Bacillibactin, a Potential Bacillus-Based Antibacterial Non-Ribosomal Peptide: In Silico Studies for Targeting Common Fish Pathogens

Abstract

Aquaculture is one of the fastest-growing sectors in food production. The widespread use of antibiotics in fish farming has been identified as a driver for the development of antibiotic resistance. One of the promising approaches to solving this problem is the use of probiotics. There are many promising aquaculture probiotics in the Bacillus genus, which produces non-ribosomal peptides (NRPs). NRPs are known as antimicrobial agents, although evidence is gradually accumulating that they may have other effects, especially at lower (subinhibitory) concentrations. The mechanisms of action of many NRPs remain unexplored, and molecular docking and molecular dynamics studies are invaluable tools for studying such mechanisms. The purpose of this study was to investigate the in silico inhibition of crucial bacterial targets by NRPs. Molecular docking analyses were conducted to assess the binding affinities of the NRPs of Bacillus for protein targets. Among the complexes evaluated, bacillibactin with glutamine synthetase, dihydrofolate reductase, and proaerolysin exhibited the lowest docking scores. Consequently, these complexes were selected for further investigation through molecular dynamics simulations. As a result, three additional potential mechanisms of action for bacillibactin were identified through in silico analyses, including the inhibition of glutamine synthetase, dihydrofolate reductase, and proaerolysin, which are critical bacterial enzymes and considered as the potential antibacterial targets. These findings were further supported by in vitro antagonism assays using bacillibactin-producing Bacillus velezensis strains MT55 and MT155, which demonstrated strong inhibitory activity against Pseudomonas aeruginosa and Aeromonas veronii.

Keywords: aquaculture probiotics; bacillibactin; fengycin; molecular docking; molecular dynamics; surfactin.

Figure 1

Binding energies (in Kcal/mol) of selected non-ribosomal peptides against target proteins calculated by molecular docking using Autodock Vina.

Figure 2

Three-dimensional diagram of the location of bacillibactin in the binding site of glutamine synthetase on the left, three-dimensional diagram of glutamine synthetase amino acid interaction with bacillibactin in the middle, and two-dimensional diagram of glutamine synthetase amino acid interaction with bacillibactin on the right.

Figure 3

Three-dimensional diagram of the location of bacillibactin in the binding site of dihydrofolate reductase on the left, three-dimensional diagram of dihydrofolate reductase amino acid interaction with bacillibactin in the middle, and two-dimensional diagram of dihydrofolate reductase amino acid interaction with bacillibactin on the right.

Figure 4

Three-dimensional diagram of the location of bacillibactin in the binding site of proaerolysin on the left, three-dimensional diagram of proaerolysin amino acid interaction with bacillibactin in the middle, and two-dimensional diagram of proaerolysin amino acid interaction with bacillibactin on the right.

Figure 5

Three-dimensional diagrams of fengycin docked in the binding site of (a) glutamine synthetase, (c) dihydrofolate reductase, and (e) proaerolysin. Two-dimensional plots of the interaction of (b) glutamine synthetase, (d) dihydrofolate reductase, and (f) proaerolysin amino acids with fengycin.

Figure 6

Three-dimensional diagrams of the surfactin docked in the binding site of (a) glutamine synthetase, (c) dihydrofolate reductase, and (e) proaerolysin. Two-dimensional plots of the interaction of (b) glutamine synthetase, (d) dihydrofolate reductase, (f) proaerolysin amino acids with surfactin.

Figure 7

(a) RMSD analysis of the molecular dynamics (MD) simulation trajectories generated using Desmond for bacillibactin and glutamine synthetase, (b) analysis of types of contacts between bacillibactin and glutamine synthetase, (c) protein–ligand contact plots for the glutamine synthetase–bacillibactin complex, and (d) protein–ligand contact plots throughout the MD simulation of bacillibactin with glutamine synthetase.

Figure 7

(a) RMSD analysis of the molecular dynamics (MD) simulation trajectories generated using Desmond for bacillibactin and glutamine synthetase, (b) analysis of types of contacts between bacillibactin and glutamine synthetase, (c) protein–ligand contact plots for the glutamine synthetase–bacillibactin complex, and (d) protein–ligand contact plots throughout the MD simulation of bacillibactin with glutamine synthetase.

Figure 8

(a) RMSD analysis of the MD simulation trajectories generated using Desmond for bacillibactin and dihydrofolate reductase, (b) analysis of types of contacts between bacillibactin and dihydrofolate reductase, (c) protein–ligand contact plots for the dihydrofolate reductase–bacillibactin complex, and (d) protein–ligand contact plots throughout the MD simulation of bacillibactin with dihydrofolate reductase.

Figure 8

(a) RMSD analysis of the MD simulation trajectories generated using Desmond for bacillibactin and dihydrofolate reductase, (b) analysis of types of contacts between bacillibactin and dihydrofolate reductase, (c) protein–ligand contact plots for the dihydrofolate reductase–bacillibactin complex, and (d) protein–ligand contact plots throughout the MD simulation of bacillibactin with dihydrofolate reductase.

Figure 8

(a) RMSD analysis of the MD simulation trajectories generated using Desmond for bacillibactin and dihydrofolate reductase, (b) analysis of types of contacts between bacillibactin and dihydrofolate reductase, (c) protein–ligand contact plots for the dihydrofolate reductase–bacillibactin complex, and (d) protein–ligand contact plots throughout the MD simulation of bacillibactin with dihydrofolate reductase.

Figure 9

(a) RMSD analysis of the MD simulation trajectories generated using Desmond for bacillibactin and proaerolysin, (b) analysis of types of contacts between bacillibactin and proaerolysin, (c) protein–ligand contact plots for the proaerolysin–bacillibactin complex, and (d) protein–ligand contact plots throughout the MD simulation of bacillibactin with proaerolysin.

Figure 9

(a) RMSD analysis of the MD simulation trajectories generated using Desmond for bacillibactin and proaerolysin, (b) analysis of types of contacts between bacillibactin and proaerolysin, (c) protein–ligand contact plots for the proaerolysin–bacillibactin complex, and (d) protein–ligand contact plots throughout the MD simulation of bacillibactin with proaerolysin.