Phd Thesis Ching-Cheng Huang Imperial College London

Development of RNA interference

in parasitic nematodes

Ching-Cheng Huang

This thesis submitted for the degree of Doctor of Philosophy in Imperial College London and for the Diploma of Imperial College

Division of Cell and Molecular Biology Imperial College London

April 2010

Abstract

Exploitation of RNA interference (RNAi) has revolutionised work on Caenorhabditis elegans, and considerable success has been recently reported with plant-parasitic nematodes. It has proven difficult to transfer this technology to animal parasitic species, and previous attempts in this laboratory by feeding Nippostrongylus brasiliensis larvae with Escherichia coli expressing double-stranded RNA (dsRNA) gave no consistent reductions in levels of target transcripts. The aim of this study was to develop methods for RNAi in N. brasiliensis, a rodent strongylid nematode which is closely related to gastrointestinal nematodes of humans and livestock, in order to explore the biological functions of parasite secreted proteins.

In order to promote uptake of exogenous macromolecules such as dsRNA and small interfering RNA (siRNA) by infective larvae, the process of activation, whereby larvae are induced to resume feeding and development, was studied in vitro. Activation could be induced solely by exposure of larvae to elevated temperature (37oC), whereas host serum or glutathione had no effect. Neither a membrane permeant analogue of cyclic GMP nor muscarinic receptor agonists promoted activation, suggesting that a cholinergic neural pathway is not involved in the process. Activation at 37oC could be blocked by inhibitors of phosphatidylinositol 3-kinase, Akt protein kinase & cytochrome P450. These data indicate that the early signalling events for larval activation in N. brasiliensis differ substantially from the hookworm Ancylostoma caninum, most probably acting through thermosensory rather than chemosensory neurons, but that they may converge downstream of a step dependent on phosphatidylinositol 3-kinase. Stimulation of protein secretion paralleled resumption of feeding, suggesting that these processes were tightly linked and regulated by similar pathways during activation.

Temperature-activated larvae were exposed to dsRNA and siRNA via electroporation or soaking in the presence of a variety of transfection reagents, but RNAi was unsuccessful. Serotonin was demonstrated to increase the rate of uptake of macromolecules, yet larvae exposed to exogenous dsRNA in the presence of serotonin still failed to show RNAi-mediated gene silencing. In addition, RNAi was also observed to be irreproducible in adult N. brasiliensis using the same methods of delivery. The use of mRNA encoding firefly luciferase identified uptake into larvae or adult worms as a major impediment in the process.

Venom Allergen Homologue/ASP-Like (VAL) molecules have been identified as major components of secreted proteins from many species of parasitic nematodes. In this study, eight N. brasiliensis VALs were identified and sequenced, and all were shown to be present in products secreted by adult parasites. NbVAL-7 and NbVAL-8 were also detected in secreted products of infective larvae. Structural similarities to other members of the Pathogenesis- Related protein superfamily are discussed, as are possible functions for these proteins in N. brasiliensis.

Acknowledgements

I would like to express my sincere gratitude to my supervisor Prof. Murray Selkirk for his excellent guidance, critical comments and continuing support along the journey towards completion of this thesis. Also, I would like to thank Dr. Niki Gounaris for her thoughtful and valuable suggestions of my research.

I deeply appreciate my best friend, Dr. Dominic Rees-Robert from Selkirk/Gounaris Lab, who not only introduced me various techniques, and provided me useful ideas, but also influenced me being familiar with the best part of Britain (pub drinking). Furthermore, I thank the past members of the lab, Drs. Lisa Mullen, David Guiliano, Holly Afferson, Emily Glyde, Glyn Ball and Min Zhao, for their greatest helps and advises on my experiments. Additionally, my thankyou also goes to the present members, Denice Chan, Emily Eleftheriou and Jennifer Lozano, for giving me so much fun over these years.

I would like to thank Ya-Fan Chang who always has been there for me through thick and thin. The major appreciation goes to my parents, to whom this thesis is dedicated, who have provided me with much support and encouragement throughout.

Finally, thanks to Imperial College Overseas Research Student (ORS) Scholarship for the financial support.

Contents

Title page 1 Abstract 2 Acknowledgements 3 Contents 4 List of figures 7 List of tables 10 Abbreviations 11

Chapter 1 General Introduction 17 1.1. Taxonomy of nematodes 18 1.2. Epidemiology and control of gastrointestinal nematode parasites 20 1.3. Nippostrongylus brasiliensis 22 1.4. Infection and invasion by parasitic nematodes 24 1.5. Modulation of the immune response by nematode secreted proteins 25 1.6. Host protective immunity to N. brasiliensis infection 30 1.7. Secreted proteins of N. brasiliensis 37 Acetylcholinesterases 37 Platelet-activating factor (PAF) acetylhydrolase 39 Cystatins 39 Globins 41 1.8. Caenorhabditis elegans as a model organism 41 1.9. Brief history of RNA interference 43 1.10. Overview of RNAi in C. elegans 44 1.11. Methods for the induction of RNAi silencing 49

Chapter 2 Materials and Methods 54 2.1. Parasites 55 Infection of rats and recovery of N. brasiliensis adult worms 55 Development and recovery of larval stages 55 In vitro culture of parasites and collection of excretory-secretory (ES) products 55 Preparation of parasite somatic extracts 56 2.2. Culture of 293 T cells and J774 macrophages 56 2.3. Proteins 56 Determination of protein concentration 56 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 57 Coomassie blue staining of polyacrylaminde gels 57 Western blotting 57 2.4. RNA extraction 58 2.5. Polymerase Chain Reaction (PCR) 59 2.6. Reverse Transcription PCR (RT-PCR) 59

2.7. Production of firefly luciferase-encoding mRNA 60 2.8. Production of double-stranded (ds)RNA 60 2.9. Production of small interfering (si)RNAs 60 2.10. Rapid amplification of 5’ and 3’ cDNA ends (5’-RACE and 3’-RACE) 61 5’-RACE 61 3’-RACE 62 2.11. Iodination of bovine serum albumin (BSA) 64 2.12. Metabolic labelling of parasite proteins in vitro 64 Secreted products 64 Parasite somatic extracts 64 2.13. Measurement of N. brasiliensis acetylcholinesterase (NbAChE) activity from parasite secreted products 65 2.14. Analysis of AChE activity in native polyacrylamide gels 65 2.15. Exsheathment of infective larvae 66 2.16. Feeding assay 66 2.17. Feeding assay using radiolabeled albumin 66 2.18. Assay of viability 67 2.19. Electroporation 67 Electroporation of 293 T cells with firefly luciferase-encoding mRNA 67 Electroporation of N. brasiliensis with firefly luciferase-encoding mRNA 67 Electroporation of N. brasiliensis with dsRNA or siRNA 68 2.20. Attempted delivery of dsRNA or siRNA into N. brasiliensis by soaking 68 Soaking of N. brasiliensis with lipofectin-encapsulated dsRNA 68 Introduction of polyethyleneimine (PEI)-complexed Cy3-labelled siRNA into J774 macrophages 68 Introduction of PEI-complexed Cy3-labelled siRNA into N. brasiliensis L3 69 Soaking of N. brasiliensis with PEI-complexed siRNA 69 Soaking of J774 macrophages with LipofectamineTM 2000 (LF2000)-encapsulated firefly luciferase-encoding mRNA 70 Soaking of N. brasiliensis with LF2000-encapsulated siRNA 70 2.21. Gel retardation assay 70 2.22. Luciferase assay 71 293 T cells/J774 macrophages 71 N. brasiliensis 71 2.23. In vivo excision of phagemid 71 2.24. Cloning of recombinant N. brasiliensis VALs for expression in E. coli 71 2.25. Recombinant protein expression in E. coli 72 2.26. Purification of recombinant proteins 73 Preparation of cleared E. coli lysates under native conditions 73 Purification of recombinant NbVAL proteins under denaturing conditions 73 2.27. Generation of polyclonal antibodies 74 2.28. Bioinformatic analysis 74 2.29. Homology modelling 75 2.30. Statistical analysis 76

Chapter 3 Activation and initiation of feeding in infective larvae of Nippostrongylus brasiliensis 77

3.1. Introduction 78 3.2. A temperature cue stimulates feeding independently of serum and exsheathment 81 3.3. Temperature-induced activation is dependent upon PI3-kinase and Akt kinase but occurs independently of a cGMP-dependent cholinergic pathway 86 3.4. Cytochrome P450 (DAF-9) is an alternative regulator for activation of infective larvae 87 3.5. Regulation of protein secretion by activated larvae 94 3.6. Discussion 102

Chapter 4 Development of RNA interference in N. brasiliensis 114 4.1. Introduction 115 RNAi in plant parasitic nematodes 115 RNAi in animal parasitic nematodes 116 4.2. Manipulating conditions for electroporation of N. brasiliensis using firefly luciferase- encoding mRNA 118 4.3. Attempted RNAi in N. brasiliensis with delivery of NbAChE via electroporation 121 4.4. Induction of RNAi in N. brasiliensis via soaking 126 4.5. Attempted knockdown of ubiquitin mRNA 127 4.6. Investigation of polyethyleneimine (PEI)-based delivery 136 4.7. A cationic liposome still could not successfully mediate RNA delivery to induce an RNAi effect in N. brasiliensis 139 4.8. Discussion 147

Chapter 5 Identification and characterization of N. brasiliensis VAH/ASP-Like proteins 156 5.1. Introduction 157 5.2. Identification of VALs from N. brasiliensis 161 5.3. Amino acid sequence analysis of N. brasiliensis VALs 162 5.4. Cloning and expression of N. brasiliensis VALs 190 5.5. Discussion 200

Chapter 6 Conclusions 210

References 217

Appendices 262 Appendix. A. 263 Appendix. B. 264 Appendix. C. 265

List of figures

Chapter 1 Fig. 1.1. Diagrammatic representation of the five clades of the phylum Nematoda 19 Fig. 1.2. The life cycle of Nippostrongylus brasiliensis 23 Fig. 1.3. Features of Th2 responses during nematode infection 34 Fig. 1.4. Life cycle of C. elegans 42 Fig. 1.5. Schematic representation of the RNAi mechanism in C. elegans 45

