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

Abstract

The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain-linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membranepermeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipidlinked disaccharide in the activity of nisin. We show here that Lipid II is not only the receptor for nisin but an intrinsic component of the pore formed by nisin, and we present a new model for the pore complex that includes Lipid II.The cell wall is an essential structure of a bacterium, providing its shape and protecting it from bursting because of the high osmotic pressures of the cytoplasm. This wall is a threedimensional network built of identical subunits consisting of two amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmu-is attached to the carboxyl group of MurNAc. These subunits are assembled in the cytosol of bacteria using UDP-activated precursors on a special lipid carrier, undecaprenyl phosphate (for a review see Ref. 1). The integral membrane protein MraY and the peripherally membraneassociated MurG that synthesize the precursors Lipid I and II, respectively, are the key enzymes in the last two cytoplasmic steps in the formation of the subunits (Fig. 1). Subsequently, Lipid II is transported across the plasma membrane via an as of yet unknown mechanism. Thereafter, the subunits are polymerized and inserted into the pre-existing cell wall by means of the penicillin-binding proteins (for review see Ref.2). Numerous antibiotics target the cell wall synthesis, including a diverse group of antibiotics that bind to Lipid II. Perhaps the best known of these antibiotics is vancomycin, the antibiotic of last resort to treat MRSA infections (3). However, there are many others, including the polypeptide nisin, that kill cells by permeabilizing bacterial membranes. Efficient membrane permeabilization by nisin requires an interaction with Lipid II (4, 5). This designates nisin as the first example of a targeted poreforming peptide antibiotic.Two recent studies have shed light on the structural requirements within nisin for the interaction with Lipid II. A genetic approach indicated that the N terminus of nisin is involved in the interaction with Lipid II (6), and more recently we could map the binding interface toward specific residues in the N terminus using 15 N-labeled nisin (7). It is not clear yet what events lead to pore formation after the...

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

“…Several groups, including ours, have used bioaffinity ESI-MS methodology previously to evaluate the [28] allows, for the first time, a more comprehensive characterization of the molecular interactions that underlie glycopeptide antibiotic efficacy. A primary assumption for the determination of association constants by mass spectrometry is that the ionization probability of the free antibiotic is equivalent to that of the antibiotic ± ligand complexes.…”

“…Nisin Z was produced by batch fermentation and purified as previously described. [43] Full-length and water-soluble lipid II was prepared by the procedure outlined in the work of Breukink et al [28] Carboxyfluorescein was purchased from Kodak and purified as described. [44] Vesicles were prepared as described in literature.…”

“…Synthesis and purification of water-soluble lipid II (LII) containing a polyprenyl chain of 3 isoprene units: Water-soluble lipid II was created according to a procedure based on the addition of farnesyl phosphate to a membrane preparation of Micrococcus flavus in the presence of suitable amounts of substrates and will be described in detail elsewhere. [28] Farnesyllipid II, water-soluble lipid II containing 3 isoprene units, was stored in methanol at À 20 8C at a concentration of 0.35 mm.…”

A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential Lipid II docking interface

“…Mersacidin forms a tightly associated 1:1 complex with Lipid II, and nisin sequesters Lipid II via encapsulating the pyrophosphate of Lipid II within a hydrogen bond network formed by nisin backbone amides (36,37). The nisin-Lipid II complex organizes into a supramolecular pore-like assembly, engendering a breach in membrane integrity and leading to cell death by osmotic lysis (38)(39)(40). Common to both mersacidin and nisin are structural changes within the antibiotic that are triggered by the encounter with the membrane environment.…”

Sustainable inhibition efficacy of liposome-encapsulated nisin on insoluble glucan-biofilm synthesis byStreptococcus mutans

“…In this in vitro study, we found that the use of naked nisin and nisin-liposome resulted in the suppression of insoluble glucan synthesis by the typical cariogenic streptococcus S. mutans 10449. The inhibition of insoluble glucan synthesis by nisin is probably a result of its inhibitory effect on streptococcal viability (Breukink et al, 2003). Nisin, a wide-spectrum bacteriocin, is used in the food industry as a preservative against harmful bacteria, its mode of antimicrobial action being interaction with target bacterial cell membranes and formation of membrane pores (Kuwano et al, 2005).…”