Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis

The FemXWv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNAAla to UDP-MurNAc-pentadepsipeptide. FemXWv is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemXWv is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemXWv depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1–C72 and G2–C71 base pairs of the acceptor stem as critical for FemXWv activity in agreement with modeling of tRNAAla in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNAAla harboring nucleotide substitutions in the G3–U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemXWv. The different modes of recognition of the acceptor stem indicate that specific inhibition of FemXWv could be achieved by targeting the distal portion of tRNAAla for the design of substrate analogues

Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases

This work is licensed under a Creative Commons Attribution 4.0 International License.As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb’s survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.NIH U19AI111143Milstein Program in Chemical Biology and Translational MedicineWilliam Randolph Hearst TrustWelch Foundation (A-0015

Nouveaux mécanismes de résistance aux b-lactamines et nouvelles cibles de ces antibiotiques chez les bactéries à Gram positif

La tuberculose est une maladie provoquée par Mycobacterium tuberculosis habituellement traitée par l association de rifampicine, d isoniazide, de pirazinamide et d éthambutol pendant 6 mois. Des problèmes d observances ont favorisé l émergence de souches ultra résistantes (XDR). Les bêta-lactamines n ont jamais été utilisées contre M. tuberculosis à cause de la production par la bactérie d une bêta-lactamase de classe A, BlaC. Nous avons montré que BlaC est capable d hydrolyser toutes les classes de bêta-lactamines, analysé l inhibition de BlaC par l acide clavulanique, le tazobactam et le sulbactam, et montré que l acide clavulanique est un inhibiteur irréversible de l enzyme. Nous avons évalué l efficacité d une association d acide clavulanique et de -lactamines contre des souches sensibles et XDR. Le méropénème, en combinaison avec l acide clavulanique, est bactéricide sur les bacilles en réplication active ainsi que dans un modèle de dormance. La cible du méropénème pourrait être une L,D-transpeptidase, l enzyme clé d une nouvelle voie de synthèse du peptidoglycane que nous avons caractérisé chez Enterococcus faecium. Cette voie implique l activation d une métallo enzyme qui fournit le substrat d une L,D transpeptidase (Ldtfm), responsable de la formation de ponts interpeptidiques de type 3-3 dans le peptidoglycane. Nous avons montré que cette enzyme était inactivée par les carbapénèmes. Comme le peptidoglycane de M. tuberculosis est majoritairement constitué de ponts 3-3 et que cinq paralogues de la famille des L,D-transpeptidases sont présent dans son génome, ces enzymes pourraient être la cible du méropénème.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Data from: Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli

The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB

Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site

International audienceβ-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldtfm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics

Peptidoglycan cross-linking activity of L,D-transpeptidases from Clostridium difficile and inactivation of theses enzymes by β-lactams

International audienc

Cloning, purification, crystallization and preliminary crystallographic analysis of a penicillin-binding protein homologue from Pyrococcus abyssi

The crystallization of a hypothetical penicillin-binding protein from the archaeon P. abyssi in space group C2 by hanging-drop vapour diffusion is reported