Specific Binding of Nisin to the Peptidoglycan Precursor Lipid II Combines Pore Formation and Inhibition of Cell Wall Biosynthesis for Potent Antibiotic Activity

Abstract

؊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...

The action mechanism of daptomycin

“…A precedent for such a dual mechanism of action is provided by the lantibiotic nisin. 87,119 In addition to the mechanism of action, much work remains to be done regarding daptomycin's structure-activity relationships. A larger and more diverse set of sequence variants is needed, which should be examined for antimicrobial activity, but also using biophysical and biochemical methods that illuminate specific steps in the action mode of daptomycin.…”

“…A precedent for a dual action mechanism is provided by the lantibiotic nisin, which binds to lipid II and, like daptomycin, permeabilizes the membrane, but at the same time also disrupts peptidoglycan synthesis. 87 We noted above that daptomycin-permeabilized cells become depleted of ions, substrates, and metabolic energy, which results in a general disruption of macromolecular biosynthesis also (see section 10.6). Therefore, to prove that daptomycin directly and specifically inhibits a given biosynthetic pathway, it is not enough to show this pathway to be obstructed in whole bacterial cells, and we must regard as inadmissible all reported evidence of this kind.…”

A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action

“…2A), indicating that VpDef may also be involved in lipid II binding (yet unidentified). Similar to Nisin Z, which combines pore-forming and lipid II-trapping activities (Wiedemann et al, 2001), VpDef may possess multiple or complementary mechanisms of action to kill bacteria. Further investigation of the ability of VpDef to disrupt membranes and inhibit peptidoglycan biosynthesis is needed to fully elucidate mechanisms of action.…”

Getting Closer to the Real Bacterial Cell Wall Target: Biomolecular Interactions of Water‐Soluble Lipid II with Glycopeptide Antibiotics

“…[48] The inhibitive effect of the glycopeptide antibiotics on the pore formation by nisin was measured by monitoring the nisin-induced leakage of carboxyfluorescein from model membrane vesicles in the presence of increasing glycopeptide antibiotic concentrations. Initially, the glycopeptide antibiotic (0 ± 50 mm) was preincubated (3 min) with CF-loaded vesicles (5 mm based on lipid-P i content).…”