.93 to 0.99), and wild type and ΔtisB were not strongly affected (fold changes of 0.94 to 1.31). Moreover, all DCF measurements were corrected for intrinsic fluorescence. In summary, the data nicely confirmed the effects seen with plasmid-borne overexpression of TisB.
Detoxification of superoxide is important for TisB-dependent persister formation and recovery
How does TisB-dependent formation of superoxide affect the persister life cycle of E. coli? To answer this question, we performed experiments with strain Δ1-41 ΔistR in comparison to wild type MG1655. Since mRNA levels of the master regulator of the superoxide response, SoxS, were strongly induced upon TisB-K12L overexpression (Fig. 2d), we tested whether the highly persistent phenotype of strain Δ1-41 ΔistR was affected by a soxS deletion. Interestingly, the Δ1-41 ΔistR ΔsoxS strain exhibited a plating defect on LB agar, which was not observed in strains with only the Δ1-41 ΔistR or the ΔsoxS mutations (Fig. 3b). The plating defect was largely suppressed upon antitoxin IstR-1 overexpression, and abolished when the ROS scavenger thiourea was added to the LB agar (Fig. 3b). Since addition of thiourea to LB agar plates had no effect on the outcome of persister assays (Fig. 3c), thiourea was routinely used in order to reliably determine perister levels of strain Δ1-41 ΔistR ΔsoxS. The soxS deletion, however, had no effect on the persister level of neither wild type nor strain Δ1-41 ΔistR after four hours of CF treatment at 1,000x MIC (Fig. 3c). It is known that SoxS shares an overlapping regulon with the transcriptional regulators MarA and Rob36, and the partial redundancy of these regulators might explain why a soxS deletion had no effect.
To further explore the role of superoxide in TisB-dependent persisters, it was tested whether directly preventing superoxide detoxification affects persistence. The SodAB− mutation (ΔsodA and ΔsodB) was constructed in strain Δ1-41 ΔistR. Persister levels after four hours of CF treatment (1,000x MIC) were reduced >20-fold relative to the parental strain (Fig. 4a). By contrast, the SodAB− mutation only caused slightly decreased (~2.7-fold) persister levels in the wild-type background (Fig. 4a). Furthermore, factor analysis (robust two-way ANOVA) revealed that the presence of sodA and sodB had a stronger contribution to persister fromation than the Δ1-41 ΔistR mutation. The ScanLag method37 was applied to monitor appearance and growth times of colonies after CF treatment (see Methods for details). If the colony growth time of a particular strain is not changed, the colony appearance time reflects the persistence time. The persistence time might be prolonged due to impaired resuscitation or recovery from the persister state35. The median colony appearance time was shifted from 1,360 to 1,840 minutes due to the SodAB− mutation in strain Δ1-41 ΔistR, while in the wild-type background the same mutation only caused a shift from 900 to 1,120 minutes (Fig. 4b). Importantly, the colony growth time was largely unaffected by the SodAB− mutation (Supplementary Fig. S8), demonstrating that the delayed colony appearance was due to failure in growth resumption. In summary, prevention of superoxide detoxification impaired both formation and recovery of persister cells, which was particularly evident for TisB-dependent persisters.
Persistence is typically revealed by biphasic killing kinetics upon treatment with antibiotics. While the susceptible subpopulation is rapidly killed during the first phase of the treatment, the persister subpopulation is only slowly eliminated during the second phase38. Killing kinetics of persisters can be affected by their wake-up kinetics, that is, how fast persisters recover and resume growth to become susceptible to antibiotics again39. Since persisters of strain Δ1-41 ΔistR SodAB− showed an impaired recovery, as judged from the 8-hour shift of the median colony appearance time in comparison to strain Δ1-41 ΔistR (Fig. 4b), the persister subpopulation might experience less killing within the second phase of long-term killing experiments. Both strains were treated with CF (1,000x MIC) for 24 hours, revealing biphasic killing kinetics (Supplementary Fig. S9). The persister level after four hours of CF treatment was chosen as reference point (set to 100%) to calculate killing of the persister subpopulation. Contrary to initial expectations, the persister subpopulation of strain Δ1-41 ΔistR SodAB− declined faster than observed for Δ1-41 ΔistR (Fig. 4c). These results indicate that killing kinetics of strain Δ1-41 ΔistR SodAB− is not determined by wake-up kinetics, but rather by the inability to detoxify superoxide.