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Synthetic serum thymic factor (FTS) and several of its analogues have been examined for immunoregulatory properties in a murine skin graft rejection model, including a syngeneic male-to-female system and an allogeneic system. In the syngeneic system control animals with and without placebo had a mean graft survival time of 29 ± 2 and 24 ± 2 days respectively. Administration of FTS [10 ng bound to a carboxymethylcellulose (CMC) vehicle three times weekly until rejection significantly delayed rejection (49 ± 3 days). Similar although less striking results were obtained across the H-2 barrier. Several FTS analogues with retarded degradation were tested in the same system without CMC. All of them were as immunosuppressive as FTS. Interesting, an analogue accelerated or delayed rejection depending on dosage indicating the multiplicity of action of thymic peptides on the various T cell subsets. [ABSTRACT FROM AUTHOR]

The misuse of antibiotics during the last decades led to the emergence of multidrug resistant pathogenic bacteria. This phenomenon constitutes a major public health issue. Consequently, the discovery of new antibacterials in the short term is crucial. Colicins, due to their antibacterial properties, thus constitute good candidates. These toxin proteins, produced by E. coli to kill enteric relative competitors, exhibit cytotoxicity through ionophoric activity or essential macromolecule degradation. Among the 25 colicin types known to date, colicin M (ColM) is the only one colicin interfering with peptidoglycan biosynthesis. Accordingly, ColM develops its lethal activity in E. coli periplasm by hydrolyzing the last peptidoglycan precursor, lipid II, into two dead-end products, thereby leading to cell lysis. Since the discovery of its unusual mode of action, several ColM orthologs have also been identified based on sequence alignments; all of the characterized ColM-like proteins display the same enzymatic activity of lipid II degradation and narrow antibacterial spectra. This publication aims at being an exhaustive review of the current knowledge on this new family of antibacterial enzymes as well as on their potential use as food preservatives or therapeutic agents.

Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS ), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway. IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C 55 -carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C 55 -carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned., (Copyright © 2020 American Society for Microbiology.)

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis . Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso -DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

Enterococcus faeciumd-aspartate ligase (Asl fm ) is a peptide bond-forming enzyme that is involved in the peptidoglycan assembly pathway. It catalyzes the ATP-dependent ligation of the β-carboxylate of D-Asp to the ε-amino group of L-Lys in the nucleotide precursor UDP- MurNAc-pentapeptide. The enzyme is of interest as a target of new, potential, narrow-spectrum antibiotics directed against multiresistant E. faecium. The kinetic mechanism of Asl fm has not been fully characterized. To determine it, a progress curve analysis of Asl fm catalytic process using pyruvate kinase/lactate dehydrogenase ATPase detection assay was performed. With an inspection of the shape of measured progress curves and the results of specific qualitative experiments, the Asl fm reaction mechanism was singled out. The proposed Asl fm kinetics reaction scheme was evaluated by fitting the parameters of the corresponding differential equations to progress curves using the computer program ENZO. The complete kinetic analysis result is consistent with the substrate binding order 1) ATP, 2) D-Asp, and 3) UDP-MurNAc-pentapeptide. The analysis suggests that slowly establishing non-productive equilibria between the free and ATP-bound enzyme with the participating pentapeptide are responsible for initial reaction burst followed by a steady-state period before the complete depletion of the reactant added in the lowest concentration., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Colicins are bacterial toxins produced by some Escherichia coli strains. They exhibit either enzymatic or pore-forming activity towards a very limited number of bacterial species, due to the high specificity of their reception and translocation systems. Yet, we succeeded in making the colicin M homologue from Pectobacterium carotovorum , pectocin M1 (PcaM1), capable of inhibiting E. coli cell growth by bypassing these reception and translocation steps. This goal was achieved through periplasmic expression of this pectocin. Indeed, when appropriately addressed to the periplasm of E. coli , this pectocin could exert its deleterious effects, i.e., the enzymatic degradation of the peptidoglycan lipid II precursor, which resulted in the arrest of the biosynthesis of this essential cell wall polymer, dramatic morphological changes and, ultimately, cell lysis. This result leads to the conclusion that colicin M and its various orthologues constitute powerful antibacterial molecules able to kill any kind of bacterium, once they can reach their lipid II target. They thus have to be seriously considered as promising alternatives to antibiotics., Competing Interests: The authors declare no conflict of interest.

The MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate (C55P), yielding C55-PP-MurNAc-pentapeptide (lipid I). A paralogue of MraY, WecA, catalyzes the transfer of the phospho-GlcNAc moiety of UDP-N-acetylglucosamine onto the same lipid carrier, leading to the formation of C55-PP-GlcNAc that is essential for the synthesis of various bacterial cell envelope components. These two enzymes are members of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, which are essential for bacterial envelope biogenesis. Despite the availability of detailed biochemical information on the MraY enzyme, and the recently published crystal structure of MraY of Aquifex aeolicus, the molecular basis for its catalysis remains poorly understood. This knowledge can contribute to the design of potential inhibitors. Here, we report a detailed catalytic study of the Bacillus subtilis MraY and Thermotoga maritima WecA transferases. Both forward and reverse exchange reactions required the presence of the second substrate, C55P and uridine monophosphate (UMP), respectively. Both enzymes did not display any pyrophosphatase activity on the nucleotide substrate. Moreover, we showed that the nucleotide substrate UDP-MurNAc-pentapeptide, as well as the nucleotide product UMP, can bind to MraY in the absence of lipid ligands. Therefore, our data are in favour of a single displacement mechanism. During this "one-step" mechanism, the oxyanion of the polyprenyl-phosphate attacks the β-phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP. The involvement of an invariant aspartyl residue in the deprotonation of the lipid substrate is discussed., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.

The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/C Vs ) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44-46°C. Its apparent K m values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.

Erysipelothrix rhusiopathiae is a Gram-positive bacterium pathogenic to many species of birds and mammals, including humans. The main feature of its peptidoglycan is the presence of l-alanine at position 3 of the peptide stem. In the present work, we cloned the murE gene from E. rhusiopathiae and purified the corresponding protein as His6-tagged form. Enzymatic assays showed that E. rhusiopathiae MurE was indeed an l-alanine-adding enzyme. Surprisingly, it was also able, although to a lesser extent, to add meso-diaminopimelic acid, the amino acid found at position 3 in many Gram-negative bacteria, Bacilli and Mycobacteria. Sequence alignment of MurE enzymes from E. rhusiopathiae and Escherichia coli revealed that the DNPR motif that is characteristic of meso-diaminopimelate-adding enzymes was replaced by HDNR. The role of the latter motif in the interaction with l-alanine and meso-diaminopimelic acid was demonstrated by site-directed mutagenesis experiments and the construction of a homology model. The overexpression of the E. rhusiopathiae murE gene in E. coli resulted in the incorporation of l-alanine at position 3 of the peptide part of peptidoglycan., (Copyright © 2015. Published by Elsevier B.V.)

A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mucosal-associated invariant T (MAIT) cells recognize microbial compounds presented by the MHC-related 1 (MR1) protein. Although riboflavin precursor derivatives from Gram-positive bacteria have been characterized, some level of ligand heterogeneity has been suggested through the analysis of the MAIT cell TCR repertoire in humans and differential reactivity of human MAIT cell clones according to the bacteria. In this study, using Gram-negative bacteria mutated for the riboflavin biosynthetic pathway, we show a strict correlation between the ability to synthesize the 5-amino-ribityl-uracil riboflavin precursor and to activate polyclonal and quasi-monoclonal mouse MAIT cells. To our knowledge, we show for the first time that the semipurified bacterial fraction and the synthetic ligand activate murine MAIT cells in vitro and in vivo. We describe new MR1 ligands that do not activate MAIT cells but compete with bacterial and synthetic compounds activating MAIT cells, providing the capacity to modulate MAIT cell activation. Through competition experiments, we show that the most active synthetic MAIT cell ligand displays the same functional avidity for MR1 as does the microbial compound. Altogether, these results show that most, if not all, MAIT cell ligands found in Escherichia coli are related to the riboflavin biosynthetic pathway and display very limited heterogeneity., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Antibiotic resistance has emerged as a real threat to mankind, rendering many compounds ineffective in the fight against bacterial infection, including for significant diseases such as plague caused by Yersinia pestis. Essential genes have been identified as promising targets for inhibiting with new classes of compounds. Previously, the gene encoding the bifunctional UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase enzyme GlmU was confirmed as an essential gene in Yersinia. As a step towards exploiting this target for antimicrobial screening, we undertook a biochemical characterisation of the Yersinia GlmU. Effects of pH and magnesium concentration on the acetyltransferase and uridyltransferase activities were analysed, and kinetic parameters were determined. The acetyltransferase activity, which is strongly increased in the presence of reducing agent, was shown to be susceptible to oxidation and thiol-specific reagents.

