The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance, and virulence in Enterococcus faecalis

Abstract

The stringent response is a global bacterial response to stress that is mediated by accumulation of the alarmone (p)ppGpp. In this study, treatment with mupirocin was shown to induce high levels of (p)ppGpp production in Enterococcus faecalis, indicating that this nosocomial pathogen can mount a classic stringent response. In addition, (p)ppGpp was found to accumulate in cells subjected to heat shock, alkaline shock, and inhibitory concentrations of vancomycin. Sequence analysis of the E. faecalis genome indicated that (p)ppGpp synthesis is catalyzed by the bifunctional synthetase/hydrolase RelA and the RelQ small synthase. The (p)ppGpp profiles of DeltarelA, DeltarelQ, and DeltarelAQ strains revealed that RelA is the major enzyme responsible for the accumulation of (p)ppGpp during antibiotic or physical stresses, while RelQ appears to be responsible for maintaining basal levels of alarmone during homeostatic growth. Compared to its parent, the DeltarelA strain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a DeltarelAQ double mutant restored many of the stress-sensitive phenotypes of DeltarelA. Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the DeltarelA strain but diminished in the DeltarelQ and DeltarelAQ strains. Finally, virulence of the DeltarelAQ strain but not of the DeltarelA or DeltarelQ strain was significantly attenuated in the Caenorhabditis elegans model. Taken together, these results indicate that (p)ppGpp pools modulate environmental stress responses, vancomycin tolerance, and virulence in this important nosocomial pathogen.

FIG. 1.

Accumulation of (p)ppGpp in E. faecalis after mupirocin treatment. (A) E. faecalis OG1RF treated with mupirocin for 15 or 30 min. (B) E. faecalis clinical isolates treated with mupirocin for 30 min. Exponentially grown cells were labeled with [³²P]orthophosphate in FMC and treated with 50 μg ml⁻¹ mupirocin for the times (minutes) indicated. Nucleotide acid extracts were spotted onto PEI-cellulose plates for TLC in 1.5 M KH₂PO₄. The identities of the radioactive spots were confirmed by comigration with GTP, GP5, and GP4 standards.

FIG. 2.

(p)ppGpp profile of E. faecalis OG1RF under selected stress conditions. Exponentially grown cells were labeled with [³²P]orthophosphate in FMC and subjected to the stress condition indicated for 60 min. Acid extracts were spotted onto PEI-cellulose plates for TLC in 1.5 M KH₂PO₄.

FIG. 3.

Accumulation of (p)ppGpp in E. faecalis OG1RF after vancomycin treatment. Exponentially grown cells were labeled with [³²P]orthophosphate in FMC and treated with 32 μg ml⁻¹ ampicillin (A) or 10 μg ml⁻¹ vancomycin (V) for 60 min. Control cells (C) were kept untreated and labeled for the duration of the experiment. Acid extracts were spotted onto PEI-cellulose plates for TLC in 1.5 M KH₂PO₄.

FIG. 4.

Accumulation of (p)ppGpp in E. faecalis OG1RF, ΔrelA, ΔrelQ, and ΔrelAPQ strains after mupirocin or vancomycin treatment. Exponentially grown cells were labeled with [³²P]orthophosphate in FMC and treated with 50 μg ml⁻¹ mupirocin for 15 min or 10 μg ml⁻¹ vancomycin for 60 min. C, untreated control cells; M, mupirocin-treated cells; V, Vancomycin-treated cells. Acid extracts were spotted onto PEI-cellulose plates for TLC in 1.5 M KH₂PO₄.

FIG. 5.

Growth curves of E. faecalis OG1RF, ΔrelA, ΔrelQ, and ΔrelAQ strains in BHI, determined using the Bioscreen growth reader monitor. The results represent the means ± standard deviations of three independent experiments.

FIG. 6.

Growth of E. faecalis OG1RF, ΔrelA, ΔrelQ, and ΔrelAQ strains under stress conditions, determined using the Bioscreen growth reader monitor. The results represent the means ± standard deviations of three independent experiments.

FIG. 7.

Growth and time-kill curves of E. faecalis OG1RF, ΔrelA, ΔrelQ, and ΔrelAQ strains in the presence of vancomycin. (A) Growth curves in BHI containing 2.5 μg ml⁻¹ vancomycin. (B) Time-kill curves of logarithmic-phase cultures treated with 40 μg ml⁻¹ vancomycin. Viable counts were determined by plating known dilutions of the samples on BHI plates every 24 h.

FIG. 8.

Killing of C. elegans by E. faecalis OG1RF and its derivatives. Exposure of C. elegans to the relA and relQ mutants did not cause a significant difference in killing from that with the wild-type strain, OG1RF (P = 0.8089 and 0.8895, respectively). However, the ΔrelAQ mutant was significantly attenuated (P < 0.0001). This experiment was repeated three times with similar results.