Sugar phosphate-mediated inhibition of peptidoglycan precursor synthesis

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 steppingstone towards antibiotic resistance. The Gram-negative pathogen Vibrio cholerae, the causative agent of cholera, is highly tolerant to {beta}-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 towards {beta}-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 {Delta}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 antibiotic resistance towards {beta}-lactams, 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 {Delta}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 PG and LPS biosynthesis. These findings identify GlmU as a critical branchpoint enzyme between central metabolism and cell envelope integrity and reveal the molecular mechanism of {Delta}pgi glucose toxicity in Vibrio cholerae. ImportanceSugar-phosphate toxicity is a well characterized phenomenon that is seen within diverse bacterial species, and yet the molecular underpinnings remain elusive. We previously discovered that disrupting Vibrio choleraes ability to eat glucose (by disrupting the pgi gene), also resulted in a damaged cell envelope. Upon deletion of pgi, glucose-phosphate levels rapidly build 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.