The synthesis of modified tripeptides (S)-Ala-γ-(R)-Glu-X, where X = (R,S) or (R,R) diastereomers of α-benzyl or α-(4-azidobenzyl)lanthionine, was carried out. The chemical strategy involved the enantioselective alkylation of a 4-MeO-phenyloxazoline. The reductive opening of the alkylated oxazolines, followed by cyclization and oxidation, led to four PMB-protected sulfamidates. Subsequent PMB removal, Boc protection and regioselective opening with cysteine methyl ester led to protected lanthionines. These compounds were further converted in a one pot process to the corresponding protected tripeptides. After ester and Boc deprotection, the four tripeptides were evaluated as potential analogues of the natural tripeptide (S)-Ala-γ-(R)-Glu-meso-A2pm. These compounds were evaluated for introduction, by means of the biosynthetic recycling pathway, into the peptidoglycan of Escherichia coli. A successful in vitro biosynthesis of UDP-MurNAc-tripeptides from the tripeptides containing α-benzyl lanthionine was achieved using purified murein peptide ligase (Mpl). Bioincorporation into E. coli W7 did not occur under different tested conditions probably due to the bulky benzyl group at the Cα carbon of the C-terminal amino acid.

The three diastereoisomers-(R,R), (S,S) and meso-of lanthionine were synthesized in aqueous solution with high diastereoselectivity (>99%). The (S) and (R) enantiomers of two differently protected sulfamidates were opened by nucleophilic attack of (R) or (S)-cysteine. Acidification and controlled heating liberated the free lanthionines. Using the same chemistry, an α-benzyl lanthionine was also prepared. The proposed method, which avoids the need of enrichment by recrystallization, opens the way to the labelling of these compounds with (35)S. Furthermore, in vivo bioincorporation into Escherichia coli W7 was studied. No incorporation of α-benzyl lanthionine was observed. In contrast, meso-lanthionine can effectively replace meso-diaminopimelic acid in vivo, while in the presence of (R,R)-lanthionine the initial increase of bacterial growth was followed by cell lysis. In the future, meso-[(35)S]lanthionine could be used to study the biosynthesis of peptidoglycan and its turnover in relation to cell growth and division., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best inhibitors displayed promising antibacterial activities against selected Gram-positive and Gram-negative strains. These results represent an important step towards the development of new antibacterial agents targeting peptidoglycan biosynthesis., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents. Escherichia coli MurD has been the first Mur ligase for which the tridimensional (3D) structure was solved. Thereafter, several co-crystal structures with different ligands or inhibitors were released. In the present review, we will deal with work performed on substrate specificity, reaction mechanism and 3D structure of E. coli MurD. Then, a part of the review will be devoted to recent work on MurD orthologs from species other than E. coli and to cellular organization of Mur ligases and in vivo regulation of the MurD activity. Finally, we will review the different classes of MurD inhibitors that have been designed and assayed to date with the hope of obtaining new antibacterial compounds.

Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Muramyl peptides (MPs) represent the building blocks of bacterial peptidoglycan, a critical component of bacterial cell walls. MPs are well characterized for their immunomodulatory properties, and numerous studies have delineated the role of MPs or synthetic MP analogs in host defense, adjuvanticity and inflammation. More recently, Nod1 and Nod2 have been identified as the host sensors for specific MPs, and, in particular, Nod2 was shown to detect muramyl dipeptide (MDP), a MP found in both Gram-positive and Gram-negative bacterial cell walls. Because mutations in Nod2 are associated with the etiology of Crohn's disease, there is a need to identify synthetic MP analogs that could potentiate Nod2-dependent immunity. Here, we analyzed the Nod2-activating property of 36 MP analogs that had been tested previously for their adjuvanticity and anti-infectious activity. Using a luciferase-based screen, we demonstrate that addition of a methyl group to the second amino acid of MDP generates a MDP derivative with enhanced Nod2-activating capacity. We further validated these results in murine macrophages, human dendritic cells and in vivo. These results offer a basis for the rational development of synthetic MPs that could be used in the treatment of inflammatory disorders that have been associated with Nod2 dysfunction, such as Crohn's disease.

The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

vanGCd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanGCd cluster to confer resistance. We showed that expression of vanGCd is inducible by vancomycin and that VanGCd , VanXYCd and VanTCd are functional, exhibiting D-Ala : D-Ser ligase, D,D-dipeptidase and D-Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans-complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP-MurNAc-pentapeptide[D-Ser], suggesting that the vanGCd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP-MurNAc-tetrapeptide, UDP-MurNAc-pentapeptide[D-Ala] and UDP-MurNAc-pentapeptide[D-Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra-Tetra is amidated on the meso-diaminopimelic acid. Taken together, our results suggest that the vanGCd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile., (© 2013 John Wiley & Sons Ltd.)

Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The murE ortholog from V. spinosum (murE Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurEVs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurEVs. Our kinetic analysis and structural model of MurEVs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.

Murein peptide ligase (Mpl) is an enzyme found in Gram-negative bacteria. It catalyses the addition of tripeptide L-Ala-γ-D-Glu-meso-diaminopimelate to nucleotide precursor UDP-N-acetylmuramic acid during the recycling of peptidoglycan. Although not essential, this enzyme represents an interesting target for antibacterial compounds through the synthesis of alternate substrates whose incorporation into peptidoglycan might be deleterious for the bacterial cell. Therefore, we have synthesised 10 tripeptides L-Ala-γ-D-Glu-Xaa in which Xaa represents amino acids different from diaminopimelic acid. Tripeptide with Xaa = ε-D-Lys proved to be an excellent substrate of Escherichia coli Mpl in vitro. Tripeptides with Xaa = p-amino- or p-nitro-L-phenylalanine were poor substrates, while tripeptides with Xaa = D- or L-2-aminopimelate, DL-2-aminoheptanoic acid, L-Glu, L-norleucine, L-norvaline, L-2-aminobutyric acid or L-Ala were not substrates at all. Although a good Mpl substrate, the D-Lys-containing tripeptide was devoid of antibacterial activity against E. coli, presumably owing to poor uptake., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP-MurNAc-pentapeptide, respectively. Synthesis of 2'-azido RNA helix starts from 2'-azido-2'-deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22-nt RNA helix mimicking the acceptor arm of Ala-tRNA(Ala) by T4 RNA ligase. For alkyne UDP-MurNAc-pentapeptide, meso-cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L-Cys is converted to dehydroalanine with O-(mesitylenesulfonyl)hydroxylamine. Reaction of but-3-yne-1-thiol with dehydroalanine affords the alkyne-containing UDP-MurNAc-pentapeptide. The Cu(I)-catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine provided the peptidyl-RNA conjugate, which was tested as an inhibitor of non-ribosomal FemX(Wv) aminoacyl transferase. The bi-substrate analogue was found to inhibit FemX(Wv) with an IC(50) of (89±9) pM, as both moieties of the peptidyl-RNA conjugate contribute to high-affinity binding., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MurF is an essential bacterial enzyme that is involved in the last intracellular stage of peptidoglycan biosynthesis, and therefore it has the potential to be exploited as a target for the development of new antibacterials. Here, we report on the expression, purification and biochemical characterization of MurF from an important pathogen, Streptococcus pneumoniae. Additionally, ligand-based virtual screening was successfully used and a new hit compound with micromolar inhibitory activities against MurF enzymes from S. pneumoniae and Escherichia coli was identified.

Colicins are proteins produced by some strains of Escherichia coli to kill competitors belonging to the same species. Among them, ColM (colicin M) is the only one that blocks the biosynthesis of peptidoglycan, a specific bacterial cell-wall polymer essential for cell integrity. ColM acts in the periplasm by hydrolysing the phosphoester bond of the peptidoglycan lipid intermediate (lipid II). ColM cytotoxicity is dependent on FkpA of the targeted cell, a chaperone with peptidylprolyl cis-trans isomerase activity. Dissection of ColM was used to delineate the catalytic domain and to identify the active-site residues. The in vitro activity of the isolated catalytic domain towards lipid II was 50-fold higher than that of the full-length bacteriocin. Moreover, this domain was bactericidal in the absence of FkpA under conditions that bypass the import mechanism (FhuA-TonB machinery). Thus ColM undergoes a maturation process driven by FkpA that is not required for the activity of the isolated catalytic domain. Genes encoding proteins with similarity to the catalytic domain of ColM were identified in pathogenic strains of Pseudomonas and other genera. ColM acts on several structures of lipid II representative of the diversity of peptidoglycan chemotypes. All together, these data open the way to the potential use of ColM-related bacteriocins as broad spectrum antibacterial agents.

