Mur ligases inhibitors with azastilbene scaffold: Expanding the structure-activity relationship.

Antibiotic resistance represents one of the biggest public health challenges in the last few years. Mur ligases (MurC-MurF) are involved in the synthesis of UDP-N-acetylmuramyl-pentapeptide, the main building block of bacterial peptidoglycan polymer. They are essential for the survival of bacteria and therefore important antibacterial targets. We report herein the synthesis and structure-activity relationships of Mur ligases inhibitors with an azastilbene scaffold. Several compounds showed promising inhibitory potencies against multiple ligases and one compound also possessed moderate antibacterial activity. These results represent a solid ground for further development and optimization of structurally novel antimicrobial agents to combat the rising bacterial resistance.

CbrA Mediates Colicin M Resistance in Escherichia coli through Modification of Undecaprenyl-Phosphate-Linked Peptidoglycan Precursors.

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.

Impact of FiuA Outer Membrane Receptor Polymorphism on the Resistance of Pseudomonas aeruginosa toward Peptidoglycan Lipid II-Targeting PaeM Pyocins.

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.

Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment.

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).

Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases.

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 µM). 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.

Can clinical practice indicator relating to long-acting benzodiazepine use in the elderly be easily generated in a hospital setting?

BACKGROUND: To improve the appropriate use of long-acting benzodiazepine (la.bzd) prescriptions in the elderly, the Haute Autorité de Santé (HAS) has developed clinical practice indicators (CPI). The alert indicator (AI) evaluates the prevalence of la.bzd prescription among older people. The mastering indicator (MI) corresponds to the prevalence of elderly with a justified, i.e., appropriate, la.bzd prescription among all the elderly with la.bzd prescriptions. OBJECTIVE: The objective of this study was to test the feasibility of routine generation of CPI regarding la.bzd prescriptions among the elderly in the hospital setting. DESIGN: This was a retrospective study. SETTING: The study was conducted in two university hospitals located in Paris. SUBJECT: Eligible cases were patients aged 65 years and older who were hospitalized in acute care units from January to June 2014. METHOD: The AI calculation was based on information extracted from medical databases from these hospitals. The appropriateness of la.bzd prescription was assessed by a physician and a pharmacist and was based on review of computerized patient records and prescriptions, using an ad hoc algorithm. The MI was then calculated. Variation in the level of indicators was explored according to the characteristics of patients and of their hospitalization using chi2 test. Factors associated with a potentially inappropriate prescription (PIP) of la.bzd were studied using univariate and multivariate logistic regression. RESULT: Among the 4167 patients included in the study, 362 had la.bzd prescriptions, i.e., the AI was 9%. Prescriptions were found to be appropriate for 83 patients, i.e., the MI was 23% and PIP was 77%. The MI varied between 13 and 31% according to characteristics of patients and of hospitalization. In multivariate analysis, factors associated with PIP were age, number of comorbidities, type of care unit, and concurrent prescription of a neuroleptic or hypnotic. CONCLUSION: Generation of the AI was routinely possible but only for acute care units with computerized prescriptions, corresponding to 78% of patients. Production of the MI has required medical record review for all patients with a la.bzd prescription and cannot be automated. However, difficulties in generating the MI have identified areas for significant improvement. Moreover, strategies to improve the care of older people with a la.bzd prescription could be targeted using characteristics of patients and of hospitalization associated with PIP. The future deployment of a single electronic medical record in all care departments would make it easier to mine the data and make possible automated production of CPI.

[Temporary approval for intranasal naloxone: Setting up in a French addiction center]. / Pratique de l'ATU de cohorte de la naloxone intranasale (Nalscue®) : mise en place dans un CSAPA parisien.

Intranasal naloxone aims at preventing opioid overdose related deaths in active drug users. In France, it has been available since July 2016 through a temporary approval which requires a hospital-based pharmacy and a nominative registration of each patient. We present the characteristics of the first patients who could receive this prescription in our hospital-based addiction center and how they used naloxone during follow-up. Results favor a larger dispensing of naloxone. Patients' as well as peers' and families' education is needed.

Discovery of new MurA inhibitors using induced-fit simulation and docking.

