The effects of development and virulence on gene expression in the protozoan parasite Neospora caninum Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Kittichai Unjit February 2018 AUTHOR’S DECLARATION Apart from help and advice acknowledged, this thesis represents the unaided work of the author …………………………………………………. Kittichai Unjit February 2018 This research was carried out in the Department of Infection Biology and School of Veterinary Science, University of Liverpool i DEDICATION I want to dedicate this thesis to my parents, brother and sister who have always supported me all my entire life especially during my PhD study. All of you are the greatest. Thank you very much for being with me all the times. ii ACKNOWLEDGEMENTS First of all, I would like to express my gratitude to Thai Government for providing me a scholarship to study aboard. I would like to thank my supervisors, Dr. Andrew Jackson and Professor Jonathan Wastling for accepting me to be a student. They always support and guide me during my PhD study to achieve my goal. Additionally, I would like to thank Dr. Nadine Randle and Dr. Dong Xia for their kindly help and support throughout my study period. Special Thanks to Dr. Nick Evans for accepting me to be his student for a certain time. I also thank to an advisory teams as Dr. Ben Makepeace and Dr.Janine Coombes for their guidance and suggestions in a research study. I would like to deeply appreciate Professor Luis Ortega-Mora for providing Neospora samples in this thesis and a good collaboration. Thanks to Prof. Andrew Hemphil, University of Bern, Switzerland for providing BAG1 antibody and Dr. Olivier Touzelet for providing a secondary antibody Alexafuor 488 for IFA test. Special thanks to Mrs.Catherine Harley, Dr. Nathifa Moyo and Mrs.Jenna Dawson for their help and support for the laboratory work. I would also like to thank Dr. Stuart Armstrong, Dr.Vicky Hunt, Dr.Gareth Weedall, Dr.Luca Lenzi, Dr. Sam Haldenby, Ross Low and Dr. Pisut Pongchaikul for suggesting me about bioinformatics. Many thanks to colleagues in Department of Infection Biology for having fun and supporting; Tonk, Poomie, Dr. Juriah, Dr. Dashty, Dr.Mariwan, Dr.Sarah, Dr.Corrado, Dr.Patrick, Dr.Fazila, Zuliza, Intan, Sanaria and the others. Finally, I would like to thank a group of Thai friends in Thai society in Liverpool and Chaba Chaba Thai restaurant for hospitality and friendships which make me feel like at home. iii ABSTRACT The effects of development and virulence on gene expression in the protozoan parasite Neospora caninum Kittichai Unjit Neospora caninum is an obligate intracellular protozoan parasite which causes abortion in cattle and neuromuscular disease in dogs. Neosporosis causes substantial economic losses in both dairy and beef cattle industries worldwide. This thesis explores changes in genes and proteins expression associated with development and virulence of N. caninum in order to gain more biological information in this parasite. To achieve the global quantification of genes and proteins expression under different phenotypic conditions, transcriptomics and proteomics approaches are used to investigate these phenomenons. Chapter 2 examines the differential protein expression during tachyzoites- bradyzoites stage conversion using a label-free proteomics approach and finds that the differential protein expression of most apical secretory proteins decrease in abundance in bradyzoites stage. In addition, most of proteins associated with parasite motility were also reduced in abundance in bradyzoites. This indicates that proteins associated with host cell adhesion, invasion and gliding motility are down-regulated in the quiescent cyst-forming stage. Chapter 3 compares the transcriptomic profiles of low- and high-virulence N. caninum using RNA-Seq and finds that protein phosphatase 2C (PP2C) was preferentially expressed in the high-virulence strain, while a group of SRS family proteins was preferentially expressed in low-virulence conditions. This finding suggests that PP2C might play a role in virulence, while a group of SRS proteins might be associated with to limiting N. caninum virulence. Chapter 4 compares protein expression in low- and high-virulence N. caninum strains at three different time points using a label-free quantitative proteomic approach and iv finds that Rop24 was preferentially expressed in high-virulence N. caninum at all time points. Proteins associated with host cell attachment such as SAG3, TgSRS35A and cathepsin C2 also showed preferentially expression throughout the time course. This finding indicates that Rop24 might be associated with the N. caninum virulence, while the discovery of proteins associated with host cell attachment in low-virulence conditions might suggest that such strains are more efficient in invading host cells than high-virulence strains. Overall, this thesis identifies genes and proteins such as the rhoptry associated proteins PP2C and Rop24, which might be associated with virulence in neosporosis. It also shows that many proteins involved in host cell adhesion, invasion and parasite movement decrease in abundance in the cyst-forming bradyzoite stage. These discoveries enhance our understanding of parasite biology, providing a basis for future research into novel ways to prevent and control the disease by inhibiting parasite survival and transmission. v TABLE OF CONTENTS AUTHOR’S DECLARATION ..................................................................................... i DEDICATION ............................................................................................................. ii ACKNOWLEDGEMENTS ........................................................................................ iii ABSTRACT ................................................................................................................ iv TABLE OF CONTENTS ............................................................................................ vi LIST OF FIGURES ................................................................................................... xii LIST OF TABLES .................................................................................................... xiii LIST OF ABBREVIATIONS .................................................................................. xvii Chapter 1: Introduction ................................................................................................ 1 1.1 Neosporosis ........................................................................................................ 2 1.2 Life cycle of N. caninum .................................................................................... 4 1.3 Mode of transmission in cattle ........................................................................... 5 1.4 N. caninum cellular structure and function ......................................................... 6 1.4.1 The tachyzoite stage ..................................................................................... 7 1.4.2 The bradyzoite stage .................................................................................... 7 1.4.3 The sporozoite stage ..................................................................................... 8 1.5 Stage conversion from tachyzoites to bradyzoites stage of N. caninum ............ 8 1.6 Host cell invasion and N. caninum apical complex ............................................ 9 1.6.1 Micronemes ................................................................................................ 10 1.6.2 Rhoptries .................................................................................................... 11 1.6.3 Dense granules ........................................................................................... 11 1.7 The virulence of Toxoplasma gondii ................................................................ 12 1.8 The virulence of Neospora caninum ................................................................ 15 1.9 Economic impact of bovine neosporosis .......................................................... 18 1.10 Omics Approaches to investigate gene and protein expression ..................... 19 vi 1.10.1 Transcriptomics ........................................................................................ 19 1.10.1.1 Real-time quantitative PCR (qPCR) ................................................. 19 1.10.1.2 Microarrays ....................................................................................... 19 1.10.1.3 RNA-Sequencing (RNA-Seq) ........................................................... 20 1.10.2 Proteomics method ................................................................................... 21 1.10.2.1 Gel-based proteomics ........................................................................ 21 1.10.2.2 Non-Gel-based proteomics................................................................ 21 1.10.2.2.1 Label-based quantitative proteomics.............................................. 22 1.10.2.2.2 Label-free quantitative proteomics ................................................. 22 1.10.3 Reverse-phase high performance liquid chromatography (LC) ............... 23 1.10.4 Tandem mass spectrometry (MS/MS) ..................................................... 24 1.10.5 Protein identification and bioinformatics analysis ................................... 24 1.10.6 Omics and apicomplexan parasites .........................................................