Genetic determinants of penicillin tolerance in Vibrio cholerae

Abstract

Many bacteria are resistant to killing (\"tolerant\") by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae. Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well-characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A Penicillin Binding Proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. TnSeq analyses revealed that LPS and cell wall biogenesis genes as well as the sigma E cell envelope stress response were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than Lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for post-antibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.