To see the world in a grain of sand And heaven in a wild flower, Hold infinity in the palm of your hand, And eternity in an hour.

Transcription

1 To see the world in a grain of sand And heaven in a wild flower, Hold infinity in the palm of your hand, And eternity in an hour. -William Blake

2 This thesis is dedicated to my family for always believing in me, and to the chicks used in these scientific studies. ii

3 ACKNOWLEDGEMENTS This work would not have been possible without the assistance from collaborators and support staff, and it is a pleasure to thank all those who made this thesis possible. I would like to thank the Husbandry Staff who have helped with the raising of thousands of birds over the years. I would especially like to thank the germ-free animal staff, in particular Sara Poe and Chriss Vowles. The microarray experiments would not have been possible without the generous help of Dr. Craig Parker at the USDA- California. Ted Huston (U-Michigan) ran the ICP-M/S analysis. The microbiota analysis, including trflp and 454-Sequencing was done in collaboration with Dr. Gary Huffnagle and Dr. Vincent Young. I would also like to thank my Campylobacter colleagues and friends, Dr. Dave Hendrixson (UT-Southwestern) and Dr. Erin Gaynor (UBC) for their support, reagents, experimental suggestions, and friendship. This thesis would not have been possible without my advisor, Dr. Victor DiRita, who has supported me and taught me invaluable life and science lessons over the years. It is not often that you find a mentor who always finds time to listen to your roadblocks, fix typing errors and share a cup of coffee. He allowed me to grow as a scientist by giving me the freedom and responsibility of independent research. I could not ask for a better mentor and friend. I would also like to thank the members of my committee for their advice, scientific suggestions, guidance, and support. I am grateful to all the members of the DiRita Lab that have encouraged, commiserated, and laughed with me over the years. I would especially like to thank Dr. iii

4 K.T. Elliot. The time she took to teach and encourage, as well as her never-ending patience, was invaluable. (Look, K.T...I m a star-fish!). Each member, past and present, (Dr. KT Elliot, Dr. Becky Weisner, Dr. Tsutomu Kakuda, Dr. Jeremy Ellermeier, Dr. Dave Hendrixson, Dr Jyl Matson, Dr. Jeff Withey, Aimee Richards, Becca Anthouard, Kevin Ginsburg, Sam Carpentier, Wei-Ping Teoh, and Andy Perault), made my time in the DiRita Lab one that I will never forget. I would especially like to thank a member of team Campy, Dr. Jeremy Ellermeier, for his banter and encouragement during the writing of this thesis. Thank you to everyone for sharing in the many frustrations and disappointments, and the few and far-between victories that come with bench-science. I would also like to thank all my friends that have helped me maintain my sanity outside of lab. I am blessed to be surrounded by great friends. I would especially like to thank Dr. Andrea Radtke, my confidant and source of endless encouragement. I would like to thank my entire family who has always supported my dream to be a microbiologist. They have encouraged and supported me over the years, never doubting my ambitions and me. I could not have done it without you. Lastly, I could not thank enough the love of my life and best friend, Ben. Thank you for making me laugh when I wanted to cry, believing in me when I had doubts, and showing me the world. Thank you for your unending love, support and encouragement. iv

5 TABLE OF CONTENTS DEDICATION... ii ACKNOWLEDGEMENTS... iii LIST OF FIGURES... vii LIST OF TABLES...x LIST OF APPENDICES... xi CHAPTER I. Introduction...1 Campylobacter jejuni Biology...1 Outcomes of Infection in Humans versus Chickens...20 Animal Models of Campylobacter jejuni Colonization and Virulence...33 Zinc Homeostasis...51 Symbiotic Relationships...55 Intestinal Microbiota and Influences on Host-Bacterial Interactions...57 Summary...65 References...66 II. III. Materials and Methods...85 Bacterial Experiments...86 Animal Use and Experiments...94 References Campylobacter jejuni Invasion of the Chick Cecum Stimulates an Inflammatory Response and Leads to Asymptomatic Colonization Introduction Results Discussion References v

6 IV. A Campylobacter jejuni znua Orthologue is Essential for Growth in Low Zinc Environments and Chick Colonization Introduction Results Discussion References V. Microbiota Influences on Intestinal Zinc in the Chick Cecum Introduction Results Discussion References VI. Conclusions and Future Directions APPENDICES vi