Chapetr 3 Fig. 3.1. Overview of the transforming growth factor-beta (TGB-β) and insulin-like signalling pathways involved in the regulation of dauer recovery in C. elegans 79 Fig. 3.2. An elevated temperature cue alone stimulates feeding in N. brasiliensis infective larvae 83 Fig. 3.3. Chemical exsheathment is unable to initiate feeding 85 Fig. 3.4. The second messenger cGMP does not initiate activation 88 Fig. 3.5. Cholinergic agonists cannot initiate activation 89 Fig. 3.6. Inhibitors of PI3 kinase and Akt kinase block activation of infective L3 90 Fig. 3.7. Serotonin promotes pharyngeal pumping in infective larvae 91 Fig. 3.8. Resorcinol inhibits feeding in infective larvae 92 Fig. 3.9. A Cytochrome P450 inhibitor blocks activation of N. brasiliensis L3 93 Fig. 3.10. Host serum does not affect the profile of proteins secreted by activated larvae 96 Fig. 3.11. Acetylcholinesterase is secreted only by activated larvae 97 Fig. 3.12. Inhibitors of PI3 kinase and Akt kinase block protein secretion of activated larvae 98 Fig. 3.13. Inhibitors of PI3 kinase and Akt kinase also block overall protein synthesis in activated larvae 99 Fig. 3.14. Cytochrome P450 inhibitor blocks protein secretion of activated larvae 100 Fig. 3.15. A gene homologous to ftt-2 in N. brasiliensis 101 Fig. 3.16. Structure of amphid neurons in C. elegans 106 Fig. 3.17. A model for regulation of larval activation in N. brasiliensis 113

Chapter 4 Fig. 4.1. Expression of luciferase in 293 T cells and N. brasiliensis electroporated with luciferase-encoding mRNA 120 Fig. 4.2. Production of three distinct dsRNA fragments from NbAChE-B 122 Fig. 4.3. RNAi-mediated suppression of NbAChE secretion in N. brasiliensis adults and L3A via electroporation of dsRNA 123 Fig. 4.4. Inconsistent knockdown of NbAChE-specific mRNA via electroporation 124 Fig. 4.5. Expression of luciferase in J774 macrophages and N. brasiliensis with Lipofectin- delivered luciferase-encoding mRNA 128 Fig. 4.6. RNAi-mediated suppression of NbAChE secretion in N. brasiliensis adults via Lipofectin-mediated delivery of dsRNA 129 Fig. 4.7. NbAChE-B specific RNAi silencing appeared not to work consistently in N. brasiliensis adults via soaking in dsRNA 130 Fig. 4.8. Production of N. brasiliensis ubiquitin-specific dsRNA and siRNA 131

Fig. 4.9. Attempted suppression of NbUbq in larval N. brasiliensis via electroporation with dsRNA 133 Fig. 4.10. Electroporation with NbUbq dsRNA or siRNA failed to induce effective RNAi in adult worms 134 Fig. 4.11. Lipofectin-mediated soaking with NbUbq dsRNA failed to induce RNAi 135 Fig. 4.12. Examination of jetPEITM complex formation and introduction of dsRNA or siRNA into macrophages 137 Fig. 4.13. Uptake of Cy3-labelled siRNA into the pharynx and intestine of larval N. brasiliensis after jetPEITM complex formation 138 Fig. 4.14. A small interfering RNA (siRNA)-jetPEITM polyplex failed to induce RNAi in N. brasiliensis 141 Fig. 4.15. LipofectamineTM 2000 (LF2000)-encapsulated siRNA also failed to silence the expression of NbUbq in N. brasiliensis 142 Fig. 4.16. Expression of AChE-B in different stages of N. brasiliensis 143 Fig. 4.17. Serotonin (5-HT) fails to promote RNAi 144 Fig. 4.18. A putative homologue of C. elegans SID-2 in N. brasiliensis 146

Chapter 5 Fig. 5.1. Expression of the N. brasiliensis VALs in different stages of the parasite life cycle 164 Fig. 5.2. Excission of the clones containing the N. brasiliensis VAL cDNAs 165 Fig. 5.3. Full-length cDNA sequence of the NbVALs 167 Fig. 5.4. Alignment of NbVALs deduced amino acid sequences 170 Fig. 5.5. Alignment of NbVALs with different SCP domain-containing proteins 172 Fig. 5.6. Amino acid sequence alignment of N. brasiliensis single SCP domain VALs with those of other nematodes 177 Fig. 5.7. Amino acid sequence alignment of N. brasiliensis double SCP domain VALs with those of other nematodes 182 Fig. 5.8. Amino acid sequence alignment of NbVALs with A. caninum platelet inhibitor (AcHPI) 184 Fig. 5.9. Amino acid sequence alignment of NbVALs with A. caninum neutrophil inhibitory factor (AcNIF) 185 Fig. 5.10. Superposition of the modelled structures of NbVAL-3 and AcNIF with NaASP-2 186 Fig. 5.11. Serine is incapable of interacting with the conserved Histidine and Glutamatic acid residues to form the putative active triad in NaASP-2 and NbVAL-3 C-terminus 187 Fig. 5.12. Putative active site residues in the GAPR-1 are conserved in NbVAL-3 188 Fig. 5.13. Putative metal-binding site residues in the snake venom toxin triflin are conserved in NbVAL-3 189 Fig. 5.14. Cloning of NbVAL-3, 4 and 5 into the pET29b expression vector 191 Fig. 5.15. Cloning of NbVAL-6, 7 and 8 into the pET29b expression vector 192 Fig. 5.16. Test expression of recombinant NbVAL-3 and NbVAL-4 in E. coli 193 Fig. 5.17. Test expression of recombinant NbVAL-5 and NbVAL-6 in E. coli 194 Fig. 5.18. Test expression of recombinant NbVAL-7 and NbVAL-8 in E. coli 195 Fig. 5.19. Purification of recombinant NbVALs from E. coli BL21 (DE3) under denaturing conditions 196 Fig. 5.20. Purified NbVAL recombinant proteins 197

Fig. 5.21a. Identification of native NbVAL proteins by western blot 198 Fig. 5.21b. Identification of native NbVAL proteins by western blot 199

List of tables

Chapter 1 Table 1.1. The impact of GI nematode infections and other major diseases in developing countries 20

Chapter 2 Table 2.1. List of primers used in this study 63

Chapetr 3 Table. 3.1. Neurotransmitters and exogenous cues do not stimulate the feeding of N. brasiliensis infective larvae at 20°C 84 Table. 3.2. Neuronal functions defined by laser ablation 107

Chapter 4 Table 4.1. Attempted delivery of dsRNA via various conditions of electroporation in N. brasiliensis 125 Table. 4.2. Comparison of core RNAi pathway genes in parasitic nematodes 145

Chapter 5 Table 5.1. Amino acid sequence identities of N. brasiliensis VALs 171

Abbreviations

°C degree Celsius 2-DE 2-demintional electrophoresis 5-HT 5-hydroxytryptamine 125I iodine isotope 125 35S sulfur isotope 35 µF microfarad µg microgramme µg ml-1 microgrammes per millilitre µl microlitre µm micrometre µM micromolar (v/v) volume to volume ratio (w/v) weight to volume ratio x g gravitational force A absorbance AAV adeno-associated virus ACh acetylcholine AChE acetylthiocholinesterase Ag5 vespid venom Antigen 5 BCR B cell receptor bp base pairs BSA bovine serum albumin Cd2+ cadmium ion cDNA complimentary deoxyribonucleic acid CEI chymotrypsin/elastase inhibitor cGMP cyclic guanosine monophosphate Ci Curie CIP calf intestinal phosphatase

CO2 carbon dioxide

11 cpm counts per minute CRD cysteine-rich domain CRISP cysteine rich secretory protein C-TL C-type lectin CuSO4 copper(II) sulfate DAF dauer formation DALY disability-adjusted life year DC dendritic cell DEPC diethyl pyrocarbonate DMEM Dulbecco’s modified eagle medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DNase deoxyribonuclease ds double stranded DTNB 5,5’-dithiobis(2-nitrobenzoic acid) DTT dithiothreitol ECF eosinophil chemotactic factor ECL enhanced chemiluminescence EDTA ethylene diamine tetra acetic acid ES excretory/secretory EST expressed sequence tag FACS fluorescent activated cell sorter FCS foetal calf serum FITC fluorescein isothiocyanate FTT fourteen-three-three GABA gamma-aminobutyric acid GAPR Golgi-associated pathogenesis related GI gastrointestinal GFP green fluorescent protein GPCR G protein-coupled receptor GSH glutathione GSP gene-specific primer

GSSs genome survey sequences

H2O2 hydrogen peroxide HDL high-density lipoprotein HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid His histidine HPI hookworm platelet inhibitor HRP horseradish peroxidase IFN interferon Ig immunoglobulin IGF insulin/insulin-like growth factor IL interleukin LDL low-density lipoprotein LPS lipopolysaccharide IPTG isopropyl β-D-1-thiogalactopyranoside kDa kilodalton KO knockout L litre LB Luria broth Luc luciferase M molar MCP mast cell proteinase mg milligram mGC transmembrane guanylyl cyclase

MgCl2 magnesium chloride MIDAS metal ion-dependent adhesion site MIF macrophage migration inhibitory factor ml millilitre mm millimetre mM millimolar M-MLV RT moloney murine leukaemia virus reverse transcriptase mRNA messenger ribonucleic acid ms millisecond

MSP major sperm protein MTP astacin-like metalloprotease MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide MyD88 myeloid differentiation primary response gene 88 NaCl sodium chloride

NaH2PO4 sodium dihydrogen phosphate NaOH sodium hydroxide

Na2EDTA disodium ethylene diamine tetra acetic acid NEM N-ethylmalemide NIF neutrophil inhibitory factor nm nanometre NMR nuclear magnetic resonance O/N over night OD optical density ORF open reading frame OVA ovalbumin PAF platelet-activating factor PAGE polyacrylamide gel electrophoresis PBMC peripheral blood mononuclear cell PBS phosphate buffered saline PCR polymerase chain reaction PEI polyethylenimine pI isoelectric points PI3-K phosphatidylinositol 3 kinase PKC protein kinase C PLA2 phospholipase A2 PMSF phenyl methyl sulphonyl fluoride PR pathogenesis-related RACE rapid amplification of cDNA ends RDE ribonucleic acid interference deficient RdRP ribonucleic acid -dependent-RNA polymerase RISC ribonucleic acid -induced silencing complex

PKC protein kinase C RELM resistin-like family of molecule RNA ribonucleic acid RNAi ribonucleic acid interference RNase ribonuclease RPMI Roswell Park Memorial Institute RS rat serum RT room temperature RT-PCR reverse transcription polymerase chain reaction SCP sperm-coating protein SDS sodium dodecyl sulphate S.E. standard error shRNA small-hairpin ribonucleic acid SID systemic ribonucleic acid interference-defective siRNA small interfering ribonucleic acid SL spliced leader SMC smooth muscle cell ssRNA single-stranded ribonucleic acid STAT6 signal transducer and activator of transcription 6 TAE Tris-acetate-EDTA TAP tobacco acid pyrophosphataes TCA trichloroacetic acid TCI trypsin/chymotrypsin inhibitor TCR T cell receptor TEMED N,N,N',N'-Tetramethylethylenediamine TGF transforming growth factor Th1 T helper lymphocyte subset 1 Th2 T helper lymphocyte subset 2 TPCK N-tosylamide-L-phenylalanine chloro-methyl ketone TLR Toll-like receptor TNF tumour necrosis factor U unit

Ubq ubiquitin UTR untranslated region VAH vespid venom antigen homologue VAL VAH/ASP-like protein V volts Ves v 5 vespid venom allergen-5 VCAM-1 vascular cell adhesion molecule-1 VIP vasoactive intestinal polypeptide YAC yeast artificial chromosome Zn2+ zinc ion

Chapter 1

General Introduction

1.1. Taxonomy of nematodes

The term helminth generally describes four diverse phyla containing parasitic worms; the Annelida, Acanthocephala, Platyhelminthes and Nematoda. Of these the two most significant phyla in terms of parasites infecting humans and animals are the Platyhelminthes and the Nematoda.

