Insights into in vivo activities of lantibiotics from gallidermin and epidermin mode-of-action studies

Abstract

The activity of lanthionine-containing peptide antibiotics (lantibiotics) is based on different killing mechanisms which may be combined in one molecule. The prototype lantibiotic nisin inhibits peptidoglycan synthesis and forms pores through specific interaction with the cell wall precursor lipid II. Gallidermin and epidermin possess the same putative lipid II binding motif as nisin; however, both peptides are considerably shorter (22 amino acids, compared to 34 in nisin). We demonstrate that in model membranes, lipid II-mediated pore formation by gallidermin depends on membrane thickness. With intact cells, pore formation was less pronounced than for nisin and occurred only in some strains. In Lactococcus lactis subsp. cremoris HP, gallidermin was not able to release K+, and a mutant peptide, [A12L]gallidermin, in which the ability to form pores was disrupted, was as potent as wild-type gallidermin, indicating that pore formation does not contribute to killing. In contrast, nisin rapidly formed pores in the L. lactis strain; however, it was approximately 10-fold less effective in killing. The superior activity of gallidermin in a cell wall biosynthesis assay may help to explain this high potency. Generally, it appears that the multiple activities of lantibiotics combine differently for individual target strains.

FIG. 1.

Primary structures of gallidermin, gallidermin mutant peptides, and epidermin (A) and the related lantibiotic nisin (B). Modifications of gallidermin mutants used in this study are indicated by arrows. Epidermin differs from gallidermin at position 6 (Ile). Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, β-methyllanthionine.

FIG. 2.

Activities of gallidermin (triangles) and nisin (squares) on unilamellar liposomes made of phospholipids with decreasing chain length. Filled symbols, liposomes with 0.1 mol% lipid II; open symbols, liposomes without lipid II. Liposomes were stabilized with 50% cholesterol and used at a final concentration of 12.5 μM phospholipid on a phosphorous basis. Carboxyfluorescein release from (A) DPoPC (C_16:1), (B) DMPC (C_14:0), and (C) DLPC (C_12:0) was determined 2.5 min after addition of peptide. The 100% leakage level was determined by addition of Triton X-100.

FIG. 3.

Potassium release from Micrococcus flavus DSM 1790 (A), Staphylococcus simulans 22 (B), and Lactococcus lactis HP (C) induced by gallidermin (triangles) and nisin (squares). Peptides were added at 30 seconds, and the potassium release was monitored with a potassium electrode. Potassium leakage is expressed relative to the total amount of potassium released after addition of 1 μM nisin (100% value). Peptides were applied at 500 nM (black symbols), 50 nM (gray symbols), or 5 nM (white symbols). Lines without symbols are baselines.

FIG. 4.

Potassium release from Micrococcus flavus cells induced by gallidermin (triangles) epidermin (circles), and [A12L]gallidermin (squares). Peptides were applied at 500 nM (black symbols) or 50 nM (gray symbols); the line without symbols is the baseline.

FIG. 5.

Inhibition of in vitro lipid II synthesis by gallidermin, epidermin, gallidermin mutant peptides, and nisin. Gallidermin and nisin were tested at molar ratios of 0.5:1 (white bars), 1:1 (gray bars), and 1.5:1 (black bars) with regard to the primary substrate C₅₅-P (10 nmol). Epidermin and gallidermin variants were tested only at a 1:1 (lantibiotic/substrate) molar ratio. The amount of lipid II synthesized by M. flavus membranes in an assay without addition of lantibiotics was taken as 100%. Inhibition of lipid II formation results from complexation of the peptides with the intermediate substrate lipid I. Mean values and standard deviations from three independent experiments are shown.

FIG. 6.

Impact of gallidermin (triangles) and nisin (squares) on the kinetics of in vitro lipid II synthesis. Peptides were added at 10 nmol to the synthesis assay in 150 μl containing 10 nmol C₅₅-P. Lipid II synthesized after 15 min of incubation in the absence of the peptides (circles) was taken as 100%. Mean values and standard deviations from three independent experiments are shown.

FIG. 7.

Inhibition of in vitro lipid II-Gly1 synthesis by gallidermin, epidermin, nisin, and teicoplanin (lanes and bars 2 to 5, respectively). (A) TLC of reaction mixtures of purified lipid II incubated with [U-¹⁴C]glycine in the presence of recombinant tRNA synthetase and purified tRNA with FemX in the absence (lane-1) or in the presence (lanes 2 to 5) of the peptides. (B) Ratio of [U-¹⁴C]glycine incorporated per lipid II as determined from the TLC presented in panel A. The results of one representative experiment are shown.