Bacillus subtilis produces (p)ppGpp in response to the bacteriostatic antibiotic chloramphenicol to prevent its potential bactericidal effect

Abstract

Antibiotics combat bacteria through their bacteriostatic (by growth inhibition) or bactericidal (by killing bacteria) action. Mechanistically, it has been proposed that bactericidal antibiotics trigger cellular damage, while bacteriostatic antibiotics suppress cellular metabolism. Here, we demonstrate how the difference between bacteriostatic and bactericidal activities of the antibiotic chloramphenicol can be attributed to an antibiotic-induced bacterial protective response: the stringent response. Chloramphenicol targets the ribosome to inhibit the growth of the Gram-positive bacterium Bacillus subtilis. Intriguingly, we found that chloramphenicol becomes bactericidal in B. subtilis mutants unable to produce (p)ppGpp. We observed a similar (p)ppGpp-dependent bactericidal effect of chloramphenicol in the Gram-positive pathogen Enterococcus faecalis. In B. subtilis, chloramphenicol treatment induces (p)ppGpp accumulation through the action of the (p)ppGpp synthetase RelA. (p)ppGpp subsequently depletes the intracellular concentration of GTP and antagonizes GTP action. This GTP regulation is critical for preventing chloramphenicol from killing B. subtilis, as bypassing (p)ppGpp-dependent GTP regulation potentiates chloramphenicol killing, while reducing GTP synthesis increases survival. Finally, chloramphenicol treatment protects cells from the classical bactericidal antibiotic vancomycin, reminiscent of the clinical phenomenon of antibiotic antagonism. Taken together, our findings suggest a role of (p)ppGpp in the control of the bacteriostatic and bactericidal activity of antibiotics in Gram-positive bacteria, which can be exploited to potentiate the efficacy of existing antibiotics.