The flatworms, Platyhelminthes, are divided into four classes including the Cestoda (tapeworms) and Trematoda (flukes), two entirely parasitic groups. The Nematoda (roundworms) are the most diverse phylum of pseudocoelomates and have been divided into five major clades (I-V) based on a molecular phylogenetic analysis of the small subunit (SSU) ribosomal RNA (rRNA) genes from various nematodes (Blaxter et al., 1998). This survey revealed that the greatest number of parasitic species relevant to humans and animals were distributed across Clades I, III and V. Trichinella and Trichuris, the human parasites, are members of the order Trichocephalida in Clade I. Interestingly, Clade III is only comprised of mammalian parasites including three genetically close orders: the ascarids (Ascaris, Toxocara), the spirurids (Brugia and Onchocerca filaria), the oxyurida (pinworms). Clade IVa has only a single mammalian parasitic nematode genus of interest, Strongyloides spp. (Blaxter et al., 1998).

This molecular phylogenetic study also showed that the nematodes in Clade V are very diverse genetically. In particular, the free-living model nematode Caenorhabditis elegans is a rhabditid, and is closely related to a major taxa of parasites, the intestinal and lung-parasitic strongyles (Fig. 1.1) (Blaxter et al., 1998).

Fig. 1.1. Diagrammatic representation of the five clades of the phylum Nematoda.

Coloured solid circles represent the trophic ecologies of the taxa. vertebrate parasites; invertebrate parasites; plant parasites; entomopathogen; fungivore; bacteriovore; algivore-omnivore-predator. Epidemiologically important parasites which infect humans and livestock are indicated. The diagram is adapted and modified from Blaxter et al. (1998).

1.2. Epidemiology and control of gastrointestinal nematode parasites

Gastrointestinal (GI) nematode parasites are important causes of disease in humans. Three major GI nematode infections (soil-transmitted helminths, STH), Necator americanus/Ancylostoma duodenale (hookworms), Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm) are amongst the most prevalent worldwide, and in particular occur widely throughout the developing countries, where nutrition and sanitation are poor (Vercruysse et al., 2008). There are approximately 3.5 billion cases of these infections, and the majority of sufferers are school age children (de Silva et al., 2003; Stepek et al., 2006). When measured in Disability-Adjusted Life Years (DALYs) lost, the number of healthy years lost to premature death or disability, STH infections are as important as malaria or tuberculosis (Table 1.1) (Vercruysse et al., 2008). Since GI nematode infections tend to be chronic, in particular causing iron-deficiency anaemia, malnutrition and stunting, one major consequence is impaired cognitive function in infected children (Awasthi and Bundy, 2007; Guyatt, 2000). In addition, infections also reduce physical fitness and productivity in adulthood (Guyatt, 2000), and such effects therefore mean that parasitic helminths inhibit economic development.

Disease DALYs lost annually Deaths Hookworm infection 22.1 million 65,000 Ascariasis 10.5 million 6,000 Trichuriasis 6.4 million 1,000 Malaria 46.5 million 112 million Tuberculosis 34.7 million 2-3 million

Table 1.1. The impact of GI nematode infections and other major diseases in developing countries. Table is adapted from Vercruysse et al. (2008).

GI nematode infections also cause a significant impact within the livestock industry globally, with Haemonchus spp., Trichostrongylus spp. and Ostertagia ostertagi all contributing significant damage and representing substantial economic concern. For instance, based on 1994 sales figures, control of GI nematodes costs the livestock industry approximately £1000 million annually (Newton and Munn, 1999).

GI nematode infections are generally considered a burden on hygiene and socioeconomics of developing countries, although Trichinellosis, a gastrointestinal nematode infection, is prevalent in some regions of Eastern and Central Europe where consumption of game meat is high (Gottstein et al., 2009). Currently, inspection for Trichinella infection in animals costs the EU approximately US$570 per year to ensure safety of human consumption (Murrell and Pozio, 2000).

Good anthelmintics are available to control nematode infections, and generally belong to three major classes of drugs, the benzimidazoles, imidothiazoles and avermectin/ macrocyclic lactones. Resistance to all three classes of drugs is now commonplace and is rising globally. In addition, there are increasing concerns about chemical residues in animal products or the environment, and these factors highlight the need to look for alternative control strategies (Geary et al., 2010; Kaplan, 2004; Newton and Munn, 1999). Benzimidazole resistance is correlated with the polymorphism of β-tubulin from Haemonchus contortus (Kwa et al., 1994), Trichuris trichiura (Diawara et al., 2009) and also Necator spp. (Geary et al., 2010), suggesting that this may be the reason behind variable efficiency of albendazole or mebendazole used to treat nematode infections in humans. Although controversial, it has also been suggested that resistance to ivermectin is appearing in human populations infected with Onchocerca volvulus, and various markers have been proposed which may underlie this potential resistance (Bourguinat et al., 2008; Bourguinat et al., 2007). Importantly, therefore, understanding the interaction between parasitic nematodes and their hosts may possibly identify parasite proteins which could be targets for developing new drugs or vaccines against infection. In addition, if improvements in hygiene, together with sanitation and health education were integrated into control programmes, then parasite transmission could be significantly reduced.

1.3. Nippostrongylus brasiliensis

Nippostrongylus brasiliensis, a GI nematode of rodents, has a simple and rapid life cycle, which involves an external free-living phase, and an internal parasitic phase, including somatic tissue migration and intestinal parasitism. The life cycle of N. brasiliensis is illustrated in Fig. 1.2. The fresh, elliptical eggs (54 to 62 µm long by 31 to 34 µm wide in size) are laid into the intestinal lumen of rodents and released into the environment with the faeces. The first-stage larvae (L1) hatch from the eggs within 18-24 hours at room temperature, with a first moult 12-24 hours later, followed by a second moult within a further 24 hours. During the free-living phase of development, the larvae carry out two moults giving rise to the infective third-stage larvae (L3, 500 to 700 µm in length) by day 5 or 6. From the external environment, infective L3 penetrate the skin of the rodent host, move deep into the epidermis, then migrate to blood vessels in the loose subcutaneous connective tissue. The circulation delivers them to the lungs by 11-20 hours after invasion, where they penetrate the pulmonary alveoli and sequentially moult to the fourth-stage larvae (L4) between 24-32 hours. The L4 are morphologically similar in appearance to infective L3; however the L4 are longer, with darker pigmentation along the length of their bodies. The final moult in the gut begins on about day 4 post-infection and completes the development to the adult stage. Adult parasites colonise the anterior half of the small intestine. The size of the male worm is from 3 to 4.5 mm in length, while the female ranges from 4 to 6 mm in length. Eggs are produced at 6 days post-infection, reaching a maximum on day 8 and 9, followed by a gradual decline in egg production until the adult parasites are expelled between days 10-13 (Camberis et al., 2003; Kassai, 1982).

Expressed sequence tag (EST) analysis and genome survey sequences (GSSs) suggest that N. brasiliensis is closely related to the extremely well characterised model organism, Caenorhabditis elegans in Clade V (Rhabditina), and also several important animal parasitic nematodes (Fig. 1.1) (Blaxter et al., 1998; Harcus et al., 2004; Mitreva et al., 2005a). This, along with the ease of laboratory passage, means that N. brasiliensis represents an ideal model parasitic nematode to manipulate and further explore aspects of parasite invasion and development, the role of secreted proteins in parasitism and the resulting immune response.

Fig. 1.2. The life cycle of Nippostrongylus brasiliensis. The diagram is adapted and modified from DeFranco et al. (2007).

1.4. Infection and invasion by parasitic nematodes

Infection by parasitic nematodes is normally achieved via the oral route by ingestion of contaminated food, by direct penetration of skin, or via injection during feeding by intermediate hosts such as insects. Migration through host tissue often occurs in order to reach a preferred niche in the body. Although some tissue transit might be achieved mechanically, the use of hydrolytic enzymes is presumed to be a common adaptation for the process of host invasion. Metalloproteases or aspartic proteases have been implicated in mediating host skin penetration (Brindley et al., 1995; Brown et al., 1999a; Hotez et al., 1990). For instance, an astacin-like metalloprotease (MTP-1) is secreted by Ancylostoma caninum infective larvae upon activation with host serum in vitro (Williamson et al., 2006). Moreover, antiserum to a recombinant AcMTP-1 reduced larval migration through tissue in an in vitro assay by up to 75%, and treatment with metalloprotease inhibitors also showed a significant reduction of penetration of L3 through skin (Williamson et al., 2006). Therefore, the involvement of AcMTP-1 has been strongly suggested in tissue migration during host invasion. In addition, infective stage larvae of Ancylostoma have been shown to release two hyaluronidases of 49 and 87 kDa, suggesting that these enzymes may serve to degrade hyaluronic acid and facilitate invasion and migration through host skin (Hotez et al., 1992).

Survival and colonization in the gastrointestinal tract is likely to require adaptations that prevent expulsion by the host, and parasite protease inhibitors may participate in this process. Serine protease inhibitors (trypsin and chymotrypsin/elastase inhibitor) from a GI nematode of humans and swine, Ascaris, which are localised in eggs, cuticle, muscle and intestine of the worm, form complexes with host-derived proteases (Martzen et al., 1985), and can inhibit proteolytic degradation by host digestive enzymes (Peanasky et al., 1984a; Peanasky et al., 1984b), suggesting that these proteases are assimilated by the parasite. In addition, two parasite secretory proteins, trypsin/chymotrypsin inhibitor (TsTCI) and chymotrypsin/elastase inhibitor (TsCEI) have been identified from adult Trichuris suis, an intestinal parasite of swine (Rhoads et al., 2000a; Rhoads et al., 2000b). TsCEI has been shown to inhibit chymotrypsin, pancreatic and neutrophil elastases, mouse mast cell proteinase-1 (mMCP-1) and cathepsin G, and it has been suggested that serine protease inhibitors may function as components of the parasite defence mechanism by modulating intestinal mucosal mast cell

24 mediators and immune responses to contribute to parasite survival in the host (Rhoads et al., 2000b). However, the primary function of these inhibitors is most likely to block the hydrolytic action of pancreatic and intestinal enzymes (e.g. trypsin, chymotrypsin and elastase), as parasites will be exposed to high concentrations of these enzymes in the intestinal tract.

