The Anti-Virulence Activities of the Antihypertensive Drug Propranolol in Light of Its Anti-Quorum Sensing Effects against Pseudomonas aeruginosa and Serratia marcescens

Abstract

The development of bacterial resistance is an increasing global concern that requires discovering new antibacterial agents and strategies. Bacterial quorum sensing (QS) systems play important roles in controlling bacterial virulence, and their targeting could lead to diminishing bacterial pathogenesis. In this context, targeting QS systems without significant influence on bacterial growth is assumed as a promising strategy to overcome resistance development. This study aimed at evaluating the anti-QS and anti-virulence activities of the β-adrenoreceptor antagonist propranolol at sub-minimal inhibitory concentrations (sub-MIC) against two Gram-negative bacterial models Pseudomonas aeruginosa and Serratia marcescens. The effect of propranolol on the expression of QS-encoding genes was evaluated. Additionally, the affinity of propranolol to QS receptors was virtually attested. The influence of propranolol at sub-MIC on biofilm formation, motility, and production of virulent factors was conducted. The outcomes of the propranolol combination with different antibiotics were assessed. Finally, the in vivo protection assay in mice was performed to assess propranolol's effect on lessening the bacterial pathogenesis. The current findings emphasized the significant ability of propranolol at sub-MIC to reduce the formation of biofilms, motility, and production of virulence factors. In addition, propranolol at sub-MIC decreased the capacity of tested bacteria to induce pathogenesis in mice. Furthermore, propranolol significantly downregulated the QS-encoding genes and showed significant affinity to QS receptors. Finally, propranolol at sub-MIC synergistically decreased the MICs of different antibiotics against tested bacteria. In conclusion, propranolol might serve as a plausible adjuvant therapy with antibiotics for the treatment of serious bacterial infections after further pharmacological and pharmaceutical studies.

Keywords: Pseudomonas aeruginosa; Serratia marcescens; bacterial virulence; drug repurposing; propranolol; quorum sensing.

Figure 1

2D- and 3D-structure of the tested β-adrenergic receptor blocker, propranolol.

Figure 2

Propranolol at sub-MIC has no significant effect on the growth of (A) P. aeruginosa or (B) S. marcescens. There were no significant differences between the viable counts in the presence or absence of propranolol, non-significant (ns): p > 0.05.

Figure 3

Sub-MIC of propranolol downregulates the expression of QS genes. RT-qPCR revealed the significant downregulation of the QS-encoding genes in P. aeruginosa. The expressions of the genes were normalized to the housekeeping gene ropD. ***: p value < 0.001.

Figure 4

Predicted binding mode of propranolol and docking controls at the P. aeruginosa QscR virulence-modulating target. (A) 3D cartoon architecture of the dimeric QscR transcription factor (protomers in cyan and orange colors) showing both its DNA binding domain and α-helix/β-sheet/α-helix sandwiched ligand binding domain. Co-crystalline autoinducer, O-C12-HSL (yellow spheres), and propranolol (magenta sticks) are shown. Letters N and C in bold correlate to the amino and carboxy terminals of the protein. (B) Zoomed image of propranolol binding pose showing surface representation of the binding site and surrounding residues within 4 Å radius as lines (cyan). Polar interactions, represented as hydrogen bonds, are illustrated as black dashed-lines (C) Overlay of both docked propranolol (green) and potent quorum sensing inhibitor chlorolactone (yellow) at the QscR binding site. (D) Overlay of co-crystalline O-C12-HSL (yellow) and its redocked pose (orange) depicting the same reported orientation/conformation and conserved polar contacts with residues.

Figure 5

Predicted binding mode of propranolol and docking controls at the P. aeruginosa LasR virulence-modulating target. (A) 3D cartoon architecture of the dimeric LasR transcription factor (protomers in cyan and orange colors) showing only the α-helix/β-sheet/α-helix sandwiched ligand binding domain. Co-crystalline autoinducer, 3-oxo-C10-HSL (yellow spheres) and propranolol (magenta sticks) are shown. Letters N and C in bold correlate to the amino and carboxy terminals of the protein. (B) Zoomed image of propranolol binding pose showing surface representation of the binding site and surrounding residues within 4 Å radius as lines (cyan). Polar interactions, represented as hydrogen bonds, are illustrated as black dashed lines (C) Overlay of both docked propranolol (green) and potent quorum sensing inhibitor, Q9 (yellow), at the LasR binding site. (D) Overlay of co-crystalline 3-oxo-C10-HSL (yellow) and its redocked pose (orange) depicting the same reported orientation/conformation and conserved polar contacts with residues.

Figure 6

Predicted binding mode of propranolol and docking controls at the P. aeruginosa LasI virulence-modulating target. (A) 3D cartoon architecture of the monomeric LasI synthase protein (cyan) showing only the ligand binding domain with its V-shaped cleft and elongated tunnel. Docked propranolol (magenta sticks) is shown. Letters N and C in bold correlate to the amino and carboxy terminals of the protein. (B) Zoomed image of propranolol binding pose showing surface representation of the binding site and surrounding residues within 4 Å radius as lines (cyan). Polar interactions, represented as hydrogen bonds, are illustrated as black dashed lines (C) Overlay of both docked propranolol (green) and potent LasI inhibitor TZD-C8 (yellow) at the LasI binding site. (D) Overlay of docked TZD-C8 (yellow) and its redocked pose (orange) depicting the same reported orientation/conformation and conserved polar contacts with residues.

Figure 7

Predicted binding mode of propranolol and docking controls at the P. aeruginosa PqsR virulence-modulating target. (A) 3D cartoon architecture of the monomeric PqsR synthase protein (cyan) showing only the co-inducer binding domain with its sub-pockets (A,B) and interconnecting anti-parallel β-sheet linker. Docked propranolol (magenta sticks) is shown. Letters N and C in bold correlate to the amino and carboxy terminals of the protein. (B) Zoomed image of propranolol binding pose showing surface representation of the binding site and surrounding residues within 4 Å radius as lines (cyan). Polar interactions, represented as hydrogen bonds, are illustrated as black dashed lines (C) Overlay of both docked propranolol (green) and potent PqsR inhibitor NV5 (yellow) at the co-inducer binding site. (D) Overlay of co-crystallized NV5 (yellow) and its redocked pose (orange) depicting the same reported orientation/conformation and conserved contacts with residues.

Figure 8

Propranolol at sub-MIC decreased the production of virulence factors in P. aeruginosa and S. marcescens. Propranolol significantly decreased the (A) biofilm formation, (B) swarming motility, (C) protease production, (D) hemolysins production, and (E) P. aeruginosa pyocyanin pigment and S. marcescens prodigiosin pigment. The results are presented as percent change from control untreated bacteria. ***: p < 0.001.

Figure 9

Propranolol at sub-MIC showed synergistic effects when combined with different antibiotics against P. aeruginosa or S. marcescens.

Figure 10

Propranolol at sub-MIC protected mice against P. aeruginosa or S. marcescens. Propranolol significantly reduced the P. aeruginosa or S. marcescens capacities to induce pathogenesis (Logrank test for trend p = 0.0023 or 0.0407, respectively). *: p < 0.05; **: p < 0.01.