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

2012 ◽  
Vol 40 (6) ◽  
pp. 1522-1527 ◽  
Author(s):  
Thierry Touzé ◽  
Hélène Barreteau ◽  
Meriem El Ghachi ◽  
Ahmed Bouhss ◽  
Aurélie Barnéoud-Arnoulet ◽  
...  
Keyword(s):  
Antibacterial Agents ◽  
Catalytic Domain ◽  
Cell Integrity ◽  
Active Site Residues ◽  
Isomerase Activity ◽  
Lipid Ii ◽  
Genes Encoding ◽  
Colicin M ◽  
Potential Use

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.

scholarly journals The ColM Family, Polymorphic Toxins Breaching the Bacterial Cell Wall

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Maarten G. K. Ghequire ◽  
Susan K. Buchanan ◽  
René De Mot
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Cytoplasmic Membrane ◽  
Catalytic Domain ◽  
Gram Negative Bacteria ◽  
Antibacterial Peptides ◽  
Gram Negative ◽  
Lipid Ii ◽  
Associated Proteins ◽  
Colicin M

ABSTRACT Bacteria host an arsenal of antagonism-mediating molecules to combat for ecologic space. Bacteriocins represent a pivotal group of secreted antibacterial peptides and proteins assisting in this fight, mainly eliminating relatives. Colicin M, a model for peptidoglycan-interfering bacteriocins in Gram-negative bacteria, appears to be part of a set of polymorphic toxins equipped with such a catalytic domain (ColM) targeting lipid II. Diversifying recombination has enabled parasitism of different receptors and has also given rise to hybrid bacteriocins in which ColM is associated with another toxin module. Remarkably, ColM toxins have recruited a diverse array of immunity partners, comprising cytoplasmic membrane-associated proteins with different topologies. Together, these findings suggest that different immunity mechanisms have evolved for ColM, in contrast to bacteriocins with nuclease activities.

scholarly journals Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

2010 ◽  
Vol 285 (16) ◽  
pp. 12378-12389 ◽  
Author(s):  
Hélène Barreteau ◽  
Ahmed Bouhss ◽  
Fabien Gérard ◽  
Denis Duché ◽  
Boubekeur Boussaid ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Degrading Enzyme ◽  
Lipid Ii ◽  
Colicin M

scholarly journals The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains

2003 ◽  
Vol 185 (15) ◽  
pp. 4418-4423 ◽  
Author(s):  
Anne Marie Di Guilmi ◽  
Andréa Dessen ◽  
Otto Dideberg ◽  
Thierry Vernet
Keyword(s):  
Streptococcus Pneumoniae ◽  
Pathogenic Bacteria ◽  
Catalytic Domain ◽  
Soluble Form ◽  
Lipid Ii ◽  
Extracellular Region ◽  
Catalytically Active ◽  
Bacterial Peptidoglycan

ABSTRACT The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat/K m were measured at 40.6 μM (± 15.5) and 1 × 10−3 M−1 s−1, respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

scholarly journals Repurposing Thiram and Disulfiram as Antibacterial Agents for Multidrug-Resistant Staphylococcus aureus Infections

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Timothy E. Long
Keyword(s):  
Staphylococcus Aureus ◽  
Combination Therapy ◽  
Antibacterial Agents ◽  
Multidrug Resistant ◽  
Positive Bacterium ◽  
Content Type ◽  
Spectrum Activity ◽  
Vancomycin Resistant ◽  
Potential Use

ABSTRACT Thiram and disulfiram were evaluated as antibacterial agents against multidrug-resistant Staphylococcus aureus. Against a 30-member panel comprised of vancomycin-susceptible, vancomycin-intermediate, and vancomycin-resistant S. aureus strains, the MIC90 values of the respective test agents were 4 and 16 μg/ml. Additional analyses revealed that thiram and disulfiram are rapid-acting bacteriostatic agents with narrow, Gram-positive-bacterium spectrum activity. Synergy studies further determined that disulfiram increases the vancomycin susceptibility of three clinical vancomycin-resistant S. aureus strains in vitro, thus establishing a potential use in combination therapy.

scholarly journals Human- and Plant-Pathogenic Pseudomonas Species Produce Bacteriocins Exhibiting Colicin M-Like Hydrolase Activity towards Peptidoglycan Precursors

2009 ◽  
Vol 191 (11) ◽  
pp. 3657-3664 ◽  
Author(s):  
Hélène Barreteau ◽  
Ahmed Bouhss ◽  
Martine Fourgeaud ◽  
Jean-Luc Mainardi ◽  
Thierry Touzé ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Hydrolase Activity ◽  
New Class ◽  
Low Concentrations ◽  
Pseudomonas Species ◽  
Genes Encoding ◽  
Colicin M ◽  
Undecaprenyl Phosphate

ABSTRACT Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae, and P. fluorescens. These genes were detected only in a restricted number of strains. In P. aeruginosa, for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit Mg2+-dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.