An aspartyl protease inhibitor, Ascaris PI-3, has been identified from Ascaris suum, and shown to inhibit pepsin A and cathepsin E in vitro, suggesting that it also might protect the parasite from digestion of host proteases and enhance the survival of the infective larvae in the host stomach (Martzen et al., 1990). Similarly, Onchocera volvulus 33 (Ov33) also has a pepsin-inhibitory activity, and is abundantly present in the hypodermis, muscle and uterus of female parasites, and embryonic microfilariae (Tume et al., 1997). Many aspartyl protease inhibitors have been identified from parasitic nematodes (De Maere et al., 2005; Delaney et al., 2005; Dissanayake et al., 1993; Shaw et al., 2003; Willenbucher et al., 1993), but the role of aspartyl protease inhibitors in parasite infection is poorly understood.

Two serine protease inhibitors, the A. caninum anticoagulant protein 5 and 6 (AcAP5 and AcAP6), have been demonstrated to directly inhibit the catalytic activity of blood coagulation factor Xa (fXa) and most likely participate in blood-feeding by the parasite (Stassens et al., 1996). Likewise, the human hookworm Ancylostoma ceylanicum makes an anticoagulant, AceAP1 which inhibits the factor VIIa/Tissue complex (fVIIa/TF) of the mammalian coagulation response (Mieszczanek et al., 2004). Intriguingly, hookworm platelet inhibitor (AcHPI), a homologue protein of A. caninum secreted proteins (AcASPs), can also block aggregation and adhesion of platelets to collagen and fibrinogen by interacting with the integrins GPIIb/IIIa and GPIa/IIa in the host (Chadderdon and Cappello, 1999; Del Valle et al., 2003).

1.5. Modulation of the immune response by nematode secreted proteins

Parasite molecules that mediate interaction with the host are expected to occur on the surface or in the excretory-secretory (ES) products which are released from the parasite. It is clear

25 that parasitic nematodes can express a range of proteins to mimic and manipulate intracellular signalling of the host immune system. The mammalian cytokine, macrophage migration inhibitory factor (MIF) is a ubiquitous protein that has been shown to play an important upstream role in both innate and adaptive immunity, and is notable for its production by a wide range of cell types, locally by macrophages, T cells and eosinophils, as well as systemically by the anterior pituitary gland (Baugh and Bucala, 2002). Intensive investigation of the function of MIF suggests that it acts in a strongly pro-inflammatory manner. In vitro, exogenous MIF promotes the release of interleukin (IL)-6, IL-8, tumour necrosis factor (TNF)-α, and endogenous MIF secretion from human monocytes and mouse macrophages. In vivo, MIF coordinates with lipopolysaccharide (LPS) to enhance toxic shock (Baugh and Bucala, 2002; Calandra and Roger, 2003). Nematode MIF homologues have been described, and were consequently suggested to mimic host MIF by delaying immune recognition by lymphocytes during parasite invasion, enabling the establishment of infection (Pennock et al., 1998; Vermeire et al., 2008).

Nematode MIFs were first discovered in Trichinella spiralis, Trichuris muris and Brugia pahangi somatic extracts (Pennock et al., 1998). The N-terminal sequences of TsMIF, TmMIF and BpMIF showed 47%, 36% and 43% identity with human MIF respectively. A MIF sequence of B. pahangi was also identical to a sequence observed in the Brugia malayi genome (Pennock et al., 1998). Subsequently, Pastrana et al. (1998) identified a 12.5 kDa B. malayi MIF with 42% identify to human MIF, and its expression was observed in L3, L4, microfilariae and most highly in adult stage parasites. No MIF homologues, however, were found in somatic extracts of N. brasiliensis, Heligmosomoides polygyrus, Hymenolepis diminuta or Schistosoma spp. (Pastrana et al., 1998).

Recombinant BmMIF reproduced the effect of live B. malayi parasite in vivo in promoting the expression of Ym1/eosinophil chemotactic factor (ECF-L) and mediating recruitment of eosinophils. Ablation of the effect was observed using an amino-terminal mutated recombinant protein (Falcone et al., 2001). Another B. malayi MIF (BmMIF-2) was subsequently identified, which was also expressed in L3, L4, microfilariae and adults (Zang et al., 2002). The protein sequence of BmMIF-2, with a predicted molecular weight of 13.1 kDa, shares 27% identity with human MIF and 26% identity with BmMIF-1. Both BmMIF-1

26 and BmMIF-2 recombinants have been shown to induce the production of IL-8, TNF-α, and human MIF. Also, determination of the crystal structure of BmMIF-2 revealed close similarity to human MIF, explaining the conserved activities (Zang et al., 2002). However, importantly, a recent study has demonstrated that Brugia MIFs synergize with IL-4 to induce the development of fully suppressive alternatively activated macrophages (AAMφs) in vitro (Prieto-Lafuente et al., 2009). One pathway for this effect may be via the induction by recombinant BmMIFs of IL-4 receptor (IL-4R) expression on macrophages, consequently amplifying the potency of IL-4 itself (Prieto-Lafuente et al., 2009).

The T. spiralis and T. trichiura MIFs were cloned, and share between 25% and 46% amino acid sequence identity with other MIFs, including all of the uniformly conserved residues, but lacking certain cysteine residues (Tan et al., 2001). TsMIF was able to inhibit monocyte migration and chemotactic activity in serum-free medium, but had no significant effect on T cell proliferation or cytokine production. The determined TsMIF crystal structure was generally similar to human MIF, but different in the putative active site, which may underlie the difference in activities between the parasite and mammalian proteins.

An A. ceylanicum MIF homologue (AceMIF) has been characterized, and was shown to be present in L3, adult worm extracts and ES proteins, but not in L1 or eggs (Cho et al., 2007). The crystal structure findings of AceMIF suggested that even though the global topology of AceMIF is similar to the human cytokine, the hookworm protein has a distinct active site and properties. Despite the resistance of AceMIF to a specific inhibitor, ISO-1, AceMIF was capable of binding to the cell surface mammalian MIF receptor, CD74, suggesting that AceMIF might engage in or modulate host inflammatory responses during infection. Recently, a MIF homologue (AsMIF) has been isolated from ES proteins of Anisakis simplex (whale worm) L3 (Park et al., 2009). AsMIF appeared to inhibit host MIF function, and significantly reduce the levels of the Th2-related cytokines IL-4, IL-5 and IL-13 in an ovalbumin (OVA) based mouse model of allergic airway inflammation, but IL-10 and transforming growth factor (TGF)-β remained high in OVA-challenged mice. In addition, CD4+CD25+Foxp3+ T cell (regulatory T cell, Treg) production in the lungs and spleen could be induced by AsMIF treatment, but was inhibited by Anti-AsMIF. Accordingly, AsMIF might be an important host immune down-regulatory protein.

The intestinal nematode T. muris has been shown to secrete a 43 kDa protein with homology to interferon-gamma (IFN-γ), as it could be detected by antibodies to both human and mouse IFN-γ (Grencis and Entwistle, 1997). The T. muris IFN-γ homologue could bind to IFN-γ receptors on host T and B lymphocytes, subsequently causing an upregulation of expression of Ly6A/E, the IFN-γ activation marker (Grencis and Entwistle, 1997). Importantly, Else et al. have shown that depletion of IFN-γ in susceptible mice resulted in expulsion of T. muris, suggesting that IFN-γ is essential for progression to chronic infection (Else et al., 1994). Therefore, possibly, a worm secreted IFN-γ homologue might be involved in interfering with the regulation of host immune response in order to potentiate its own survival. A component of secreted products from adult Necator americanus was shown to selectively bind to mouse and human natural killer cells, stimulating the secretion of IFN-γ in an IL-2 and IL-12- dependent, and endotoxin (LPS)-independent manner (Hsieh et al., 2004). Similarly, a component of secreted products from the larval Taenia crassiceps of tapeworms, p66, has been characterized, which mimicked some characteristics of murine IFN-γ. It was found that p66 could enhance splenic T cell proliferation and also upregulated the level of IL-10 and IFN-γ production. Increased nitric oxide production in macrophages by p66 stimulation was also observed (Spolski et al., 2002). Consequently, this kind of IFN-γ signalling interaction may be a significant means of contributing to parasite persistence.

Worms also express members of the TGF-β and TGF-β receptor superfamilies. B. malayi and B. pahangi secrete TGF-β homologues, termed BmTGH-1 and BpTGH-1 respectively (Gomez-Escobar et al., 1998). The transcripts of tgh-1s were found to be expressed in the L3 and adult but not the microfilarial stage of both Brugia species. Gomez-Escobar et al. (1997) have also identified a TGF-β receptor, named BpTRK-1, in the L3, adults and microfilariae of B. pahangi. Due to these data it has been suggested that the tgh-1s have non-immune roles and are possibly involved in Brugia development. However, another homologue of human TGF-β, TGH-2 was identified in the microfilarial and adult stages of B. malayi, its sequence showing most similarity to the C. elegans developmental protein, DAF-7 (Gomez-Escobar et al., 2000). BmTGH-2 was detected in adult parasite secreted products, and its recombinant protein bound to the mammalian TGF-β receptor, suggesting it may participate in promoting the generation of regulatory T cells during infection. Nevertheless, BmTGH-2 was secreted at

28 very low levels, under the limitation of detection for proteomics, and it is unclear whether this is sufficient for biological activity (Gomez-Escobar et al., 2000; Hewitson et al., 2009).

ES-62, a dominant secreted protein of Acanthocheilonema viteae, was discovered in 1989 (Harnett et al., 1989). It is a leucine aminopeptidase and heavily conjugated with phosphorylcholine via N-type glycans (Harnett et al., 1999). Phosphorylcholine (PC) is a molecular pattern associated with pathogenic products of various prokaryotic and eukaryotic pathogens (Harnett and Harnett, 1999). ES-62 modified with PC has been demonstrated to impair the proliferation of CD4+ T cells through the T cell receptor (TCR) and the activation of B cells following B cell receptor (BCR) ligation in vivo, and also reduced IL-4 and IFN-γ production by CD4+ cells (Marshall et al., 2005; Wilson et al., 2003a). On the other hand, ES-62 has been shown to cause an upregulation of production of IL-10 from B1 cells in vivo (Wilson et al., 2003b).

ES-62 also induced bone marrow-derived dendritic cells (DCs) to stimulate Th2 responses, resulting in a significant increase of IL-4 and suppression of IFN-γ production in vitro (Whelan et al., 2000). Moreover, the production of IL-12p70 by pre-stimulated macrophages and DCs was inhibited by exposure to ES-62 (Goodridge et al., 2004; Goodridge et al., 2001). Importantly, the effects of ES-62 on macrophages and DCs were abolished in myeloid differentiation primary response gene (88) (MyD88) knockout mice and required Toll-like receptor (TLR) 4 (Goodridge et al., 2005). Likewise, ES-62 interacted with TLR4 in mast cells resulting in the degradation of intracellular protein kinase C-a (PKC-a), which caused direct inhibition of the release of inflammatory mediators (Melendez et al., 2007).

