Peptide Antibiotic Nisin Research Articles

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

Activity and Export of Engineered Nisin-(1-22) Analogs

The pentacyclic peptide antibiotic nisin, produced by Lactococcus lactis is ubiquitously applied as a food preservative. We previously demonstrated that the truncated nisin-(1-22) has only 10-fold lower activity than nisin. Here we aimed at further developing this tricyclic nisin analog to reach activity comparable to that of nisin. Our data demonstrate that: (1) ring A has a large mutational freedom; (2) the composition of residues 20–22 strongly affects production levels of nisin-(1-22); (3) a positively charged C-terminus of nisin-(1-22) significantly enhances its antimicrobial activity; (4) nisin-(1-22) inhibits in vitro growth of a target strain using different dynamics than nisin.

Oxidation of lanthionines renders the lantibiotic nisin inactive.

The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the beta-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.

Ring-Closing Alkyne Metathesis Approach toward the Synthesis of Alkyne Mimics of Thioether A-, B-, C-, and DE-Ring Systems of the Lantibiotic Nisin Z

[reaction: see text] Ring-closing alkyne metathesis toward the synthesis of the alkyne-brigded A-, B-, C-, and (D)E-ring mimics of the peptide antibiotic nisin Z is described. We have successfully synthesized alkyne-bridged cyclic peptides containing 4-7 amino acid residues in yields ranging from 18 to 82%.

Mammalian defensins: structures and mechanism of antibiotic activity

Antibiotic peptides are important effector molecules in host-parasite interactions throughout the living world. In vertebrates, they function in first-line host defense by antagonizing a wide range of microbes including bacteria, fungi, and enveloped viruses. The antibiotic activity is thought to be based on their cationic, amphipathic nature, which enables the peptides to impair vital membrane functions. Molecular details for such activities have been elaborated with model membranes; however, there is increasing evidence that these models may not reflect the complex processes involved in the killing of microbes. For example, the overall killing activity of the bacterial peptide antibiotic nisin is composed of independent activities such as the formation of target-mediated pores, inhibition of cell-wall biosynthesis, formation of nontargeted pores, and induction of autolysis. We studied the molecular modes of action of human defense peptides and tried to determine whether they impair membrane functions primarily and whether additional antibiotic activities may be found. We compared killing kinetics, solute efflux kinetics, membrane-depolarization assays, and macromolecular biosynthesis assays and used several strains of Gram-positive cocci as test strains. We found that membrane depolarization contributes to rapid killing of a significant fraction of target cells within a bacterial culture. However, substantial subpopulations appear to survive the primary effects on the membrane. Depending on individual strains and species and peptide concentrations, such subpopulations may resume growth or be killed through additional activities of the peptides. Such activities can include the activation of cell-wall lytic enzymes, which appears of particular importance for killing of staphylococcal strains.

Assembly and stability of nisin-lipid II pores.

The peptide antibiotic nisin was the first reported example of an antibiotic that kills bacteria via targeted pore formation. The specific target of nisin is Lipid II, an essential intermediate in the bacterial cell-wall synthesis. High-affinity binding of the antibiotic to Lipid II is followed by rapid permeabilization of the membrane. Here, we investigated the assembly and stability of nisin-Lipid II pore complexes by means of pyrene fluorescence and circular dichroism. We demonstrated that nisin uses all available Lipid II molecules in the membrane to form pore complexes. The pore complexes have a uniform structure and consist of 8 nisin and 4 Lipid II molecules. Moreover, the pores displayed a remarkable stability, because they were able to resist the solubilization of the membrane environment by mild detergents. Similar experiments with [N20P/M21P]nisin showed that the hinge region is essential for the assembly into stable pore complexes. The new insights were used to propose a refined model for nisin pore formation.

Lipid II-mediated pore formation by the peptide antibiotic nisin: a black lipid membrane study.

The antibiotic peptide nisin is the first known lantibiotic that uses a docking molecule within the bacterial cytoplasmic membrane for pore formation. Through specific interaction with the cell wall precursor lipid II, nisin forms defined pores which are stable for seconds and have pore diameters of 2 to 2.5 nm.

Structure-activity relationships in the peptide antibiotic nisin: role of dehydroalanine 5

A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized. It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis. These observations, which parallel those of W. Liu and J. N. Hansen (Appl. Environ. Microbiol. 59:648-651, 1993) on the corresponding mutant of the related antibiotic subtilin, are discussed in terms of the mechanism(s) of action of these antibiotics.

Structure-activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin

The post-translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth-inhibition activity. Removal of the C-terminal five residues leads to approximately a 10-fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100-fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin 1−12 is inactive itself, and specifically antagonises the growth-inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.

Synthetic Study on Peptide Antibiotic Nisin. V. Total Synthesis of Nisin

Abstract Total synthesis of a lanthionine peptide nisin was achieved by the successive condensations of four segments including cyclic lanthionine peptide parts and the C-terminal linear segment including dehydroalanine, followed by the deprotection procedure with anhydrous HF. Modified reaction conditions for the Hofmann degradation were applied to prepare the dehydroalanine residue from the 2,3-diaminopropionic acid residue. The final deprotection with anhydrous HF proceeded without decomposition of the dehydroamino acid residues which are generally labile in acidic media. The synthetic nisin was completely identical with the natural one in all respects, resulting in a confirmation of the proposed structure.

