Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa

Abstract

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.

Keywords: (p)ppGpp stringent response; Pseudomonas aeruginosa; antibiotic tolerance; stationary phase; superoxide dismutase.

Fig. 1.

SR inactivation impairs multidrug tolerance in stationary phase P. aeruginosa. (A) Stationary phase (STAT) or (B) exponential phase (EXP) cells of (●) WT, (□) ΔSR, and (▲) +SR challenged with 50 µg/mL gentamicin, 5 µg/mL ofloxacin, or 300 µg/mL meropenem in antibiotic killing assays. Note the different time scale in A and B. Results are mean ± SEM (n = 6). **P < 0.01 vs. WT.

Fig. 2.

SOD activity and sodB expression are induced during stationary phase in the WT but not the ΔSR mutant. (A) Total SOD activity of exponential (EXP) or stationary phase (STAT) WT, ΔSR, +SR, and sodB strains. (B) SodA and SodB specific activities in stationary phase cells measured by in-gel SOD activity assays, with a representative gel image shown. SodB activity in the sodA mutant and SodA activity in the sodB mutant were confirmed as control. (C) sodB-lacZ reporter activities in the WT (●) or ΔSR (□) cells during growth in LB medium. (D) Total SOD activity in stationary-phase WT, ΔSR, rpoS, and ΔSR rpoS strains measured by in-solution assay. All results are shown as mean ± SEM (n ≥ 5). **P < 0.01 vs. WT in the corresponding growth phase.

Fig. 3.

Inactivation of the SR is associated with increased superoxide levels and paraquat killing. (A) Relative intracellular superoxide levels using the DHE/EtBr fluorescence ratio. (B) Paraquat (PQ) killing of exponential (EXP) or stationary-phase (STAT) cells, calculated as percent bacterial survival after 6-h challenge with 10 mM PQ, compared with similar conditions without PQ. Results are shown as mean ± SEM (n ≥ 6). **P < 0.01 vs. WT.

Fig. 4.

Complementation of SOD activity restores superoxide levels and rescues ofloxacin tolerance in the ΔSR mutant. Stationary-phase WT and ΔSR cells carrying the pBAD-sodB (+sodB) or control (+vc) vector were assayed for (A) total SOD activity, (B) relative superoxide levels (DHE/EtBr fluorescence ratio), or (C) killing by 5 µg/mL ofloxacin. (D) Stationary-phase ΔSR cells pretreated ± the SOD mimetic Mn^IIITMPyP before challenge with 5 µg/mL ofloxacin. All results are shown as mean ± SEM (n ≥ 6). *P < 0.05 and **P < 0.01 vs. WT +vc (in A to C) or vs. “+ofloxacin” (in D). (E) Correlation between bacterial viability of stationary-phase cells after ofloxacin challenge and SOD activity. The SOD activity was measured in stationary-phase cultures before ofloxacin challenge, and bacterial viability of the same culture was measured after 10-h challenge of 5 µg/mL ofloxacin. Data from different strains (WT, ΔSR, ΔSR +sodB, ΔSR +sodA, +SR) were combined and each data point represents an independent replicate (n ≥ 40). The correlation coefficient R² was calculated using linear regression.

Fig. 5.

The SR and SOD activity confers membrane impermeability, a key determinant of stationary-phase tolerance. EtBr internalization in the presence of CCCP in A of exponential (EXP) or stationary-phase (STAT) cells, (B) stationary-phase WT and ΔSR cells carrying the pBAD-sodB (+sodB) or control (+vc) vector, or in stationary-phase WT cells ±50 µg/mL PMBN. (C) Killing of stationary-phase WT with 5 µg/mL ofloxacin ± PMBN pretreatment. All results are shown as mean ± SEM (n ≥ 6). **P < 0.01 vs. “WT+vc” or vs. “+PMBN”. (D) Correlation between bacterial viability of stationary-phase cells after ofloxacin challenge and membrane permeability, as measured by the EtBr internalization assay of the same culture before ofloxacin challenge (5 µg/mL for 10 h). Data from different strains (WT, ΔSR, ΔSR +sodB, ΔSR +sodA, +SR) noted as ● or WT +50 µg/mL PMBN noted as □, were combined and each data point (n ≥ 40) represents an independent replicate. The correlation coefficient R² was calculated using linear regression.

Fig. 6.

Loss of (p)ppGpp signaling and SOD activity increase ofloxacin internalization and abrogates emergence of genotypic resistance to ofloxacin. Intracellular ofloxacin levels (RFU, Ex/Em 292/496 nm) in (A) stationary-phase WT, ΔSR, +SR, and sodB strains or (B) WT and ΔSR expressing the pBAD-sodB (+sodB) or control (+vc) vector. The number of ofloxacin-resistant colonies that arose between 3 and 5 d after incubation of 10¹¹ cells of each strain on 12 µg/mL ofloxacin LB agar plates in (C) WT, ΔSR, +SR, and sodB strains and (D) WT and ΔSR expressing the pBAD-sodB (+sodB). Results are mean ± SEM (n ≥ 6). **P < 0.01 vs. WT (for A and C) or WT +vc (for B and D).