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

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.

The stringent response modulates 4-hydroxy-2-alkylquinoline biosynthesis and quorum-sensing hierarchy in Pseudomonas aeruginosa.

As a ubiquitous environmental organism and an important human pathogen, Pseudomonas aeruginosa readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow P. aeruginosa to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp. P. aeruginosa produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (ΔSR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated pqsA and pqsR expression and reduced repression by the rhl system. We also found that (p)ppGpp is required for full expression of both rhl and las AHL (acyl-homoserine lactone) quorum-sensing systems, since the ΔSR mutant has reduced rhlI, rhlR, lasI, and lasR expression, butanoyl-homoserine lactone (C4-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the las system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.

The stringent response controls catalases in Pseudomonas aeruginosa and is required for hydrogen peroxide and antibiotic tolerance.

Pseudomonas aeruginosa, a human opportunistic pathogen, possesses a number of antioxidant defense enzymes under the control of multiple regulatory systems. We recently reported that inactivation of the P. aeruginosa stringent response (SR), a starvation stress response controlled by the alarmone (p)ppGpp, caused impaired antioxidant defenses and antibiotic tolerance. Since catalases are key antioxidant enzymes in P. aeruginosa, we compared the levels of H2O2 susceptibility and catalase activity in P. aeruginosa wild-type and ΔrelA ΔspoT (ΔSR) mutant cells. We found that the SR was required for optimal catalase activity and mediated H2O2 tolerance during both planktonic and biofilm growth. Upon amino acid starvation, induction of the SR upregulated catalase activity. Full expression of katA and katB also required the SR, and this regulation occurred through both RpoS-independent and RpoS-dependent mechanisms. Furthermore, overexpression of katA was sufficient to restore H2O2 tolerance and to partially rescue the antibiotic tolerance of ΔSR cells. All together, these results suggest that the SR regulates catalases and that this is an important mechanism in protecting nutrient-starved and biofilm bacteria from H2O2- and antibiotic-mediated killing.