Lantibiotic Immunity: Inhibition of Nisin Mediated Pore Formation by NisI

Abstract

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC50 of 73±10 nM, an 8–10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.

The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin

“…Surprisingly, only three of 28 residues affected by nisin binding are located in the C-terminal 22 amino acids. This is in contrast to previous proposals that these C-terminal residues are very important for nisin binding and specificity (26,27) (see below). To confirm that the residues of the C-terminal domain, which show chemical shift changes upon nisin addition are functionally important for nisin binding we constructed a double mutant including two of these residues located in ␤-strand ␤4a in the center of the putative binding site.…”

“…Stein et al (24) also showed that NisI does not bind the structurally closely related subtilin. In vivo studies with truncated NisI variants (26,27) implied the C terminus of NisI and in particular the last 22 amino acids in nisin binding. To further elucidate the NisI/nisin interaction we titrated NisI with purified nisin in 15 N-HSQC experiments.…”

“…A deletion of 21 amino acids and even further truncated constructs resulted in a remaining immunity of only 14%. Later Al-Khatib et al (27) showed that a deletion of the last 22 amino acids at the C terminus of NisI resulted in a decrease of the IC 50 value for nisin by two-thirds. Our structural studies on NisI reveal that the 22 C-terminal amino acids are part of the structured core of the C-terminal domain of NisI.…”

“…A nisin binding affinity in the low micromolar range was determined for lipid-free NisI by surface plasmon resonance (25). Furthermore, experiments with C-terminal truncation variants of NisI suggested that the C-terminal 22 amino acids of NisI are involved in nisin immunity (26,27). For a better understanding of the specificity of NisI and SpaI and the lantibiotic immunity mechanism in general a detailed structural analysis of NisI is required.…”

Development and Characterization of Nisin Nanoparticles as Potential Alternative for the Recurrent Vaginal Candidiasis Treatment

“…This peptide is a bacterioc in produced by Gram-positive microorganisms which has bactericidal and bacteriostatic properties, as well as antifungal activity (7). Its effect consists in membrane pore formation of microorganisms (8). Previous reports describe the effectiveness of the NIS against C. albicans strains.…”