Engineering quorum-quenching enzymes

Biofilms are structured communities of bacteria that adhere to surfaces and shield bacteria from hostile surroundings. They are implicated in 75% of all infections in humans and are difficult to treat. New therapies are being developed to combat biofilm infections, such as quorum sensing inhibition (quorum quenching). Quorum sensing is a process where bacteria use chemical signals to communicate with individuals in a group for co-operative processes such as biofilm formation. I investigated how quorum quenching can be carried out using enzymes to degrade quorum-sensing signals, particularly acyl-homoserine lactones (AHLs) used by Gram-negative bacteria. AHL acylases are enzymes known to be involved in quorum quenching. However, they are expressed poorly in native and heterologous hosts and difficult to purify for further study, limiting their use. Therefore, I chose a related acylase as a scaffold to engineer AHL acylase activity using structure-based mutagenesis. As this scaffold did not have prior activity against AHLs, I developed a sensitive high-throughput screen to detect engineered AHL acylase variants. From 637 variants screened, this is the first time a variant of this scaffold was found to be active against AHLs. This engineered AHL acylase is active against 3-oxododecanoyl homoserine lactone (3-oxo-C12-HSL), the quorum-sensing signal of the clinical pathogen Pseudomonas aeruginosa PAO1. When tested against P. aeruginosa biofilms, the acylase reduced the extracellular matrix surrounding the cells, indicating potential for further clinical application. The position mutated in this engineered AHL acylase was found to be critical not only for substrate specificity but also for autocatalytic processing, providing new insight for this family of enzymes