Aggregates of nisin with various bactoprenol-containing cell wall precursors differ in size and membrane permeation capacity |
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Authors: | Katharina Scherer Imke Wiedemann Corina Ciobanasu Hans-Georg Sahl Ulrich Kubitscheck |
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Affiliation: | 1. Institute for Physical and Theoretical Chemistry, Wegeler Str. 12, Rheinische Friedrich-Wilhelms-University Bonn, 53115 Bonn, Germany;2. Institute for Medical Microbiology, Immunology and Parasitology, Pharmaceutical Microbiology Unit, Meckenheimer Allee 168, Rheinische Friedrich-Wilhelms-University Bonn, 53115 Bonn, Germany |
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Abstract: | Many lantibiotics use the membrane bound cell wall precursor Lipid II as a specific target for killing Gram-positive bacteria. Binding of Lipid II usually impedes cell wall biosynthesis, however, some elongated lantibiotics such as nisin, use Lipid II also as a docking molecule for pore formation in bacterial membranes. Although the unique nisin pore formation can be analyzed in Lipid II-doped vesicles, mechanistic details remain elusive. We used optical sectioning microscopy to directly visualize the interaction of fluorescently labeled nisin with membranes of giant unilamellar vesicles containing Lipid II and its various bactoprenol precursors. We quantitatively analyzed the binding and permeation capacity of nisin when applied at nanomolar concentrations. Specific interactions with Lipid I, Lipid II and bactoprenol-diphosphate (C55-PP), but not bactoprenol-phosphate (C55-P), resulted in the formation of large molecular aggregates. For Lipid II, we demonstrated the presence of both nisin and Lipid II in these aggregates. Membrane permeation induced by nisin was observed in the presence of Lipid I and Lipid II, but not in the presence of C55-PP. Notably, the size of the C55-PP–nisin aggregates was significantly smaller than that of the aggregates formed with Lipid I and Lipid II. We conclude that the membrane permeation capacity of nisin is determined by the size of the bactoprenol-containing aggregates in the membrane. Notably, transmitted light images indicated that the formation of large aggregates led to a pinch-off of small vesicles, a mechanism, which probably limits the growth of aggregates and induces membrane leakage. |
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Keywords: | AF647, Alexa Fluor 647 N-hydroxysuccinimide hydrazide Atto488-NHS, Atto 488 N-hydroxysuccinimide CF, 5(6)-carboxyfluorescein CLSM, confocal laser scanning microscopy C55-PP, undecaprenyl-diphosphate (bactoprenol-diphosphate) C55-P, undecaprenyl-phosphate (bactoprenol-phosphate) DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1&prime -rac-glycerol) (sodium salt) EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide GlcNAc, N-acetylglucosamine GUV, giant unilamellar vesicle LI, Lipid I LII, Lipid II LSM, laser scanning microscope LUV, large unilamellar vesicle LY, Lucifer yellow MES, 2-(N-morpholino)ethanesulfonic acid MIC, minimal inhibitory concentration MurNAc, N-acetylmuramic acid nisin&ndash AF647, nisin coupled to Alexa Fluor 647 SUV, small unilamellar vesicle TFA, trifluoroacetic acid UDP, uridine diphosphate |
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