PROTEOMIC AND TRANSCRIPTOMIC ANALYSIS OF 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 Sarah J Vermont September 2012 AUTHOR’S DECLARATION Apart from help and advice acknowledged, this thesis represents the unaided work of the author ..................................... Sarah J Vermont September 2012 ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisors, Professor Jonathan Wastling and Dr. Andy Jones, not only for accepting me as a student, but for all their expertise, advice and support along the way. They have not been the only people to whom I have turned for help: to everyone (past and present) in Infection Biology; Nadine, Sanya, Beccy, Sophie, Stu, Dong, Andy, Hos, Sarah T, Corrado, Mariwan and Poom; a massive thank you. Similarly, to members of the PFG group and especially Duncan, thank you for your patience and understanding in mass spectrometry assistance! Without Gianluca, Achuchuthan, Kat, Ritesh and the rest of the Bioinformatics group I could never had understood my data. A special thank you is due to Dr. Iain Young, not only for inspiring me to attempt a PhD in the first place, but also for being a fantastic and supportive boss in my three years as a halls tutor. My PhD has involved collaborations on a number of projects: Professor Arnab Pain, Adam, Peter, Professor Andrew Hemphill, Thierry; thank you all for your involvement and fruitful discussions. To Steffi and Professor Jonathan Howard, thank you for making me welcome in your labs in Cologne and for all your help with the ROP18 work. Thank you also to the BBSRC and Intervet for providing the funding that allowed me to carry out this work and to the University of Liverpool for 4 more brilliant years. My partners in crime, Emma and Jenna, along with Alex and Monika, have made studying for my PhD especially enjoyable and entertaining; Maz and Luke have been so generous in letting me invade their home these last few weeks - thank you all. Finally, my parents have provided unending support and encouragement and have provided a home to my continually expanding family of horses and other animals, without all of whom to distract me at times and provide escapism; I would surely never have managed. So, Mum and Dad, I can never thank you enough. Proteomic and transcriptomic analysis of the protozoan parasite Neospora caninum Sarah J Vermont ABSTRACT Neospora caninum is an economically significant parasitic protozoan causing the disease neosporosis in cattle and dogs. Although a close relative of the zoonotic apicomplexan Toxoplasma gondii, the two organisms exhibit differing host ranges and infection dynamics. T. gondii is a model organism that has been much studied, and a great deal is known about the genes and proteins involved when it invades a host cell. This thesis explores protein expression in the proliferative and invasive tachyzoite stage of N. caninum, in particular the expression of proteins pertaining to the apical complex of organelles; those responsible for entry and establishment within a host cell. Almost 20 % of the predicted proteome has been identified by this analysis to be expressed in the tachyzoite stage, with approximately 50 % of the predicted repertoire of apical proteins being detected. The discovery of differences between these two parasites’ highly syntenic genomes could lead to a better understanding of the process by which T. gondii is able to cause disease in humans, while N. caninum has not been observed to do so. One finding of the recent genome sequencing and annotation project in N. caninum was that a key T. gondii virulence determinant, rhoptry gene 18 (ROP18) was pseudogenised in N. caninum. This finding was investigated further in this thesis to demonstrate that the pseudogenisation of ROP18 was conserved across a range of N. caninum isolates and that in vitro, N. caninum was not able to subvert the murine interferon-gamma (IFN-γ) immune response using ROP18 in the way that virulent T. gondii tachyzoites do. The tissue-dwelling Coccidia have a multi-stage life cycle which includes a latent tissue cyst- encapsulated stage called the bradyzoite. Tachyzoites convert to this more quiescent form when induced by cellular stress, and are able to remain as such for long periods, even years. At times of weakened host immunity, bradyzoites can recrudesce to produce an active infection, which can cross the placenta in a pregnant animal to infect the foetus. This a major route by which N. caninum infection is maintained within cattle herds, therefore the biology of stage conversion from tachyzoite to bradyzoite and vice-versa is of interest to researchers. An