Arul Marie

Identification of the l,d-Transpeptidases Responsible for Attachment of the Braun Lipoprotein to Escherichia coli Peptidoglycan

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.

Identification of the l,d-Transpeptidases for Peptidoglycan Cross-Linking in Escherichia coli

Three active-site cysteine L,D-transpeptidases can individually anchor the Braun lipoprotein to the Escherichia coli peptidoglycan. We show here that two additional enzymes of the same family form peptide bonds between the third residues of peptidoglycan stems, generating meso-DAP(3)-->meso-DAP(3) unusual cross-links. This activity partially replaces the D,D-transpeptidase activity of penicillin-binding proteins.

Inactivation of Mycobacterium tuberculosis l,d-Transpeptidase LdtMt1 by Carbapenems and Cephalosporins

ABSTRACT The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 cross-links formed by the d,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l,d-transpeptidase LdtMt1 by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of LdtMt1, which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited LdtMt1, with a binding rate constant of 0.08 μM−1 min−1. The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (<0.1 min−1) in comparison to the acylation rate constant (3.1 min−1). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10−3 min−1. Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient LdtMt1 inactivator. Cephalosporins also formed covalent adducts with LdtMt1, although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis.

Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the l,d-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldtfm from Enterococcus faecium, was previously shown to bypass the d,d-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and β-lactam antibiotics. Ldtfm homologues from Bacillus subtilis (LdtBs) and E. faecalis (Ldtfs) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP3) and l-Lys3-l-Ala-l-Ala at the third position of the stem peptide, respectively, instead of l-Lys3-d-iAsn in E. faecium. Ldtfs differed from Ldtfm and LdtBs by its capacity to hydrolyze the l-Lys3-d-Ala4 bond of tetrapeptide (l,d-carboxypeptidase activity) and pentapeptide (l,d-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldtfs, which was also specific for its cognate acyl donor, Ldtfm tolerated substitution of l-Lys3-d-iAsn by l-Lys3-l-Ala-l-Ala. Likewise, LdtBs tolerated substitution of mesoDAP3 by l-Lys3-d-iAsn and l-Lys3-l-Ala-l-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the l,d-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving l,d-transpeptidases.

Evolution of Nacre: Biochemistry and Proteomics of the Shell Organic Matrix of the Cephalopod Nautilus macromphalus

Matrix evolutions: We have biochemically characterized the nacre matrix of the cephalopod Nautilus macromphalus, in part by a proteomic approach applied to the acetic acid‐soluble and ‐insoluble shell matrices, as well as to spots obtained after 2D gel electrophoresis. Strikingly, most of the obtained partial sequences are entirely new, whereas a few correspond only partly with bivalvian nacre proteins. Our findings shed new light on the macroevolution of nacre matrix proteins.

Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR ☆

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

In Vitro Cross-Linking of Mycobacterium tuberculosis Peptidoglycan by l,d-Transpeptidases and Inactivation of These Enzymes by Carbapenems

ABSTRACT The Mycobacterium tuberculosis peptidoglycan is cross-linked mainly by l,d-transpeptidases (LDTs), which are efficiently inactivated by a single β-lactam class, the carbapenems. Development of carbapenems for tuberculosis treatment has recently raised considerable interest since these drugs, in association with the β-lactamase inhibitor clavulanic acid, are uniformly active against extensively drug-resistant M. tuberculosis and kill both exponentially growing and dormant forms of the bacilli. We have purified the five l,d-transpeptidase paralogues of M. tuberculosis (Mt1 to -5) and compared their activities with those of peptidoglycan fragments and carbapenems. The five LDTs were functional in vitro since they were active in assays of peptidoglycan cross-linking (Mt5), β-lactam acylation (Mt3), or both (Mt1, Mt2, and Mt4). Mt3 was the only LDT that was inactive in the cross-linking assay, suggesting that this enzyme might be involved in other cellular functions such as the anchoring of proteins to peptidoglycan, as shown in Escherichia coli. Inactivation of LDTs by carbapenems is a two-step reaction comprising reversible formation of a tetrahedral intermediate, the oxyanion, followed by irreversible rupture of the β-lactam ring that leads to formation of a stable acyl enzyme. Determination of the rate constants for these two steps revealed important differences (up to 460-fold) between carbapenems, which affected the velocity of oxyanion and acyl enzyme formation. Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenem, indicating that modification of the carbapenem side chain could be used to optimize their antimycobacterial activity.

Proteomic study of the effects of microcystin-LR on organelle and membrane proteins in medaka fish liver

The microcystin-leucine-arginine toxin (MC-LR) is produced by cyanobacteria that sometimes bloom in water reservoirs. It targets the liver, thus posing potential health risks to human and animals. Microcystin inhibits the protein phosphatases PP1 and PP2A, leading to diverse cellular deregulation processes. A proteomic approach was applied to the medaka fish (Oryzias latipes) to obtain an overview of the effects of MC-LR on the liver. As membrane and organelle proteins are major structural and functional components of several cell signalling pathways, we decided to investigate here the membrane and organelle-enriched fractions from the livers of control and MC-LR treated medaka fish. Seventeen proteins were identified by proteomic analysis as being modulated in response to MC-LR treatment. This is the first time for eight of them to be reported as being involved in MC-LR effects: prohibitin, fumarylacetoacetase, protein disulfide isomerase A4 and A6, glucose regulated protein 78kDa, 40S ribosomal protein SA, cytochrome b5, and ATP synthase mitochondrial d subunit. These proteins are involved in protein maturation or in the response to oxidative stress highlighting the role of organelles in protein processing and the complex cooperation associated with oxidative stress.

The differential spatial distribution of secondary metabolites in Arabidopsis leaves reacting hypersensitively to Pseudomonas syringae pv. tomato is dependent on the oxidative burst

Secondary metabolites (SMs) play key roles in pathogen responses, although knowledge of their precise functions is limited by insufficient characterization of their spatial response. The present study addressed this issue in Arabidopsis leaves by non-targeted and targeted metabolite profiling of Pseudomonas syringae pv. tomato (Pst-AvrRpm1) infected and adjacent uninfected leaf tissues. While overlap was observed between infected and uninfected areas, the non-targeted metabolite profiles of these regions differed quantitatively and clustering analysis underscores a differential distribution of SMs within distinct metabolic pathways. Targeted metabolite profiling revealed that infected tissues accumulate more salicylic acid and the characteristic phytoalexin of Arabidopsis, camalexin, than uninfected adjacent areas. On the contrary, the antioxidant coumarin derivative, scopoletin, was induced in infected tissues while its glucoside scopolin predominated in adjacent tissues. To elucidate the still unclear relationship between the accumulation of SMs and reactive oxygen species (ROS) accumulation and signalling, a catalase-deficient line (cat2) in which ROS signalling is up-regulated, was used. Metabolic analysis of cat2 suggests that some SMs have important interactions with ROS in redox homeostasis during the hypersensitive response to Pst-AvrRpm1. Overall, the study demonstrates that ROS availability influences both the amount and the pattern of infection-induced SM accumulation.

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.