Arabidopsis fructokinase-like protein associations are regulated by ATP

2017 ◽  
Vol 474 (11) ◽  
pp. 1789-1801 ◽  
Author(s):  
John W. Riggs ◽  
Judy Callis
Keyword(s):  
Kinase Activity ◽  
Active Site Residues ◽  
Large Protein ◽  
Dominant Form ◽  
Sequence Comparisons ◽  
X Ray ◽  
Sequence Identity ◽  
Large Protein Complex ◽  
Genes Encoding

The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequence comparisons, and mutational analyses suggests that FLN proteins may bind their substrates differently from other pfkB proteins. We provide evidence that purified recombinant FLN1 undergoes an ATP-mediated change in binding affinity with both itself and recombinant FLN2. The ATP-mediated change in the affinity of FLN1 for FLN2 is not affected by mutations in conserved active-site residues known to affect catalysis in active pfkB enzymes. In contrast, recombinant FLN2 hetero-oligomerizes independently of ATP concentration. At ATP concentrations that promote FLN1 homomeric interactions, the FLN1–FLN2 hetero-oligomer is the dominant form in vitro. We further present evidence that FLN1 associates with a large protein complex in chloroplasts independently of ATP. Given that ATP levels fluctuate between light–dark cycles in the 1–5 mM range, we propose that changes in FLN1 and FLN2 interactions are biologically meaningful.

scholarly journals Bioactive Enterococci Isolated from Slovak Ewes’ Lump Cheese

2016 ◽  
Vol 47 (4) ◽  
pp. 187-193 ◽  
Author(s):  
A. Lauková ◽  
V. Strompfová ◽  
R. Szabóová ◽  
A. Slottová ◽  
M. Tomáška ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Listeria Monocytogenes ◽  
Enterococcus Faecium ◽  
Dairy Industry ◽  
Inhibition Zone ◽  
Listeria Innocua ◽  
Inhibition Activity ◽  
Genes Encoding ◽  
Potential Use

Abstract Enterococci are widespread organisms; some of their properties are desired in dairy industry. They can produce antimicrobial proteinaceous substances (enterocins) linked to food biopreservation. This study focused on bioactive Enterococcus faecium and Enterococcus faecalis strains from Slovak ewes’ lump cheeses to check genes encoding enterocins production and inhibition activity. The total counts of enterococci in ewes’ lump cheeses reached 5.95 ± 2.44 log CFU/g on average. Genotypization by PCR and identification by MALDI-TOF mass spectrometry alloted 12 strains to the species Enterococcus faecium and 18 strains to the species E. faecalis. Enterococci were hemolytic phenotype free. Gelatinase negative strains were tested for the presence of enterocins genes. E. faecium and E. faecalis strains from Slovak ewes’ lump cheeses possessed mostly genes for enterocins P and A. Enterocin gene free E. faecalis EE29E3 inhibited indicator Enterococcus avium EA5 (inhibition zone > 10 mm); EE36E1inhibited Listeria innocua LMG 13568 (inhibiton zone 12 mm). Among E. faecium possessing enerocins genes, inhibition activity was only noted in EF27E4 strain (against E. avium EA5, Listeria monocytogenes CCM4699; inhibiton zone 10–22 mm). E. faecium EF27E4 was selected for more detailed studies in vitro aimed at its potential use in dairy industry.

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

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Hélène Barreteau ◽  
Delphine Patin ◽  
Ahmed Bouhss ◽  
Didier Blanot ◽  
Dominique Mengin-Lecreulx ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cleavage Site ◽  
Resistance Mechanism ◽  
Content Type ◽  
E Coli ◽  
Lipid Ii ◽  
Colicin M ◽  
Undecaprenyl Phosphate

ABSTRACT 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.

scholarly journals Toxicity of the Colicin M Catalytic Domain Exported to the Periplasm Is FkpA Independent

2010 ◽  
Vol 192 (19) ◽  
pp. 5212-5219 ◽  
Author(s):  
Aurélie Barnéoud-Arnoulet ◽  
Hélène Barreteau ◽  
Thierry Touzé ◽  
Dominique Mengin-Lecreulx ◽  
Roland Lloubès ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Catalytic Domain ◽  
Periplasmic Protein ◽  
Inner Membrane ◽  
Toxic Activity ◽  
Immunity Protein ◽  
Outer Leaflet ◽  
Lipid Ii ◽  
Colicin M ◽  
Peptidylprolyl Isomerase

ABSTRACT 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.

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