Investigation of the Gallbladder Host Environment and Small Rnas in the Pathobiology of Campylobacter Jejuni Sheep Abortion Clon
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Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2016 Investigation of the gallbladder host environment and small RNAs in the pathobiology of Campylobacter jejuni sheep abortion clone IA 3902 Amanda Jo Kreuder Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Microbiology Commons, and the Veterinary Medicine Commons Recommended Citation Kreuder, Amanda Jo, "Investigation of the gallbladder host environment and small RNAs in the pathobiology of Campylobacter jejuni sheep abortion clone IA 3902" (2016). Graduate Theses and Dissertations. 14982. https://lib.dr.iastate.edu/etd/14982 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigation of the gallbladder host environment and small RNAs in the pathobiology of Campylobacter jejuni sheep abortion clone IA 3902 by Amanda Jo Kreuder A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Veterinary Microbiology Program of Study Committee: Paul J. Plummer, Co-Major Professor Qijing Zhang, Co-Major Professor F. Chris Minion Eric Burrough Micheal Yaeger Iowa State University Ames, Iowa 2016 Copyright © Amanda Jo Kreuder, 2016. All rights reserved. ii TABLE OF CONTENTS Page NOMENCLATURE .............................................................................................. iv ACKNOWLEDGMENTS ..................................................................................... vi ABSTRACT .......................................................................................................... vii CHAPTER 1 LITERATURE REVIEW AND INTRODUCTION ..................... 1 Specific aims and significance ......................................................................... 1 Organization of the dissertaton ....................................................................... 2 Literature review ............................................................................................. 2 CHAPTER 2 HISTOPATHOLOGYAND SPATIAL DISTRIBUTION OF PUTATIVE GROWTH FACTORS IN THE OVINE GALLBLADDER FOLLOWING DIRECT INOCULATION WITH CAMPYLOBACTER JEJUNI SHEEP ABORTION CLONE IA 3902 ......................................... 21 Abstract .................................................................................................... 21 Introduction .................................................................................................... 22 Materials and methods .................................................................................... 24 Results .................................................................................................... 34 Discussion .................................................................................................... 41 Figures and tables ............................................................................................ 49 CHAPTER 3 THE TRANSCRIPTOME OF CAMPYLOBACTER JEJUNI SHEEP ABORTION CLONE IA 3902 FOLLOWING IN VIVO EXPOSURE TO THE OVINE GALLBLADDER ..................................... 58 Abstract .................................................................................................... 58 Introduction .................................................................................................... 59 Materials and methods .................................................................................... 62 Results .................................................................................................... 72 Discussion .................................................................................................... 79 Figures and tables ............................................................................................ 99 CHAPTER 4 THE TRANSCRIPTOME OF MUTANTS ∆CjNC110, ∆LUXS, AND ∆CjNC110∆LUXS IN THE CAMPYLOBACTER JEJUNI SHEEP ABORTION CLONE IA 3902 ................................................................... 146 Abstract .................................................................................................... 146 Introduction .................................................................................................... 147 Materials and methods .................................................................................... 150 Results .................................................................................................... 157 iii Discussion .................................................................................................... 165 Figures and tables ............................................................................................ 178 CHAPTER 5 PHENOTYPIC CHANGES ASSOCIATED WITH INACTIVATION OF THE CjNC110 SMALL RNA IN CAMPYLOBACTER JEJUNI IA 3902........................................................ 205 Abstract .................................................................................................... 205 Introduction .................................................................................................... 206 Materials and methods .................................................................................... 208 Results .................................................................................................... 211 Discussion .................................................................................................... 214 Figures and tables ............................................................................................ 226 CHAPTER 6 SUMMARY AND FUTURE DIRECTIONS ............................... 233 Summary ......................................................................................................... 233 Future directions .............................................................................................. 240 REFERENCES...................................................................................................... 240 iv NOMENCLATURE 6-PPK 6-phosphofructokinase YLD years lived with disability CDC Centers for Disease Control PFGE Pulsed-field gel electrophoresis MLST Multi-locus sequence typing SA Sheep abortion ST Sequence type RNA Ribonucleic acid DNA Deoxyribonucleic acid RNAseq High-throughput RNA sequencing TSS Transcriptional start sites mRNA Messenger RNA sRNA Small non-coding RNA ncRNA Small non-coding RNA asRNA Antisense RNA UTRs 5’ or 3’ untranslated regions MH Mueller-Hinton BAP Blood agar plates MOMP Major outer membrane protein PAS Periodic acid-Schiff reaction MALDI-TOF Matrix assisted laser desorption ionization-time of flight v IACUC Institutional Animal Care and Use Committee CFU Colony forming unit ANOVA Analysis of variance H & E Hematoxylin and eosin PBS Phosphate buffered saline RAP RNA adapter plate IGV Integrated Genome Viewer COG Clusters of Orthologous Groups MCP Methyl-accepting chemotaxis protein TAT Twin arginine targeting secretion system NO Nitric oxide LB Luria-Bertani SAM S-adenosylmethionine AB Autoinducer broth CFS Cell-free supernatant AI-2 Autoinducer-2 RLU Relative light units SNPs Single nucleotide polymorphisms AAG Autoagglutination vi ACKNOWLEDGMENTS I would like to thank my co-mentors, Drs. Paul Plummer and Qijing Zhang, and my committee members, Drs. Eric Burrough, Mike Yaeger, and Chris Minion for their guidance and support throughout the course of this research. Thank you to Dr. Jennifer Schleining and Dr. Mike Yaeger for their invaluable assistance in developing the sheep gallbladder inoculation model, as well as Dr. Victoria Lashley and Dr. Mike Yaeger for assistance with the histopathology related to that project. Thank you to Dr. Andrew Severin for his assistance with bioinformatics related to RNA sequencing. Thank you also to Kathy Mou and the members of the Zhang lab for invaluable assistance in working with Campylobacter. In addition, I would also like to thank my friends, colleagues, the VDPAM and VMPM department faculty and staff for making my doctoral studies at Iowa State University an enjoyable and educational experience. vii ABSTRACT Campylobacter jejuni is an important zoonotic agent that is the leading cause of both human foodborne bacterial gastroenteritis worldwide, as well as ovine abortion in the United States. In particular, a single C. jejuni sheep abortion clone, of which IA 3902 is a prototypical isolate, has recently emerged as the dominant causative agent of sheep abortion due to Campylobacter sp. in the U.S. and has been increasingly identified in human outbreaks of disease. Multi-omics approaches to studying this hypervirulent strain have shown that it is remarkably similar to other common strains of C. jejuni such as 11168 that do not show the same ability to cause systemic clinical disease. Further work to elucidate the molecular mechanisms that allow for small changes in genomic structure to lead to large changes in virulence ability in this important zoonotic agent is warranted. A number of studies have demonstrated that the gallbladder of ruminants, as well as other domestic animal species, is often positive on culture for Campylobacter sp. following oral exposure, suggesting that this environment may serve as a chronic nidus of infection for maintenance