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The Relationship Between Phenylacetic Acid Catabolism And THE RELATIONSHIP BETWEEN PHENYLACETIC ACID CATABOLISM AND PATHOGENICITY OF BURKHOLDERIA CENOCEPACIA K56-2 IN THE CAENORHABDITIS ELEGANS HOST MODEL by Robyn Jamie Law A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology University of Manitoba Winnipeg Copyright © 2009 by Robyn Jamie Law Library and Archives Bibliothgque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'6dition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-63924-5 Our file Notre r6f6rence ISBN: 978-0-494-63924-5 NOTICE: AVIS: The author has granted a non- L'auteur a accorde une licence non exclusive * exclusive license allowing Library and permettant a la Bibliothdque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par ('Internet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada THE UNIVERSITY OF MANITOBA FACULTY OF GRADUATE STUDIES COPYRIGHT PERMISSION The Relationship Between Phenylacetic Acid Catabolism and Pathogenicity of Burkholderia Cenocepacia K56-2 in the Caenorhabditis Elegans Host Model By Robyn Jamie Law A Thesis/Practicum submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirement of the degree Of Master of Science Robyn Jamie Law©2009 Permission has been granted to the University of Manitoba Libraries to lend a copy of this thesis/practicum, to Library and Archives Canada (LAC) to lend a copy of this thesis/practicum, and to LAC's agent (UMI/ProQuest) to microfilm, sell copies and to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. The Burkholderia cepacia complex (Bcc) is a group of genetically similar strains that have been identified as environmental pathogens with the ability to colonize various hosts. B. cenocepacia is a member of the Bcc capable of establishing devastating infections associated with severe necrotizing pneumonia and death. Currently, it is unclear as to what virulence factors have contributed to the development of pathogenicity of the Bcc across its diverse host range. In particular, metabolic versatility of these bacteria could be a reason for their ability to colonize diverse hosts. In this work, we characterize gene clusters involved in the phenylacetic acid (PA) degradation pathway of B. cenocepacia K56-2 in relation to pathogenicity of the organism in the Caenorhabditis elegans infection model. We use C. elegans to characterize B. cenocepacia infection and the relevance of PA catabolism to this infection. B. cenocepacia mutants defective in the activation, ring hydroxylation, ring opening and P-oxidation stages of PA degradation were tested for their ability to infect and kill C. elegans. By generating survival curves for C. elegans fed on each mutant, we have determined that inhibition of the activation and ring hydroxylation steps of the pathway result in reduced nematode killing. Further analysis of these mutations has confirmed that while activation of PA and a functional ring hydroxylation system are required for full pathogenicity, interruption of p-oxidation and ring opening actually increases the virulence of B. cenocepacia in C. elegans. Nematode survival was adversely affected by the presence of wild type and P-oxidation mutant metabolites in comparison to that of the ring hydroxylation mutant, which showed no significant effect iii on survival. Bacterial titre of the nematode intestine shows no appreciable difference between accumulation of PA pathway mutants and K56-2. Microscopic examination of worms feeding on wild type and mutant strains expressing a fluorescent reporter, reveal similar intestinal distension, characteristic of this type of infection. In addition, nematode innate immune mechanisms employed in response to B. cenocepacia infection were investigated using RNA interference. Through specific gene inactivation by ingested double stranded RNA, we have found that worms with compromised immunity due to inhibition of the p38 mitogen-activated protein kinase pathway gene, pmk-1, no longer display the reduced killing phenotype; suggesting, that the diminished virulence seen in upper PA pathway mutants is dependent on the p38 MAPK pathway. The requirement