Abstract
Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.
Highlights
Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation
Whereas the wedge model may illustrate results obtained with model membranes, a number of effects observed with intact living cells remain unexplained; in particular, the fact that nisin acts on model membranes at micromolar concentrations whereas in vivo minimal inhibitory concentration (MIC)1 values are in the nanomolar range
In this report we have analyzed the first known case of a pore formation mechanism by an amphiphilic antibiotic peptide that is based on specific interaction with a defined integral component of bacterial cytoplasmic membranes
Summary
Chemicals and Materials—All chemicals were of analytical grade or better. The phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) were purchased from Avanti Polar Lipids, Inc. and stored at Ϫ20 °C in a 9:1 chloroform/methanol solution. Vesicles (160 M, on a phosphorus base) made of DOPC with 4% DOPG or with 0.5 mol % lipid II were incubated at room temperature with 14C-labeled peptide in 0.5 ml of buffer (25 mM Mes, pH 6.0, 50 mM K2SO4) for 5 min. Membranes were collected by centrifugation (40,000 ϫ g), washed and resuspended in buffer (50 mM Tris HCl, pH 7.5), to give a protein concentration of 8 mg/ml. For cell wall biosynthesis assays [35], it was applied in 0.2 mM concentrations together with membranes (160 g of protein), 0.05 mM [14C]UDP-GlcNAc and 15– 60 M nisin peptides in a total volume of 60 l of membrane suspension buffer (50 mM Tris-HCl, pH 8, 10 mM MgCl2, 1 mM mercaptoethanol). Polymeric peptidoglycan does not migrate from the starting point, and the respective area was cut out and counted in a -scintillation counter (1900 CA Tri-Carb scintillation counter, Packard, Zurich)
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