Secreted proteins in microsporidian parasites: a functional and evolutionary perspective on host-parasite interactions. Submitted by Scott Edward Campbell to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Biological Science. In September 2013 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from this thesis may be published without proper acknowledgment. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. Signature ……………………………………. Page| 1 Abstract The Microsporidia form a phylum of obligate intracellular parasites known to cause disease in humans and a diverse range of economically important animal species. Once classified as ‘primitive’ eukaryotes, it is now recognised that the peculiarities of microsporidian genomics and cell biology are, in fact, the consequence of extreme reduction allowed by an intimate relationship with the host cell. Excluding survival as an extracellular spore, microsporidia are in direct contact with the host throughout their developmental lifecycle, from entry to egress. Host cell manipulations have been described in morphological terms, but despite this, characterisation of such processes at the molecular level remains challenging. The logistics of the microsporidian lifecycle suggest secreted proteins and membrane proteins with extracellular domains may be involved in virulence and implicated in host cell manipulation. This study employs bioinformatic tools to predict secreted proteins in diverse microsporidia and comparative genomics to identify conserved proteins which may be required for host cell manipulation, pathogenicity and lifecycle progression. The protein complement secreted into the extracellular environment during microsporidian spore germination, a lifecycle stage required for host cell invasion, is identified experimentally. This analysis suggests that novel microsporidian specific hypothetical proteins, that is, proteins with no functional annotation or domain, play a significant role during parasite invasion of the host and provides the first identification of potential microsporidian effector proteins. Aiming to address microsporidian pathogenicity during intracellular stages, candidate virulence factor proteins, namely a hemolysin and a protein tyrosine phosphatase are also characterised and localised in situ. Lastly, an animal-derived horizontal gene transfer event is used in conjunction with both the fossil record and molecular dating approaches to add timescale to the microsporidian diversification. This work suggests that microsporidia radiated recently, achieving extreme cellular diversity, acquiring a novel infection mechanism and undergoing vast speciation in a short evolutionary timescale, likely within the last 200 million years. Page| 2 Table of contents List of Figures | page 8 List of Tables | page 11 Acknowledgements| page 13 Chapter 1| Introduction – page 14 1.1| An introduction to the microsporidia – 15 1.2| The microsporidian lifecycle: how is life on the inside? – 23 1.3| The microsporidian mitosome – 30 1.4| Microsporidian genomics: why less is sometimes more – 34 1.5| The impact of horizontal gene transfer on microsporidian evolution - 38 1.6| Microsporidian-host interactions and manipulations – 43 1.7| The eukaryotic secretory pathway – 46 1.8| Non-classical protein secretion – 52 1.9| Secreted proteins as virulence factors in parasitic protists – 53 1.10| Introduction to the current study – 58 Chapter 2| General Methods – page 61 2.1| Microsporidian spore purification – 62 2.2| DNA extraction – 62 2.3| RNA extraction – 63 2.4| Protein extraction – 64 2.5| Polymerase chain reaction (PCR) and plasmid ligation – 64 2.6| Bacterial transformation – 65 2.7| Plasmid purification and restriction digest – 66 2.8| Fungal transformation – 66 2.9| Tissue culture – 67 Page| 3 2.10| Transient transfection of mammalian cells – 68 2.11| Recombinant protein expression and purification – 68 2.12| SDS-PAGE and western blot – 69 2.13| Immuno-localisation – 70 2.14| In vitro spore germination – 71 Chapter 3| Dissection of the microsporidian secretory pathway: a minimal and unconventional model for protein export – page 73 3.1| Introduction – 74 3.2| Methods – 77 3.2.1| KEGG pathway analysis and database mining – 77 3.3| Results – 78 3.3.1| Protein secretion machinery in the microsporidia – 78 3.3.2| Retrograde protein transport and the fate of misfolded proteins – 84 3.4| Discussion – 88 Chapter 4| An investigation of protein secretion during Spraguea lophii germination – page 93 4.1| Introduction – 94 4.2| Methods – 98 4.2.1| Database mining and ortholog clustering – 98 4.2.2| RNA extraction and transcriptomic analysis of germinated spores – 99 4.2.3| Whole cell protein analysis of germinated and non-germinated spores – 99 4.2.4| Identification of proteins secreted extracellularly during spore germination – 100 4.3| Results – 102 4.3.1| The microsporidian secretome – 102 4.3.2| The transcriptome