Lipodepsipeptide Empedopeptin Inhibits Cell Wall Biosynthesis through Ca2+-dependent Complex Formation with Peptidoglycan Precursors

Müller, Anna; Münch, Daniela; Schmidt, Yvonne; Reder-Christ, Katrin; Schiffer, Guido; Bendas, Gerd; Gross, Harald; Sahl, Hans‐Georg; Schneider, Tanja; Brötz‐Oesterhelt, Heike

doi:10.1074/jbc.m112.369561

Cited by 28 publications

(18 citation statements)

References 43 publications

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“…In Vitro Synthesis of Lipid II-Lipid II was synthesized as described with minor modifications (21). In brief 100 nmol of UDP-GlcNAc in 50 mM Bistris propane, pH 8.…”

Section: Methodsmentioning

confidence: 99%

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Henrich

Engels

et al. 2016

Journal of Biological Chemistry

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Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cellfree expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were cotranslationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY⅐nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.Proteins involved in bacterial cell wall biosynthesis are highly conserved and often essential. In stage I, the cytoplasmic proteins MurA-F are responsible for the formation of the soluble peptidoglycan precursor (Park's nucleotide). In stage II, the integral membrane protein MraY catalyzes the ligation of Park's nucleotide with the membrane-bound undecaprenylphosphate (C 55 -P) 4 resulting into lipid I. The membrane-associated globular protein MurG catalyzes subsequent glycosyl transfer forming lipid II (Fig. 1). As the final monomeric building block in bacterial cell wall biosynthesis, lipid II will be transported through the membrane into the periplasmic space for further polymerization in stage III of cell wall biosynthesis (1).The bacterial cell wall biosynthetic pathway is an important and traditional target for antibiotics and numerous drugs directed against key catalysts for clinical applications have been developed. However, most known drugs such as the penicillin derivatives affect the periplasmic or extracellular steps of peptidoglycan synthesis catalyzed by soluble enzymes. In contrast, almost no clinical drugs have been developed that are targeted against the integral membrane proteins involve...

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“…In Vitro Synthesis of Lipid II-Lipid II was synthesized as described with minor modifications (21). In brief 100 nmol of UDP-GlcNAc in 50 mM Bistris propane, pH 8.…”

Section: Methodsmentioning

confidence: 99%

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Henrich

Engels

et al. 2016

Journal of Biological Chemistry

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“…In depth analysis on the mechanism of action revealed that empedopeptin interferes with peptidoglycan biosynthesis by sequestering the cell wall building block lipid II as its primary target in a calcium-dependent fashion (Müller et al, 2012). Calcium ions increase the antibacterial activity of empedopeptin by strengthening the interaction of the net-negative antibiotic with its target and with phospholipids in the cytoplasmic membrane.…”

Section: Empedopeptin Tripropeptins and Plusbacinsmentioning

confidence: 99%

Cyclic lipopeptides as antibacterial agents – Potent antibiotic activity mediated by intriguing mode of actions

Schneider¹,

Müller²,

Miess³

et al. 2014

International Journal of Medical Microbiology

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“…Although it contains a 14‐carbon myristic tail, empedopeptin has a net negative charge and remains water soluble. Empedopeptin may have eventual clinical considerations, as preclinical studies suggest significant in vitro and in vivo activity against antibiotic‐resistant pathogens, low overall toxicity, and a favorable pharmacologic profile 19 . Using membrane preparations and lipid II precursor components, empedopeptin was shown to bind to lipid II with a 2:1 stoichiometry.…”

Section: Basic Structure and Function Of Microbial Cell Wallsmentioning

confidence: 99%

“…Notably, Ca 2+ ions are essential for the interaction between this net negatively charged peptide and lipid II. Empedopeptin does not appear to cause membrane depolarization 19 . Given their structural similarities, it is possible that the tripropeptins and plusbacins may also target lipid II in a similar fashion 19 .…”

Section: Basic Structure and Function Of Microbial Cell Wallsmentioning

confidence: 99%

Peptide antimicrobials: cell wall as a bacterial target

Yount

Yeaman

2013

Annals of the New York Academy of Sciences

127

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Endogenous host defense peptides (HDPs) are among the most ancient immune mediators, constituting a first line of defense against invading pathogens across the evolutionary continuum. Generally, HDPs are small (<10 kDa), cationic, and amphipathic polypeptides, often broadly classified based on structure. In eukaryotes, major HDP classes include disulfide-stabilized (e.g., defensins), and α-helical or extended (e.g., cathelicidins) peptides. Prokaryote HDPs are generally referred to as bacteriocins, colicins, or lantibiotics, many of which undergo extensive posttranslational modifications. One target for prokaryotic and eukaryotic HDPs is the bacterial cell wall, an essential structural feature conserved among broad classes of bacteria. A primary building block of the cell wall is peptidoglycan, a macromolecular complex that arises through a series of reactions including membrane translocation, extracellular anchoring, and side chain cross-linking. Each of these steps represents a potential target for HDP inhibition, leading to bacteriostatic or bactericidal outcomes. Thus, understanding the relationships between HDPs and cell wall targets may shed light on new peptide antimicrobial agents and strategies to meet the daunting challenge of antibiotic resistance.

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Lipodepsipeptide Empedopeptin Inhibits Cell Wall Biosynthesis through Ca2+-dependent Complex Formation with Peptidoglycan Precursors

Cited by 28 publications

References 43 publications

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Cyclic lipopeptides as antibacterial agents – Potent antibiotic activity mediated by intriguing mode of actions

Peptide antimicrobials: cell wall as a bacterial target

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