The potential therapeutic benefits of rhPON2 against Pseudomonas aeruginosa infections

Cystic fibrosis is an autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR protein channel is responsible for chloride and sodium transport primarily in epithelial cells, and insufficient or dysfunctional CFTR channels cause accumulation of thick and sticky mucus in the airways of people with CF, predisposing the airway to colonisation by microbial respiratory pathogens. In particular, airway colonisation and subsequent chronic infection by Pseudomonas aeruginosa is responsible for much of the morbidity and mortality in people with CF. Current antibiotic therapeutic regimes cannot eradicate P. aeruginosa infections once they are established, in part at least because this organism is able to form complex and antibiotic resistant multicellular structures called biofilms. Biofilm formation by P. aeruginosa and expression of many of its virulence genes is regulated by a cell-to-cell signalling network called quorum sensing, involving the transcriptional effector N-3-oxododecanoyl homoserine lactone (3-oxo-C\\(_{12}\\)-HSL). Importantly, the P. aeruginosa 3-oxo-C\\(_{12}\\)-HSL can also adversely modulate diverse functions in mammalian host cells, in ways that are important for promoting successful establishment of persistent infection. Our laboratory has been developing a therapy for people with CF, that specifically degrades and inactivates the 3-oxo-C\\(_{12}\\)-HSL signalling molecule, which could be expected to attenuate P. aeruginosa pathogenesis by diminishing its virulence and biofilm formation, as well as protecting host cells against adverse effects of 3-oxo-C\\(_{12}\\)-HSL. In the present study, a recombinant form of the native human lactonase, paraoxonase 2 (rhPON2), was produced in insect cells. When applied extracellularly to stationary-phase cultures of P. aeruginosa, rhPON2 diminished expression of two key bacterial quorum sensing-related genes (lasI and lasR), and significantly reduced biofilm formation. The effect of 3-oxo-C\\(_{12}\\)-HSL on gene expression in human airway epithelial cells was investigated in the absence and presence of rhPON2 using whole-transcriptome RNA-seq analysis. When epithelial cells were treated with 3-oxo-C\\(_{12}\\)-HSL alone, the expression of a diverse set of genes was up-regulated compared to non-treated control cells, including genes whose products are involved in maintaining epithelial barrier integrity and cytoskeletal remodelling and promoting inflammatory responses and apoptosis. In sharp contrast, the expression level of many of these same genes in cells treated with rhPON2 before exposure to 3-oxo-C\\(_{12}\\)-HSLwere similar to levels measured in non-treated controls. Additionally, rhPON2 treatment significantly reduced the release of pro-inflammatory cytokines (IL-8 and IL-6) and diminished apoptosis-related caspase3/7 activity in airway epithelial cells following exposure to 3-oxo-C\\(_{12}\\)-HSL. The rhPON2 also blocked the 3-oxo-C\\(_{12}\\)-HSL induced hyper-inflammatory response and corresponding higher unfolded protein response (UPR) gene expression in CF cells containing the F508del mutation compared to non-CF cells. Interestingly, the expression of certain genes whose products are involved in redox homeostasis were also differentially regulated in 3-oxo-C\\(_{12}\\)-HSL-treated epithelial cells, highlighting a possible role(s) for oxidative stress in the pathogenesis of P. aeruginosa; in turn, this phenomenon was also abrogated by rhPON2. Collectively, these results suggest that rhPON2 can protect epithelial cells from the detrimental effects of 3-oxo-C\\(_{12}\\)-HSL. Next, rhPON2 therapy was trialled in an in vivo acute P. aeruginosa infection pulmonary mouse model. Delivery of rhPON2 at the time of infection led to reduced expression of the pro-inflammatory gene IL-6 and its transcriptional regulator EGR1 but had little effect on bacterial load 12 hours post infection. These preliminary findings are encouraging but require further in-depth investigation, possibly using a combination of rhPON2 and CF-relevant antibiotic(s) to definitively demonstrate the therapeutic efficacy of rhPON2 in vivo. In summary, the experimental work presented in this thesis demonstrates proof-of-principle that rhPON2 can rapidly hydrolyse and inactivate 3-oxo-C\\(_{12}\\)-HSL, reduce P. aeruginosa biofilm formation and expression of some of its key biofilm-related genes. Importantly, rhPON2 prevented many of the detrimental effects of 3-oxo-C\\(_{12}\\)-HSL on mammalian airway epithelial cells, further demonstrating its potential therapeutic usefulness for P. aeruginosa infections and people with CF.