Periplasmic maturation and modification of LdtB, an L,D-transpeptidase from Escherichia coli

Details

Supervisors

Soumillion, Patrice

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

The use of antibiotics has become common and daily to treat bacterial infections. The most frequently used antibiotics belong to the β-lactam antibiotic family. This family targets and inhibits the proper development of the bacterial envelope by preventing the synthesis of the peptidoglycan (PG) matrix. However, the global use of such antibiotics is correlated with an increase in the emergence of bacterial strains resistant to these antibiotics. The search for new compounds and targets is therefore essential. To identify such molecules, it is important to understand all aspects of bacterial envelope biosynthesis and regulation. In Escherichia coli, the primary mechanisms for cross-linking and transpeptidation of peptidoglycan involve D,D-transpeptidases, which facilitate the formation of bonds between peptide chains. Another class of enzymes, L,D-transpeptidases, plays a crucial role in transpeptidation by linking two peptidoglycan chains through their meso-diaminopimelic acid (mDAP). Additionally, specific L,D-transpeptidases are responsible for attaching peptidoglycan to the outer membrane (OM) of Gram-negative bacteria, through a bridging reaction, for example with Braun's lipoprotein (Lpp) in E. coli. Unlike D,D-transpeptidases, L,D-transpeptidases are not inhibited by most β-lactam antibiotics. This property is significant in the context of antibiotic development, as L,D-transpeptidases can contribute to specific antibiotic resistances. Not much is known about the six L,D-transpeptidases (LdtA to LdtF) of Escherichia coli. Given the oxidizing nature of the periplasm and the presence of a nucleophilic cysteine in the catalytic site of L,D-transpeptidases, we investigated the possibility that it could provide an opportunity for activity regulation through covalent-reversible modification of the active site. In this work, we focus our attention on LdtB, the main Ldt involved in PG-OM bridging. From periplasmic extracts containing overexpressed LdtB, we were able to trap a covalent complex between the enzyme and DsbG. Interestingly, experiments also revealed an alternative cleavage of the signal peptide of LdtB, generating two isoforms in comparable amounts and prompting a deeper investigation into its maturation within the periplasm. While all E. coli L,D-transpeptidases are predicted to be soluble in the periplasm, our results suggest that a significant fraction of LdtB is cleaved before Cys19 and may undergo a maturation into a lipoprotein while the remaining fraction is cleaved before Val25 as predicted. Experiments with a Strep-tagged LdtB expressed from its chromosomal locus further indicate that LdtB is distributed between a soluble fraction and a membrane associated fraction in comparable amounts. Additionally, mass spectrometry analyses revealed two stable conformations of the N-terminus tryptic peptide (residue 25 to 35). We hypothesise that cis-trans isomerisation of prolines at positions 28 or 30 may be responsible for these conformations and may determine the cleavage site of the signal peptide. Surprisingly, we also observed the acetylation of the N-terminal residue (Val25) in the short isoform of LdtB. To our knowledge, such modification was never reported for a periplasmic protein of E. coli.