Brevibacillin 2V, a Novel Antimicrobial Lipopeptide With an Exceptionally Low Hemolytic Activity

Abstract

Bacterial non-ribosomally produced peptides (NRPs) form a rich source of antibiotics, including more than 20 of these antibiotics that are used in the clinic, such as penicillin G, colistin, vancomycin, and chloramphenicol. Here we report the identification, purification, and characterization of a novel NRP, i.e., brevibacillin 2V (lipo-tridecapeptide), from Brevibacillus laterosporus DSM 25. Brevibacillin 2V has a strong antimicrobial activity against Gram-positive bacterial pathogens (minimum inhibitory concentration = 2 mg/L), including difficult-to-treat antibiotic-resistant Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Notably, brevibacillin 2V has a much lower hemolytic activity (HC₅₀ > 128 mg/L) and cytotoxicity (CC₅₀ = 45.49 ± 0.24 mg/L) to eukaryotic cells than previously reported NRPs of the lipo-tridecapeptide family, including other brevibacillins, which makes it a promising candidate for antibiotic development. In addition, our results demonstrate that brevibacillins display a synergistic action with established antibiotics against Gram-negative bacterial pathogens. Probably due to the presence of non-canonical amino acids and D-amino acids, brevibacillin 2V showed good stability in human plasma. Thus, we identified and characterized a novel and promising antimicrobial candidate (brevibacillin 2V) with low hemolytic activity and cytotoxicity, which can be used either on its own or as a template for further total synthesis and modification.

Keywords: Brevibacillus; Enterococcus; NRPS; Staphylococcus; antimicrobial activity; brevibacillin; lipopeptide.

FIGURE 1

The structures of brevibacillins and the predicted biosynthetic gene cluster. (A) The non-ribosomal peptide synthetase 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; KR, ketoreductase; T, thiolation; C, condensation; E, epimerization; TD, terminal reductase; TE, thioesterase. (C) Structures of brevibacillins. The red rectangles indicate the more hydrophobic amino acid residues of brevibacillin V, brevibacillin, and brevibacillin I relative to brevibacillin 2V.

FIGURE 2

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) spectra and the proposed structures of brevibacillin 2V (A) and brevibacillin I (B). Fragment ions are indicated. LC–MS/MS analysis was performed using a Q-Exactive mass spectrometer fitted with an Ultimate 3000 UPLC, an ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 μm particle size, 200 Å; Waters), a HESI ion source, and a Orbitrap detector. MS/MS was performed in a separate run in parallel reaction monitoring mode, selecting the doubly and triply charged ion of the peptides. The structures of brevibacillin 2V and brevibacillin I were elucidated by using known lipo-tridecapeptides as reference templates.

FIGURE 3

Brevibacillins activities against eukaryotic cells. (A) Human erythrocytes were incubated with brevibacillins at concentrations ranging from 1 to 128 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. ^#HC₅₀ is the concentration that causes 50% hemolysis of human red blood cells. N.D., not determined. ^a,b,cSignificant differences exist when not the same letters are indicated in the HC₅₀ column (p < 0.05). *No hemolytic activity was observed for brevibacillin 2V at 128 mg/L and its HC₅₀> 128 mg/L. (B) Cytotoxicity of brevibacillins against HepG2 cells. HepG2 cells were treated with brevibacillins at concentrations ranging from 2 to 64 mg/L. Cells treated without a tested compound were used as untreated control. After 24 h of treatment, cell viability was determined by an XTT kit. The data are representative of three independent experiments. ^a,b,c,dSignificant differences exist when not the same letters are indicated in the CC₅₀ column (p < 0.05).

FIGURE 4

Antimicrobial activity of brevibacillin 2V brevibacillin against Staphylococcus aureus (MRSA) after exposure to human plasma. The starting concentration was 200 mg/L for both brevibacillin 2V and brevibacillin. Samples were collected at 0, 1, 2, 4, and 8 h, and 6 μl of each sample was loaded to a square plate containing S. aureus (MRSA). Bre 2V, brevibacillin 2V; Bre, brevibacillin.

FIGURE 5

Pipeline for the development of novel non-ribosomally produced peptide (NRP)-based antimicrobials. Following the arrows, the process starts with the identification of NRP clusters. Next, the predicted NRPs have to be produced, isolated, and characterized. Finally, the antimicrobial activity of the purified NRPs will be determined.