The Lantibiotic Nisin Induces Lipid II Aggregation, Causing Membrane Instability and Vesicle Budding

Abstract

The antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane.

Carboxyl Analogue of Mutacin 1140, a Scaffold for Lead Antibacterial Discovery

“…Class 1 lantibiotics are known to bind to lipid II by forming a cage around the pyrophosphate residue using rings A and B (the lipid II binding domain) (47,48). Furthermore, the latter half of the peptide is believed to help in the lateral assembly of the lantibiotic-lipid II complexes to form islands (17,49). In nisin, these islands form a pore complex, but the epidermin group primarily sequesters lipid II without forming a pore, as has been reported in fluorescently labeled lipid II vesicle experiments (17,50).…”

A Chemical Biology Approach to Understanding Molecular Recognition of Lipid II by Nisin(1–12): Synthesis and NMR Ensemble Analysis of Nisin(1–12) and Analogues

“…[7] As econd effect of this binding is the inhibition of peptidoglycan biosynthesis, caused by the large-scale sequestration and aggregation of lipid II. [8,9] The importance of the interaction with lipid II in the antibacterial action of nisin has also been demonstrated in functional studies, in which it wass hown that nisin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)i sa ble to antagonize the activity of WT nisin. [10] NMR studies have provedt ob eav aluable method in the study of lantibiotic conformation and lipid II binding.…”

“…[46][47][48][49] We have previously reported the solid-phase synthesis of the individual Aa nd Br ings of nisin and of the related lantibiotic, mutacin I, [25] and have investigated the conformationalp roperties of these isolated rings ands ynthetic analogues by NMR. We have now extended this work to prepares ynthetic analogues of WT nisin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), which can be compared with WT nisin(1-12) itself (1)( Figure 2). (Thr2, Ser5) analogue 2 was designed using the amino acids in the 2-and5 -positions that would be present in the biosynthetic precursor peptide, and that would undergo dehydration by the enzyme NisB in the producing organism.…”

“…[34] As parto ft his work the authors also investigated the effect of replacing both Dha and Dhb with Ala, and found that the presence of the dehydro residues increased the affinity of the dicarbab ridged peptides for lipid II. Introduction of at hirdc yclic constraint in dicarbab ridged analogueso fn isin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), by creating al actam bridge between the N-terminus and the Br ing, has also been investigated by Harmsen et al [35] The resulting reduction in flexibility increased the affinity of the peptide for lipid II over the bicyclic dicarba analogue, but was still five-fold less active than WT nisin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”

“…The first aim of this research was to furtherd evelopo ur existing solid-phase peptides ynthesis (SPPS) methodology to enable the synthesis of two analogues of nisin(1-12) bearing dehydro residue replacementsa nd aM eLan!Lans ubstitution. Our second aim was to examine in detail the solution conformations of these peptides using the NAMFIS method and compare them to the conformations of WT nisin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)i no rder to explore the effect of residue mutation on ring conformation and pre-organization for lipid II binding. Our third aim was to carry out ad etailed conformational analysiso fW Tn isin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) itself, in order to elucidatef urtherh ow this peptides equence achievesh ighly selectiveb inding to lipid II, and to compare the conformations to the previously published structures of full length nisin, both alone and bound to lipid II.…”