BrevicidineB, a New Member of the Brevicidine Family, Displays an Extended Target Specificity

Abstract

The group of bacterial non-ribosomally produced peptides (NRPs) has formed a rich source for drug development. Brevicidine, a bacterial non-ribosomally produced cyclic lipo-dodecapeptide, displays selective antimicrobial activity against Gram-negative pathogens. Here, we show that brevicidineB, which contains a single substitution (Tyr2 to Phe2) in the amino acid sequence of the linear part of brevicidine, has a broadened antimicrobial spectrum, showing bactericidal activity against both Gram-negative (with a MIC value of 2 to 4 mg/L) and Gram-positive (with a MIC value of 2 to 8 mg/L) pathogens. Compared with an earlier reported member of the brevicidine family, the broadened antimicrobial spectrum of brevicidineB is caused by its increased membrane disruptive capacity on Gram-positive pathogens, which was evidenced by fluorescence microscopy assays. In addition, DiSC3(5) and resazurin assays show that brevicidine and brevicidineB exert their antimicrobial activity against Gram-negative bacteria via disrupting the proton motive force of cells. Notably, as a brevicidine family member, brevicidineB also showed neither hemolytic activity nor cytotoxicity at a high concentration of 64 mg/L. This study provides a promising antibiotic candidate (brevicidineB) with a broad antimicrobial spectrum, and provides novel insights into the antimicrobial mode of action of brevicidines.

Keywords: Brevicidine; NRPs; antimicrobial activity; cyclic peptide; lipopeptide.

FIGURE 1

The structures of brevicidines and the predicted biosynthetic gene cluster. (A) The non-ribosomal peptide synthetases genes harbored by the Brevibacillus laterosporus DSM 25 genome. (B) the catalytic domains encoded by the gene cluster, and the substrates incorporated by the respective modules. Domains: A, adenylation; T, thiolation; C, condensation; E, epimerization; TE, thioesterase. (C) structures of brevicidines. The red color indicates the different structure of brevicidines.

FIGURE 2

BrevicidineB acts as a bactericidal antibiotic against both Gram-negative and Gram-positive pathogenic bacteria. (A) time killing curve of brevicidines (10 × MIC) against Escherichia coli. Each experiment was performed in triplicate. (B) time killing curve of brevicidines (10 × MIC) against Staphylococcus aureus (MRSA). Each experiment was performed in triplicate.

FIGURE 3

BrevicidineB acts as a potent antibiotic against Gram-positive bacteria by disruption of the cellular membrane. (A) Fluorescence microscopy image of Staphylococcus aureus cells after being exposed to nisin (6 mg/L; 1 × MIC), brevicidine (8 mg/L, 4 mg/L, or 2 mg/L), and brevicidineB [8 mg/L (2 × MIC), 4 mg/L (1 × MIC) or 2 mg/L (0.5 × MIC)] for 15 min. (B) Fluorescence microscopy image of E. coli cells after being exposed to polymyxin B (2 mg/L; 1 × MIC), brevicidine [4 mg/L (2 × MIC), 2 mg/L (1 × MIC) or 1 mg/L (0.5 × MIC)], and brevicidineB [4 mg/L (2 × MIC), 2 mg/L (1 × MIC), or 1 mg/L (0.5 × MIC)] for 15 min. Green denotes a cell with an intact membrane, whereas red denotes a cell with a compromised membrane.

FIGURE 4

Brevicidines disrupt the proton motive force of Gram-negative bacteria. (A) DiSC₃(5) fluorescence in E. coli upon exposure to polymyxin B [4 mg/L (2 × MIC), 2 mg/L (1 × MIC) or 1 mg/L (0.5 × MIC)], Bre [4 mg/L (2 × MIC), 2 mg/L (1 × MIC) or 1 mg/L (0.5 × MIC)], and BreB [4 mg/L (2 × MIC), 2 mg/L (1 × MIC) or 1 mg/L (0.5 × MIC)]. Representative examples from three technical replicates are shown. (B) Resorufin fluorescence in E. coli upon exposure to polymyxin B [4 mg/L (2 × MIC), 2 mg/L (1 × MIC) or 1 mg/L (0.5 × MIC)], Bre [4 mg/L (2 × MIC), 2 mg/L (1 × MIC) or 1 mg/L (0.5 × MIC)], and BreB [4 mg/L (2 × MIC), 2 mg/L (1 × MIC) or 1 mg/L (0.5 × MIC)]. Representative examples from three technical replicates are shown.

FIGURE 5

Brevicidines show exceptional low cytotoxicity and hemolytic activity. (A) Human erythrocytes were incubated with brevicidines at concentrations ranging from 2 to 64 mg/L. Their hemolytic activity was assessed by the release of hemoglobin. Cells treated without a tested compound were used as no-lysis control. Cells treated with 10% Triton X-100 were used as complete lysis control. The data are representative of three independent experiments. Correlation analyses were evaluated by Pearson r2, ns: p > 0.05, *p < 0.05, and ***p < 0.001 vs. 10% Triton X-100-treated cells. (B) Cytotoxicity of brevibacillins against HepG2 cells. The data are representative of three independent experiments. Correlation analyses were evaluated by Pearson r2, ns: p > 0.05, *p < 0.05, and ***p < 0.001 vs. Untreated cells.