Abstract
Antibiotic tolerance, the widespread ability of diverse pathogenic bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied stepping stone toward antibiotic resistance. The gram-negative pathogen Vibrio cholerae, the causative agent of cholera, is highly tolerant to β-lactam antibiotics. We previously found that the disruption of glycolysis, via deletion of pgi (vc0374, glucose-6-phosphate isomerase), resulted in significant cell wall damage and increased sensitivity toward β-lactam antibiotics. Here, we uncover the mechanism of this resulting damage. We find that glucose causes growth inhibition, partial lysis, and a damaged cell envelope in ∆pgi. Supplementation with N-acetylglucosamine, but not other carbon sources (either from upper glycolysis, TCA cycle intermediates, or cell wall precursors) restored growth, re-established wild-type β-lactam resistance, and recovered cellular morphology of a pgi mutant exposed to glucose. Targeted metabolomics revealed the cell wall precursor synthetase enzyme GlmU (vc2762, coding for the bifunctional enzyme that converts glucosamine-1P to UDP-GlcNAc) as a critical bottleneck and mediator of glucose toxicity in ∆pgi. In vitro assays of GlmU revealed that sugar phosphates (primarily glucose-1-phosphate) inhibit the acetyltransferase activity of GlmU (likely competitively), resulting in compromised peptidoglycan and lipopolysaccharide biosynthesis. These findings identify the molecular mechanism of Δpgi glucose toxicity in V. cholerae.IMPORTANCESugar-phosphate toxicity is a well-characterized phenomenon that is seen within diverse bacterial species, and yet the molecular underpinnings often remain elusive. We previously discovered that disrupting Vibrio cholerae's ability to eat glucose (by disrupting the pgi gene) resulted in a damaged cell envelope and enabled us to kill V. cholerae more easily using antibiotics like penicillin. Upon deletion of pgi, glucose-phosphate levels rapidly build up and inhibit the enzymatic activity of GlmU, a key step of bacterial peptidoglycan precursor synthesis. GlmU inhibition causes enhanced killing by antibiotics and a pronounced cell envelope defect. Thus, GlmU serves as a prime target for novel drug development. This research opens new routes through which central metabolism and sugar-phosphate toxicity modulate antibiotic susceptibility.