Poly (ε-lysine) dendrons as effective modulators of virulence and biofilm formation in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a significant contributor to multiple-drug resistant nosocomial and respiratory tract infections. In a vigorous search for alternative therapeutic approaches, innovative strategies that aim to modulate functions essential for infection have come to prominence. Natural and synthetic molecules capable of modulating virulence have been described. However, little is known regarding hyperbranched peptide-based macromolecules. The overall aim of this work was to determine the anti-virulence potential of poly (e-lysine) dendrons against P. aeruginosa; their suitability as novel modulators of biofilm formation and virulence factor production, and their efficiency in an in vitro co-culture infection model. Using N-Fmoc solid-phase peptide synthesis, dendrons (consisting of an arginine root and branching points of lysine) were assembled onto a solid support to attain an asymmetrical hyperbranched structure. Dendrons with up to three branching generations (G) were successfully synthesised, as confirmed by high-performance liquid chromatography, mass spectrometry and Fourier transform infrared spectroscopy. The cationic charge of poly (e- lysine) dendrons, exhibited as 16 terminal amine functionalities at G3, was predicted to enable dendron-bacteria interactions. In order to identify changes in cellular functions post-exposure to G3 dendrons, the metabolic activity of P. aeruginosa strain PAO1 was investigated. G3 dendrons induced a reduction in total RNA and protein synthesis, which caused a temporary (6h) growth arrest but did not affect the endpoint cell density. The anti-biofilm potential of G3 dendrons was assessed against PAO1 grown on three surfaces. G3 dendrons demonstrated a dosedependent effect against phenotypes associated with biofilm formation. As such, inhibition of swarming/swimming motility, reduction in surface coverage and microcolony formation, and attenuation of extracellular polymeric substance (EPS) matrix production reduced the bacterium’s biofilm forming capacity. The lack of EPS, which serves as a physical barrier when present, increased susceptibility to ciprofloxacin. Fluorescent labelling of G3 dendrons revealed their intracellular localisation and cell-penetrating capacity. Within 2h of incubation, G3 dendrons presented high uptake efficiency in PAO1 biofilms, and their accumulation at the inner poles of the cells was demonstrated using confocal microscopy. Free dendrons were rapidly released from the biofilm within 2-3 h. Using transcriptional and translational lacZ reporters, the regulation of virulence factors controlled by the las, rhl, and pqs networks of quorum sensing was investigated. G3 dendrons reduced the expression of two autoinducer synthases, lasI and rhlI, independent of changes to growth kinetics. Subsequently, the expression of genes that encode for elastase (lasB) and rhamnolipid (rhlA) production, and PQS (pqsA) signalling was downregulated. Complementation studies, using a lasI rhlI deficient mutant and exogenous autoinducers, demonstrated the ability of G3 dendrons to prevent biofilm formation, lascontrolled LasA protease and rhl-/pqs-controlled pyocyanin production. Using an optimised in vitro co-culture model of human bronchial epithelial (16HBE 14o-) cells and PAO1, the efficiency of G3 dendrons was evaluated. At a concentration non-toxic to 16HBE 14o- cells (0.8 mg/mL), G3 dendrons exhibited bacterial density-dependent protective properties. Specifically, a reduction in P. aeruginosa-induced cytotoxicity impaired the adherence and invasion of PAO1, and delayed the negative impact of PAO1 on the airway epithelial barrier function and integrity. In conclusion, this research highlights the anti-virulence effects of hyperbranched peptidebased macromolecules and constitutes a novel potential therapeutic. This may provide a non-antibiotic alternative for control and prevention of P. aeruginosa infections, and an effective approach to challenge the increasing resistance of bacteria to antibiotics.