Potential of human PON2 as an anti-pseudomonal therapy
Creators: name: Ali, NM
Publisher: name: University Of Tasmania
Year: 2023

description: Cystic Fibrosis (CF) is the most common life-limiting single gene disorder in Caucasian populations. CF results from mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which leads to accumulation of thick and sticky mucus in the airways of people with CF, ultimately dampening immune clearance of potential respiratory pathogens. In fact, chronic airway infection by the opportunistic bacterial pathogen Pseudomonas aeruginosa is the major cause of mortality and morbidity in individuals with CF. The ability of P. aeruginosa to chronically infect the airways of people with CF stems, in part at least, from its remarkable capacity to grow as complex multi-cellular communities called biofilms, which offer protection from host immune responses and eradication by conventional antibiotics. Biofilm formation by P. aeruginosa is co-ordinated by a cell density-dependent signalling system called quorum sensing (QS). While P.aeruginosa has multiple QS systems, the major system involves the diffusible signallingmolecule N-3-oxo-dodecanoyl-L-homoserine lactone (C12HSL). C12HSL not only acts an important biofilm signalling molecule but also regulates bacterial virulence factor production. In addition, C12HSL has gene modulatory and cytotoxic effects in host cells. Collectively, these properties have stimulated much research interest in developing novel antimicrobial therapies that specifically target QS. The present study investigated the potential of using the human enzyme, Paraoxonase-2 (PON2), to target and inhibit biofilm formation by, and virulence of, P. aeruginosa. Human PON2 is usually produced intracellularly and has previously been demonstrated to have highly specific hydrolytic activity towards C12HSL, and thereby seems to represent an innate mechanism by which host cells might attempt to counter P. aeruginosa QS, and hence infection by this organism. Here, it was hypothesized that the virulence of, and biofilm-formation by, P. aeruginosa could be diminished by manipulating human PON2 (hPON2) levels, either by augmenting native PON2 expression, or by supplementing lactonase activity using a recombinant form of human PON2 (rhPON2) exogenously. Thus, the aim of the first part of this thesis was to determine the gene modulatory and cytotoxic effects of C12HSL on airway epithelia, and investigate the regulation of PON2 expression in mammalian cells. The second part of this thesis was aimed at producing rhPON2 and investigating whether exogenously supplied rhPON2 could disrupt P. aeruginosa QS, and thereby increase its susceptibility to the clinically relevant antibiotic tobramycin. C12HSL was found to alter gene expression in cultured airway epithelial cells, even at low concentrations. This included the down-regulation of genes involved in immune signalling and the unfolded protein response. C12HSL also induced apoptosis and affected epithelial cell viability and metabolic activity in a concentration dependent manner. The results also show that while C12HSL itself did not significantly alter the expression of native PON2 in airway epithelial cells, PON2 gene expression and PON2 protein levels rapidly increased in response to agonism of the major mammalian C12HSL receptor PPAR˜í‚â•; in turn, these results would seem to link the regulation of human PON2 to a known major regulator of the inflammatory response. Recombinant hPON2 was able to hydrolyse almost all detectable C12HSL produced in P. aeruginosa cultures with no effect on bacterial growth. Treatment of P. aeruginosa cultures with rhPON2 tended to decrease expression of all three P. aeruginosa QS systems and associated virulence genes. Importantly, rhPON2-dependent hydrolysis of C12HSL during bacterial growth, significantly increased the susceptibility of P. aeruginosa cells to the bactericidal effects of the antibiotic tobramycin. In contrast, treatment of P. aeruginosa with rhPON2 combined with a high concentration of tobramycin, whilst hydrolysing the C12HSL and increasing bacterial killing by over an order of magnitude, significantly up-regulated the expression levels of a number of important QS and QS-dependent virulence genes of P. aeruginosa including, lasI, lasB and algD. One interpretation of this result is that bacterial cells treated with this combination up-regulate lasI, the gene that catalyses C12HSL synthesis, and other virulence encoding genes, presumably due to antibiotic-induced stress, via a C12HSL-independent mechanism. Taken together, the results of this thesis demonstrate that low-level C12HSL can influence gene expression in human airway cells, and reveal for the first time that native human PON2 expression is regulated via a major mammalian C12HSL receptor. In addition, rhPON2-treatment of P. aeruginosa leads to reduction of C12HSL levels and the reduction of bacterial virulence gene expression. The efficacy of rhPON2 in conjunction with antibiotics however, requires further investigation, using lower-concentrations (clinically achievable levels) of antibiotic and antibiotics with alternative mechanisms of action. The results of this thesis strongly support rhON2 being a novel and useful anti-Pseudomonal therapy.