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Oncorhynchus Mykiss) INVESTIGATING SURVIVAL MECHANISMS OF YERSINIA RUCKERI IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by INDERVESH In partial fulfilment of requirements for the degree of Doctor of Philosophy May, 2008 © Indervesh, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-42562-6 Our file Notre reference ISBN: 978-0-494-42562-6 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and 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 Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou 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 et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada ABSTRACT INVESTIGATING SURVIVAL MECHANISMS OF YERSINIA RUCKERI IN RAINBOW TROUT (ONCHORHYNCHUS MYKISS) Indervesh Advisor: University of Guelph, 2008 Dr. R.M.W. Stevenson Yersinia ruckeri causes hemorrhagic septicemia of salmonid fish. We hypothesized that the ability to persist inside infected fish was a key component of virulence strategy of Y. ruckeri and aimed to identify survival-essential genes of Y. ruckeri serotype 1 strain, RSI 154. After two rounds of screening of 1056 miniTn5Km2 signature-tagged mutants, 25 mutants that did not survive in kidney at 7 days post­ infection in immersion infected rainbow trout were selected for further study. Sequencing of interrupted genes in selected mutants identified genes homologous to znuA, which encodes a component of a zinc transporter in Escherichia coli, and uvrY, which encodes the response regulator of BarA-UvrY two-component system (TCS) in E. coli. The uvrY mutant was hypersensitive to EkC^-mediated killing and was less invasive to Epithelioma papulosum cyprini fish cells than wild type (WT) bacteria, but was not affected in serum sensitivity or growth under iron-limiting conditions. In a competitive infection with WT, the uvrY mutant had lower infection loads in rainbow trout kidney. When present in a low-copy plasmid, the znuACB locus of Y. ruckeri fully restored growth of a zinc-transport deficient kznuACB mutant of E. coli in Luria-Bertani (LB) medium supplemented with 2.0 mM ethylenediamine tetraacetic acid (EDTA), indicating that znuACB locus of Y. ruckeri is likely involved in zinc transport. Unlike AznuACB mutants of E. coli, AznuACB mutant of Y. ruckeri did not show poor growth in zinc-deficient M9 medium and LB medium supplemented with metal chelators, EDTA and tetrakis-(2-pyridylmethyl)-ethylenediamine, suggesting presence of additional zinc transporters in Y. ruckeri. The znuA mutant of Y. ruckeri was out-competed by WT in rainbow trout kidney. Survival of Y. ruckeri in rainbow trout was also reduced with mutations in gene homologs encoding an O-antigen polymerase (wzy), peptidoglycan deacetylase (pdaA), protease (ptrA), bundle-forming pili (rcpA), ATPase of DNA segregation (parA) and transposase of Tn7, bacteriophage tail fiber-like protein and genes of unknown functions. Characterization of survival-essential genes in fish helps identify new virulence genes and contributes to understanding survival strategies and pathogenic mechanisms of Y. ruckeri. ACKNOWLEDGMENTS Financial support for this study was provided by the Natural Sciences and Engineering Research Council of Canada and the Fish Culture section of Ontario Ministry of Natural Resources through grants to Dr. R.M.W. Stevenson. I sincerely thank my advisor Dr. R.M.W. Stevenson for her constant help, constructive criticism, and excellent mentorship throughout my thesis research. Besides learning principles of science from her, I learned so many things as a person that I will cherish throughout life. I would like to thank members of my advisory committee, Dr. J. Wood, Dr. J. Prescott and Dr. M. Ferguson for their guidance, constructive criticism and critical evaluation of my thesis. Thank you to members of my examination committee, Dr. J. Nash, Dr. P. Krell, Dr. J. Wood, Dr. J. Prescott, and R. Lo for reading my thesis and suggestions. Special thanks to Dr. L. Mutharia, Dr. Reggie Lo and Dr. Jerry King for