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

TLDR

Nisin achieves nanomolar antibacterial potency by binding the lipid‑bound cell‑wall precursor lipid II, which serves as a docking site for pore formation. The study aimed to determine the structural features of nisin required for its interaction with lipid II by using genetically engineered variants. Engineered variants with mutations in the N‑terminal rings A–C and the flexible hinge region were tested for lipid II binding and pore‑forming activity. Mutations in rings A–C lowered binding affinity and raised the pore‑forming concentration, while hinge‑region mutations abolished pore formation yet preserved some in‑vivo activity through continued lipid II binding, demonstrating that nisin simultaneously blocks peptidoglycan synthesis and forms specific pores, yielding nanomolar MICs.

Abstract

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, <i>e.g.</i>[S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 × 10⁷m⁻¹ compared with 2 × 10⁷m⁻¹ for the wild-type 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, <i>e.g.</i> [ΔN20/ΔM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their <i>in vivo</i> activity. We found the remaining <i>in vivo</i> 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.