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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 C55-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 C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.

International audience; A halophilic organism, SWO25T, was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25T grew optimally at 35–45 °C, at pH 6.0–8.0 and 0.05–0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3–5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25T to the genus Haloarcula. The DNA G+C content was 61.1mol%. DNA–DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB′ genes showed that strain SWO25T is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarculasebkhae sp. nov. is proposed. The type strain is SWO25T (=CIP 110583T=JCM 19018T).

International audience; The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 mu M). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.

Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination.IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

WOS:000450383500008; International audience; Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors. (C) 2018 Elsevier Masson SAS. All rights reserved.

International audience; 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.

International audience; 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.

International audience; Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four β strands and four α helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated.

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His6-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l -Ala, l -Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l -Ala. S. aureus MurE was very specific for l -lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l -lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms ( l -lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l -lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.

International audience; Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

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. Given that urgency, the finding of new antibacterials in the short term is crucial.Colicins, due to their antimicrobials properties, constitute good candidates. They are protein toxins produced by E. coli to kill competitors belonging to the same or related species. In most cases, they exhibit their cytotoxic activity through an ionophoric or nucleasic activity. Among the twenty colicins known to date, colicin M (ColM) is the only one known to interfere with peptidoglycan biosynthesis. It develops its lethal activity in the E. coli periplasm, in three steps deeply linked to its structural organization in three domains. Once in the periplasm, ColM degrades the lipid II, i.e. the last precursor in the peptidoglycan biosynthesis pathway, in two products that cannot be reused, thereby leading to cell lysis. Several ColM homologues have been identified in other bacterial genera, such as Pseudomonas, Pectobacterium and Burkholderia, but no cross activity has been shown to date, explaining the narrow antibacterial spectrum displayed by the members of this new family of antibacterial enzymes.This work deals with the structural and biochemical study of ColM and some of its homologues. Structural studies on several variants of PaeM, the ColM homologue from P. aeruginosa, led to identify a conserved water molecule in the active site, probably playing a central role in the catalytic mechanism of this enzyme family. Moreover, expression of ColM homologues from Pseudomonas or Pectobacterium species directly in the E. coli periplasm showed that all these homologues were able to induce E. coli cell lysis, thus demonstrating the great potential of these bacteriocins as an alternative to antibiotics. Following these results, several chimera colicins were created between ColM and its homologues, which were shown to degrade lipid II in vitro and to induce E. coli cell lysis after their periplasmic expression, opening the way to future new therapeutic options.; L’émergence de bactéries multirésistantes aux antibiotiques est la conséquence de leur utilisation à mauvais escient au cours de ces dernières décennies. Ce phénomène constitue un problème de santé publique majeur, et face à cette urgence sanitaire, il est nécessaire de trouver rapidement de nouveaux agents antibactériens.Les colicines, au regard de leurs propriétés antimicrobiennes intrinsèques, constituent des candidats intéressants. Naturellement produites par E. coli dans le but de tuer des souches compétitrices de la même espèce ou d’espèces apparentées, elles exercent en général leur activité cytotoxique par le biais d’une activité ionophorique ou nucléasique. Parmi les nombreuses colicines connues à ce jour, la colicine M (ColM) est la seule à interférer avec la voie de biosynthèse du peptidoglycane, macromolécule essentielle et spécifique au monde bactérien. En effet, une fois dans le périplasme de E. coli, la ColM clive le lipide II, dernier précurseur de la voie de biosynthèse du peptidoglycane, conduisant de ce fait à la lyse bactérienne. Plusieurs homologues de la ColM ont été identifiés chez d’autres genres bactériens (Pseudomonas, Pectobacterium et Burkholderia) mais aucune cytotoxicité croisée n’a été mise en évidence à ce jour, d’où un spectre d’action restreint pour les membres de cette nouvelle famille d’enzymes antibactériennes.Ce travail traite de l’étude structurale et biochimique de la ColM et de certains de ses homologues. L’étude structurale de différents variants de la PaeM, homologue issu de P. aeruginosa, a permis d’identifier une molécule d’eau conservée au sein du site actif qui joue probablement un rôle central dans le mécanisme catalytique de cette famille d’enzyme. L’expression des homologues de la ColM issus de Pseudomonas et de Pectobacterium, directement dans le périplasme de E. coli, a permis de démontrer leur activité lytique, prouvant ainsi le grand potentiel de ces bactériocines en tant qu’alternatives aux antibiotiques. Enfin, la construction de plusieurs colicines chimères entre la ColM et ses homologues, capables de dégrader le lipide II in vitro et d’induire la lyse d’E. coli suite à leur expression périplasmique, ouvre la voie à de futurs espoirs thérapeutiques.