RNA- Seq analysis of cultured tachyzoites and bradyzoites identified a marked reduction in rhoptry gene expression, and differing expression profiles of other invasion-related genes from the micronemes and dense granules. Overall, these data identify proteins released from the apical organelles in N. caninum and give an insight into the different repertoires expressed by the tachyzoite and bradyzoite life stages. Furthermore, a comparison between N. caninum and T. gondii predicted apical proteomes indicates that although most genes are shared in a one-to-one orthologous relationship between the two organisms, there are a small number of differences which may turn out to be important to the biology of the parasite, as in the case of ROP18. CONTENTS List of figures List of tables List of abbreviations CHAPTER 1: INTRODUCTION 1.1 Neospora caninum 1 1.2 Lifecycle and epidemiology 1 1.3 Economic impact of neosporosis 3 1.4 Host cell invasion and the apical complex 4 1.4.1 Micronemes 6 1.4.2 Rhoptries 7 1.4.3 Dense granules 8 1.5 Neospora and Toxoplasma – similarities and differences 8 1.6 Host immune response and pathology 10 1.7 Proteomics 11 1.7.1 Electrophoresis 11 1.7.2 Reverse-Phase High Performance Liquid Chromatography (LC) 12 1.7.3 Tandem mass spectrometry (MS/MS) 12 1.7.4 Multidimensional protein identification technology (MudPIT) 13 1.7.5 Bioinformatics – Peptide/Protein Identification 13 1.8 Transcriptomics 14 1.8.1 RNA-Seq 14 1.9 Current status of the N. caninum genome, proteome and transcriptome 15 1.10 Aims and objectives 15 CHAPTER 2: AN ANALYSIS OF THE TACHYZOITE PROTEOME 2.1 Introduction 18 2.1.1 Global proteomic analyses on N. caninum and related Apicomplexa 18 2.1.2 Peptide data as an aid to genome annotation 19 2.1.3 Aims and objectives 20 2.2 Materials and methods 21 2.2.1 Cell culture 21 2.2.1.1 Host cell passage 21 2.2.1.2 Parasite passage 21 2.2.1.3 Parasite isolation 21 2.2.2 Proteomic analyses 22 2.2.2.1 Preparation of tachyzoite lysates 22 2.2.2.3 One dimensional electrophoresis (1-DE) 22 2.2.2.4 In-gel tryptic digestion 23 2.2.3 Reverse-phase high performance liquid chromatography and tandem mass spectrometry (LC MS/MS) 23 2.2.4 Multidimensional protein identification technology (MudPIT) 24 2.2.5 Analysis of tachyzoite lysate on an Orbitrap Velos mass spectrometer 25 2.2.5.1 In-solution digestion and analysis 25 2.2.5.2 1-DE and in-gel digestion 25 2.2.5.3 Orbitrap Velos mass spectrometry 26 2.2.6 Protein identification using multiple search engines 26 2.2.7 Assignment of proteins to MIPS Functional Catalogue categories 26 2.2 Results 28 2.3.1 One dimensional gel electrophoresis (1-DE) and mass spectrometry (LC MS/MS) analysis of whole tachyzoite lysate 28 2.3.2 Multidimensional proteomic identification technology (MudPIT) analysis of whole tachyzoite lysate 30 2.3.3 Analysis of tachyzoite lysate using an Orbitrap Velos mass 32 spectrometer 2.3.3.1 In-solution analysis 32 2.3.3.2 1-DE separation prior to LC MS/MS 33 2.3.4 OFFGEL separation 34 2.4 Discussion 35 CHAPTER 3: BIOINFORMATIC MINING OF THE GENOME FOR PREDICTED APICAL PROTEINS 3.1 Introduction 39 3.1.1 Definitions 39 3.1.2 ToxoDB 39 3.1.3 The Basic Local Alignment Search Tool (BLAST) 40 3.1.4 Aims and objectives 42 3.2 Materials and methods 43 3.2.1 Identification of potential apical genes 43 3.3 Results and discussion 44 3.3.1 Microneme genes 44 3.3.2 Rhoptry genes 49 3.3.3 Dense granule genes 55 3.3.4 Discussion of bioinformatic resources 59 3.3.5 Requirement for experimental validation 59 CHAPTER 4: PROTEOMIC ANALYSIS OF THE APICAL ORGANELLES 4.1 Introduction 60 4.1.1 Excretory/secretory antigen (ESA) analysis 60 4.1.2 Rhoptry/dense granule - enriched fraction analysis 62 4.1.3 Aims and objectives 62 4.2 Materials and methods 63 4.2.1 Excretory/secretory antigen analysis 63 4.2.1.1 Preparation of ESA material from parasite cultures 63 4.2.1.2 Tricarboxylic acid (TCA) precipitation of ESA 63 4.2.1.3 One dimensional gel electrophoresis (1-DE) of ESA 63 4.2.2 Rhoptry (R) and rhoptry/dense granule (R/DG) fractions 64 4.2.2.1 Preparation of rhoptry and rhoptry/dense granule fractions 64 4.2.2.2 TCA precipitation of R and R/DG fractions 65 4.2.2.3 1-DE of R and R/DG fractions 65 4.2.3 Trypsin digestion of samples for mass spectrometry 65 4.2.4 Liquid chromatography and tandem mass spectrometry analysis (LC MS/MS) 66 4.2.5 Bioinformatic Analyses 66 4.2.5.1 Protein identification using Mascot 66 4.2.5.2 Assignment of proteins to MIPS Functional Catalogue categories 66 4.2.5.3 Signal peptide predictions 67 4.3 Results 68 4.3.1 Analysis of excretory/secretory antigens from N.