Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

Abstract

Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this unprecedented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

Genome-wide identification of genes required for alternative peptidoglycan cross-linking in Escherichia coli revealed unexpected impacts of β-lactams

“…PG recycling is stimulated in the +CRO condition 49 . This implies that the rescue of PBPs by YcbB reduces but does not abolish the high anabolic demand necessary to feed the PG assembly pathway in the presence of the drug.…”

“…In contrast, the inactivation of SltY favors YcbB-mediated resistance to β-lactams, with uncross-linked glycan strands formed by the elongasome complex serving as suitable substrates for the assembly of a functional PG polymer 7 . Pulse-labeling experiments indicated that YcbB supported PG polymerization via a two-step mechanism involving (i) assembly of 3 → 3 cross-linked glycan strands followed by (ii) insertion of the resulting neo-synthesized polymer into the existing PG 49 . In the present work, we provide direct evidence that this mode of peptidoglycan polymerization requires a functional elongasome, supporting glycan strand polymerization by catalytically active RodA (Fig.…”