The Experts below are selected from a list of 2238 Experts worldwide ranked by ideXlab platform

Eefjan Breukink - One of the best experts on this subject based on the ideXlab platform.

  • An Engineered Double Lipid II Binding Motifs-Containing Lantibiotic Displays Potent and Selective Antimicrobial Activity against Enterococcus faecium.
    Antimicrobial Agents and Chemotherapy, 2020
    Co-Authors: Xinghong Zhao, Eefjan Breukink, Gert N. Moll, Oscar P Kuipers
    Abstract:

    : Lipid II is an essential precursor for bacterial cell wall biosynthesis and thereby an important target for various antibiotics. Several lanthionine-containing peptide antibiotics target Lipid II with lanthionine-stabilized Lipid II binding motifs. Here, we used the biosynthesis system of the lantibiotic nisin to synthesize a two-Lipid II binding motifs-containing lantibiotic, termed TL19, which contains the N-terminal Lipid II binding motif of nisin and the distinct C-terminal Lipid II binding motif of one peptide of the two-component haloduracin (i.e., HalA1). Further characterization demonstrated that (i) TL19 exerts 64-fold stronger antimicrobial activity against Enterococcus faecium than nisin(1-22), which has only one Lipid II binding site, and (II) both the N- and C-terminal domains are essential for the potent antimicrobial activity of TL19, as evidenced by mutagenesis of each single and the double domains. These results show the feasibility of a new approach to synthesize potent lantibiotics with two different Lipid II binding motifs to treat specific antibiotic-resistant pathogens.

  • Fluorescence anisotropy assays for high throughput screening of compounds binding to Lipid II, PBP1b, FtsW and MurJ.
    Scientific Reports, 2020
    Co-Authors: Adrien Boes, Eefjan Breukink, Samir Olatunji, Tamimount Mohammadi, Mohammed Terrak
    Abstract:

    : Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent Lipid II) and monitored direct binding between Lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between Lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled Lipid II, four Lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with Lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with Lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to Lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

  • Towards the Native Binding Modes of Antibiotics that Target Lipid II
    ChemBioChem, 2019
    Co-Authors: João Medeiros-silva, Eefjan Breukink, Shehrazade Jekhmane, Markus Weingarth
    Abstract:

    The alarming rise of antimicrobial resistance (AMR) imposes severe burdens on health care systems and the economy worldwide, urgently calling for the development of novel antibiotics. Antimicrobial peptides could be ideal templates for next-generation antibiotics due to their low propensity to develop resistance. An especially promising branch of antimicrobial peptides target Lipid II, the precursor of the bacterial peptidoglycan network. In order to develop these peptides into clinically applicable compounds, detailed information on their pharmacologically relevant modes of action is of critical importance. Here, we review the current understanding on the binding mode of a selection of Lipid II targeting peptides, and we highlight shortcomings in our molecular understanding that, at least partly, relate to the widespread use of artificial membrane mimics for structural studies of membrane-active antibiotics. In particular, we showcase on the example of the antimicrobial peptide nisin that the native cellular membrane environment can be critical to understand the physiologically relevant binding mode.

  • De novo identification of Lipid II binding lipopeptides with antibacterial activity against vancomycin-resistant bacteria
    Chemical Science, 2017
    Co-Authors: Peter 't Hart, Kamaleddin Haj Mohammad Ebrahim Tehrani, Eefjan Breukink, Antoni P. A. Hendrickx, Thomas M Wood, Rob J. L. Willems, Roel M. Van Harten, Małgorzata Śleszyńska, Inmaculada Rentero Rebollo, Nathaniel I. Martin
    Abstract:

    Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor Lipid II. In doing so we identified unique Lipid II binding peptides that upon Lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a Lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides.

  • De novo identification of Lipid II binding lipopeptides with antibacterial activity against vancomycin-resistant bacteria
    Chemical Science, 2017
    Co-Authors: Peter Hart, Kamaleddin Haj Mohammad Ebrahim Tehrani, Roel M. Van Harten, Małgorzata Śleszyńska, Inmaculada Rentero Rebollo, Eefjan Breukink, Antoni P. A. Hendrickx, Thomas M Wood, Rob J. L. Willems, Nathaniel I. Martin
    Abstract:

    Lipid II binding lipopeptides discovered via bicyclic peptide phage display exhibit promising antibacterial activity. Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor Lipid II. In doing so we identified unique Lipid II binding peptides that upon Lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a Lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides.