helpful discussion on various research problems pertaining to this work. I would like to acknowledge help of my friend, Dr. A. Singh for statistical analyses. Many thanks go to S. Lord, V. Harper, M. Raymond, R. Quinn and R. Jones for their nice company and help with experiments. I enjoyed wonderful company and help of many friends throughout this study. No words can express my thankfulness to my parents, grandparents and wife for everything they have done and are doing for me. Their constant help and support made this difficult task possible. You people matter the most for me. i TABLE OF CONTENTS Page# TABLE OF CONTENTS i LIST OF TABLES v LIST OF FIGURES vi CHAPTER 1 INTRODUCTION 1 LITRATURE REVIEW 3 1.1. Yersinia ruckeri 3 1.2. Plasmids of Y. ruckeri 4 1.3. Serotypes of Y. ruckeri 6 1.4. Enteric redmouth disease 8 1.5. Stress and ERM disease 9 1.6. Pathogenic mechanisms of Y. ruckeri 10 1.6.1. Environmental survival of Y. ruckeri 10 1.6.2. Attachment and entry into fish 12 1.6.3. Factors involved in resistance to fish defenses 14 1.6.4. Toxins causing host damage 18 1.7. Regulation of virulence genes expression 21 1.8. Model of Y. ruckeri pathogenesis 23 1.9. Signature-tagged mutagenesis (STM) and identification 25 of virulence genes 1.9.1. Animal model for screening ST mutants 29 1.9.2. Genes identified in STM screens 32 1.10. Research objectives and approach 37 n CHAPTER 2 MATERIALS AND METHODS 38 2.1. Bacterial strains and growth conditions 38 2.2. Recombinant DNA techniques 41 2.3. Competent cell preparation 41 2.4. Polymerase chain reaction 44 2.5. Generation of signature-tagged (ST) mutants 44 2.6. Randomness of transposon insertion 49 2.7. Rainbow trout infection experiments 50 2.8. Screening of ST mutants in rainbow trout 52 2.9. Number of transposon insertions in the genome of mutants 53 2.10. Sequence characterization of mutants 53 2.11. Screening for auxotrophs 54 2.12. Identification of barA homolog of Y. ruckeri RSI 154 54 2.13. PCR amplification of barA and uvrY genes in Y. ruckeri strains 55 2.14. Growth of uvrY mutant in Luria-Bertani medium and 55 under iron-limiting conditions 2.15. Fish cell invasion by uvrY mutant 56 2.16. Serum sensitivity of uvrY mutant 57 2.17. Ultraviolet light (UV) sensitivity of uvrY mutant 57 2.18. H2O2 sensitivity of uvrY mutant 58 2.19. Sequence characterization of znuACB locus 58 2.20. PCR amplification and cloning of znuACB locus 59 2.21. Generation of AznuACB mutant 59 2.22. In vitro growth of AznuACB mutant 62 2.23. Transcomplementation of AznuACB mutant of E. coli 64 2.24. The znuAv.lacZ and znuCBv.lacZ transcriptional fusion analysis 64 2.25. Generation of isogenic mutants of Y. ruckeri 68 2.26. Statistical analyses 68 iii CHAPTER 3 GENERATION AND SCREENING OF SIGNATURE-TAGGED 69 MUTANTS RESULTS 69 3.1. Generation of ST mutants 69 3.2. Primary screening of ST mutants in rainbow trout 69 3.3. Secondary screening of ST mutants in rainbow trout 79 3.4. One-to-one competitive challenge of mutants with WT strain 81 3.5. Effect of miniTn5Km2 cassette on survival of mutants 85 3.6. Number of transposon insertions in ST mutants 85 DISCUSSION 85 CHAPTER 4 CHARACTERIZATION OF MUTANTS UNABLE TO 92 SURVIVE IN FISH RESULTS AND DISCUSSION 92 4.1. Screening for auxotrophy 92 4.2. Identification of mutated genes in ST mutants 92 4.3. Gene encoding bundle-forming pili (Bfp) 99 4.4. O-antigen polymerase 100 4.5. Polysaccharide deacetylase 101 4.6. PtrA protease 103 4.7. Genes involved in cellular transport 106 4.8. Insertion sequence (IS) /transposon proteins 106 4.9. Bacteriophage-like genes 109 4.10. ATPase for DNA segregation 111 CHAPTER 5 CHARACTERIZATION OF THE BarA-UvrY 113 TWO-COMPONENT SYSTEM RESULTS 114 5.1. In silico analysis of UvrY response regulator 114 5.2. Non-polarity of the uvrY mutation 119 IV 5.3. Growth of uvrY mutant in Luria-Bertani medium 123 5.4. H2O2 sensitivity of uvrY mutant 123 5.5. Serum sensitivity of uvrY mutant 126 5.6. Growth of uvrY mutant under iron-limiting conditions 126 5.7.
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