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 undocumented 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.
MS/MS spectra have been provided in Supplementary data file.The software developments required to predict and analyze the labeled/unlabeled muropeptide ions isotopic clusters either in MS or MS/MS experiments are hosted at https://gitlab.com/kantundpeterpan/masseltof and published under a Free Software license.
- Heiner Atze
- Michel Arthur
- Yucheng Liang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Bavesh D Kana, University of the Witwatersrand, South Africa
© 2022, Atze et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.
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