Hans-georg Sahl - One of the best experts on this subject based on the ideXlab platform.

  • Docking on Lipid II-A Widespread Mechanism for Potent Bactericidal Activities of Antibiotic Peptides.
    Journal of Molecular Biology, 2019
    Co-Authors: Fabian Grein, Tanja Schneider, Hans-georg Sahl
    Abstract:

    Abstract Natural product antibiotics usually target the major biosynthetic pathways of bacterial cells and the search for new targets outside these pathways has proven very difficult. Cell wall biosynthesis maybe the most prominent antibiotic target, and s-lactams are among the clinically most relevant antibiotics. Among cell wall biosynthesis inhibitors, glycopeptide antibiotics are a second group of important drugs, which bind to the peptidoglycan building block Lipid II and prevent the incorporation of the monomeric unit into polymeric cell wall. However, Lipid II acts as a docking molecule for many more naturally occurring antibiotics from diverse chemical classes and likely is the most targeted molecule in antibacterial mechanisms. We summarize current knowledge on Lipid II binding antibiotics and explain, on the levels of mechanisms and resistance development, why Lipid II is such a prominent target, and thus provide insights for the design of new antibiotic drugs.

  • The lantibiotic nisin induces Lipid II aggregation, causing membrane instability and vesicle budding.
    Biophysical Journal, 2015
    Co-Authors: Katharina Scherer, Hans-georg Sahl, Jan-hendrik Spille, Fabian Grein, Ulrich Kubitscheck
    Abstract:

    The antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane.

  • structural variations of the cell wall precursor Lipid II and their influence on binding and activity of the lipoglycopeptide antibiotic oritavancin
    Antimicrobial Agents and Chemotherapy, 2015
    Co-Authors: Daniela Munch, Hans-georg Sahl, Gabriele Bierbaum, Fabian Grein, Gerd Bendas, Ina Engels, Anna Muller, Katrin Rederchrist, Hildegard Falkensteinpaul, Tanja Schneider
    Abstract:

    Oritavancin is a semisynthetic derivative of the glycopeptide antibiotic chloroeremomycin with activity against Gram-positive pathogens, including vancomycin-resistant staphylococci and enterococci. Compared to vancomycin, oritavancin is characterized by the presence of two additional residues, a hydrophobic 4′-chlorobiphenyl methyl moiety and a 4-epi-vancosamine substituent, which is also present in chloroeremomycin. Here, we show that oritavancin and its des-N-methylleucyl variant (des-oritavancin) effectively inhibit Lipid I- and Lipid II-consuming peptidoglycan biosynthesis reactions in vitro. In contrast to that for vancomycin, the binding affinity of oritavancin to the cell wall precursor Lipid II appears to involve, in addition to the d-Ala-d-Ala terminus, other species-specific binding sites of the Lipid II molecule, i.e., the crossbridge and d-isoglutamine in position 2 of the Lipid II stem peptide, both characteristic for a number of Gram-positive pathogens, including staphylococci and enterococci. Using purified Lipid II and modified Lipid II variants, we studied the impact of these modifications on the binding of oritavancin and compared it to those of vancomycin, chloroeremomycin, and des-oritavancin. Analysis of the binding parameters revealed that additional intramolecular interactions of oritavancin with the peptidoglycan precursor appear to compensate for the loss of a crucial hydrogen bond in vancomycin-resistant strains, resulting in enhanced binding affinity. Augmenting previous findings, we show that amidation of the Lipid II stem peptide predominantly accounts for the increased binding of oritavancin to the modified intermediates ending in d-Ala-d-Lac. Corroborating our conclusions, we further provide biochemical evidence for the phenomenon of the antagonistic effects of mecA and vanA resistance determinants in Staphylococcus aureus, thus partially explaining the low frequency of methicillin-resistant S. aureus (MRSA) acquiring high-level vancomycin resistance.

