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Abstract
Pseudomonas aeruginosa is a significant pathogen of immunocompromised individuals, and the main mechanism by which it mediates virulence is through the coordination of gene expression by an intricate quorum sensing system. One of the signalling molecules of this system, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to have immunomodulatory capabilities, discrete to its quorum sensing role.
While the general focus of research in this area is on the physiological outcomes of this interaction on cell function, there is currently little concrete evidence identifying the receptor(s) for OdDHL in mammalian cells, and hence the biochemical mechanism behind the immunomodulation caused by OdDHL remains largely unknown. This study identifies peroxisome proliferator-activated receptor γ (PPARγ) as a mammalian target for OdDHL. PPARγ is a mammalian transcription factor involved in fatty acid metabolism and is heavily involved in the inflammatory response, being a negative regulator of inflammation.
It is shown here that OdDHL is able to instigate signalling through PPARγ by activation of the ligand binding domain (LBD), suggesting that OdDHL may act as a PPARγ agonist. OdDHL is able to compete with the PPARγ agonist, rosiglitazone, causing a relative antagonism of PPARγ activity when given in tandem with the agonist. The bacterial signalling molecule is unable to displace the irreversible PPARγ antagonist GW9662. This effect on PPARγ is specific to OdDHL, as the smaller P. aeruginosa signalling molecule, N-butyryl-L-homoserine lactone had no significant effect on PPARγ activation.
In order to confirm PPARγ as a putative receptor for OdDHL in mammalian cells, initial experiments were undertaken to optimise conditions to produce PPARγ LBD protein for binding interaction studies. The fidelity of the protein sequence was established and expression of the protein in an appropriate vector was confirmed. The protein produced was insoluble and hence not functional for binding studies, suggesting that additional optimisation of expression conditions, or manipulation and refolding of the protein is necessary before further experimentation can take place.
The identification of PPARγ as a receptor for OdDHL in mammalian cells is an important step in understanding the nature and scope of the interaction between OdDHL and host cell physiology, especially the significance of this interaction during P. aeruginosa infection. Continuation of this research, in particular completion of protein-ligand binding studies between OdDHL and PPARγ has the potential to clarify the significance of the immunomodulation caused by OdDHL, while providing us with a platform from which we may exploit it.