of germinated S. lophii spores – 107 Page| 4 4.3.3| Proteomic analysis of germination and secretion – 113 4.4| Discussion – 123 Chapter 5| E. cuniculi hemolysin III: Do pores mean prizes for an intracellular parasite? – page 127 5.1| Introduction – 128 5.2| Methods – 133 5.2.1| Phylogenetic analysis – 133 5.2.2| Cloning of E. cuniculi HlyIII for expression in E. coli – 134 5.2.3| Cloning of E. cuniculi HlyIII for expression in S. cerevisiae Izh2Δ – 134 5.2.4| Cloning of E. cuniculi HlyIII for transient mammalian expression – 135 5.2.5| Hemolysis assays – 135 5.2.6| Reverse Transcription PCR (RT-PCR) – 136 5.2.7| Functional complementation of EcHlyIII in S. cerevisiae Izh2Δ – 137 5.2.8| Recombinant expression and purification of E. cuniculi HlyIII – 137 5.2.9| Determining anti-HlyIII serum specificity – 139 5.2.10| Immuno-localisation of E. cuniculi HlyIII in situ – 139 5.2.11| Transient expression and immuno-localisation of E. cuniculi HlyIII in IMCD-3 cells – 140 5.2.12| List of oligonucleotides – 141 5.3| Results – 142 5.3.1| Evolutionary diversity of EcHlyIII orthologs – 142 5.3.2| Generation of EcHlyIII, S. cerevisiae Izh1 and S. cerevisiae Izh2 expression constructs – 147 5.3.3| Is EcHlyIII a divergent zinc transporter? – 150 5.3.4| EcHlyIII is capable of erythrocyte lysis in vitro – 153 5.3.5| EcHlyIII displays a nuclear localisation both in situ and in mammalian cells – 158 Page| 5 5.4| Discussion – 168 Chapter 6| Chitin and the origin of the Microsporidia – page 173 6.1| Introduction – 174 6.2| Methods – 180 6.2.1| Cloning of E. cuniculi endochitinase Cht1 in S. cerevisiae – 180 6.2.2| Over expression screen of E. cuniculi Cht1 in S. cerevisiae – 180 6.2.3| Chitin staining of E. cuniculi spores, germinated spores and meronts – 181 6.2.4| Database searching – 182 6.2.5| Phylogenetic analysis – 183 6.2.6| Molecular dating – 184 6.2.7| Prediction of protein localisation and domain searching – 185 6.2.8| List of oligonucleotides – 185 6.3| Results – 186 6.3.1| Chitin and morphological development of microsporidia - 186 6.3.2| E. cuniculi Cht1 displays chitin substrate specificity – 191 6.3.3| Horizontal gene transfer of Cht1 and the microsporidian radiation – 196 6.3.4| Prediction of functional domains and secretion of Cht1 orthologs – 203 6.4| Discussion – 205 Chapter 7| Characterisation of E. cuniculi tyrosine phosphatase Ppt1 and the impact of infection on the host phosphoproteome – page 209 7.1| Introduction – 210 7.2| Methods – 215 7.2.1| Cloning of EcPpt1 and S. cerevisiae Phs1 for expression in S. cerevisiae – 215 7.2.2| Cloning of EcPpt1 for expression in IMCD-3 cells – 216 Page| 6 7.2.3| Functional complementation and GFP localisation of EcPpt1 in S. cerevisiae – 216 7.2.4| Anti-EcPpt1 polyclonal antibody production and verification of serum specificity - 217 7.2.5| Localisation of EcPpt1 in situ in E. cuniculi infected RK-13 cells – 218 7.2.6| Localisation of EcPpt1 in IMCD-3 cells – 219 7.2.7| Phosphoproteomic analysis of E. cuniculi infected IMCD-3 cells – 219 7.2.8| List of oligonucleotides – 220 7.3| Results – 221 7.3.1| Generation of EcPpt1 and S. cerevisiae Phs1 expression constructs - 221 7.3.2| Functional complementation and GFP localisation of EcPpt1 in S. cerevisiae - 223 7.3.3| EcPpt1 localises to the cell membrane in situ and in mammalian cells - 227 7.3.4| The impact of E. cuniculi infection on the host cell phosphoproteome - 234 7.4| Discussion – 241 Chapter 8| General discussion – page 245 References – page 253 Appendix 1| Recipes for standard reagents and media - 291 Appendix 2| A full list of proteins identified in whole cell protein extracts from germinated and non-germinated spores – page 295 Appendix 3| E. cuniculi infection and the host phosphoproteome: a quantitative comparison of protein phosphorylation between non- infected IMCD-3 cells and E. cuniculi infected IMCD-3 cells – page 298 Appendix 4| Campbell, S. E., T. A. Williams, A. Yousuf, D. M. Soanes, K. H. Paszkiewicz, B. A. P. Williams (2013). The genome of Spraguea lophii and the basis of host-microsporidian interactions. PLoS Genet 9(8) – page 313 Page| 7 List of Figures Chapter 1| Introduction – page 14 Figure 1.1| Taxonomic reclassification of the microsporidia across time - 22 Figure 1.2| Overview of the microsporidian lifecycle – 29 Figure 1.3| Metabolism and import in the microsporidian mitosome – 33 Figure 1.4| Comparison of microsporidian genome size to model and parasitic eukaryotes and examples of microsporidian genome reduction mechanisms – 37 Figure 1.5| Summary of horizontal gene transfer in microsporidia – 42 Figure 1.6| Structure and properties of the eukaryotic signal peptide – 51 Chapter 4| An investigation of protein secretion during Spraguea lophii germination – page 93 Figure 4.1| Predicted secretome size in diverse microsporidia – 105 Figure 4.2| Number of species-specific secreted proteins – 106 Figure 4.3| Aggregations of individual S.