  • Requirement of Lipid II biosynthesis for cell division in cell wall-less Wolbachia, endobacteria of arthropods and filarial nematodes.
    International Journal of Medical Microbiology, 2013
    Co-Authors: Jennifer Vollmer, Andrea Schiefer, Tanja Schneider, Kelly L Johnston, Hans-georg Sahl, Karen Jülicher, Mark J. Taylor, Achim Hoerauf, Kenneth Pfarr
    Abstract:

    Obligate Wolbachia endobacteria have a reduced genome and retained genes are hypothesized to be crucial for survival. Although intracellular bacteria do not need a stress-bearing peptidoglycan cell wall, Wolbachia encode proteins necessary to synthesize the peptidoglycan precursor Lipid II. The activity of the enzymes catalyzing the last two steps of this pathway was previously shown, and Wolbachia are sensitive to inhibition of Lipid II synthesis. A puzzling characteristic of Wolbachia is the lack of genes for l-amino acid racemases essential for Lipid II synthesis. Transcription analysis showed the expression of a possible alternative racemase metC, and recombinant Wolbachia MetC indeed had racemase activity that may substitute for the absent l-Ala racemase. However, enzymes needed to form mature peptidoglycan are absent and the function of Wolbachia Lipid II is unknown. Inhibition of Lipid II biosynthesis resulted in enlargement of Wolbachia cells and redistribution of Wolbachia peptidoglycan-associated lipoprotein, demonstrating that Lipid II is required for coordinated cell division and may interact with the lipoprotein. We conclude that Lipid II is essential for Wolbachia cell division and that this function is potentially conserved in the Gram-negative bacteria.

  • ring a of nukacin isk 1 a Lipid II binding motif for type a II lantibiotic
    Journal of the American Chemical Society, 2012
    Co-Authors: Mohammad Riazul Islam, Hans-georg Sahl, Jun-ichi Nagao, Takeshi Zendo, Mami Nishie, Sandro Keller, Jiro Nakayama, Daisuke Kohda, Kenji Sonomoto
    Abstract:

    Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a Lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to Lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating Lipid II precursor inside the cell, and the peptide activity can be repressed by Lipid I and Lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with Lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (KD = 0.17 and 0.19 μM, respectively) for Lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the st...

Ben De Kruijff - One of the best experts on this subject based on the ideXlab platform.

  • specificity of the transport of Lipid II by ftsw in escherichia coli
    Journal of Biological Chemistry, 2014
    Co-Authors: Tamimount Mohammadi, Ben De Kruijff, Nathaniel I. Martin, Mandy Lutters, Robert Sijbrandi, Jolanda Verheul, Tanneke Den Blaauwen, Eefjan Breukink
    Abstract:

    Synthesis of biogenic membranes requires transbilayer movement of Lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to obtain insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II, the important building block of the bacterial cell wall, and its inner membrane-localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (Arg145 and Lys153) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants, as manifested by their inability to complement a temperature-sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.

  • Potential scorpionate antibiotics: targeted hydrolysis of Lipid II containing model membranes by vancomycin-TACzyme conjugates and modulation of their antibacterial activity by Zn-ions.
    Bioorganic & Medicinal Chemistry Letters, 2009
    Co-Authors: H. Bauke Albada, Eefjan Breukink, Ben De Kruijff, Christopher J. Arnusch, Hilbert M. Branderhorst, Anne-marie Verel, Wouter T.m. Janssen, Roland J. Pieters, Rob M. J. Liskamp
    Abstract:

    The antibiotic vancomycin—that binds Lipid II in the bacterial cell membrane—was conjugated to a mono- and tetravalent mimic of the tris-histidine catalytic triad of metalloenzymes. Targeted hydrolysis by the conjugate was observed using model membranes containing Lipid II, and in vitro MIC-values of the targeted mimic constructs could be modulated by Zn-ions.

