Deciphering the Antibacterial Role of Peptide From Bacillus subtilis subsp. spizizenii Ba49 Against Staphylococcus aureus

Abstract

An increase in antibiotic resistance has led to escalating the need for the development of alternate therapy. Antimicrobial peptides (AMPs) are at the forefront of replacing conventional antibiotics, showing slower development of drug resistance, antibiofilm activity, and the ability to modulate the host immune response. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that jeopardize most conventional antibiotics are known to be involved in severe respiratory tract, bloodstream, urinary tract, soft tissue, and skin infections. Among them, S. aureus is an insidious microbe and developed resistance against conventional antibiotics. In the present study, an AMP (named as peptide-Ba49) isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa (the common onion) exhibited strong antibacterial efficacy against S. aureus ATCC 25923. The mode of action of this peptide-Ba49 on S. aureus was deciphered through various sensitive probes, i.e., DiSC₃ (5) and H₂DCFDA, suggesting the peptide-Ba49 to be acting upon through change in membrane potential and by triggering the production of reactive oxygen species (ROS). This induced disruption of the cell membrane was further supported by morphological studies using scanning electron microscopy (SEM). Investigations on a possible post-antibiotic effect (PAE) of peptide-Ba49 showed prolonged PAE against S. aureus. Furthermore, the peptide-Ba49 prevented the formation of S. aureus biofilm at low concentration and showed its potential to degrade the mature biofilm of S. aureus. The peptide-Ba49 also exhibited intracellular killing potential against S. aureus ATCC 25923 in the macrophage cells, and moreover, peptide-Ba49 was found to bolster the fibroblast cell migration in the scratch assay at low concentration, exhibiting a wound healing efficacy of this peptide. These studies demonstrated that peptide-Ba49 isolated from the strain B. subtilis subsp. spizizenii could be a therapeutic candidate to combat the pathogenic S. aureus infections.

Keywords: PAE; ROS; Staphylococcus aureus; antimicrobial peptides; biofilm; intracellular activity; scratch assay.

FIGURE 1

Post-antibiotic effect of peptide-Ba49. (A) Post 2 h and (B) post 4 h of exposure of peptide-Ba49. The bacteria showed suppression for approximately 7 and 10 h at post 2 and 4 h exposure to peptide-Ba49 at a concentration of 1 × MIC and 2 × MIC, respectively. The bacterial repair was also reduced at post 4 h exposure to peptide-Ba49 at a concentration of 2 × MIC.

FIGURE 2

Intracellular activity of peptide-Ba49 against S. aureus-infected RAW 264.7 cells: The figure showed the intracellular killing efficacy of peptide-Ba49 against S. aureus ATCC 25923 infected macrophage cells. After 24 h of incubation with the peptide-Ba49, there was a significant reduction in the log₁₀ CFU/ml, as compared to the untreated one. The analysis was carried out in two-way ANOVA with Tukey’s multiple comparisons test. ****p < 0.0001, ***p < 0.0001, **p < 0.01 indicated the significant difference. The values “mean ± SD” were representative of two independent experiments done in triplicate.

FIGURE 3

Spectrometric analysis of ROS level alteration in S. aureus. Following treatment of S. aureus cells with varied concentrations of peptide-Ba49, the cells were stained with ROS marker 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA). The histogram represents fluorescence of H₂DCFDA dye normalized with untreated cells. One-way ANOVA followed by Holms–Sidak multiple comparisons test showed *p < 0.05 significant difference between polymyxin B and the untreated one. Intriguingly, we observed a significant difference at 2 × MIC (with ****p < 0.0001) as compared to untreated.

FIGURE 4

Spectrometric analysis of membrane potential of S. aureus cells. Cell membrane permeability of S. aureus ATCC 25923 was evaluated with the release of voltage-sensitive dye DiSC₃-(5) during peptide-Ba49 treatment. Fluorescence was measured spectroscopically at 620–670 nm excitation and emission wavelengths. S. aureus cells were treated with peptide-Ba49 at concentrations of 4 μM (1 × MIC) and 8 μM (2 × MIC). Significant increase in fluorescence signal was observed in peptide-treated cells as compared to untreated cells. Polymyxin B-treated cells were taken as a positive control. The analysis was carried out in two-way ANOVA with Dunnett’s multiple comparisons test. ****p < 0.0001 indicated the significant difference between control and treatment groups. The values “mean ± SD” were representative of two independent experiments done in triplicate.

FIGURE 5

Scanning electron microscopy of S. aureus cells treated with purified peptide-Ba49. (A,B) Untreated S. aureus cells from the control group at 2.5 and 10 K resolution. (C,D) Purified peptide-Ba49-treated S. aureus cells at 2.5 and 10 K resolution. A white arrow shows ruptured cells after peptide treatment for a duration of 240 min.

FIGURE 6

Effects of peptide-Ba49 on inhibition of S. aureus biofilm formation. (A) S. aureus biofilm biomass was measured using crystal violet assay. (B) Viability was measured by using XTT assay. One-way ANOVA followed by Dunnett’s test for multiple comparisons, N = 2 independent experiments with triplicates, **p < 0.01, *****p < 0.00001, and p > 0.05 were considered as non-significant (ns).

FIGURE 7

Confocal imaging of live/dead stained S. aureus biofilm [left—green channel (live), middle—red channel (dead), and right—merged channel]. 2D and 3D images showing the S. aureus biofilm inhibition effect of peptide-Ba49; untreated S. aureus biofilm (A–D) and Peptide-Ba49-treated biofilm (E–H) were post stained with Syto 9 (live stain, green), CFW (EPS stain, blue), and PI (dead stain, red).

FIGURE 8

Effects of peptide-Ba49 treatment on inhibition of pre-formed biofilm of S. aureus. (A) Pre-formed S. aureus biofilm biomass after peptide-Ba49 treatment was estimated using crystal violet assay. (B) Viability was evaluated using XTT assay. One-way ANOVA followed by Dunnett’s test for multiple comparisons, N = 2 independent experiments with triplicates, ****p < 0.0001.

FIGURE 9

Cell migration assay. The fibroblast cells L929 were treated with peptide-Ba49 and observed under microscope until 36 h to evaluate the cell migration: (A) Untreated L929 cells. (B) Peptide-Ba49-treated L929 cells.

FIGURE 10

Proposed mechanism of action of peptide-Ba49 isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa against S. aureus.