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Comparative Genomic Analyses of Corynebacterium Pseudotuberculosis

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Comparative Genomic Analyses of Corynebacterium Pseudotuberculosis Comparative genomic analyses of Corynebacterium pseudotuberculosis Florence Elizabeth Pethick B.Sc. (Hons) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy at the Faculty of Veterinary Medicine, University of Glasgow May 2013 © Florence Elizabeth Pethick 2013 To Mum and Dad with love and thanks... ...& to Grandpa, whom I know has been beside me through every page. ii Abstract This study set out to sequence the genome of Corynebacterium pseudotuberculosis (Cp) 3/99-5, an ovine strain isolated from a naturally-occurring case of caseous lymphadenitis (CLA) in Scotland. The isolate was sequenced and assembled by 454 Life Sciences, and then gap closure performed by ‘PCR bridging’. The resulting sequence consisted of three contigs with a length of 2,319,079 bp and a G+C content of 52.18%. The genome was then annotated and predicted to contain 2,153 coding sequences. Analysis of the coding sequences revealed the presence of several putative virulence factors, including four sortases with multiple sortase target proteins containing LPXTG motifs. A further two Cp strains, an Australian ovine and a North American equine isolate, as well as C. ulcerans NCTC 12077 were sequenced for comparison. Comparative genomics, both intra- and inter-species showed all the genomes to be highly homologous. However, the C. ulcerans genome is larger than the Cp genomes and is more distinct; it was found to be more similar to the equine Cp 1/06-A isolate which is the most diverged of the Cp isolates. Phylogenetic analyses of the Corynebacterium genus were performed using house-keeping loci but also secreted protein loci from Cp 3/99-5. Bayesian analysis of house-keeping loci distinguished the bacteria to a species level. Inclusion of secreted protein loci did not distinguish the isolates any further. The main objective of this work was to utilise the Cp genome sequence to identify potential diagnostic targets which could be used to augment the available ELITEST CLA or replace it. The ELITEST CLA is the only diagnostic test for CLA that exists on the commercial market in the UK. However, due to low specificity and sensitivity, it is only operated on a flock/group basis. Analyses of the Cp 3/99-5 genome identified several potential diagnostic candidates and seven protein targets were investigated further. Attempts were made to express these candidates as recombinant proteins, however, only two recombinants were successfully expressed and purified, Cp3995_0570 and CP40. The seroreactivity of these were then assessed by IgG ELISA using a panel of ten positive and ten negative CLA ovine sera. The sera were previously defined as positive or negative by PLD and whole cell ELISAs; both of which showed a significant difference between sera types. However, neither Cp3995_0570 nor CP40 distinguished between sera originating from Cp-infected and Cp-naïve animals. iii Declaration The work reported in this thesis was carried out under the supervision of Dr Michael C. Fontaine and Dr Alex F. Lainson at the Moredun Research Institute and Professor David G. E. Smith at the Moredun Research Institute and the Institute for Infection, Immunity & Inflammation, University of Glasgow. All results presented, unless otherwise stated, are the sole work of this author, as is the composition of this thesis. Signed: Date: iv Acknowledgments First and foremost I would like to thank my supervisors, Prof David G.E Smith, Dr Alex Lainson and Dr Mike Fontaine for their guidance throughout this project. In particular thanks go to Alex but also Raja Yaga and Dushyanth Jyothi for introducing me to the ever- expanding world of bioinformatics. I would like to thank Dr Frank Wright for phylogenetics assistance and for helpful and informative discussions. Thanks must also go to Quality Meat Scotland, The English Beef and Lamb Executive and Hybu Cig Cymru (Meat Promotion Wales) who funded this work. Importantly I would like to thank both past and present members of lab. 3.060, particularly Colin and Allen for their technical help, but also Sabrina, Anita, Tammy, Eleanor, Pam, Scott, Craig and Chris for making those long days in the lab much more enjoyable. Thanks also go to my fellow PhD students who were always a valuable source of encouragement and advice especially Sam, Heather and Cassie. More recently I would like to thank my colleagues at the University of Strathclyde for their endless support. Huge thanks especially go to Gillian, Mark, Richard and Vartul for their encouragement at a time when completion of the thesis seemed unreachable. I owe thanks to all the girls at Edinburgh Civil Service Ladies Hockey Club for their support, and helping me to vent when it was much needed. Heartfelt thanks must go to my friends and family, particularly my Mum and Dad, who have always been incredibly supportive and understanding, not to mention patient. Their numerous phone calls have helped through the troublesome stages and they have constantly seen the potential in me to achieve something which at times, I did not think I could, but which I am now proud of. Last but not least I would like to say a massive thank you to John for his support, understanding, love and kindness. But most of all, I thank him for keeping me calm amd strong and pushing me to stick with it when things got tough, coining the term ‘TUP’ in the process. v Contents Abstract ........................................................................................................................... iii Declaration ...................................................................................................................... iv Acknowledgments ............................................................................................................ v Contents .......................................................................................................................... vi List of Figures ................................................................................................................. ix List of Tables .................................................................................................................. xi Abbreviations................................................................................................................. xii 1. Chapter One: General Introduction ...................................................... 1 1.1 The genus Corynebacterium .................................................................................. 2 1.1.1 General characteristics of the genus Corynebacterium .................................................................. 2 1.1.2 Notable Corynebacteria .................................................................................................................. 3 1.2 Corynebacterium pseudotuberculosis .................................................................... 5 1.2.1 Phenotypic characterisation ............................................................................................................ 5 1.2.2 Virulence factors of C. pseudotuberculosis .................................................................................... 7 1.2.3 Diseases caused by Corynebacterium pseudotuberculosis ............................................................. 9 1.3 Caseous lymphadenitis ........................................................................................ 11 1.3.1 Introduction .................................................................................................................................. 11 1.3.2 Distribution and prevalence of CLA ............................................................................................ 11 1.3.3 Economic significance of CLA in sheep ...................................................................................... 12 1.3.4 Pathogenesis and transmission ..................................................................................................... 13 1.4 Treatment of CLA in sheep ................................................................................ 16 1.5 Vaccines against CLA ......................................................................................... 17 1.5.1 Bacterin vaccines .......................................................................................................................... 17 1.5.2 Toxoid vaccines ............................................................................................................................ 18 1.5.3 Combined vaccines ....................................................................................................................... 18 1.5.4 Live vaccines ................................................................................................................................ 19 1.5.5 DNA vaccines ............................................................................................................................... 20 1.6 Diagnosis of CLA ................................................................................................. 21 1.6.1 Conventional identification .......................................................................................................... 21 1.6.2 Analytical Profile Index (API) Identification ............................................................................... 22 1.6.3 Serological tests ............................................................................................................................ 23 1.7 Population biology ..............................................................................................
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