  • Lipid II a central component in bacterial cell wall synthesis and a target for antibiotics
    Prostaglandins Leukotrienes and Essential Fatty Acids, 2008
    Co-Authors: Ben De Kruijff, Eefjan Breukink
    Abstract:

    The bacterial cell wall is mainly composed of peptidoglycan, which is a three-dimensional network of long aminosugar strands located on the exterior of the cytoplasmic membrane. These strands consist of alternating MurNAc and GlcNAc units and are interlinked to each other via peptide moieties that are attached to the MurNAc residues. Peptidoglycan subunits are assembled on the cytoplasmic side of the bacterial membrane on a polyisoprenoid anchor and one of the key components in the synthesis of peptidoglycan is Lipid II. Being essential for bacterial cell survival, it forms an attractive target for antibacterial compounds such as vancomycin and several lantibiotics. Lipid II consists of one GlcNAc-MurNAc-pentapeptide subunit linked to a polyiosoprenoid anchor 11 subunits long via a pyrophosphate linker. This review focuses on this special molecule and addresses three questions. First, why are special Lipid carriers as polyprenols used in the assembly of peptidoglycan? Secondly, how is Lipid II translocated across the bacterial cytoplasmic membrane? And finally, how is Lipid II used as a receptor for lantibiotics to kill bacteria?

  • Lipid II: A central component in bacterial cell wall synthesis and a target for antibiotics
    Prostaglandins Leukotrienes and Essential Fatty Acids, 2008
    Co-Authors: Ben De Kruijff, Vincent Van Dam, Eefjan Breukink
    Abstract:

    The bacterial cell wall is mainly composed of peptidoglycan, which is a three-dimensional network of long aminosugar strands located on the exterior of the cytoplasmic membrane. These strands consist of alternating MurNAc and GlcNAc units and are interlinked to each other via peptide moieties that are attached to the MurNAc residues. Peptidoglycan subunits are assembled on the cytoplasmic side of the bacterial membrane on a polyisoprenoid anchor and one of the key components in the synthesis of peptidoglycan is Lipid II. Being essential for bacterial cell survival, it forms an attractive target for antibacterial compounds such as vancomycin and several lantibiotics. Lipid II consists of one GlcNAc-MurNAc-pentapeptide subunit linked to a polyiosoprenoid anchor 11 subunits long via a pyrophosphate linker. This review focuses on this special molecule and addresses three questions. First, why are special Lipid carriers as polyprenols used in the assembly of peptidoglycan? Secondly, how is Lipid II translocated across the bacterial cytoplasmic membrane? And finally, how is Lipid II used as a receptor for lantibiotics to kill bacteria? ?? 2008 Elsevier Ltd. All rights reserved.

  • an alternative bactericidal mechanism of action for lantibiotic peptides that target Lipid II
    Science, 2006
    Co-Authors: Hester E Hasper, Ben De Kruijff, Naomi E Kramer, Oscar P Kuipers, James Leif Smith, Jeffrey D Hillman, Cherian Zachariah, Eefjan Breukink
    Abstract:

    Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component Lipid II to form pores and thereby lyse the cells. Yet several members of these Lipid II–targeted lantibiotics are too short to be able to span the Lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing Lipid II from the cell division site (or septum) and thus block cell wall synthesis.

Tanja Schneider - One of the best experts on this subject based on the ideXlab platform.

  • Docking on Lipid II-A Widespread Mechanism for Potent Bactericidal Activities of Antibiotic Peptides.
    Journal of Molecular Biology, 2019
    Co-Authors: Fabian Grein, Tanja Schneider, Hans-georg Sahl
    Abstract:

    Abstract Natural product antibiotics usually target the major biosynthetic pathways of bacterial cells and the search for new targets outside these pathways has proven very difficult. Cell wall biosynthesis maybe the most prominent antibiotic target, and s-lactams are among the clinically most relevant antibiotics. Among cell wall biosynthesis inhibitors, glycopeptide antibiotics are a second group of important drugs, which bind to the peptidoglycan building block Lipid II and prevent the incorporation of the monomeric unit into polymeric cell wall. However, Lipid II acts as a docking molecule for many more naturally occurring antibiotics from diverse chemical classes and likely is the most targeted molecule in antibacterial mechanisms. We summarize current knowledge on Lipid II binding antibiotics and explain, on the levels of mechanisms and resistance development, why Lipid II is such a prominent target, and thus provide insights for the design of new antibiotic drugs.