Host lectins participate in a variety of cellular immune processes, such as antigen uptake and presentation, apoptosis and T cell proliferation. Interestingly, C-type lectins (C-TLs) and S- type lectins (galectins) are abundantly expressed in secreted products of parasitic nematodes, and are suggested to play a role in immunomodulation (Loukas and Maizels, 2000). The parasite C-TLs TES-32 and TES-70 have been identified in the ES products of larval Toxocara canis (Loukas et al., 2000; Loukas et al., 1999). The C-terminal domain of TES-32 was found to have great similarities to mammalian C-TLs including macrophage mannose receptor, E-selectin and low affinity IgE receptor, CD23 (Loukas et al., 1999). Furthermore,

TES-70 was shown to share similar sequence characteristics with TES-32, which bound the surface of mammalian epithelial cells in a calcium-dependent manner, suggesting that parasite C-TLs possibly combat the immune response by inhibiting host immune cell lectins from recognizing their ligands, thus interfering with signalling pathways (Loukas et al., 2000).

C-TLs have also been found from N. brasiliensis (Harcus et al., 2004), N. americanus (Daub et al., 2000), A. caninum (Mulvenna et al., 2009) and Haemonchus contortus (Loukas and Maizels, 2000), suggesting that parasite C-TLs might play an important role in invasion and parasitism. However, a non-secreted C-TL from A. ceylanicum (AceCTL-1) has been identified, and demonstrated to have 26% and 32% identities to TES-32 and TES-70 respectively (Brown et al., 2007). AceCTL-1 was the first protein of the C-TL family shown to be expressed in sperm cells of the nematode, suggesting a potential role in nematode sperm-egg recognition and reproduction (Brown et al., 2007).

Secreted S-type lectins or galectins are apparent in H. contortus (Newlands et al., 1999) and B. malayi (Hewitson et al., 2008). A recombinant B. malayi galectin (BmGAL-1) has been reported which was able to bind to host immune cells in a carbohydrate dependent manner (Hewitson et al., 2009), but did not behave like an eosinophil chemoattractant reported for a H. contortus galectin (Turner et al., 2008). A galectin (Di33) was identified in somatic extracts of the dog heartworm Dirofilaria immitis, and is a target for IgE responses of humans exposed to D. immitis (Pou-Barreto et al., 2008). In addition, two-dimensional electrophoresis (2-DE) of D. immitis somatic extracts has identified more galectins, one of which shared high sequence identity with one from B. malayi ES (Oleaga et al., 2009). Therefore, it is suggested that parasite galectins are possibly common mediators of survival in parasitic nematodes.

1.6. Host protective immunity to N. brasiliensis infection

Parasite infections and the corresponding host immunity are products of a prolonged dynamic co-evolution between the host and parasite. It is advantageous for parasites to trick the host

30 into developing ineffective immune responses, to find a suitable niche for maturation and propagation without killing or harming the host. By contrast, the host has to produce an effective immune response coping with the infection to expel the parasite, therefore minimizing its harmful effects. It is clear that the protective immune response against many GI nematodes has been demonstrated to be what is referred to as the type-2 (Th2)-mediated response (Anthony et al., 2007). CD4+ T cells are functionally programmed to differentiate into two distinct T helper cell subsets, Th1 and Th2 cells. The Th2 response (Fig. 1.3) is typically characterized by elevated levels of IL-4 and other Th2 cytokines including IL-5 (Ovington et al., 1998), IL-9 (Faulkner et al., 1998), IL-13 (McKenzie et al., 1998) and IL-21 (Pesce et al., 2006), as well as activation and expansion of plasma cells secreting IgE, and high levels of eosinophils, mast cells and basophils. IL-25 (also known as IL-17E) is associated with the Th2 response and can promote Th2-cell differentiation and expulsion of GI nematodes, although its precise biological function remains unclear (Fallon et al., 2006). Conversely, IFN-γ-dominant Th1-type responses are typically elicited by microbial infections, and result in an increase of cytotoxic T cells, neutrophils and macrophages. Although IL-10 was formerly assumed to be the major means by which Th2 cells downregulate Th1 cells, recent studies have revealed that IL-10 is also generated by Th1 and regulatory T cells during protozoan infection (Jankovic et al., 2007); and it can suppress both Th1 and Th2 cytokine response development in the pathogenesis of schistosomiasis (Hoffmann et al., 2000). Immunity to GI nematode infections appears to have the Th2-mediated response as a common signature, however the components of the immune response that mediate protection and expulsion are dependent on the particular parasite.

N. brasiliensis L3 infect rodent hosts by penetrating through the skin, migrating to the lungs, and maturing into lumen-dwelling adults in the intestine (Fig. 1.2). It has been shown that host immune responses to infection are polarised to a Th2 cytokine response, eventually expelling adult parasites by about two weeks after initiation of the infection (Jacobson and Reed, 1974). Treatment of infected mice with recombinant IL-12, inducing production of IFN-γ, inhibited parasite expulsion (Finkelman et al., 1994). Similarly, treatment with recombinant IFN-γ caused an increase in parasite egg production and delayed adult expulsion by 48-72 hr in infected mice (Urban et al., 1993). This suggests that host protection against N. brasiliesis can be eliminated by administration of Th1-type cytokines.

The importance of Th2 cytokines has been intensively investigated and further defined via the use of transgenic mice. While both IL-4 and IL-13 are required for the expulsion of the intestinal nematode Trichuris muris (Bancroft et al., 1998; Else et al., 1994), it has become apparent that IL-13 is a critical component of the protective response to N. brasiliensis, and IL-4 seems to be not so important (Urban et al., 1998). Urban et al. (1998) demonstrated that the IL-4 knockout (KO) mice retained the capacity to expel the parasite; however, interestingly, IL-4 receptor α (IL-4Rα) and signal transducer and activator of transcription 6 (STAT6) KO mice failed to expel N. brasiliensis adult worms. Hence, IL-13 was suggested as the only cytokine other than IL-4 that efficiently activates STAT6 during expulsion of the parasite.

Treatment of infected mice with an IL-13 antagonist, a soluble IL-13 receptor α2- immunoglobulin G (IgG) Fc recombinant protein (sIL-13Rα2-Fc) has been shown to result in a significant suppression of egg production and parasite clearance. Similar findings were also observed in IL-4, IL-13 and IL-4/IL-13-double KO mice (McKenzie et al., 1998; McKenzie et al., 1999). It was found that STAT6 signalling which resulted in expulsion of Trichinella spiralis occurred through IL-4Rα expressed on both bone marrow-derived and non-bone marrow-derived cells (Finkelman et al., 2004). Unlike T. spiralis, clearance of N. brasiliensis and also Strongyloides venezuelensis was mediated by signalling through IL-4 receptors expressed on non-bone marrow-derived cells alone (Negrao-Correa et al., 2006; Urban et al., 2001). In addition, investigators noted that IL-13 induced a significant increase in intestinal mucus production which coincided with N. brasiliensis expulsion (McKenzie et al., 1998). STAT6-dependent IL-4 and IL-13 have been demonstrated to increase mucosal permeability, decrease glucose-stimulated fluid absorption, and promote contractility of intestinal longitudinal smooth muscle in Heligmosomoides polygyrus infected mice (Goldhill et al., 1997; Madden et al., 2002). Therefore, it has been suggested that production of IL-4 and IL- 13 by T cells may act directly on cells in the small intestine to create an unfavourable environment, preventing N. brasiliensis adult worms from maintaining contact with the jejunal mucosa (Finkelman et al., 2004; Khan et al., 1995).

Mast cells are considered to be crucial effector cells for expulsion of some GI nematodes, and this role has been intensively studied in T. spiralis (Artis and Grencis, 2008; Grencis, 1997).

However, mast cells appear to be unnecessary for expulsion of N. brasiliensis (Crowle and Reed, 1981; Madden et al., 1991). W/Wv mice, with a mutation in the stem cell factor (SCF) receptor (c-kit) affecting mast-cell development, or mice treated with antibodies against IL-3 or IL-4 which inhibit mastocytosis, expel N. brasiliensis with the same dynamics as wild type or untreated control mice (Crowle and Reed, 1981; Madden et al., 1991). Conversely, Urban et al. (1998) have demonstrated that STAT6-defective, N. brasiliensis infected mice developed a considerably stronger mucosal mast cell response reflected in a high level of mouse mast cell protease-1 (mMCP-1) production, and yet expulsion of adult parasites is delayed.

IL-9, a Th2 cytokine was described as inducing the differentiation of mast cells in vitro (Hultner et al., 1990). Studies on infected IL-9 transgenic mice or IL-9 treated mice, demonstrated a promotion of T. spiralis or T. muris expulsion, which was associated with an increase of intestinal mastocytosis and IgE production. IL-9 transgenic mice also exhibited an increased Th2-type response to parasite challenge (Faulkner et al., 1997; Faulkner et al., 1998). These studies suggested that IL-9 plays an important role in generation of the Th2 response, and also may be involved in generation of mast cells resulting in worm expulsion. However, the subsequent use of anti-c-kit antibodies to block mast cell expansion suggested that they do not appear to be crucial in resistance to T. muris (Betts and Else, 1999). In N. brasiliensis infected mice, IL-9 deficiency did not affect the dynamics of adult worm expulsion (Townsend et al., 2000).

Goblet cell hyperplasia is a feature of GI nematode infections (Miller, 1987; Miller et al., 1981). The protective function which is associated with goblet cells is increased mucus production which traps and excludes worms from the gastrointestinal mucosa, and promotes expulsion by preventing their attachment and feeding (Miller, 1987; Miller et al., 1981). Indeed, an increase in goblet cell numbers during N. brasiliensis and also T. spiralis infection has been noted, which was regulated by Th2-type cells, but not Th1-type cells (Ishikawa et al., 1994; Ishikawa et al., 1997). Goblet cell size also increased, and in N. brasiliensis

Fig. 1.3. Features of Th2 responses during nematode infection.

AAM, alternatively activated macrophage; ChaFFs, chitinase and FIZZ family members; RELMβ, resistin-like molecules. Diagram taken from Anthony et al. (2007).

34 infection, there were alterations in the mucin glycoproteins present in these cells (Garside et al., 1992; Ishikawa et al., 1993; Ishikawa et al., 1997). In addition, as animals infected with N. brasiliensis, the glycosylation alternations of gastrointestinal mucins were also observed, suggesting the modulation of intestinal mucin glycoslations would be part of host defence mechanisms to affect infectious pathogens (Holmen et al., 2002; Karlsson et al., 2000). Although goblet cell hyperplasia is influenced by Th2 cytokines, it appears to be IL-4 independent, with instead IL-13/IL-4Rα/STAT6 signalling required. It has been shown that KO of IL-13 in mice resulted in the failure of elimination of N. brasiliensis adults, and this result was observed in neither IL-4 KO mice nor wild-type mice (McKenzie et al., 1998). Importantly, infected IL-13 KO recipients also failed to generate goblet-cell hyperplasia. Therefore, this suggests that IL-13 may promote the production of goblet cell hyperplasia, and secreted intestinal mucus of goblet cells facilitates clearance of N. brasiliensis (McKenzie et al., 1998).