7 LIST OF FIGURES 1. Sources and Outcomes of C. jejuni Infection Uptake and Activity of Cytolethal Distending Toxin O- and N-Linked Protein Glycosylation Molecular and Cellular Features of the Innate Immune Response to C. jejuni in Humans and Chickens Signaling Cascades and Outcomes of Infection Model of C. jejuni Infection in Humans Colonization and Heterophil Response to 6 C. jejuni Strains H&E Staining of Infected Tissue virb11 Detection by PCR Colonization and Heterophil Response to ΔvirB11 and ΔvirD4 Infections Colonization and Heterophil Response to ΔcdtB Infection Cell Culture Invasion of INT407 and Primary Chicken Intestinal Cells In vivo Gentamicin Protection Assay Electron Microscopy Immunofluorescent Microscopy Colonization and Heterophil Response to C. jejuni Strain Str R C. jejuni Recovery From Systemic Sites Colonization and Heterophil Response During Low-Dose Infection Colonization and Heterophil Response During Day-7 Inoculation vii

8 20. Colonization and Heterophil Response to Cecal Material Colonization and Heterophil Response to S. typhimurium Quantitative RT-PCR of Cytokine Transcripts from Infected Chicks Alignment of znua and Operon Structure RT-PCR of znuabc Operon Quantitative RT-PCR of znua in EDTA Quantitative RT-PCR of znua in Defined Media Growth of ΔznuA in Defined Media Growth of ΔznuA in EDTA Glycosylation of ZnuA Localization of ZnuA ZnuA Stabilizes with Increasing Amounts of EDTA ZnuA Binds Zinc Chick Colonization by ΔznuA ΔznuA Mutant Early Colonization Events trflp Chromatogram of Conventional and Limited-flora Chicks DAPI-Stained Cecal Tissue from Conventional and Limited-flora Chicks Phyla Composition from 454-Sequencing of Conventional and Limited-flora Chicks C. jejuni Colonization in Germ-Free Mice and Limited-flora Chicks Enumeration of Heterophils in Limited-flora Chick Ceca Conventionalization of C. jejuni-colonizing Limited-Flora Chicks ICP-HRMS of Chick Cecal Contents Growth of C. jejuni in Chicken Cecal Media viii

9 43. C. jejuni Growth with Zinc-Binding Proteins from Conventional Chick Cecal Contents Growth of C. jejuni with Zinc-Binding Proteins and ZnSO Heat Treatment of Zinc-Binding Proteins Growth of C. jejuni in the Presence of Ovotransferrin Model of C. jejuni Colonization of the Chick Cecum Chick Cecal Colonization of the ΔpglB Mutant Motility of the ΔpglB Mutant and Variants Arylsulphatase Assay for flaa Expression in Wild-type and ΔpglB Backgrounds C. jejuni ΔcprS Mutant Chick Colonization C. jejuni cproe Chick Colonization Western-Blot of C. jejuni-infected Chick Sera Detection of siga in the Cecal Mucus of C. jejuni Infected Chicks Wild-type Colonization of Germ-Free Mice C. jejuni Mutant Colonization of Germ-Free Mice Cecal Colonization and Mouse Survival of C. jejuni Infected IL-10 -/- Germ-Free Mice ΔznuA Mutant Colonization of Germ-Free IL-10 -/-Mice Recovery of C. jejuni from 5-FU Treated Chicks Heterophil Recruitment in 5-FU Treated Chicks Growth of C. jejuni in the Presence of 5-FU ix

10 LIST OF TABLES 1. Strains and Plasmids Used Phyla Identities from 454 Sequencing of Conventional and Limited Flora Chicks Genus Identities from 454 Sequencing of Conventional and Limite4ed Flora Chicks Zinc-Binding Protein Identification Genes Down-regulated in ΔpglB Mutant Compared to Wild-type C. jejuni Genes Up-regulated in ΔpglB Mutant Compared to Wild-type C. jejuni Genes Down-regulated in ΔpglB- motile Variant Mutant Compared to Wild-type C. jejuni Genes Up-regulated in ΔpglB- motile Variant Mutant Compared to Wild-type C. jejuni x

11 LIST OF APPENDICES A. Disruption of the N-glycosylation system in C. jejuni Inhibits Chick Colonization and Increases Flagellar Phase Variation B. The Two-Component Regulatory System CprRS Controls Aspects of Campylobacter jejuni Pathogenesis by Mediating the Switch Between Planktonic and Biofilm Lifestyles C. Antibody Production Against C. jejuni in the Day-of-hatch Chick Colonization Model D. Murine Models of C. jejuni Colonization and Pathogenesis E. 5-Flurouracil Depletion of Heterophils and Its Effects on C. jejuni Colonization xi