Recombinant human Paraoxonase-2 as a therapy to diminish <i>Pseudomonas aeruginosa</i> virulence and protect host cells from infection
Creators: name: Page, SA
Publisher: name: University of Tasmania
Year: 2024

description: Cystic fibrosis (CF) is caused by the absence or dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) ion transport channel, which is usually localised to the surface of cells, including epithelial and immune cells. Dysregulated ion transport in the CF lung leads to the build-up of persistent thick and dehydrated mucus predisposing people with CF to chronic infection by the opportunistic pathogenic Gram-negative bacterium, Pseudomonas aeruginosa, which is responsible for much of the morbidity and mortality associated with CF. When establishing infection of the airway, <i>P. aeruginosa</i> uses an intricate cell-cell signalling mechanism called quorum sensing (QS), which involves the production and secretion of small signalling molecules called acyl homoserine lactones (HSLs). HSLs usually accumulate in the extracellular environment, and upon re-entry into the bacterial cells regulates growth behaviours relative to population density, as well as the production of key virulence factors, including various adhesion proteins, toxins and antibiotic resistant biofilm formation. In addition, the major <i>P. aeruginosa</i> HSL, 3OC<sub>12-</sub>HSL, has been demonstrated to be a potent host immune cell response disruptor that dysregulates immune clearance mechanisms, thereby promoting bacterial infection and persistence. Thus, for therapies to be effective they should perhaps be broadly acting and involve those that can simultaneously target the <i>P. aeruginosa</i> 3OC<sub>12-</sub>HSL to reduce this organism's biofilm formation and virulence, as well as protect host cells from 3OC<sub>12-</sub>HSL-mediated host dysfunction during infection.In this context, our laboratory has been attempting to develop an effective therapeutic strategy to mitigate <i>P. aeruginosa</i> infections by targeting its production of 3OC<sub>12-</sub>HSL in people with CF; and has successfully produced a recombinant form of the human lactonase, paraoxonase 2 (rhPON2). Previous works by members of our group showed that rhPON2 efficiently hydrolysed extracellular bacterial 3OC<sub>12-</sub>HSL and thereby was able to i) protect mammalian respiratory epithelial cells from 3OC<sub>12-</sub>HSL-mediated disruption, and ii) strongly inhibit <i>P. aeruginosa</i> biofilm formation. However, the effectiveness of rhPON2 in downregulating bacterial virulence and increasing bacterial cell susceptibility to antibiotics, as well as protecting against the adverse effects of bacterial 3OC<sub>12-</sub>HSL on host immune cell function(s), remained to be investigated.In the first part of this study, the effects of rhPON2 on <i>P. aeruginosa</i> biofilm formation and virulence were explored using various phenotypic assays. When <i>P. aeruginosa</i> was cultured in the presence of rhPON2 for up to 18 hours, the overall viability of the bacterial cells was not affected, but analysis on collected supernatants fractions revealed that rhPON2 hydrolysed 3OC<sub>12-</sub>HSL and prevented its extracellular accumulation in the growth medium. In addition, rhPON2 inhibited 3OC<i>12-</i>HSL-mediated biofilm formation (by 66%,), bacterial swarming (by 67%) and pyoverdine production (by 60%), but not swimming motility that is usually regulated by QS-independent mechanisms. Together, these initial results demonstrated that the rhPON2 was active and a potent inhibitor of some specific 3OC<sub>12-</sub>HSL-regulated growth and virulence behaviours of <i>P. aeruginosa</i>. To gain broader insight into the potential effects of rhPON2 on the production of virulence factors by <i>P. aeruginosa</i>, a proteomic analysis was performed on bacterial cells cultured in the presence and absence of rhPON2. Exposing mid-exponential growth bacterial cultures to active rhPON2 for four hours significantly altered the abundance levels of over 150 proteins compared to control and reduced the levels of many proteins associated with quorum sensing, phenazine biosynthesis, and cationic antimicrobial peptide resistance, all of which have been reported to contribute to the virulence of <i>P. aeruginosa</i>. To verify a subset of the proteomic data a limited number of phenotypic assays were performed. Levels of elastase and protease were diminished in <i>P. aeruginosa</i> culture by 60% and 50% respectively after treatment with rhPON2. Collectively, these phenotypic and proteomic data provide important insight on the potential efficacy of extracellular rhPON2 as a treatment against <i>P. aeruginosa</i> biofilm formation and overall virulence.Bacterial cells within biofilms have been demonstrated to be