Moreover, recently, IL-13/IL-4Rα/STAT6 transduction has been shown, not only, to be required for effective goblet cell proliferation, but also for longitudinal smooth muscle cell (SMC) contraction in the intestine (Horsnell et al., 2007). N. brasiliensis infected SMC-IL- 4Rα-deficient mice showed a delayed worm expulsion and also a reduction in the expression of the M3 muscarinic receptor, which is the principal acetylcholine receptor in smooth muscle and drives 75% of the contractile response in the small intestine (Matsui et al., 2002). This suggests that the mechanism of IL-4Rα-responsive SMC is correlated with the Th2 cytokine response, goblet-cell hyperplasia, and acetylcholine responsiveness. Thus, goblet cell hyperplasia and increased SMC contraction of the intestine play important roles in immune clearance of N. brasiliensis. In addition, one of the members of the resistin-like family of molecules, RELMβ (also known as FIZZ2), a goblet cell-specific immune effector molecule, expression of which is believed to be under the control of IL-13, was found to be a common response in the GI tract following infection with N. brasiliensis, T. spiralis and T. muris (Artis et al., 2004). A possible role for RELMβ has been described in T. muris and Strongyloides stercoralis infection, in which it bound to structures on the lateral alae, thereby interfering with worm chemotaxis towards host tissue (Artis et al., 2004; Tilney et al., 2005).

Therefore, intestinal goblet cell-derived RELMβ may be a novel Th2 cytokine-induced effector molecule in resistance to GI nematode infections.

Eosinophilia, as well as a markedly elevated level of IL-5, the Th2-type cytokine responsible for the generation of eosinophils, is a prominent response during nematode parasite infections. Eosinophils have been shown to adhere to N. brasiliensis L3 in vitro and release their granule contents (Mackenzie et al., 1981; McLaren et al., 1977). This can harm the parasites causing impaired movement of tissue migratory larvae in host (Daly et al., 1999; Shin et al., 2001). Infection of IL-5 transgenic mice with N. brasiliensis has shown that tissue eosinophilia is associated with a potent and early resistance, which drastically reduced the number of lung- stage larvae (L4), intestinal adults and faecal eggs (Dent et al., 1999; Shin et al., 1997). Equally, when IL-5 transgenic mice were infected with Angiostrongylus cantonensis (Sugaya et al., 1997), A. costaricensis (Sugaya et al., 2002), S. stercoralis (Herbert et al., 2000) or Litomosoides sigmodontis (Martin et al., 2000) they showed much lower parasite burdens compared to wild-type mice. In contrast, IL-5 transgenic mice infected with Toxocara canis or T. spiralis tended to harbour more parasites (Dent et al., 1997; Dent et al., 1999; Fabre et al., 2009; Hokibara et al., 1997).

Studies on ablation of eosinophilia by anti-IL-5 monoclonal antibody treatment indicated diminished resistance of immune mice challenged with S. stercoralis (Rotman et al., 1997), and increased numbers of tissue larvae recovered from a primary infection of mice with the rat lung parasite A. cantonensis (Sasaki et al., 1993) or with the filarial parasite Onchocerca lienalis (Folkard et al., 1996). Furthermore, eosinophils were found to provide a potent resistance to secondary N. brasiliensis infection, as IL-5-deficient mice and eosinophilopoiesis-deficient mice presented higher L4 burdens in lungs compared to wild- type mice after 2 days post-secondary challenge (Knott et al., 2007). Recently, Giacomin et al. (2008) demonstrated that a high number of N. brasiliensis L4s were recovered from both complement Factor B-deficient (defective in the alternative pathway of complement activation) and C3-deficient (defective in general complement activation) animals, indicating that an inhibition of larval parasite migration is associated with complement activation. Also, PMX53, an antagonist of the C5a receptor, was shown to suppress eosinophil and neutrophil

36 recruitment to the skin in the early stage of N. brasiliensis primary infection, indicating an important role for complement activation in this process (Giacomin et al., 2008).

In contrast to N. brasiliensis, eosinophils and IL-5 do not appear to play a significant role in resistance to T. spiralis, as anti-IL-5 monoclonal antibody treatment or infection of IL-5- deficient mice had little effect on establishment of the parasite in the intestine and muscle (Herndon and Kayes, 1992; Vallance et al., 2000). However, IL-5 is essential for intestinal eosinophilia, which plays a role in protecting the host against secondary T. spiralis infection (Vallance et al., 2000). Intriguingly, Fabre et al. have shown that eosinophils can promote the survival of T. spiralis muscle-stage larvae by altering cytokine production and inhibiting nitric oxide synthesis, and proposed that eosinophils may influence host immunity in a manner that would sustain chronic infection and ensure parasite survival in the host population (Fabre et al., 2009). Accordingly, the function of eosinophils during nematode parasite clearance is still unclear and controversial with a need to be comprehensively characterized.

1.7. Secreted proteins of N. brasiliensis

As previously illustrated, parasitic nematodes secrete biologically active molecules to mimic or manipulate intracellular interactions during host invasion in order to survive immune attack. The GI nematode of rodents, N. brasiliensis drives dominant Th2 responses in the host (Urban et al., 1992), and this bias can be reproduced with secreted proteins from N. brasiliensis adult worms in vitro (Holland et al., 2000). Relatively few secreted proteins from N. brasiliensis have been characterised in any detail, and thus far there has been no systematic proteomic analysis for this species, although many of the proteins are likely to be similar to those secreted by other parasitic nematodes. What follows is a description of several classes of proteins known to be secreted by N. brasiliensis.

Acetylcholinesterases Acetylcholinesterase (AChE) has been identified in the ES of many parasitic nematodes, including B. malayi (Rathaur et al., 1987), Heligmosomoides polygyrus (Lawrence and

Pritchard, 1993), Dictyocaulus viviparus (McKeand, 2000) and also N. brasiliensis (Hussein et al., 2002a). It has been suggested that their secretion may hydrolyse acetylcholine released from the enteric nervous system of the host (Selkirk et al., 2005a).

AChE secretion by N. brasiliensis was initially discovered via cytochemical staining of sections of the parasite by Lee (Lee, 1970), confirmed by analysis of proteins secreted by adult worms in vitro (Sanderson, 1972) and extended to a wide range of species (Ogilvie et al., 1973). Genes for three isoenzymes of NbAChE, NbAChE-A, -B and -C have been cloned, the biological characteristics of which indicate they are monomeric and hydrophilic, with molecular weights and acid pIs estimated at 74 kDa and 4.0 for form A, 69 kDa and 3.8 for form B, and 71 kDa and 3.6 for form C (Hussein et al., 1999a; Hussein et al., 1999b; Hussein et al., 2000). Sanderson (1972) showed that N. brasiliensis adult can secrete large amounts of NbAChE, with up to 11% of the total body content of enzyme being released per hour in vitro. However, the biological functions of secreted AChEs have not yet been systematically investigated and addressed. Secreted products of N. brasiliensis significantly decreased the amplitude of concentrations of segments of uninfected rat intestine (Foster et al., 1994). In contrast, testing of AChE from the electric eel did not show the same phenomenon, suggesting that AChE in the ES products was not responsible for the reduction in motility of the host intestine (Foster et al., 1994). In subsequent investigations, the result could be reproduced with a 30-50 kDa fraction of the N. brasiliensis adult secretions, which contained a 30 kDa protein recognized by rabbit antibody to vasoactive intestinal polypeptide (VIP) (Foster and Lee, 1996). Interestingly, the inhibitory effect of N. brasiliensis secretory products on contraction of rat intestine was significantly reduced when integrated with anti- VIP. It would appear therefore that the ES products of the parasite contains a VIP-like protein, which in mammals directly inhibits contraction of intestinal smooth muscle (Foster and Lee, 1996).

In addition, it is well known that the enteric nervous system does not simply regulate smooth muscle contraction, but also participates intimately in the control of transport processes in intestinal absorptive cells. According to the model of cholinergic signalling in the intestinal mucosa, acetylcholine released from the enteric cholinergic motor neurons stimulates chloride secretion from intestinal epithelial cells (Cooke, 1984), and mucus secretion from

38 goblet cells (Specian and Neutra, 1980). It is assumed that these events are involved in the expulsion of pathogens. Nematode parasites can initiate and stimulate fluid and mucus secretion during primary infection, and the secreted AChEs may engage in the inhibition of these secretory responses by hydrolyzing acetylcholine released from the enteric nervous system (Selkirk et al., 2001). It was demonstrated that the expression of muscarinic acetylcholine receptors (mAChRs) on cells in the lamina propria was upregulated after entry of N. brasiliensis into the jejunum (Russell et al., 2000). Recently, rats immunized with recombinant NbAChE isoform B, showed a significant decrease in egg output of between 23% and 48% (Ball et al., 2007). These data could possibly provide a lead to understanding the role of AChE secretion by parasitic nematodes.

Platelet-activating factor (PAF) acetylhydrolase The proteins secreted by N. brasiliensis adult worms contain an acetylhydrolase activity which functionally inhibits platelet-activating factor (PAF)-mediated platelet aggregation (Blackburn and Selkirk, 1992). The enzyme has been indentified as a heterodimer composed of two subunits with molecular weights of 25 and 38 kDa, the properties of which are more closely related to an acetylhydrolase than a phospholipase A2 (PLA2), a protein which can also inactive PAF. As PAF has potent pro-inflammatory activities, this suggests that N. brasiliensis secreted PAF hydrolase may act as an anti-inflammatory molecule to promote parasite infection (Grigg et al., 1996).

Cystatins The cystatins are tightly and reversibly binding inhibitors of papain-like cysteine proteases, which form a superfamily subdivided into three major families: stefins, single-chain proteins of approximately 11 kDa molecular weight, which lack disulfide bonds and carbohydrates; cystatins, which have two disulfide bonds located close to the carboxyl terminus with a molecular weight of about 13 kDa, and kininogens, glycoproteins with a relatively high molecular weight ranging from 68 to 120 kDa (Turk and Bode, 1991). There are high levels of sequence homology among members of these families, particularly within a Gln-Val-Val- Ala-Gly region.

A 14 kDa protein secreted by adult stage N. brasiliensis has been identied as a cystatin and named nippocystatin. It has been demonstrated to inhibit antigen processing by antigen presenting cells, specifically the in vitro processing of ovalbumin (OVA) by cathepsin B and L, which resulted in down-regulation of OVA-specific IgE production in treated mice (Dainichi et al., 2001a; Dainichi et al., 2001b). These studies suggest that nippocystatin may modulate antigen processing and presentation during infection.