We report on the successful application of ProBiS-CHARMMing web server in the discovery of new inhibitors of MurA, an enzyme that catalyzes the first committed cytoplasmic step of bacterial peptidoglycan synthesis. The available crystal structures of Escherichia coli MurA in the Protein Data Bank have binding sites whose small volume does not permit the docking of drug-like molecules. To prepare the binding site for docking, the ProBiS-CHARMMing web server was used to simulate the induced-fit effect upon ligand binding to MurA, resulting in a larger, more holo-like binding site. The docking of a filtered ZINC compound library to this enlarged binding site was then performed and resulted in three compounds with promising inhibitory potencies against MurA. Compound 1 displayed significant inhibitory potency with IC50 value of 1µM. All three compounds have novel chemical structures, which could be used for further optimization of small-molecule MurA inhibitors.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

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.

Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex.

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 Šresolution 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.

Inhibitor design strategy based on an enzyme structural flexibility: a case of bacterial MurD ligase.

Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound 3 from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound 3 were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process.

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents.

Functional and structural characterization of PaeM, a colicin M-like bacteriocin produced by Pseudomonas aeruginosa.

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.

Biochemical characterization of MurF from Streptococcus pneumoniae and the identification of a new MurF inhibitor through ligand-based virtual screening.

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.

Colicin M, a peptidoglycan lipid-II-degrading enzyme: potential use for antibacterial means?

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.

Towards discovery of inhibitors of the undecaprenyl-pyrophosphate phosphatase BacA by virtual high-throughput screening.

Increasing resistance to common antibiotics is becoming a major challenge that requires the development of new antibacterial agents. Peptidoglycan is an essential heteropolymer of the bacterial envelope that ensures the integrity and shape of all bacteria and is also an important target for antibiotics. The biosynthesis of peptidoglycan depends on a lipid carrier, undecaprenyl phosphate. As a byproduct of peptidoglycan polymerization, the lipid carrier is released as undecaprenyl pyrophosphate, which must be recycled to allow new polymerization cycles. To this end, it undergoes a dephosphorylation process catalyzed by the membrane phosphatase BacA, which is specific and highly conserved in bacteria. In the present study, we identified small molecules displaying inhibitory potency towards BacA. We began by preparing a commercial compound library, followed by high-throughput virtual screening by ensemble docking using the 3D structure of BacA and molecular dynamics snapshots to account for the flexibility of the protein. Of 83 compounds computationally selected and tested in a biochemical assay, one sulfamoylthiophene molecule showed significant inhibition of the undecaprenyl pyrophosphate dephosphorylation activity catalyzed by BacA. Subsequently, an additional 33 scaffold analogs were selected and acquired, of which 6 compounds exhibited BacA inhibition. The IC50 values of these compounds ranged from 42 to 366 µM. In addition, significant antibacterial activity against Escherichia coli was observed in TolC/PAP2-depleted strains. We believe that the overall strategy followed in this study and the identified class of inhibitors provide a solid foundation for the further development of potent BacA-targeted inhibitors and the discovery of novel antibacterial compounds.

X-ray structure and site-directed mutagenesis analysis of the Escherichia coli colicin M immunity protein.

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 ß 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.

Deciphering the catalytic domain of colicin M, a peptidoglycan lipid II-degrading enzyme.

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 Biology of Colicin M and Its Orthologs.

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.

Toxicity of the colicin M catalytic domain exported to the periplasm is FkpA independent.

Colicin M (ColM) is a bactericidal protein that kills sensitive cells by hydrolyzing lipid II, involved in the biosynthesis of cell wall peptidoglycan. It recognizes FhuA on the outer leaflet, and its translocation through the outer membrane depends on the energized Ton complex in the inner membrane. To be active in the periplasm, ColM must be translocated through the outer membrane and then interact with FkpA, a periplasmic protein that exhibits both cis- and trans-peptidylprolyl isomerase (PPiase) and chaperon activities. In an attempt to directly target ColM to the periplasm of the producing bacteria, we fused the presequence of OmpA to ColM (sp-ColM). We found that expression of this hybrid protein in an Escherichia coli strain devoid of ColM immunity protein (Cmi) was bactericidal. We showed that sp-ColM was correctly expressed, processed, and associated with the inner membrane. sp-ColM toxicity was related to its enzymatic activity and did not rely on the TonB import proteins or the FhuA receptor. The presence of both activity domains of FkpA was still required for sp-ColM activity. Analyses of deletion mutants of sp-ColM show that the domain required for toxicity corresponds to the C-terminal last 153 amino acids of ColM. Like the full-length protein, this domain is not active in the presence of the immunity protein Cmi. On the other hand, it does not require FkpA for toxic activity.