Structural modification of the Pseudomonas aeruginosa alkylquinoline cell-cell communication signal, HHQ, leads to benzofuranoquinolines with anti-virulence behaviour in ESKAPE pathogens

Abstract

Microbial populations have evolved intricate networks of negotiation and communication through which they can coexist in natural and host ecosystems. The nature of these systems can be complex and they are, for the most part, poorly understood at the polymicrobial level. The Pseudomonas Quinolone Signal (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ) are signal molecules produced by the important nosocomial pathogen Pseudomonas aeruginosa . They are known to modulate the behaviour of co-colonizing bacterial and fungal pathogens such as Bacillus atropheaus, Candida albicans and Aspergillus fumigatus. While the structural basis for alkyl-quinolone signalling within P. aeruginosa has been studied extensively, less is known about how structural derivatives of these molecules can influence multicellular behaviour and population-level decision-making in other co-colonizing organisms. In this study, we investigated a suite of small molecules derived initially from the HHQ framework, for anti-virulence activity against ESKAPE pathogens, at the species and strain levels. Somewhat surprisingly, with appropriate substitution, loss of the alkyl chain (present in HHQ and PQS) did not result in a loss of activity, presenting a more easily accessible synthetic framework for investigation. Virulence profiling uncovered significant levels of inter-strain variation among the responses of clinical and environmental isolates to small-molecule challenge. While several lead compounds were identified in this study, further work is needed to appreciate the extent of strain-level tolerance to small-molecule anti-infectives among pathogenic organisms.

Keywords: ESKAPE pathogens; HHQ; Pseudomonas aeruginosa; anti-virulence; antimicrobial resistance; biofilm.

Fig. 1.

Substrate scope of benzofuroquinoline structures used in this study.

Fig. 2.

Activity analysis of benzofuroquinoline compounds (30 µM) against P. aeruginosa PA14. (a) Biofilm assay presented as crystal violet biomass Abs_595nm. (b) Growth curve analysis in the presence of compounds and DMSO carrier control. (c) Swarming motility on Eiken agar plates measured as the swarm distance from the tips of the outermost tendrils characteristic of this strain and normalized to the untreated control. In all cases, data represent the mean (±sem) of at least three independent biological replicates. Statistical significance (one-way ANOVA with Dunnett’s Multiple Comparison test) is presented relative to the DMSO carrier control (*P≤0.05, **P≤0.005, ***P≤0.001).

Fig. 3.

(a) Biofilm assay normalized to untreated control. (b) Pyocyanin extraction normalized to the untreated control. Data presented are the mean (±sem) of at least three independent biological replicates. Significant differences were determined by one-way ANOVA with Dunnett’s multiple comparison test (*P≤0.05, **P≤0.005).

Fig. 4.

Biofilm formation in (a) S. aureus , (b) S. haemolyticus and (c) B. atropheaus strains as measured in 96-well plates with crystal violet staining. All data presented are the mean (±sem) of at least five independent biological replicates. Statistical analysis was performed by one-way ANOVA with Dunnett’s Multiple Comparison test (*P≤0.05, ***P≤0.001). In each panel, Unt refers to the untreated control.

Fig. 5.

Biofilm formation in E. faecalis and ESKAPE pathogens in the presence of lead compounds (30 µM) 4j, 4o and 5. All data presented are the mean (±sem) of at least three independent biological replicates. Statistical analysis was performed by one-way ANOVA with Dunnett’s Multiple Comparison testing (**P≤0.005).

Fig. 6.

Strain-level divergence of compound efficacy against (i) biofilm formation and (ii) growth kinetics of Staphylocccus species, with model and clinical isolates: (a-c) S. aureus , (d–f) S. haemolyticus , (g–i) S. epidermidis , (j–l) S. equorum and (m) S. hominis . All data presented are the mean (±sem) of at least three independent biological replicates performed on 24-well plates. Statistical analysis was performed by one-way ANOVA with Dunnett’s Multiple Comparison test (*P≤0.05, **P≤0.005, ***P≤0.001).

Fig. 7.

Swarming interference by derivative compounds. (a) Swarm distance in B. atropheaus (n=2 independent biological replicates); (b) visual representation of B. atropheaus swarming motility; (c) swarm distance in P. aeruginosa PA14 (n=3 independent biological replicates); (d) visual representation of P. aeruginosa swarming motility. Data are normalized relative to the untreated control (±sem). Significant differences were determined by one-way ANOVA with Dunnett’s Multiple Comparison test. Asterisks represent statistically significant differences relative to the untreated control (*P≤0.05).