Lipid II-based antimicrobial activity of the lantibiotic plantaricin C

Abstract

We analyzed the mode of action of the lantibiotic plantaricin C (PlnC), produced by Lactobacillus plantarum LL441. Compared to the well-characterized type A lantibiotic nisin and type B lantibiotic mersacidin, which are both able to interact with the cell wall precursor lipid II, PlnC displays structural features of both prototypes. In this regard, we found that lipid II plays a key role in the antimicrobial activity of PlnC besides that of pore formation. The pore forming activity of PlnC in whole cells was prevented by shielding lipid II on the cell surface. However, in contrast to nisin, PlnC was not able to permeabilize Lactococcus lactis cells or to form pores in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes supplemented with 0.1 mol% purified lipid II. This emphasized the different requirements of these lantibiotics for pore formation. Using cell wall synthesis assays, we identified PlnC as a potent inhibitor of (i) lipid II synthesis and (ii) the FemX reaction, i.e., the addition of the first Gly to the pentapeptide side chain of lipid II. As revealed by thin-layer chromatography, both reactions were clearly blocked by the formation of a PlnC-lipid I and/or PlnC-lipid II complex. On the basis of the in vivo and in vitro activities of PlnC shown in this study and the structural lipid II binding motifs described for other lantibiotics, the specific interaction of PlnC with lipid II is discussed.

FIG. 1.

Primary structures of PlnC, mersacidin, and nisin. The putative lipid II binding motifs of these peptides are shaded. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, β-methyllanthionine.

FIG. 2.

Impact of PlnC and nisin on the integrity of the cytoplasmic membrane of Micrococcus flavus (A) and Lactococcus lactis subsp. lactis HP (B). Peptides were added after 30 s, and potassium release was monitored with a potassium-sensitive electrode. PlnC was added at different concentrations: 0.1 μM, white triangles; 0.5 μM, gray triangles; 1 μM, black triangles; 5 μM, diamonds; no peptide addition, asterisks. Cells were preincubated with A12L gallidermin (1 μM) prior addition of PlnC (1 μM) (circles). Potassium leakage is expressed relative to the total amount of potassium (100% value) released after addition of 1 μM nisin (squares).

FIG. 3.

Activity of PlnC (triangles) and nisin (squares) against unilamellar liposomes made of DOPC supplemented with 0.1 mol% lipid II. Peptide-induced marker release from liposomes (25 μM phospholipid on a phosphorous basis) with entrapped CF was determined 2.5 min after peptide addition. The 100% leakage level was determined by addition of Triton X-100.

FIG. 4.

Inhibition of in vitro lipid II synthesis by PlnC. Lipid II synthesized in the absence of peptides was taken as the 100% level (black column). PlnC at concentrations of 5 nmol (dark gray) and 10 nmol (light gray) and nisin at 10 nmol (white) were added to the synthesis assay mixture containing 10 nmol C₅₅-P. Mean values from three independent experiments are shown.

FIG. 5.

Inhibition of in vitro lipid II-Gly₁ synthesis by PlnC, mersacidin, and nisin. (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 (control) or in the presence of nisin, mersacidin, and PlnC. Samples were applied at the origin. The arrow indicates the position of lipid II-Gly₁. (B) [U-¹⁴C]glycine/lipid ratios of the analyzed glycine lipids. Results from one representative experiment are shown.