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Potential of human PON2 as an anti- Pseudomonal therapy by Naseem Mohammad Ali School of Medicine Submitted in fulfilment of the requirements for the Doctor of Philosophy (Medical Studies) University of Tasmania June, 2015 DECLARATION OF ORIGINALITY This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Signature Date - 02/11/15 [i] AUTHORITY OF ACCESS This thesis may be made available for loan. Copying and communication of any part of this thesis is prohibited for two years from the date this statement was signed; after that time limited copying and communication is permitted in accordance with the Copyright Act 1968. Signature Date - 02-11-15 [i] STATEMENT OF ETHICAL CONDUCT The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University [i] Abstract ABSTRACT 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 signalling molecule 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. [ii] Abstract 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 PPARγ; 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. [iii] Acknowledgements ACKNOWLEDGEMENTS Firstly, I would like to extend my deepest thanks and gratitude to my supervisory team. They have been the most knowledge, inspiring, insightful and caring team I could have asked for. They have nurtured my growth as a scientist and as a person, providing support beyond what I expected. It was a privilege to work with them. This thesis is as much a testament to their efforts as it is to mine. I thank my supervisor Louise Roddam for all the help and guidance over the last five to six years. I also thank her for accepting me as a PhD. student, it has been a pleasure and a distinct highlight of my life so far. Her ability to impart wisdom and calm my nerves has been instrumental in helping me to complete this work. I thank my co-supervisors Margaret Cooley, Phoebe Griffin and Mark Ambrose for being available to give advice whenever required, provide an outlet for intellectual debate and their insightful input into my research. I would also like to thank all the members of the Host Pathogen Interactions group, past and present for their friendship over the years, particularly Joanne Pagnon and Emily Mulcahy. In addition, I need to thank my peers here at the University of Tasmania. I have made many life-long friends during my years as an honours and PhD. student, friendships that I will cherish for a very long time. I thank in particular, Cesar Tovar, Nick Blackburn, Stan Mitew and Emma Cazaly for sharing successes and failures and for the help and advice along the way. You guys, have made my PhD an amazing experience. Importantly, I need to acknowledge the tireless contribution of my family. My parents and siblings for their support and most importantly my wife, Shahad, for her understanding, love, support and unwavering belief in me even when I found it hard to believe in myself. I would like to thank the Clifford Craig Medical Research Trust and the Royal Hobart Hospital Research Foundation for funding these studies and finally, I want to acknowledge the Australian government for my IPRS scholarship and the Australian Cystic Fibrosis Research Trust for their studentship grant. Without these scholarships, this degree would not have been feasible for me. iv Table of contents TABLE OF CONTENTS Declaration of Originality ........................................................................................................................... i Authority of Access .................................................................................................................................... i Statement of Ethical Conduct .................................................................................................................... i Abstract .................................................................................................................................................... ii Acknowledgements ................................................................................................................................
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  • Supplementary Table 2

    Supplementary Table 2

    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942