  • structural variations of the cell wall precursor Lipid II and their influence on binding and activity of the lipoglycopeptide antibiotic oritavancin
    Antimicrobial Agents and Chemotherapy, 2015
    Co-Authors: Daniela Munch, Hans-georg Sahl, Gabriele Bierbaum, Fabian Grein, Gerd Bendas, Ina Engels, Anna Muller, Katrin Rederchrist, Hildegard Falkensteinpaul, Tanja Schneider
    Abstract:

    Oritavancin is a semisynthetic derivative of the glycopeptide antibiotic chloroeremomycin with activity against Gram-positive pathogens, including vancomycin-resistant staphylococci and enterococci. Compared to vancomycin, oritavancin is characterized by the presence of two additional residues, a hydrophobic 4′-chlorobiphenyl methyl moiety and a 4-epi-vancosamine substituent, which is also present in chloroeremomycin. Here, we show that oritavancin and its des-N-methylleucyl variant (des-oritavancin) effectively inhibit Lipid I- and Lipid II-consuming peptidoglycan biosynthesis reactions in vitro. In contrast to that for vancomycin, the binding affinity of oritavancin to the cell wall precursor Lipid II appears to involve, in addition to the d-Ala-d-Ala terminus, other species-specific binding sites of the Lipid II molecule, i.e., the crossbridge and d-isoglutamine in position 2 of the Lipid II stem peptide, both characteristic for a number of Gram-positive pathogens, including staphylococci and enterococci. Using purified Lipid II and modified Lipid II variants, we studied the impact of these modifications on the binding of oritavancin and compared it to those of vancomycin, chloroeremomycin, and des-oritavancin. Analysis of the binding parameters revealed that additional intramolecular interactions of oritavancin with the peptidoglycan precursor appear to compensate for the loss of a crucial hydrogen bond in vancomycin-resistant strains, resulting in enhanced binding affinity. Augmenting previous findings, we show that amidation of the Lipid II stem peptide predominantly accounts for the increased binding of oritavancin to the modified intermediates ending in d-Ala-d-Lac. Corroborating our conclusions, we further provide biochemical evidence for the phenomenon of the antagonistic effects of mecA and vanA resistance determinants in Staphylococcus aureus, thus partially explaining the low frequency of methicillin-resistant S. aureus (MRSA) acquiring high-level vancomycin resistance.

  • Requirement of Lipid II biosynthesis for cell division in cell wall-less Wolbachia, endobacteria of arthropods and filarial nematodes.
    International Journal of Medical Microbiology, 2013
    Co-Authors: Jennifer Vollmer, Andrea Schiefer, Tanja Schneider, Kelly L Johnston, Hans-georg Sahl, Karen Jülicher, Mark J. Taylor, Achim Hoerauf, Kenneth Pfarr
    Abstract:

    Obligate Wolbachia endobacteria have a reduced genome and retained genes are hypothesized to be crucial for survival. Although intracellular bacteria do not need a stress-bearing peptidoglycan cell wall, Wolbachia encode proteins necessary to synthesize the peptidoglycan precursor Lipid II. The activity of the enzymes catalyzing the last two steps of this pathway was previously shown, and Wolbachia are sensitive to inhibition of Lipid II synthesis. A puzzling characteristic of Wolbachia is the lack of genes for l-amino acid racemases essential for Lipid II synthesis. Transcription analysis showed the expression of a possible alternative racemase metC, and recombinant Wolbachia MetC indeed had racemase activity that may substitute for the absent l-Ala racemase. However, enzymes needed to form mature peptidoglycan are absent and the function of Wolbachia Lipid II is unknown. Inhibition of Lipid II biosynthesis resulted in enlargement of Wolbachia cells and redistribution of Wolbachia peptidoglycan-associated lipoprotein, demonstrating that Lipid II is required for coordinated cell division and may interact with the lipoprotein. We conclude that Lipid II is essential for Wolbachia cell division and that this function is potentially conserved in the Gram-negative bacteria.

  • identification and in vitro analysis of the gatd murt enzyme complex catalyzing Lipid II amidation in staphylococcus aureus
    PLOS Pathogens, 2012
    Co-Authors: Daniela Munch, Hans-georg Sahl, Terry Roemer, Marianne Engeser, Tanja Schneider
    Abstract:

    The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors Lipid I, Lipid II and Lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo Lipid II and/or Lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished Lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

  • Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus
    PLOS Pathogens, 2012
    Co-Authors: Daniela Munch, Hans-georg Sahl, Terry Roemer, Marianne Engeser, Tanja Schneider
    Abstract:

    The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors Lipid I, Lipid II and Lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo Lipid II and/or Lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished Lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

Nathaniel I. Martin - One of the best experts on this subject based on the ideXlab platform.

