Abstract
Many clinically relevant bacteria use a population-dependent communication network - known as quorum sensing - to regulate the processes essential for the establishment of infections. Quorum sensing enables pathogens to restrict the expression of specific genes until a population capable of overwhelming the host defences is reached. Due to its importance in bacterial virulence and biofilm formation, the disruption of this system (quorum quenching) is a promising strategy to treat bacterial infections.
Quorum sensing involves the synthesis, secretion, and detection of chemical signals, termed autoinducers. Disrupting any one of these steps can inhibit quorum sensing. This project investigated two different approaches for quorum quenching: the enzymatic degradation of autoinducers, and the inhibition of autoinducer synthesis. The model system used to investigate the effectiveness of these approaches was Pseudomonas aeruginosa PAO1. P. aeruginosa is an ubiquitous environmental bacterium and opportunistic pathogen.
The first approach investigated the degradation of the two N-acyl-homoserine lactone (AHL) autoinducers produced by P. aeruginosa; N-butyryl-L-homoserine lactone (C4-HSL) and N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL). The P. aeruginosa AHL acylase HacB was selected as a scaffold for mutagenesis to introduce acylase activity towards C4-HSL and generate a broad specificity acylase. Although mutagenesis of HacB successfully introduced C4-HSL activity, the formation of P. aeruginosa PAO1 biofilms was not disrupted. Therefore, an alternative quorum quenching method that focused on inhibiting AHL production was investigated.
During the synthesis of AHLs, the by-product 5'-methylthioadenosine (MTA) is also generated. MTA is rapidly recycled to prevent product inhibition. To determine if this pathway was a viable target for drug development, the effects of deleting it in P. aeruginosa PAO1 were characterised. Deletion of the MTA recycling pathway reduced C4-HSL accumulation, pyocyanin production, and swarming motility, however 3-oxo-C12-HSL accumulation and biofilm formation were not affected.
The work presented here shows that targeting P. aeruginosa quorum sensing is a viable approach to reducing virulence, and provides the foundation for further development of effective quorum quenching methods.