The Molecular Alarmone (p)ppGpp Mediates Stress Responses, Vancomycin Tolerance, and Virulence inEnterococcus 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 ⌬relA, ⌬relQ, and ⌬relAQ 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 ⌬relA strain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a ⌬relAQ double mutant restored many of the stress-sensitive phenotypes of ⌬relA. Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the ⌬relA strain but diminished in the ⌬relQ and ⌬relAQ strains. Finally, virulence of the ⌬relAQ strain but not of the ⌬relA or ⌬relQ 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.

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

“…Specifically, the (p)ppGpp 0 strain displays increased sensitivity to macrophages, impaired growth and survival in serum or whole blood, and attenuated virulence in animal models (6)(7)(8). In this report, we show that these phenotypes are intimately associated with metal homeostasis.…”

“…3B). Even though the Δrel Ef strain displays intrinsically higher (p)ppGpp levels due to the absence of (p)ppGpp hydrolase activity (6,18), the lack of pppGpp accumulation in this strain indicated that Rel Ef is responsible for (p)ppGpp accumulation in response to metal starvation.…”

“…In E. faecalis, (p)ppGpp metabolism is carried out by the bifunctional Rel Ef enzyme and the small alarmone synthetase RelQ Ef (6). To determine which enzyme is responsible for (p)ppGpp accumulation during metal limitation, we also quantified (p)ppGpp pools in the Δrel Ef and ΔrelQ Ef strains grown under metal-depleted conditions (Fig.…”

“…Bacterial strains used in this study are listed in Table 1. The parent E. faecalis OG1RF strain and the corresponding derivative Δrel, ΔrelQ, and Δrel ΔrelQ [(p)ppGpp 0 ] strains have been previously described (6). The E. faecalis V583 wild-type and (p)ppGpp 0 strains were generously provided by Axel Hartke at the University of Caen (22).…”

“…Previous characterization of E. faecalis strains defective in (p)ppGpp production revealed that a complete lack of (p)ppGpp [(p)ppGpp 0 strain] led to attenuated virulence in two invertebrate animal models and in a rabbit subdermal abscess model (6)(7)(8). In addition, the (p)ppGpp 0 strain displayed impaired survival within macrophages as well as a growth and survival defect in whole blood and serum ex vivo (7,8).…”