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5'-TTTTTG-3'. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.

Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes.

The interaction of the peptide antibiotic nisin with liposomes has been studied. The effect of this interaction was analyzed on the membrane potential (inside negative) and the pH gradient (inside alkaline) in liposomes made from Escherichia coli phosphatidylethanolamine and egg phosphatidylcholine (9:1, wt/wt). The membrane potential and pH gradient were generated by artificial ion gradients or by the oxidation of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and cytochrome c by the beef heart cytochrome c oxidase incorporated in the liposomal membranes. Nisin dissipated the membrane potential and the pH gradient in both types of liposomes and inhibited oxygen consumption by cytochrome c oxidase in proteoliposomes. The dissipation of the proton motive force in proteoliposomes was only to a minor extent due to a decrease of the oxidase activity by nisin. The results in these model systems show that a membrane potential and/or a pH gradient across the membrane enhances the activity of nisin. Nisin incorporates into the membrane and makes the membrane permeable for ions. As a result, both the membrane potential and pH gradient are dissipated. The activity of nisin was found to be influenced by the phospholipid composition of the liposomal membrane.

Synthetic Approach to the B-Ring of the Peptide Antibiotic Nisin

Synthetic Approach to the B-Ring of the Peptide Antibiotic Nisin

Synthetic Study on Peptide Antibiotic Nisin. IV. Synthesis of Ring D–E

Abstract In the series of synthetic study on peptide antibiotic nisin, a bicyclic sulfide part ring D–E in this molecule was successfully synthesized. Conjunctive sulfide rings were constructed either by one step or stepwise desulfurization reaction from corresponding disulfides by use of hexaethylphosphorus triamide. Thus, all of the ring moieties in nisin were prepared for total synthesis of this antibiotic.

Synthetic Study on Peptide Antibiotic Nisin. III. Synthesis of Ring C

Abstract A cyclic sulfide moiety corresponding to ring C in peptide antibiotic nisin was successfully synthesized from a disulfide compound prepared from a heptapeptide involving l-cysteine and threo-3-methyl-d-cysteine through desulfurization reaction with hexaethylphosphorus triamide.

ChemInform Abstract: Confirmation of the Structure of Nisin by Complete 1H NMR Resonance Assignment in Aqueous and Dimethyl Sulfoxide Solution.

AbstractThe chemical structure of the peptide antibiotic nisin is confirmed by the unambiguous sequence‐specific assignment of its 1H NMR spectrum using a range of 2D‐NMR techniques.

Analysis of the Genetic Determinant for Production of the Peptide Antibiotic Nisin

The structural gene for the precursor of the peptide antibiotic nisin was isolated and characterized. As with other lanthionine-containing antibiotics, nisin is synthesized as a pre-propeptide which undergoes post-translational modification to generate the mature antibiotic. The sequence data obtained agreed with those of precursor nisin genes isolated by other workers from different Lactococcus lactis strains. Analysis of regions flanking the precursor nisin gene revealed the presence of a downstream open reading frame that may be involved in maturation of the precursor molecule. Nucleotide sequences characteristic of an IS element were located upstream of the nisin determinant. This element, termed IS904, is present in multiple copies in the genome of L. lactis. The nisin determinant of L. lactis is a component of a large transmissible gene block that also encodes nisin resistance and sucrose-metabolizing genes. Gene probe experiments indicated that the nisin/sucrose gene block was located in the chromosome. Furthermore, the copy of IS904 identified adjacent to the precursor nisin gene lies at, or very close to, one end of this transmissible DNA segment and may play a role in mediating its transfer between strains.

Isolation and characterisation of two degradation products derived from the peptide antibiotic nisin

Two degradation products of nisin have been isolated and their structures have been determined by 1H NMR. Nisin 1–32 [(des-ΔAla33-Lys34; Val32-NH 2)nisin] and (des-ΔAla5)nisin 1–32 [(des-ΔAla5, ΔAla33-Lys34; Ile4-NH 2, pyruvyl-Leu6, Val32-NH 2)nisin] are formed on storage or by acid treatment. Contrary to previous reports, nisin 1–32 showed potent antimicrobial activity against Gram-positive organisms comparable to that of nisin itself. (des-ΔAla5)Nisin 1–32, however, was biologically inactive, thus demonstrating the importance of ΔAla5 and/or ring A for biological activity.

Confirmation of the structure of nisin by complete 1H n.m.r. resonance assignment in aqueous and dimethyl sulphoxide solution

The chemical structure of the peptide antibiotic nisin has been confirmed by the unambiguous sequence-specific assignment of its 1H n.m.r. spectrum using a range of 2-D n.m.r. techniques. Nisin was examined in both aqueous (sodium phosphate buffer, pH 2.25) and dimethyl sulphoxide–trifluoroacetic acid solution. Amino acid spin systems were identified from observation of relayed connectivity patterns in the HOHAHA spectrum. Sequential assignments were accomplished with the aid of NOESY and relayed-NOESY spectra. In addition, the locations of the five thioether ring systems were confirmed. The detailed n.m.r. analysis provides strong evidence for the structure originally proposed on the basis of chemical degradation studies.

Total synthesis of peptide antibiotic nisin

Total synthesis of a lanthionine peptide nisin was successfully achieved for the first time by application of new methods for preparations of dehydroalanine and lanthionine moieties, resulting in a confirmation of the proposed structure.