Pseudomonas aeruginosa is a ubiquitous bacterium that can cause severe and chronic lung infections in patients with cystic fibrosis, particularly in establishing a resistant structured form referred to as biofilm, which is very difficult to eliminate. Biofilm formation is mainly regulated by the communication system of the Quorum Sensing (QS), which is controlled by the natural N-acyl homoserine lactones (HSL) molecules: C4-HSL and 3-oxo-C12-HSL. P. aeruginosa is an example of a human opportunistic pathogen for which HSL-related compound have been described as potent inhibitors of biofilm formation and virulence factors, given their similarity to the natural QS autoinducers.
Thus, the first aim of our project is to design potent analogs of natural HSL and to screen them for their antibiofilm activity in order to inhibit the QS mechanisms, both the formation and/or development of the biofilm of P. aeruginosa and the secretion of virulence factors associated. The second objective is to determine the best combination of effective molecules (associated or not with antibiotics) and then evaluate its activity during a chronic lung infection in a murine model.
Among various original analogs of the C4-HSL synthesized and then screened for their ability to impair biofilm formation in an innovative in vitro model that allow to analyze the impact of compounds on different stages of biofilm formation, three compounds (includind C11, N-pymimidyl butanamide) showed a significant inhibition of biofilm formation when added from the early stages of biofilm formation, in a dose-dependent manner, coupled with an absence of cytotoxicity on lung cells.
To determine the best combination of effective molecules against the QS mechanisms and the biofilm formation, the active analogs will be in a first step analyzed in combination with antibiotics in the objective to view a potent synergy activity. The combination of compounds will then be studied on the screening model placed under anaerobic conditions to approximate the in vivo colonization conditions of the bacterium, and its lack of cytotoxicity on lung cells will be controlled.
Meanwhile, the effect of active analogs on the expression of virulence genes of P. aeruginosa will be tested to confirm the impact of the compounds on all of the QS mechanisms and thus the pathogenicity of the bacterium.
Finally, the experimental murine model of chronic lung infection will be developed to obtain a biofilm with a similar structure to that observed in patients with cystic fibrosis, and then to assess the in vivo activity of the pre-selected best combination of molecules.
Thus, this project should allow us to identify one or more active compounds (analogs ± antibiotics) with optimal activity on the different steps of the biofilm formation, and with effects observed in vitro and validated in mice, and of course an absence of toxicity.
The research of original synthetic molecules capable of interfering with biofilm development and expression of virulence factors associated represents an interesting alternative to the massive use of antibiotics and one promising new approaches for the prevention and/or treatment of infections associated with cystic fibrosis.
This project is part of a process whose purpose is a real therapeutic application with establishment of new therapeutic protocols, and therefore experiments should then be continued in order to define the optimal conditions for further investigations in humans.