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg(2+) ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

D-Glutamic acid-adding enzyme (MurD ligase) catalyses the addition of D-glutamic acid to UDP-N-acetylmuramoyl-L-alanine, an essential cytoplasmic step in the pathway for bacterial cell-wall peptidoglycan synthesis. As such, it represents an important antibacterial drug-discovery target enzyme. Recently, several series of compounds have been synthesised and found to inhibit MurD from Escherichia coli, the best one having an IC(50) value of 8 μM. In the present work, we have tested 20 of these compounds against the MurD enzymes from Staphylococcus aureus, Streptococcus pneumoniae, Borrelia burgdorferi and Mycobacterium tuberculosis. Most of the E. coli MurD inhibitors appeared less efficient against the four other orthologues. This divergent result can be explained by the differences in amino acid sequences and topologies of the active sites of the MurD ligases studied., (Copyright © 2012 Elsevier Inc. All rights reserved.)

MurD and MurE ligases, consecutive enzymes participating in the intracellular steps of bacterial peptidoglycan biosynthesis, are important targets for antibacterial drug discovery. We have designed, synthesized, and evaluated the first d-glutamic acid-containing dual inhibitor of MurD and MurE ligases from Escherichia coli and Staphylococcus aureus (IC50 values between 6.4 and 180 μM) possessing antibacterial activity against Gram-positive S. aureus and its methicillin-resistant strain (MRSA) with minimal inhibitory concentration (MIC) values of 8 μg/mL. The inhibitor was also found to be noncytotoxic for human HepG2 cells at concentrations below 200 μM.

For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

Lipids II found in some Gram-positive bacteria were prepared in radioactive form from l-lysine-containing UDP-MurNAc-pentapeptide. The specific lateral chains of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus (di-L-alanine, D-isoasparagine, and pentaglycine, respectively) were introduced by chemical peptide synthesis using the Fmoc chemistry. The branched nucleotides obtained were converted into the corresponding lipids II by enzymatic synthesis using the MraY and MurG enzymes. All of the lipids were hydrolysed by Escherichia coli colicin M at approximately the same rate as the meso-diaminopimelate-containing lipid II found in Gram-negative bacteria, thereby opening the way to the use of this enzyme as a broad spectrum antibacterial agent., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Mur ligases are bacterial enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have performed virtual screening for potential ATP-competitive inhibitors targeting MurC and MurD ligases, using a protocol of consecutive hierarchical filters. Selected compounds were evaluated for inhibition of MurC and MurD ligases, and weak inhibitors possessing dual inhibitory activity have been identified. These compounds represent new scaffolds for further optimisation towards multiple Mur ligase inhibitors with improved inhibitory potency.

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective ΔAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic β-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.

Muramyl peptides derived from bacterial peptidoglycan have long been known for their ability to trigger host innate immune responses, including inflammation and antimicrobial defense. Muramyl peptides have also been widely studied for their role as immune adjuvants. In mammals, the nucleotide-binding oligomerization domain (Nod) proteins Nod1 and Nod2 detect distinct muramyl peptide structures and mediate their biological activity. Because of the poor immunogenicity of these small peptidoglycan derivatives, research in this field is currently limited by the lack of reagents to track or immobilize specific muramyl peptides. We present here the generation and initial biological characterization of synthetic muramyl peptides covalently coupled to dansyl or biotinyl derivatives and demonstrate that biotinyl coupling on the muramyl moiety results in derivatives that can be tracked by immunofluorescence and maintain full biological activity, as observed by their capacity to trigger Nod signaling. Moreover, using digitonin-mediated permeabilization techniques on live cells, we also demonstrate that biotinylated muramyl peptides efficiently reach the host cytosol, where they activate Nod signaling. Therefore, these derivatives represent useful probes to study the cell biology and the biochemistry of host responses to muramyl peptides., (© 2011 John Wiley & Sons A/S.)

A series of optimized sulfonamide derivatives was recently reported as novel inhibitors of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD). These are based on naphthalene-N-sulfonyl-D-glutamic acid and have the D-glutamic acid replaced with rigidified mimetics. Here we have defined the binding site of these novel ligands to MurD using (1)H/(13)C heteronuclear single quantum correlation. The MurD protein was selectively (13)C-labeled on the methyl groups of Ile (δ1 only), Leu and Val, and was isolated and purified. Crucial Ile, Leu and Val methyl groups in the vicinity of the ligand binding site were identified by comparison of chemical shift perturbation patterns among the ligands with various structural elements and known binding modes. The conformational and dynamic properties of the bound ligands and their binding interactions were examined using the transferred nuclear Overhauser effect and saturation transfer difference. In addition, the binding mode of these novel inhibitors was thoroughly examined using unrestrained molecular dynamics simulations. Our results reveal the complex dynamic behavior of ligand-MurD complexes and its influence on ligand-enzyme contacts. We further present important findings for the rational design of potent Mur ligase inhibitors.

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)