(p)ppGpp and DksA play crucial role in reducing the efficacy of b-lactam antibiotics by modulating bacterial membrane permeability

Abstract

The key signaling molecules in the bacterial stress sensing pathway, the alarmone (p)ppGpp and transcription factor DksA, help in survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. At threshold or elevated concentrations of (p)ppGpp, the level of cellular metabolites and proteins in the presence and absence of DksA in V. cholerae and other bacteria has not yet been comprehensively studied. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0{Delta}dksA V. cholerae mutant to different -lactam antibiotics compared to the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA V. cholerae strains. Further, the mutant strains displayed enhanced inner and outer membrane permeability in comparison to the WT strains. These results directly correlate with the tolerance and survival of V. cholerae to -lactam antibiotics. These findings may help in the development of adjuvants for -lactam antibiotics by inhibiting the functions of stringent response modulators. ImportanceThe (p)ppGpp biosynthetic pathway is widely conserved in bacteria. Intracellular levels of (p)ppGpp and the transcription factor DksA play crucial roles in bacterial multiplication and viability in the presence of antibiotics and/or other xenobiotics. The present findings have shown that (p)ppGpp and DksA significantly reduces the efficacy of -lactam and other antibiotics by modulating the availability of peptidoglycan and cell membrane-associated metabolites by reducing membrane permeability. Nevertheless, the whole-cell proteome analysis of (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA strains identified the biosynthetic pathways and associated enzymes that are directly modulated by the stringent response effector molecules. Thus, the (p)ppGpp metabolic pathways and DksA could be a potential target for increasing the efficacy of antibiotics and developing antibiotic adjuvants.