Cobalt Complex with Thiazole-Based Ligand as New Pseudomonas aeruginosa Quorum Quencher, Biofilm Inhibitor and Virulence Attenuator

Abstract

Pseudomonas aeruginosa is one of the most dreaded human pathogens, because of its intrinsic resistance to a number of commonly used antibiotics and ability to form sessile communities (biofilms). Innovative treatment strategies are required and that can rely on the attenuation of the pathogenicity and virulence traits. The interruption of the mechanisms of intercellular communication in bacteria (quorum sensing) is one of such promising strategies. A cobalt coordination compound (Co(HL)₂) synthesized from (E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)-4-(p-tolyl)thiazole (HL) is reported herein for the first time to inhibit P. aeruginosa 3-oxo-C12-HSL-dependent QS system (LasI/LasR system) and underling phenotypes (biofilm formation and virulence factors). Its interactions with a possible target, the transcriptional activator protein complex LasR-3-oxo-C12-HSL, was studied by molecular modeling with the coordination compound ligand having stronger predicted interactions than those of co-crystallized ligand 3-oxo-C12-HSL, as well as known-binder furvina. Transition metal group 9 coordination compounds may be explored in antipathogenic/antibacterial drug design.

Keywords: antibacterial resistance; antivirulence/antipathogenic compounds; biofilm prevention; cobalt complex; furvina; pyocyanin; pyoverdine; quorum sensing inhibition; transcriptional activator protein LasR.

Scheme 1

Structures of the hydrazonyl-thiazole based compound, (E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)-4-(p-tolyl)thiazole (HL) (a) and its Co(III) complex; Co(HL)₂ (b).

Figure 1

Effect of increasing concentrations of Co(HL)₂ (6.25 to 1000 µg mL⁻¹) on P. aeruginosa 3-oxo-C12-HSL-dependent QS system (bars) in general and on growth inhibition (dashed line) (based on the co-culture of wild-type/biosensor). Bioluminescence emissions were normalized to the cell density of the bacterial culture and expressed as percentages with respect to untreated controls (cells + DMSO at 6%; relative bioluminescence). Mean values ± standard deviations for at least three replicates are illustrated.

Figure 2

Effect of increasing concentrations of Co(HL)₂ (6.25 to 1000 µg mL⁻¹) on the production of 3-oxo-C12-HSL by P. aeruginosa PA14 (wild-type strain) (bars) and quantification of the 3-oxo-C12-HSL produced levels (dashed line). Bioluminescence emissions were normalized to the cell density of the bacterial culture and expressed as percentages with respect to untreated controls (cells + DMSO at 6%; relative bioluminescence). 3-oxo-C12-HSL levels were expressed as percentages with respect to untreated controls (cells + DMSO at 6%; relative 3-oxo-C12-HSL). Mean values ± standard deviations for at least three replicates are illustrated.

Figure 3

Effect of increasing concentrations of Co(HL)₂ (6.25 to 1000 µg mL⁻¹) on the detection of 3-oxo-C12-HSL by P. aeruginosa PA14-R3 (biosensor strain). Bioluminescence emissions were normalized to the cell density of the bacterial culture and expressed as percentages with respect to untreated controls (cells + DMSO at 6%; relative bioluminescence). Mean values ± standard deviations for at least three replicates are illustrated.

Figure 4

Preventive action of Co(HL)₂ at MIC (800 µg mL⁻¹) and sub-inhibitory concentrations (6.25 to 400 µg mL⁻¹) on biomass productivity of P. aeruginosa PA14 (wild-type strain) biofilm. Mean values ± standard deviations for at least three replicates are illustrated.

Figure 5

Influence of Co(HL)₂ on pyocyanin production (bars) and on cell growth (A_600nm) (dashed line) of P. aeruginosa PA14 (wild-type strain) as a function of the different concentrations (6.25 to 1000 µg mL⁻¹). The levels of pyocyanin were measured in cell-free supernatants from cultures of P. aeruginosa PA14 (wild-type strain). The total amount of protein was calculated (μg mL⁻¹), normalized per cell density (A_600nm values) and expressed as relative pyoverdine production. Mean values ± standard deviations for at least three replicates are illustrated.

Figure 6

Influence of Co(HL)₂ on pyoverdine production (bars) and on cell growth (A_600nm) (dashed line) of P. aeruginosa PA14 (wild-type strain) as a function of the different concentrations (6.25 to 1000 µg mL⁻¹). The levels of pyoverdine were measured in cell-free supernatants from cultures of P. aeruginosa PA14 (wild-type strain), normalized per cell density (A_600nm values) and expressed as relative pyoverdine production. Mean values ± standard deviations for at least three replicates are illustrated.

Figure 7

Docked ligands HL in cyan, docked known inhibitor furvina in magenta, docked known binder 3-oxo-C12-HSL in slate (HET-ID OHN), co-crystallized (X-ray) known binder 3-oxo-C12-HSL in yellow, in the binding site of transcriptional activator protein LasR (3IX3) in white, hydrogen bonds as dashes, sulphur in yellow, oxygen in red, nitrogen in blue, and halogen in salmon.