up to 1000-times more resistant to antibiotics compared to freely growing planktonic cells. It was reasoned that since rhPON2 was effective at inhibiting <i>P. aeruginosa</i> biofilm formation that combining rhPON2 with a relevant antibiotic might be able to increase the antibiotic susceptibility of the bacterial cells. To test this idea, mid-exponential <i>P. aeruginosa</i> cultures were treated with rhPON2 with or without a sub-minimal inhibitory concentration (MIC) of the model antibiotic tobramycin and biofilm formation was assessed. Interestingly, while rhPON2 inhibited biofilm formation, co-treatment with tobramycin did not cause a further reduction in biofilm and cell viability, in turn suggesting that the rhPON2+tobramycin treatment did little, if anything, to increase the susceptibility of the bacterial cells to the antibiotic. Consistent with these findings, the results of experiments that involved co-cultured <i>P. aeruginosa</i> cells and human respiratory epithelial cells exposed to rhPON2, tobramycin or else rhPON2+tobramycin, showed that rhPON2 dampened expression of the inflammatory cytokine <i>IL-6</i> by CF respiratory epithelial cells, however, tobramycin did little to protect against <i>P. aeruginosa</i>-mediated inflammation, compared to control. The impact of rhPON2 and/or tobramycin on <i>P. aeruginosa</i> virulence during co-culture with respiratory epithelial cells was also investigated. Overall, expression of <i>lasB</i> (encoding elastase) and <i>phz1</i> (involved in the production of phenazine) genes tended to be decreased by rhPON2, but the rhPON2+tobramycin combination did not synergistically reduce bacterial virulence. Furthermore, rhPON2 treatment protected against <i>P. aeruginosa</i> induction of apoptosis in normal but not CF respiratory epithelial cells when co-cultured with <i>P. aeruginosa</i>. Thus, these data together suggest that rhPON2 alone provided moderate protection to CF respiratory cells against <i>P. aeruginosa</i> and neither this protection, nor bacterial killing was enhanced by the inclusion of sub-MIC tobramycin.3OC<sub>12-</sub>HSL is detectable in plasma from people with CF who have a <i>P. aeruginosa</i> lung infection, and<i> in vitro</i> has also been shown to suppress the secretion of proinflammatory cytokines by immune cells. Against this background, it seemed reasonable to suspect that rhPON2 would prove effective at inhibiting 3OC<sub>12-</sub>HSL-mediated immunosuppression i<i>n vitro</i>. To investigate whether rhPON2 could be immunoprotective, peripheral blood mononuclear cells (PBMCs) collected from people with and without CF were stimulated with <i>P. aeruginosa</i> lipopolysaccharide (LPS) in the presence and absence of 3OC<sub>12-</sub>HSL and with or without rhPON2 and host cell cytokine secretion and apoptosis was measured. LPS induced the secretion of TNFα by PBMCs and this induction was significantly inhibited by the addition of 3OC<sub>12-</sub>HSL, compared to controls. Importantly, and for the first time, this study demonstrated that when PBMCs were co-treated with LPS, 3OC<sub>12-</sub>HSL and rhPON2, the 3OC<sub>12-</sub>HSL mediated suppression of TNF-α secretion was inhibited. Furthermore, rhPON2 treatment protected both CF and non-CF derived PBMCs from 3OC<sub>12-</sub>HSL-mediated induction of apoptosis <i>in vitro</i>, suggesting that rhPON2 effectively mitigated the immunomodulatory and pro-apoptotic effects of 3OC<sub>12-</sub>-HSL on immune cells.To determine whether rhPON2 could confer protection against the cellular effects of 3OC<sub>12-</sub>HSL <i>in vivo</i>, a mouse model was employed. <i>P. aeruginosa</i> LPS with and without 3OC<sub>12-</sub>HSL and/or rhPON2 was injected into the tail vein of mice and after two hours treatment the levels of several plasma cytokines were measured. As expected, LPS induced secretion of TNF-α, <i>IL-6</i> and MCP-1 secretion. However, in these experiments 3OC<sub>12-</sub>HSL did not appear to suppress the LPS-mediated induction of these proinflammatory cytokines, which may possibly be due to poor 3OC<sub>12-</sub>HSL solubility, preventing the administration of an adequate dose of 3OC<sub>12-</sub>HSL. Despite these negative findings, injection of rhPON2 alone or in combination with LPS nonetheless did not affect the levels of proinflammatory cytokines in the plasma, demonstrating at least that rhPON2 had few or no adverse effects <i>in vivo</i>.In summary, data presented in this thesis provides deeper insight into the potential benefits of using extracellular rhPON2 as a therapy against <i>P. aeruginosa</i> infection by demonstrating its efficacy in reducing bacterial biofilm formation and virulence and protecting host cells from some of the adverse effects of the 3OC<sub>12-</sub>HSL quorum-sensing molecule.