Cilostazol is a promising anti-pseudomonal virulence drug by disruption of quorum sensing

Abstract

Resistance to antibiotics is a critical growing public health problem that desires urgent action to combat. To avoid the stress on bacterial growth that evokes the resistance development, anti-virulence agents can be an attractive strategy as they do not target bacterial growth. Quorum sensing (QS) systems play main roles in controlling the production of diverse virulence factors and biofilm formation in bacteria. Thus, interfering with QS systems could result in mitigation of the bacterial virulence. Cilostazol is an antiplatelet and a vasodilator FDA approved drug. This study aimed to evaluate the anti-virulence activities of cilostazol in the light of its possible interference with QS systems in Pseudomonas aeruginosa. Additionally, the study examines cilostazol's impact on the bacterium's ability to induce infection in vivo, using sub-inhibitory concentrations to minimize the risk of resistance development. In this context, the biofilm formation, the production of virulence factors and influence on the in vivo ability to induce infection were assessed in the presence of cilostazol at sub-inhibitory concentration. Furthermore, the outcome of combination with antibiotics was evaluated. Cilostazol interfered with biofilm formation in P. aeruginosa. Moreover, swarming motility, biofilm formation and production of virulence factors were significantly diminished. Histopathological investigation revealed that liver, spleen and kidney tissues damage was abolished in mice injected with cilostazol-treated bacteria. Cilostazol exhibited a synergistic outcome when used in combination with antibiotics. At the molecular level, cilostazol downregulated the QS genes and showed considerable affinity to QS receptors. In conclusion, Cilostazol could be used as adjunct therapy with antibiotics for treating Pseudomonal infections. This research highlights cilostazol's potential to combat bacterial infections by targeting virulence mechanisms, reducing the risk of antibiotic resistance, and enhancing treatment efficacy against P. aeruginosa. These findings open avenues for repurposing existing drugs, offering new, safer, and more effective infection control strategies.

Keywords: Pseudomonas aeruginosa; Healthcare; Anti-virulence; Antimicrobial resistance; Cilostazol; Quorum sensing.

Fig. 1

Cilostazol diminished the PAO1 motility and ability to form biofilms. (A) Growth of PAO1 with and without sub-inhibitory concentrations of cilostazol. No statistically significant differences were found in turbidity of overnight cultures of PAO1 in the presence or absence of cilostazol (ns: non-significant p> 0.05). (B) Cilostazol at sub-MIC significantly inhibited biofilm formed by PAO1 (p < 0.01). (C) Cilostazol at sub-MIC significantly reduced swarming zones formed in swarming agar plates (p < 0.001)

Fig. 2

Cilostazol significantly reduced the production of virulence factors. (A) Anti-proteolytic activity of cilostazol. Cilostazol significantly reduced clearance zones around the wells in skim milk agar plates (p <0.05). (B) Cilostazol reduced the pyocyanin production to a significant extent (*p < 0.05; *** p< 0.001)

Fig. 3

Augmentation of oxidative stress exerted by hydrogen peroxide. Cilostazol significantly potentiated hydrogen peroxide as seen by the increase in the diameter of inhibition zone (p < 0.001)

Fig. 4

Downregulation of QS genes by cilostazol. Cilostazol significantly reduced the expression of QS genes (p < 0.001)

Fig. 5

Cilostazol alleviates pathogenesis of PAO1 in liver tissues. PAO1 caused infiltration of inflammatory cells, severe congestion of hepatic blood vessel and diffuse vacuolation of hepatocytes (A–C). With cilostazol, normal parenchyma and cellular details are maintained with only mild cellular infiltration (D–F).

Fig. 6

Cilostazol alleviates pathogenesis of PAO1 in renal tissues. PAO1 caused renal congestion, renal tubules degeneration, cystic dilation of renal tubules and atrophy of some glomeruli (A–D). The renal tissues exhibited normal renal cortex with normal renal tubules and glomeruli and normal parenchyma with both white and red pulps (E and F)

Fig. 7

Effect of cilostazol on histology of spleen tissues. PAO1 caused congestion of splenic blood, hemorrhage and depletion of lymphocytes from white pulp (A–C). Normal spleen parenchyma with mild depletion of lymphocytes from white pulps when injected with cilostazol treated PAO1 (D and F)

Fig. 8

The putative binding mode (2D&3D) of Cilostazol (upper panel) versus the internal ligand (lower panel) against the crystal structure of P. aeruginosa LasRquorum-sensing receptor, (PDB: 6MVN)

Fig. 9

The putative binding mode (2D&3D) of Cilostazol (upper panel) versus the native autoinducer C4-HSL ligand (lower panel) against the crystal structure of P. aeruginosa PROSS optimized variant of RhlR (75 mutations), (PDB: 7R3H)

Fig. 10

The putative binding mode (2D&3D) of Cilostazol (upper panel) versus the native HHQ ligand (lower panel) against the crystal structure of P. aeruginosa PqsR (MvfR) ligand-binding domain (PDB: 6Q7U)