The C-Terminal Region of Nisin Is Responsible for the Initial Interaction of Nisin with the Target Membrane

Abstract

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.

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

“…CF release started at about 1 nM of the lantibiotic, whereas more than 1 M nisin was necessary to affect DOPC liposomes without lipid II. In accordance with previous studies in the absence of lipid II (24), membranes containing the negatively charged lipids DOPG were considerably more sensitive (Fig. 1B) than the DOPC liposomes.…”

“…2; 90% K ϩ release after 1 min with 100 nM nisin). The absence of ion selectivity of nisin pores formed in lipid II-doped membranes stands in contrast to data obtained without lipid II (24) and suggests that lipid II not only enhances the pore formation activity of nisin but also has an influence on the pore architecture.…”

“…Preparation of Vesicles-Large unilamellar vesicles for the binding, CF efflux, and potassium efflux experiments were prepared by the extrusion technique (23) and treated as described previously (24). Vesicles were made of DOPC with or without 0.065 mol % lipid II (referring to the total amount of lipids).…”

“…Radioactive Labeling-Radioactive labeling of nisin and binding experiments were performed as described previously by Breukink (24). ]nisin Z and variants was determined under equilibrium conditions.…”

Lipid II Is an Intrinsic Component of the Pore Induced by Nisin in Bacterial Membranes

“…For control purposes, nisin was first preincubated with DOPC vesicles lacking Lipid II. In this case, a maximum amount of leakage (about 60%) is obtained as expected, considering the low affinity of nisin for DOPC membranes (22,23). Hardly any activity of nisin was detected if nisin was preincubated with Lipid II variants with prenyl chains of 7 isoprene units or longer, indicating that pores formed by nisin with these variants are stable.…”