New strain Brevibacillus laterosporus TSA31-5 produces both brevicidine and brevibacillin, exhibiting distinct antibacterial modes of action against Gram-negative and Gram-positive bacteria

Abstract

The growing prevalence of antibiotic resistance has made it imperative to search for new antimicrobial compounds derived from natural products. In the present study, Brevibacillus laterosporus TSA31-5, isolated from red clay soil, was chosen as the subject for conducting additional antibacterial investigations. The fractions exhibiting the highest antibacterial activity (30% acetonitrile eluent from solid phase extraction) were purified through RP-HPLC. Notably, two compounds (A and B) displayed the most potent antibacterial activity against both Escherichia coli and Staphylococcus aureus. ESI-MS/MS spectroscopy and NMR analysis confirmed that compound A corresponds to brevicidine and compound B to brevibacillin. Particularly, brevicidine displayed notable antibacterial activity against Gram-negative bacteria, with a minimum inhibitory concentration (MIC) range of 1-8 μg/mL. On the other hand, brevibacillin exhibited robust antimicrobial effectiveness against both Gram-positive bacterial strains (MIC range of 2-4 μg/mL) and Gram-negative bacteria (MIC range of 4-64 μg/mL). Scanning electron microscopy analysis and fluorescence assays uncovered distinctive morphological alterations in bacterial cell membranes induced by brevicidine and brevibacillin. These observations imply distinct mechanisms of antibacterial activity exhibited by the peptides. Brevicidine exhibited no hemolysis or cytotoxicity up to 512 μg/mL, comparable to the negative control. This suggests its promising therapeutic potential in treating infectious diseases. Conversely, brevibacillin demonstrated elevated cytotoxicity in in vitro assays. Nonetheless, owing to its noteworthy antimicrobial activity against pathogenic bacteria, brevibacillin could still be explored as a promising antimicrobial agent.

Fig 1. Neighbor-joining phylogenetic tree of the TSA31-5 strain.

The tree was generated based on analysis of the 16S rRNA gene sequence using MEGA ver. 11.0.8. The neighbor-joining method was employed for tree construction.

Fig 2. RP-HPLC analysis and antibacterial activity of the 30% acetonitrile eluent obtained from solid phase extraction of TSA31-5 culture media.

(a) RP-HPLC chromatogram. (b) Antibacterial activity of compound A and B in the 30% eluent against E. coli and S. aureus.

Fig 3

Sequence determination of compound A (a) and compound B (b) using ESI-MS/MS analysis. B-type fragment ions are highlighted in gray, and y-type fragment ions are indicated in black. The mass values that are visible in the spectra are enclosed in boxes.

Fig 4

The HN-Hα region of the NOESY and DQF-COSY spectra of compound A (a) and compound B (b). Sequential dαN(i, i + 1) NOE connectivity for the peptides was determined using ¹H 2D NOESY. Intra-residue NH-CαH cross peaks are labeled with the corresponding residue name and number.

Fig 5

Time-dependent killing kinetics of brevicidine and brevibacillin against Escherichia coli (a) and Staphylococcus aureus (b) with 1× MIC peptides.

Fig 6

CD spectra of brevicidine (a) and brevibacillin (b) were recorded in different conditions: 10 mM sodium phosphate buffer (pH 6.8), 30 mM sodium dodecyl sulfate (SDS), 50% (v/v) 2,2,2-trifluoroethanol (TFE), and 0.1% (w/v) lipopolysaccharide of E. coli. The peptide concentration was maintained at 100 μM.

Fig 7. The stability of brevicidine and brevibacillin under different pH and high temperature condition.

Brevicidine (a) and brevibacillin (b) at different pH 2, 7, and 10. Brevicidine (c) and brevibacillin (d) with 80°C incubation for 120 min.

Fig 8. Cytotoxicity of brevicidine and brevibacillin.

(a) Hemolytic activity assessed using red blood cells from sheep. Cell viability of brevicidine (b) and brevibacillin (c) measured using the MTT assay for RAW264.7 cells.

Fig 9. Membrane depolarization of E. coli and S. aureus induced by brevicidine and brevibacillin.

(a) E. coli treated with brevicidine, (b) E. coli treated with brevibacillin, (c) S. aureus treated with brevicidine and (d) S. aureus treated with brevibacillin.

Fig 10. SEM images of E. coli treated with the peptides.

Control (a), brevicidine at 0.5× MIC (b), 1× MIC (c), and 2× MIC (d), as well as brevibacillin at 0.5× MIC (e). SEM images of S. aureus treated with brevibacillin. Control (f), brevibacillin at 0.5× MIC (g), 1× MIC (h), and 2× MIC (i). The left column presents images at 25,000× magnification. The right column features enlarged sections from the left images.