for a functional activation and ring hydroxylation complex in relation to B. cenocepacia pathogenesis is not completely understood; however, our findings, taken together with other analyses relating similar metabolic pathways to pathogenicity create a potential link between B. cenocepacia metabolism and its ability to colonize multiple hosts. Eventually, studies of this nature may provide new insights into Bcc pathogenesis in humans. iv Acknowledgements Foremost, I would like to thank my graduate supervisor, Dr. Silvia Cardona, for sharing with me her research insight and expertise and whose encouragement and guidance has been invaluable throughout my development. I would like to express my gratitude to my thesis committee members, Dr. Ivan Oresnik and Dr. Steve Whyard for their advice and suggestions. I would also like to thank other members of the Department of Microbiology for the use of equipment and general assistance as well as the Natural Sciences and Engineering Research Council (NSERC), The Faculty of Science and The Faculty of Graduate Studies at The University of Manitoba for providing funding for this research. Thank you to Jason Hamlin, Ruhi Bloodworth and other members of the Cardona laboratory for their friendship and support. V Table of Contents Title Page Abstract ii Acknowledgements iv Table of Contents v List of Tables ix List of Figures x List of Copyrighted Material xii List of Abbreviations xiii 1 - Introduction 1.0 The Burkholderia cepacia complex (Bcc) 1 1.1 B. cenocepacia K56-2 2 1.1.1 Genome of B. cenocepacia 3 1.1.2 Ecology of B. cenocepacia 4 1.1.3 Biotechnological Potential 5 1.2 Pathogenesis of the Bcc 6 1.2.1 Virulence Factors of B. cenocepacia 6 1.2.2 Cystic Fibrosis 8 1.2.3 Cystic Fibrosis Lung Infections 9 1.2.4 'Cepacia Syndrome' in Cystic Fibrosis Patients 10 vi 1.3 Host Models for the Bcc 11 1.3.1 Caenorhabditis elegans 11 1.3.2 C. elegans as an Infection Model 12 1.3.3 C. elegans Innate Immunity 14 1.3.4 The p38 Mitogen Activated Protein Kinase (MAPK) Pathway 16 1.3.5 Inactivation of C. elegans Genes by RNA Interference 17 1.4 Metabolism and Pathogenesis in the Cystic Fibrosis Lung Environment 19 1.4.1 Metabolism of Aromatic Compounds 20 1.4.2 Properties of Phenylacetic Acid (PA) 21 1.4.3 Phenylacetic acid Degradation in Bacteria 22 1.4.4 Phenylacetyl-Coenzyme A Ligase 27 1.4.5 Properties of Experimentally Confirmed PA-CoA Ligases 29 2 - Rationale and Hypothesis 30 3 - Materials and Methods 3.0 Bacterial Strains, Nematode Strains and Culture Conditions 31 3.1 Bacterial Growth 31 3.2 Oxidative Stress Assays 33 3.3 Hypochlorite Synchronization of C. elegans DH26 L4 Larvae 33 3.4 Nematode Infection Assays 34 3.5 Quantification of Nematode Intestinal Colonization 35 3.6 Fluorescent Microscopy 36 vii 3.7 Metabolite Filter Diffusion Assays 36 3.8 RNAi Knockdown and Survival 37 3.9 Molecular Biology Techniques 37 3.10 Tri-parental Mating with B. cenocepacia 38 3.11 Colony PCR Screening 38 3.12 Complementation of STC199-pooF 39 3.13 Construction of B. cenocepacia Deletion Mutants 39 3.14 Phylogenetic Analysis of the Putative B. cenocepacia PA-CoA Ligases 41 4- Results 4.0 Mutants in the ring-hydroxylation step of the B. cenocepacia K56-2 PA 44 catabolic pathway display diminished virulence in comparison to mutants of the ring opening and (3-oxidation steps in the C. elegans model of infection 4.1 Wild type Burkholderia cenocepacia K56-2 and the ring-hydroxylation 52 mutant STC155-poof accumulate to similar levels in the C. elegans intestine 4.2 Complementation of STC155-poof with the paaE gene in trans 58 restores wild type pathogenicity of B. cenocepacia K56-2, while the complemented strain STC199-pooF/pRLl remains slightly more pathogenic in the C. elegans host model 4.3 Attenuation of pathogenicity in the paaA and paaE mutants is 64 dependent on a functional p38 mitogen-activated protein kinase (MAPK) pathway in C. elegans 4.4 Burkholderia cenocepacia ring-hydroxylation mutants are not more 65 susceptible to reactive oxygen species (ROS) than wild type K56-2 4.5 Survival of Caenorhabditis elegans is reduced in the presence of B.
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