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. Given that urgency, the finding of new antibacterials in the short term is crucial.Colicins, due to their antimicrobials properties, constitute good candidates. They are protein toxins produced by E. coli to kill competitors belonging to the same or related species. In most cases, they exhibit their cytotoxic activity through an ionophoric or nucleasic activity. Among the twenty colicins known to date, colicin M (ColM) is the only one known to interfere with peptidoglycan biosynthesis. It develops its lethal activity in the E. coli periplasm, in three steps deeply linked to its structural organization in three domains. Once in the periplasm, ColM degrades the lipid II, i.e. the last precursor in the peptidoglycan biosynthesis pathway, in two products that cannot be reused, thereby leading to cell lysis. Several ColM homologues have been identified in other bacterial genera, such as Pseudomonas, Pectobacterium and Burkholderia, but no cross activity has been shown to date, explaining the narrow antibacterial spectrum displayed by the members of this new family of antibacterial enzymes.This work deals with the structural and biochemical study of ColM and some of its homologues. Structural studies on several variants of PaeM, the ColM homologue from P. aeruginosa, led to identify a conserved water molecule in the active site, probably playing a central role in the catalytic mechanism of this enzyme family. Moreover, expression of ColM homologues from Pseudomonas or Pectobacterium species directly in the E. coli periplasm showed that all these homologues were able to induce E. coli cell lysis, thus demonstrating the great potential of these bacteriocins as an alternative to antibiotics. Following these results, several chimera colicins were created between ColM and its homologues, which were shown to degrade lipid II in vitro and to induce E. coli cell lysis after their periplasmic expression, opening the way to future new therapeutic options.; L’émergence de bactéries multirésistantes aux antibiotiques est la conséquence de leur utilisation à mauvais escient au cours de ces dernières décennies. Ce phénomène constitue un problème de santé publique majeur, et face à cette urgence sanitaire, il est nécessaire de trouver rapidement de nouveaux agents antibactériens.Les colicines, au regard de leurs propriétés antimicrobiennes intrinsèques, constituent des candidats intéressants. Naturellement produites par E. coli dans le but de tuer des souches compétitrices de la même espèce ou d’espèces apparentées, elles exercent en général leur activité cytotoxique par le biais d’une activité ionophorique ou nucléasique. Parmi les nombreuses colicines connues à ce jour, la colicine M (ColM) est la seule à interférer avec la voie de biosynthèse du peptidoglycane, macromolécule essentielle et spécifique au monde bactérien. En effet, une fois dans le périplasme de E. coli, la ColM clive le lipide II, dernier précurseur de la voie de biosynthèse du peptidoglycane, conduisant de ce fait à la lyse bactérienne. Plusieurs homologues de la ColM ont été identifiés chez d’autres genres bactériens (Pseudomonas, Pectobacterium et Burkholderia) mais aucune cytotoxicité croisée n’a été mise en évidence à ce jour, d’où un spectre d’action restreint pour les membres de cette nouvelle famille d’enzymes antibactériennes.Ce travail traite de l’étude structurale et biochimique de la ColM et de certains de ses homologues. L’étude structurale de différents variants de la PaeM, homologue issu de P. aeruginosa, a permis d’identifier une molécule d’eau conservée au sein du site actif qui joue probablement un rôle central dans le mécanisme catalytique de cette famille d’enzyme. L’expression des homologues de la ColM issus de Pseudomonas et de Pectobacterium, directement dans le périplasme de E. coli, a permis de démontrer leur activité lytique, prouvant ainsi le grand potentiel de ces bactériocines en tant qu’alternatives aux antibiotiques. Enfin, la construction de plusieurs colicines chimères entre la ColM et ses homologues, capables de dégrader le lipide II in vitro et d’induire la lyse d’E. coli suite à leur expression périplasmique, ouvre la voie à de futurs espoirs thérapeutiques.