Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus

Abstract

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Figure 1

Hemolytic activity of S. aureus strains isolated from cows with mastitis on sheep blood agar plates. (A) Percentage of strains showing alpha, beta, and non-hemolytic activity. (B) Number of beta-hemolytic strains categorized as showing low (< 10 mm), medium (between 10 and 13 mm), and high hemolytic (> 13 mm) activity based on the diameter of hemolysis zones.

Figure 2

Effect of Bacillus spp. supernatants on S. aureus hemolysis (A) and growth (B). The optical density (OD) was normalized in comparison with the controls (set to 1—dotted line). The floating bars show the minimum, maximum, and mean OD of all the replicates of the three S. aureus strains analyzed (S. aureus 4051, S. aureus 4347, and S. aureus 4628) represented by gray dots. The line inside the bars represents the mean normalized OD and the asterisks indicate a significant difference in the treatments compared to the control at a 95% confidence level.

Figure 3

Effect of cell-free crude extracts containing lipopeptides from Bacillus spp. on the hemolytic activity of S. aureus 4051 (A), S. aureus 4347 (B), S. aureus 4628 (C), and S. aureus O11 (D). Each strain of Bacillus is color-coded as indicated in the figure legend. Error bars show the standard error of the mean. S. aureus cultures without treatment (data set to 100%) were used as controls for data normalization. Models contained fixed effect of lipopeptide extract, concentration, their interaction, and random effect of run. Main effects of concentration and lipopeptide type (Bacillus strains) were assessed using two-way ANOVA.

Figure 4

Chromatogram of lipopeptides produced by (A) B. velezensis 87 and (B) B. velezensis TR47II analyzed in this study. Numbers 1, 2, and 3 show the two major peaks of iturins, fengycins, and surfactins, respectively. The peptides were analyzed in a C12 chromatography column and eluted using a gradient of acetonitrile + TFA 0.1%.

Figure 5

Hemolytic activity of S. aureus O11 supernatants in the presence of purified lipopeptides from B. velezensis strains. The panel shows the activity of iturins (IT—A) and bacillomycins (BL—D), fengycins (FG—B and E), and surfactins (SF—C and F), produced by B. velezensis 87 (A–C) and B. velezensis TR47II (D–F). The lipopeptides were purified using RP-HPLC and their masses were confirmed using MALDI-TOF MS. The hemolytic activity of the samples was normalized by setting the hemolytic activity of non-treated S. aureus O11 supernatant to 100% (dotted lines). Models contained fixed effect of lipopeptide extract, concentration, their interaction, and random effect of run. Main effects of concentration and lipopeptide type (Bacillus strains) were assessed using two-way ANOVA.

Figure 6

Best molecular models of the interaction between hemolysin and iturins categorized by AutoDock Vina. Top panels (1A–5A) show the complete heptameric structure of hemolysin interacting with iturins; Bottom panels (1B–5B) show interactions between the compounds with hydrogen bonds highlighted in light blue. Panels 1, 2, 3, 4, and 5 represent, respectively, the following iturin variants: iturin A, iturin A1, iturin A2, iturin A4, iturin A C-15. The docking analyses were performed using AutoDock Vina v.1.1.2 and images were generated using UCSF Chimera.

Figure 7

Molecular docking model proposed for hemolysin and iturin A1. Panel (A) shows the entire structure while Panel (B) highlights the interaction between hemolysin and Iturin A1 through hydrogen bonds is highlighted in light blue. The docking analyses were performed using AutoDock Vina v.1.1.2 and images were generated using UCSF Chimera.

Figure 8

Molecular dynamics results of the complexes composed by the heptameric structure of hemolysin and the iturin variants. In (A) is shown the RMSD of the α-carbons present in the protein, and in (B) is shown the RMSD of iturins after fitting to the same selection. Panel (C) represents the RMSF of protein residues fit to the average structure. Panel (D) demonstrates the variation from the mean RMSF of each protein residue in relation to the average structure. Panel (E) shows the mass-weighted radius of gyration for the complexes. The complexes were named LG1, LG2, LG3, LG4, LG5 and LG6, corresponding to PubChem CIDs: 9988651, 11062109, 101589794 (poses 1 and 2), 101589795 and 102287549, respectively.