؊1 for the wildtype 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. [⌬N20/⌬M21]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.The antimicrobial peptide nisin is produced by numerous strains of Lactococcus lactis and inhibits a broad range of Gram-positive bacteria (1, 2). It belongs to the lantibiotics, a group of antimicrobial peptides that is characterized by the presence of intramolecular rings formed by the thioether amino acids lanthionine and 3-methyllanthionine (3, 4). Nisin has had a long history as a potent and safe food preservative, although recent insight into the molecular mechanism of its bactericidal activity also make it interesting for biomedical applications (5, 6). Generally, the nisin-type subgroup of lantibiotics comprises elongated cationic peptides that have the capacity to adopt amphiphilic structures. Such peptides are assumed to kill microbes by disturbing the integrity of the energy-transducing membrane. Indeed, early experiments demonstrated that nisin or related lantibiotics induced rapid efflux of ions or cytoplasmic solutes such as amino acids and nucleotides. The concomitant depolarization of the cytoplasmic membrane resulted in an instant termination of all biosynthetic processes (7,8). Structural analysis in the presence of micelles indicated that the hydrophilic groups of the peptide interact with the phospholipid headgroups, and the hydrophobic side chains are immersed in the hydrophobic core of the membrane (9, 10). The wedge model as proposed by Driessen et al. (11) takes into account such structural data and proposes that the peptides insert into the membrane without losing contact with the membrane surface, resulting in the formation of a short-lived pore.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. The discrepancies were explained by the finding that nisin and epidermin use lipid II, the bactoprenol-bound precursor of the bacterial cell wall as a docking molecule for subsequent pore formation (12). The specificity of the nisin-lipid II interaction a...

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).

The interaction of nisin Z and a nisin Z mutant carrying a negative charge in the C-terminus ([Glu-32]-nisin Z) with anionic lipids was characterized in model membrane systems, and bacterial membrane systems. We focused on three possible steps in the mode of action of nisin, i.e., binding, insertion, and pore formation of nisin Z. Increasing amounts of anionic lipids in both model and natural membranes were found to strongly enhance the interaction of nisin Z with the membranes at all stages. The results reveal a good correlation between the anionic lipid dependency of the three stages of interaction, of which the increased binding is probably the major determinant for antimicrobial activity. Maximal nisin Z activity could be observed for negatively charged lipid concentrations exceeding 50-60%, both in model membrane systems as well as in bacterial membrane systems. We propose that the amount of negatively charged lipids of the bacterial target membrane is a major determinant for the sensitivity of the organism for nisin. Nisin Z induced leakage of the anionic carboxyfluorescein was more efficient as compared to the leakage of the potassium cation. This lead to the conclusion that an anion-selective pore is formed. In contrast to the results obtained for nisin Z, the binding of [Glu-32]-nisin Z to vesicles remained low even in the presence of high amounts of negatively charged lipids. The insertion and pore-forming ability of [Glu-32]-nisin Z were also decreased. These results demonstrate that the C-terminus of nisin is responsible for the initial interaction of nisin, i.e., binding to the target membrane.

Nisin is a 34 residue long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The antimicrobial activity is based on pore formation in the cytoplasmic membrane of target organisms. The mechanism which leads to pore formation remains to be clarified. We studied the orientation of nisin via site-directed tryptophan fluorescence spectroscopy. Therefore, we engineered three nisin Z variants with unique tryptophan residues at positions 1, 17, and 32, respectively. The activity of the tryptophan mutants against Gram-positive bacteria and in model membrane systems composed of DOPC or DOPG was established to be similar to that of wild type nisin Z. The tryptophan fluorescence emission maximum showed an increasing blue-shift upon interaction with vesicles containing increased amounts of DOPG, with the largest effect for the 1W peptide. Studies with the aqueous quencher acrylamide showed that all tryptophans became inaccessible from the aqueous phase in the presence of negatively charged lipids in the vesicles. From these results it is concluded that anionic lipids mediate insertion of the tryptophan residues in at least three positions of the molecule into the lipid bilayer. The depth of insertion of the tryptophan residues was determined via quenching of the tryptophan fluorescence by spin-labeled lipids. The results showed that the depth of insertion was dependent on the amount of negatively charged lipids. In membranes containing 50% DOPG, the distances from the bilayer center were determined to be 15.7, 15.0, and 18.4 A for the tryptophan at position 1, 17, and 32, respectively. In membranes containing 90% DOPG, these distances were calculated to be 10.8, 11.5, and 13.1 A, respectively. These results suggest an overall parallel average orientation of nisin in the membrane, with respect to the membrane surface, with the N-terminus more deeply inserted than the C-terminus. These data were used to model the orientation of nisin in the membrane.

Introduction 1.1 Discovery and structure of lantibiotics 2 Biosynthesis of lantibiotics 2.1 Organization of the gene clusters 2.2 Modification and transport 2.3 Processing of precursor peptides 2.4 Immunity of producer organisms 2.5 Regulation of biosynthesis 3 Protein engineering of lantibiotics 3.1 Expression systems for modified lantibiotic structural genes 3.2 Site-directed mutants of lantibiotics 4 Mode of action of lantibiotics 4.1 Bacteriocidal activity towards Gram-positive bacteria 4.2 Binding to membranes 4.3 Insertion into membranes 4.4 The orientation of nisin in membranes 4.5 Permeabilisation of membranes 4.6 Model for the mode of action of lantibiotics 5 Applications and prospects 5.1 Pharmaceutical applications 5.2 Food applications 6 Concluding remarks 7 Acknowledgements 8 References

Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.

Three mutants of the lantibiotic nisin Z , in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 'H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus jlavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z , [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3 -5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.