  • Dissecting the Binding Interactions of Teixobactin with the Bacterial Cell‐Wall Precursor Lipid II
    ChemBioChem, 2019
    Co-Authors: Sorina Chiorean, Nathaniel I. Martin, Stephen A Cochrane, Isaac Antwi, Daniel W. Carney, Ioli Kotsogianni, Andrew M. Giltrap, Francesca M. Alexander, Richard J. Payne, Antoine Henninot
    Abstract:

    : The prevalence of life-threatening, drug-resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered "resistance-proof" antimicrobial peptide that targets the bacterial cell wall precursor Lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram-positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding Lipid II from both Gram-positive and Gram-negative bacteria. Furthermore, we show that when combined with known outer membrane-disrupting peptides, teixobactin is active against Gram-negative organisms.

  • De novo identification of Lipid II binding lipopeptides with antibacterial activity against vancomycin-resistant bacteria
    Chemical Science, 2017
    Co-Authors: Peter 't Hart, Kamaleddin Haj Mohammad Ebrahim Tehrani, Eefjan Breukink, Antoni P. A. Hendrickx, Thomas M Wood, Rob J. L. Willems, Roel M. Van Harten, Małgorzata Śleszyńska, Inmaculada Rentero Rebollo, Nathaniel I. Martin
    Abstract:

    Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor Lipid II. In doing so we identified unique Lipid II binding peptides that upon Lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a Lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides.

  • De novo identification of Lipid II binding lipopeptides with antibacterial activity against vancomycin-resistant bacteria
    Chemical Science, 2017
    Co-Authors: Peter Hart, Kamaleddin Haj Mohammad Ebrahim Tehrani, Roel M. Van Harten, Małgorzata Śleszyńska, Inmaculada Rentero Rebollo, Eefjan Breukink, Antoni P. A. Hendrickx, Thomas M Wood, Rob J. L. Willems, Nathaniel I. Martin
    Abstract:

    Lipid II binding lipopeptides discovered via bicyclic peptide phage display exhibit promising antibacterial activity. Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor Lipid II. In doing so we identified unique Lipid II binding peptides that upon Lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a Lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides.

  • hit em where it hurts the growing and structurally diverse family of peptides that target Lipid II
    Biochimica et Biophysica Acta, 2016
    Co-Authors: Sabine F. Oppedijk, Nathaniel I. Martin, Eefjan Breukink
    Abstract:

    Understanding the mode of action of antibiotics is becoming more and more important in the time that microorganisms start to develop resistance. One very well validated target of several classes of antibiotics is the peptidoglycan precursor Lipid II. In this review different classes of Lipid II targeting antibiotics will be discussed in detail, including the lantibiotics, human invertebrate defensins and the recently discovered teixobactin. By hitting bacteria where it hurts, at the level of Lipid II, we expect to be able to develop efficient antibacterial agents in the future. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  • New Insights into Nisin's Antibacterial Mechanism Revealed by Binding Studies with Synthetic Lipid II Analogues.
    Biochemistry, 2015
    Co-Authors: Peter 't Hart, Eefjan Breukink, Sabine F. Oppedijk, Nathaniel I. Martin
    Abstract:

    Nisin is the preeminent lantibiotic, and to date its antibacterial mechanism has been investigated using a variety of techniques. While nisin’s Lipid II-mediated mode of action is well-established, a detailed analysis of the thermodynamic parameters governing this interaction has not been previously reported. We here describe an approach employing isothermal titration calorimetry to directly measure the affinity of nisin for Lipid II and a number of synthetic Lipid II precursors and analogues. Our measurements confirm the pyrophosphate unit of Lipid II as the primary site of nisin binding and also indicate that the complete MurNAc moiety is required for a high-affinity interaction. Additionally, we find that while the pentapeptide unit of the Lipid II molecule is not required for strong binding by nisin, it does play an important role in stabilizing the subsequently formed nisin–Lipid II pore complex, albeit at an entropic cost. The anchoring of Lipid II in a membrane environment was also found to play a ...