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RNA interference in parasitic nematodes – from genome to control Thomas Tzelos Thesis submitted for the degree of Doctor of Philosophy to The College of Medicine and Veterinary Medicine The University of Edinburgh Research was carried out at Moredun Research Institute Doctor of Philosophy – The University of Edinburgh – 2014 Contents Declaration ................................................................................................................... i Acknowledgements ..................................................................................................... ii Abstract ...................................................................................................................... iii Chapter 1: Review of the literature .......................................................................... 1 1.1 Parasites ........................................................................................................ 1 1.2 The parasitic nematode Teladorsagia (formerly Ostertagia) circumcincta 1 1.2.1 Taxonomical classification and life cycle ................................................ 1 1.2.2 Clinical implications and pathophysiology .............................................. 4 1.2.3 Control of the parasitism .......................................................................... 6 1.3 Host immunity ............................................................................................ 12 1.4 Vaccination as an alternative control strategy and vaccine development strategies ................................................................................................................. 16 1.4.1 Radiation attenuated worms as vaccines ................................................ 17 1.4.2 ‘Hidden’ antigens as vaccine targets ...................................................... 19 1.4.3 Extracellular microvesicles as novel vaccine targets ............................. 20 1.4.4 Excretory/Secretory molecules and surface antigens as vaccine targets 22 1.5 Activation-associated Secreted Proteins in parasitic nematodes ............... 24 1.5.1 Features of ASPs .................................................................................... 25 1.5.2 ASPs of nematodes ................................................................................ 26 1.5.3 ASPs as vaccine candidates ................................................................... 28 1.5.4 Function of the ASPs ............................................................................. 30 1.6 RNA interference ....................................................................................... 31 1.6.1 History and discovery of RNA interference .......................................... 31 1.6.2 RNAi pathway ........................................................................................ 32 1.6.3 RNAi in parasitic nematodes ................................................................. 35 1.7 Aims of the project ..................................................................................... 37 Chapter 2: Marker genes that indicate the activation of the RNA interference pathway ..................................................................................................................... 38 2.1 Introduction ................................................................................................ 38 2.2 Materials and methods ............................................................................... 40 2.2.1 In vitro culture of Caenorhabditis elegans ............................................ 40 2.2.2 RNAi target gene selection, dsRNA preparation and establishing a successful RNAi protocol .................................................................................. 41 2.2.3 Development of the RNAi pathway marker genes ................................ 44 2.3 Results ........................................................................................................ 48 2.3.1 Establishing a successful RNAi protocol ............................................... 48 2.3.2 Assessment of the potential RNAi pathway marker genes .................... 51 2.4 Discussion .................................................................................................. 56 Chapter 3: An analysis of the Activation-associated Secreted Proteins in T. circumcincta .............................................................................................................. 60 3.1 Introduction ................................................................................................ 60 3.2 Materials and methods ............................................................................... 63 3.2.1 Searching for SCP-like extracellular proteins in parasitic nematodes’ transcriptome ...................................................................................................... 63 3.2.2 SCP-like extracellular proteins in T. circumcincta ................................ 63 3.2.3 Comparison of exsheathment methods: sodium hypochlorite (NaClO) vs. CO2 ............................................................................................................... 64 3.2.4 Stage-specific end-point PCR to determine the transcriptional pattern of the ASPs with a signal peptide ........................................................................... 65 3.3 Results ........................................................................................................ 68 3.3.1 Searching for SCP-like extracellular proteins in the parasitic nematodes 68 3.3.2 SCP-like extracellular proteins in T. circumcincta ................................ 70 3.3.3 Comparison of exsheathment methods (NaClO and CO2) and their effect on detection of Tci-asp-1transcript by RT-PCR ................................................ 76 3.3.4 Stage specific PCR ................................................................................. 76 3.4 Discussion .................................................................................................. 78 Chapter 4: RNA interference in the parasitic nematode T. circumcincta ........... 82 4.1 Introduction ................................................................................................ 82 4.2 Materials and methods ............................................................................... 84 4.2.1 T. circumcincta material for RNAi experiments .................................... 84 4.2.2 Design and production of gene specific dsRNA .................................... 85 4.2.3 RNAi in T. circumcincta ........................................................................ 87 4.2.4 Transcriptional pattern of Tci-asp-1 and immunoblotting of somatic extracts using anti-rTci-ASP-1 sera after exsheathment by CO2 ....................... 89 4.2.5 Tci-asp-1 transcript recovery after successful RNAi ............................. 90 4.2.6 Searching for the essential RNAi pathway genes in T. circumcincta’s genome ............................................................................................................... 91 4.2.7 Testing dsRNA delivery by soaking larvae in fluorescently labeled RNA (siRNA) and dsRNA .......................................................................................... 92 4.2.8 Testing the efficacy of RNAi after soaking xL3 to heterogeneous small interfering RNA (siRNA) ................................................................................... 92 4.2.9 Testing the effect of storage period of the sL3 on the RNAi efficacy ... 93 4.2.10 Effect of RNAi with hsiRNA on ‘fresh’ larvae ................................. 94 4.3 Results ........................................................................................................ 94 4.3.1 RNAi in T. circumcincta ........................................................................ 94 4.3.2 Transcriptional pattern of Tci-asp-1 and immunoblotting of somatic extracts using anti-rTci-ASP-1 sera after exsheathment by CO2 ....................... 95 4.3.3 Tci-asp-1 transcript recovery after successful RNAi ............................. 98 4.3.4 Searching for potential reasons for the RNAi inconsistencies ............. 100 4.3.5 Effect of RNAi with hsiRNA in ‘fresh’ larvae .................................... 109 4.4 Discussion ................................................................................................ 110 Chapter 5: Characterization of exosomes in T. circumcincta ............................ 115 5.1 Introduction .............................................................................................. 115 5.2 Materials and methods ............................................................................