The first cystatin described from nematode parasites was onchocystatin from the human filarial nematode Onchocera volvulus (Lustigman et al., 1992). This protein was initially thought to regulate parasite proteases during moulting of the nematode. However, additional functions outside the moulting process may be possible, given that cystatin is secreted by male parasites and blood-stage microfilariae of Acanthocheilonema vitae, which do not moult (Hartmann et al., 1997). Cystatins from both filarial and GI nematodes were described to inhibit the cysteine proteases cathepsin S and L that are implicated in the proteolytic processing of polypeptides (Newlands et al., 2001; Schonemeyer et al., 2001). Additionally, Brugia malayi cystatin, Bm-CPI-2, was shown to inhibit legumain-like proteases. Bm-CPI-2 contains an additional protein motif that is required to inhibit the distinct legumain cysteine proteases and thereby inhibits the processing and presentation of class II MHC restricted antigens by B cells (Manoury et al., 2001).

Furthermore, filarial cystatins appear to have an effect on cytokine production resulting in an anti-inflammatory responses (Hartmann et al., 1997; Schonemeyer et al., 2001). Onchocera volvulus cystatin induced the release of TNF-α in antigen specific and polyclonally stimulated peripheral blood mononuclear cells (PBMCs), followed by a downregulation of IL-12 production and massive increase of IL-10 production, ie a Th-2 response (Schonemeyer et al., 2001). On the other hand, when PBMC were treated with C. elegans cystatin, this induced only production of Th1 cytokines (Schierack et al., 2003). While filarial cystatins interfered with proliferation of mammalian T cells, C. elegans cystatin did not (Schierack et al., 2003). Thus, this inhibition of antigen presentation or T cell proliferation by filarial cystatins might contribute to parasite survival in the host.

Globins Adult N. brasiliensis adult can colonize an anoxic environment, ie the intestinal tract, due to the fact that N. brasiliensis globins have oxygen affinities 100-fold higher than the rodent host’s haemoglobins, thus enabling them to obtain sufficient oxygen for its metabolic needs. Secreted and intracellular globins have been characterized in this nematode (Blaxter et al., 1994). The secreted globin (cuticular globin) is an 18 kDa protein, which is located in the cuticle and expressed at a high level only in stages of the parasite which reside in the jejunum. The intracellular globin (body wall globin) isoform is a 17.5 kDa protein that lacks a signal peptide, and is expressed in lung stage L4, continuing through residence in the gut, although the exact tissue in which it is expressed is unknown. These two globins have been expressed in E. coli as functional holoenzymes, providing a way to understand the basis for increased oxygen affinity which is presumably necessary for gastrointestinal parasitism (Blaxter et al., 1994).

1.8. Caenorhabditis elegans as a model organism

The free-living soil nematode Caenorhabditis elegans is a small (1.5 mm long adult), rapidly growing (approximate 3 day life cycle under optimal conditions) organism which is easy to manipulate in the laboratory, the entire genome sequence of which became available in 1998 (Consortium, 1998). The similarity of genes between this nematode and humans is remarkable, with about 42% of genes associated with human diseases having orthologues in the C. elegans genome (Culetto and Sattelle, 2000). Due to these properties, C. elegans became not only a model system for probing general biological functions, but also for understanding the pathogenesis of disease.

The C. elegans life cycle is illustrated in Fig. 1.4. Juvenile worms hatch and develop through a serious of four larval stages (L1-L4) before reaching adulthood. When worms encounter a harsh or food-limited environment, they may subsequently enter an alternative developmental stage at the second moult and become dauer larvae, which do not feed and can survive at least four to eight times the normal 2-week life span without further development. When favourable environmental conditions arise, the dauer larvae resume normal development and

41 progress to become L4, the adult worms (Riddle and Albert, 1997). Interestingly, this characteristic of dauer developmental arrest is also found in a variety of parasitic nematodes (Hotez et al., 1993). It is believed that the infective third-stage larva (L3) of parasitic nematodes may share some behavioural and biological similarities with dauer larva of C. elegans, which can be used as a basis for comparison and ultimately as a paradigm for investigating the role of infection and development of the infective stage larvae of parasites (Hotez et al., 1993).

Fig. 1.4. Life cycle of C. elegans (The diagram is adapted from http://www.scq.ubc.ca/wpcontent/uploads/2006/07/wormcycle.gif).

Since Sydney Brenner began to use C. elegans as a model organism to explore animal development and behaviour (Brenner, 1973), this nematode has been broadly used to comprehend various aspects of biological problems, and one of the most important findings was RNA interference (RNAi) (Fire et al., 1998). Gene silencing via the RNAi strategy provides a straightforward method of understanding function, expression, regulation and interaction of genes in C. elegans and also other organisms (Hannon, 2002). One such example is the GI nematode N. brasiliensis, a phylogenetically close relative to C. elegans in Clade V of Nematoda (Fig. 1.1), upon which the RNAi technique has been successfully performed to knock down expression of a parasite protein (Hussein et al., 2002b).

C. elegans shares a number of common characteristics with other organisms, and thus has been providing insights for many basic biological processes over decades. In the meanwhile, a comprehensive amount of genome information has being generated and made available from C. elegans and other eukaryotic species. Therefore, this nematode could be considered a window to open for understanding in-depth knowledge of distinct biological phenomena.

1.9. Brief history of RNA interference

In 1990, Napoli et al. were the first to report the phenomenon of RNA interference, known as “posttranscriptional gene silencing (PTGS)” in plants (Napoli et al., 1990). In the following years, Romana and Macino published data describing a similar phenomenon in the fungi Neurospora crassa, noting that introduction of homologous RNA sequences caused “quelling” of the endogenous gene (Romano and Macino, 1992). RNA silencing was first documented in animals by Guo and Kemphues, who had found that the introduction of antisense RNA dramatically downregulated target mRNA resulting in degradation of message in the nematode C. elegans (Guo and Kemphues, 1996). However, importantly, Fire and Mello revealed in a seminal experiment in C. elegans a response to double-stranded RNA (dsRNA), which resulted in 10 to 100-fold effective, sequence specific, post-translational silencing, which was then named “RNA interference” (Fire et al., 1998). In order to address a variety of basic biological questions in worms and other various species, RNAi has now been

43 used extensively as a means to manipulate gene expression and probe gene function on a whole genome scale experimentally.

1.10. Overview of RNAi in C. elegans

The basic RNAi process is understood to involve three main steps (Fig. 1.5). Firstly, a long double-stranded RNA (dsRNA) that is expressed in, or introduced into, the cell is processed to small RNA duplexes (21-23 nucleotides) by a ribonuclease III (RNaseIII) enzyme known as Dicer. Secondly, these small RNA duplexes are unwound, and one strand is loaded into the RNA-induced silencing complex (RISC). Thirdly, this complex binds to the target messenger RNA (mRNA) in the transcriptome. The loaded single-stranded RNA (ssRNA) guide strand subsequently leads an endonuclease (silencer) present in the RISC to degrade the mRNA transcribed from the targeted gene. The effect of RNAi silencing was initially considered to act only via mRNA degradation, but transcriptional blocking (Grishok et al., 2005; Robert et al., 2004) and translational inhibiting effects (Gu and Rossi, 2005; Tomari and Zamore, 2005) were discovered later.

One fascinating feature of RNAi in C. elegans is the systemic spread of induced silencing. The original RNAi experiments were performed by injecting dsRNA into the gonad of worms (Fire et al., 1998). However, Fire et al. (1998) observed that injections into the intestine, instead of the germline, still induced RNAi and appeared to be more effective. Spreading of the injected dsRNA into most cells of the worm may lead to inheritance of the exogenous material and establishment of the RNAi phenomenon in the progeny of the injected worms (Fire et al., 1998).

Genetic screens of C. elegans have identified several genes that are crucial in the RNAi process. Systemic RNAi-defective (sid) mutants were isolated as worms that could not transport or systemically spread silencing information between cells, even though silencing was observed at the site where dsRNA was injected or expressed (Winston et al., 2002). The gene sid-1 is expressed in all non-neuronal cells and encodes a protein with nine predicted transmembrane domains that accumulates in the peripheral cell membrane, and is a key

Fig. 1.5. Schematic representation of the RNAi mechanism in C. elegans. dsRNA, double-stranded RNA; siRNA, small interfering RNA; DRH-1, dicer related helicase-1; RDE-1, RNAi defective-1; RDE-4, RNAi defective-4; RISC, RNA-induced silencing complex. The diagram is based on figures from Grishok (2005) and Hannon (2002).

45 component, required as a passive dsRNA channel, for the uptake of dsRNA into all cells sensitive to RNAi (Feinberg and Hunter, 2003; Winston et al., 2002). More recently, Shih et al. (2009) showed that the accumulation of short dsRNA in C. elegans tissues was more rapid than long dsRNA, and that uptake through the SID-1 channel was dependent upon the concentration of exogenous dsRNA. Although SID-1 participates in importing silencing signals into C. elegans tissues, it is not involved in exporting the signal from the gut lumen to internal tissues. Exported silencing signals are suggested to be generated in the nucleus or in the cytoplasm (Jose et al., 2009).

The gene sid-2 encodes a 311 amino acid single-pass transmembrane protein localizing to the intestinal cell apical membrane, which is required for the initial import of dsRNA from the environment into the intestinal cells of C. elegans (Winston et al., 2002; Winston et al., 2007). Sid-2 was initially identified in a genetic screen for C. elegans mutants defective in systemic RNAi (Winston et al., 2002; Winston et al., 2007). Caenorbabditis briggsae cannot import dsRNA from the environment, but transgenic expression of C. elegans SID-2 in C. briggsae is sufficient to facilitate sensitivity to environmental dsRNA (Winston et al., 2007). However, transgenically expressed SID-2 in sid-1 mutants is unable to facilitate take up of environmental dsRNA from the intestinal lumen (Winston et al., 2007). Presumably, SID-2 may cooperate with SID-1 at the lumen or function in series with SID-1, internalizing environmental dsRNA for SID-1 transport across the membrane (Whangbo and Hunter, 2008).

The C. elegans RNAi pathway has been viewed as a mechanism comprised of small interfering (si)RNA production from long dsRNA and subsequent utilization (Meister and Tuschl, 2004). The RNAi deficient (rde) genes, rde-1 and rde-4, play an important role in the formation of this second, interference component of the pathway. The PAZ-PIWI family protein, RDE-1, and the dsRNA binding protein, RDE-4 were found in a complex with the RNase III family ribonuclease Dicer, DCR-1 and a conserved DexH-box helicase, DRH-1 (Tabara et al., 2002). The exogenous dsRNA is initially processed into siRNA by DCR-1 in the tissue, and RDE-1 is then bound to the siRNA bringing it to the downstream RNAi pathway. Structural studies of PAZ-PIWI have revealed that the PAZ domain is important for siRNA interaction and can perform endonucleolytic cleavage of target mRNA (Song et al.,

2003). However, RDE-1 and RDE-4 are dispensable in the following mechanism. Genes rde- 2 and mut-7 act downstream of rde-1 and rde-4 and represent an intermediate step between the Dicer complex and RISC, which mediate accumulation of siRNA in vivo and the heritablity of RNAi in C. elegans (Grishok et al., 2000).

