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Host Pathogen Responses of Chicken Cells in Vitro and in Vivo to a Diverse Population of Campylobacter Strains

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Host Pathogen Responses of Chicken Cells in Vitro and in Vivo to a Diverse Population of Campylobacter Strains _________________________________________________________________________Swansea University E-Theses Host Pathogen responses of chicken cells in vitro and in vivo to a diverse population of Campylobacter strains John, Daniel A. How to cite: _________________________________________________________________________ John, Daniel A. (2018) Host Pathogen responses of chicken cells in vitro and in vivo to a diverse population of Campylobacter strains. Doctoral thesis, Swansea University. http://cronfa.swan.ac.uk/Record/cronfa50200 Use policy: _________________________________________________________________________ This item is brought to you by Swansea University. Any person downloading material is agreeing to abide by the terms of the repository licence: copies of full text items may be used or reproduced in any format or medium, without prior permission for personal research or study, educational or non-commercial purposes only. The copyright for any work remains with the original author unless otherwise specified. The full-text must not be sold in any format or medium without the formal permission of the copyright holder. Permission for multiple reproductions should be obtained from the original author. Authors are personally responsible for adhering to copyright and publisher restrictions when uploading content to the repository. Please link to the metadata record in the Swansea University repository, Cronfa (link given in the citation reference above.) http://www.swansea.ac.uk/library/researchsupport/ris-support/ Host pathogen responses of chicken cells in vitro and in vivo to a diverse population of Campylobacter strains Daniel John Submitted to Swansea University in fulfilment of the requirements for the Degree of Doctor of Philosophy Swansea University 2018 i Abstract Campylobacter is the leading cause of bacterial food-borne diarrheal disease worldwide, with the bacteria being pathogens in humans and chickens. There are differences in the mechanistic understanding between Campylobacter strains, due, in part, to genomic and phenotypic strain diversity, which leads to inconsistent findings. This study aimed to understand underlying infection biology in host-pathogen interactions between populations of Campylobacter and the chicken host. A collection of Campylobacter jejuni strains were used to investigate the genotypic and phenotypic differences which yielded a diverse pan-geome of 2715 genes with genome wide association studies uncovering genes related to flagella assocaited with strains isolated from both humans and chickens. The C. jejuni strains were then used with a new commercially available chicken cell line to investigate the chicken systems with host responses measured in comparison to human systems. The pathogenic diversity of C. jejuni was measured through their inflammatory, cytotoxicity, adhesion, invasion and signalling responses in a high-throughput model using avian and human intestinal epithelial cells. C. jejuni induced IL-8 and CXCLi1/2 in human and avian epithelial cells, respectively, in a MAP kinase-dependent manner. In contrast, IL-10 responses in both cell types were PI 3- kinase/Akt-dependent. C. jejuni strains showed diverse levels of invasion with high invasion dependent on MAP kinase signaling in both cell lines. C. jejuni induced diverse cytotoxic responses in both cell lines with cdt-positive isolates showing significantly higher toxicity. Blockade of endocytic pathways suggested that invasion by C. jejuni was clathrin- and dynamin-dependent but caveolae- independent in both cells. In contrast, IL-8 (and CXCLi1/2) production was dependent on clathrin, dynamin, and caveolae. Strong correlations were found between IL-8 with invasion and toxicity responses with GWAS being able to associated genes to expression of IL-8,-10 and level of invasion. Campylobacter strains were isolated from the caeca, ileum and liver and a pan-genome created with GWAS analysis discovering genes relating to iron transport and heat shock proteins in strains from the liver. Subsequently, caecal tonsils were extracted from chickens uninfected and chickens infected with a cocktail of six Campylobacter strains with a Campylobacter effect found where infected chickens produced significantly higher Th1, Th2 and Th17 cytokines. The work in this thesis contributes invaluable knowledge on the chicken innate immune system in response to Campylobacter and how similar host defence pathways to humans are utlised. The diversity found strongly indicates that a „one strain fits all‟ approach to in vitro and in vivo experimental infections may not give accurate results on Campylobacter pathogenesis. Campylobacter isolates can use host immune responses to its advantage and to move from the gut of chickens to infect edible tissues such as the liver. ii Declaration This work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature for any degree. Signed............ .............. Date 27/03/2019 This thesis is the result of my own investigations, except where otherwise stated. Where correction services have been used, the extent and nature of the correction is clearly marked. Other sources are acknowledged by footnotes giving explicit references. A bibliography is appended. Signed........................................................................Date 27/03/2019 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations following the expiry of the bar on access. Signed....................... ...... Date 27/03/2019. iii Contents Abstract .......................................................................................................................................... ii Declaration .................................................................................................................................... iii Acknowledgments .......................................................................................................................... x List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii Abbreviations ............................................................................................................................... xv Chapter 1: Introduction ................................................................................................................ 1 1.1 History of Campylobacter ................................................................................................... 1 1.2 Campylobacter .................................................................................................................... 1 1.3 Campylobacteriosis ............................................................................................................. 2 1.3.1 Human infections ......................................................................................................... 2 1.3.1 Chickens and Campylobacter ....................................................................................... 3 1.4 Campylobacter in different hosts ....................................................................................... 4 1.5 Campylobacter Genomics ................................................................................................... 4 1.6 Isolation and identification of Campylobacter .................................................................... 5 1.7 Virulence ............................................................................................................................. 6 1.7.1 Motility ......................................................................................................................... 6 1.7.2 Adhesion ...................................................................................................................... 8 1.7.3 Invasion and internalisation ......................................................................................... 9 1.7.4 Intracellular survival ................................................................................................... 13 1.7.6 Endotoxin and exotoxin production .......................................................................... 14 1.8 The Epithelial cell .............................................................................................................. 15 1.9 Mucosal Immunity ............................................................................................................ 16 1.9.1 The human gut ........................................................................................................... 16 1.9.2 The chicken gut .......................................................................................................... 17 1.10 Toll-Like Receptors and signalling ................................................................................... 20 1.10.1 Toll-like receptors and ligands ................................................................................. 20 1.10.2 Toll-like receptor signalling ...................................................................................... 21 1.11 Cytokines ........................................................................................................................
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