RNA-dependent-RNA polymerases (RdRPs) are critical for RNAi of C. elegans, and function in an amplification step synthesizing more secondary siRNA using the antisense strand of the siRNA as a primer. Two well-characterized RdRPs, ego-1 and rrf-1 are implicated in the mechanism, and are required for RNAi in the germline and somatic tissues respectively (Sijen et al., 2001; Smardon et al., 2000).

C. elegans mutants in the gene encoding the putative RdRP, rrf-3, were the first ones in which an enhanced RNAi response was demonstrated (Simmer et al., 2002). It is thought that rrf-3 acts as endogenous inhibitor competing with rrf-1 and ego-1 function in the RNAi pathway. Meanwhile, rrf-3 mutant worms have been used for a genome-wide RNAi screen, as a tool to identify more phenotypes associated with gene knockdown (Simmer et al., 2003). Furthermore, Kennedy et al. (2004) have described the isolation of a mutant with enhanced RNAi sensitivity (eri), and cloned the underlying gene eri-1, which encodes a protein with two conserved domains, a DEDDh-like 3’5’ exonuclease domain found in RNases (involved in the turnover process of tRNAs or mRNAs) (Zuo and Deutscher, 2001) and a SAP DNA binding domain (involved in transcription, replication, DNA repair or DNA- protein interactions) (Aravind and Koonin, 2000). The eri-1 mutant was shown to accumulate more siRNAs than wild-type worms. ERI-1 is predominantly found in the cytoplasm and expressed preferentially in the neurons and gonad, implying that ERI-1 downregulates RNAi more intensely in these tissues (Kennedy et al., 2004). Both rrf-3 and eri-1 mutants present the phenotype of temperature-sensitive sterility and defects in sperm development, suggesting the possibility that these genes act in the same “RNAi opposing” pathway in C. elegans (Grishok, 2005).

Recently, class B synthetic multivulva (synMuv B) genes have been identified as inhibitors of the RNAi pathway (Grishok et al., 2008; Lehner et al., 2006; Wang et al., 2005). SynMuv B genes seem to act in parallel to the putative pathway shared by rrf-3 and eri-1, as

47 combinations of mutations in these two pathways result in enhanced hypersensitivity to RNAi in C. elegans. Notably, the loss of synMuv B gene, lin-35, the worm orthologue of the mammalian retinoblastoma (Rb) tumour suppressor, has been shown to significantly enhance RNAi sensitivity in the nervous system and germline genes, and its mutants are more sensitive than either rrf-3 or eri-1 mutant animals to RNAi (Lehner et al., 2006). In addition, out of 10953 genes targeted in the ORFeome RNAi library, 523 showed an enhanced RNAi phenotype in lin-35 mutants including 57 genes that are putative synthetic lin-35 interacting genes (Ceron et al., 2007). In the same report, the synthetic lin-35 interacting gene, zfp-2 was demonstrated to interact with lin-35 in somatic gonad development, and zfp-2 mutants were also hypersensitive to RNAi.

Although RNAi predominantly appears in cytoplasm of the cell, nuclear-localized RNAs can be targeted by siRNA leading to RNA degradation. Recently, Guang et al. (2008) reported the identification of a PAZ-PIWI family protein, NRDE-3 (nuclear RNAi-defective-3) in C. elegans that is required for nuclear siRNA import and is therefore essential for nuclear RNAi. NRDE-3 encodes a nuclear localization signal (NLS), and requires siRNA binding. NRDE-3 most likely binds to siRNA generated by RdRP acting on mRNA templates in the cytoplasm and is then imported into the nucleus, suggesting that NRDE-3-mediated nuclear RNAi occurs downstream of events in the cytoplasm. Generally, PAZ-PIWI family proteins are important for cleavage activity, but NRDE-3 is involved in nuclear RNAi and has no catalytic triad normally associated with these proteins. Presumably, NRDE-3 is required only for nuclear import of siRNA in the worm (Guang et al., 2008).

Besides these mechanisms, RNAi can also be induced by endogenous expression of small regulatory RNAs known as microRNAs (miRNAs) in C. elegans (Bartel, 2004). MiRNAs are genomically-encoded, untranslated RNA molecules. The generation of miRNAs occurs via sequential processing and maturation of long primary transcripts (pri-miRNA). Pri-miRNA is further processed by the RNase III endonuclease drsh-1 (Drosha in human) to the precursor miRNA (pre-miRNA), a 60-70 nucleotide molecule that can fold and form a hairpin loop structure. The pre-miRNA is then transported out of the nucleus into cytoplasm by means of the export receptor Exportin-5 (Yi et al., 2003). In the cytoplasm, pre-miRNA is recognized by Dcr-1, which processes the pre-miRNA into mature miRNA. Mature miRNA

48 subsequently incorporates into imperfect complementary sequences in the 3' untranslated regions (3'-UTRs) of target mRNA to negatively regulate target gene expression. miRNA is thus believed to function as a guide to recruit a silencing complex to target mRNA, but the exact mechanism used by miRNA to down-regulate gene expression is unclear (Vella and Slack, 2005).

1.11. Methods for the induction of RNAi silencing

In C. elegans, exogenous dsRNA eliciting systemic RNA silencing can be simply delivered into extracellular spaces via four methods; (a) microinjection of dsRNA into any site in the worm (Fire et al., 1998; Grishok et al., 2000), (b) feeding animals with bacteria engineered to express dsRNA (Timmons et al., 2001), (c) soaking animals in dsRNA (Tabara et al., 1998), and (d) in vivo transcription of dsRNA from transgene promoters (Tabara et al., 1999; Tavernarakis et al., 2000). Following uptake, processing of dsRNA by Dicer yields RNA duplexes of about 21-23 nucleotides in length, siRNAs, which lead to downregulation of targeted RNA in all cells of the treated worm and its offspring afterwards.

Due to RNAi representing a powerful, naturally specific strategy for gene silencing, the potential use of RNAi as a therapeutic agent has gained great attention as a novel approach for the treatment of various severe and chronic diseases. Elbashir et al. (2001) revealed that synthetic siRNA could successfully mediate sequence-specific gene knockdown in mammalian cell lines. The first successful use of synthetic siRNA and small-hairpin RNA (shRNA) for gene silencing in mice was achieved against a hepatitis C target shortly thereafter (McCaffrey et al., 2002). These breakthroughs seemed to offer the promise of RNAi-based therapy, but at the same time the problem of efficiency of RNAi required to elicit a sufficient gene knockdown response in target cells has arisen. There are many tissues that require an additional delivery system to aid transfection due to degradation of naked siRNA by endogenous enzymes, and in addition dsRNA is too large and too negatively charged a molecule to cross the cellular membrane. Consequently, it is important to determine the most appropriate conditions for the effectiveness of siRNA uptake, and

49 optimization of the response in target cells depending on the delivery system (De Paula et al., 2007; Whitehead et al., 2009).

A number of groups have developed plasmid vector-based siRNA expression constructs in mammalian cells, which are designed to pair perfectly with the target mRNA to induce RNAi (Brummelkamp et al., 2002; Paddison et al., 2002). In this expression system, generally, the sense and antisense strands of the gene-specific sequence are expressed as a single transcript separated by a short loop (4-10 nucleotides) of sequence. The transcript forms a hairpin structure, hence short hairpin RNA or shRNA that can be processed by Dicer into functional siRNA inside the cell being targeted for gene silencing (De Paula et al., 2007). Although the plasmid DNA of this system can be rapidly generated and the duration of silencing can be extended, it is limited in several aspects, as it is not easy to transfect and cannot be grown in culture for long periods of time in culture (e.g. primary cells) (Dykxhoorn et al., 2003).

In order to overcome the limitations of plasmid vector-based RNAi expression, viral vectors encoding shRNA, including lentiviral and adeno-associated viral (AAV) vectors, have been demonstrated to mediate an efficient and stable siRNA expression (Tiscornia et al., 2003; Wu et al., 2003). Lentiviral vectors can efficiently integrate into the genome of nondividing cells or terminally differentiated cells. It was firstly proposed that a lentiviral vector encoding shRNA could successfully silence GFP expression in 293T-GFP cell lines, and impressively the reduction of GFP fluorescence in transgenic mice introduced with lentivirus-expressing siGFP virus was also observed (Tiscornia et al., 2003). Lentiviral vector backbone-expressed siRNAs have been shown to potentially inhibit human immunodeficiency virus type 1 (HIV- 1) infection in primary haematopoietic stem cells and human CD4+ T cells (Li et al., 2003a; Nishitsuji et al., 2006). Meanwhile, the use of adeno-associated virus (AAV) vectors was also adopted for siRNA delivery into both dividing and nondividing cells (Wu et al., 2003). Tomar et al. (2003) presented the use of an AAV modified vector to efficiently introduce siRNA leading to downregulation of p53 and caspase 8 expression in HeLa S3 cells. In addition, the AAV stereotype 8 vector (AAV-8) expressing shRNA is reported to transduce almost 100% of liver cells after injection into mice, which provides an ideal tool for clinical RNAi therapy (Grimm and Kay, 2006; Nakai et al., 2005).

Synthetic materials have demonstrated potential as effective non-viral siRNA delivery carriers (Whitehead et al., 2009). Unilamellar and multilamellar liposomes are frequently used as pharmaceutical delivery vehicles (Torchilin, 2005). In an aqueous environment, certain materials have the ability to form liposomes, in which a lipid bilayer forms a sphere with an aqueous core. Liposomes can be composed of single or multiple types of lipid that allows for additional flexibility when optimizing the physical or chemical properties of the nanoparticle (Torchilin, 2005; Whitehead et al., 2009). The transfection reagent Lipofectin, the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), was originally studied to deliver both DNA and RNA into mouse, rat and human cell lines (Felgner et al., 1987; Malone et al., 1989). Small interfering RNAs interact electrostatically with cationic liposomes to form particles that are able to introduce RNA into cells. However, electrostatic interactions between siRNA and cationic liposomes pose two potential problems: firstly, a relatively uncontrolled interaction process leading to lipid/siRNA complexes of excessive size and poor stability; secondly, incomplete encapsulation of siRNA molecules, which consequently expose siRNA to potential enzymatic or physical degradation prior to introducion into the cell (De Paula et al., 2007). Zhang et al. (2006) successfully demonstrated siRNA delivery into lung tumour cells via packaging of siRNA into liposomes bearing the arginine octamer (R8), a type of membrane permeant peptide (MPP), attached to the liposome surface. This complex was formulated using 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP)/siRNA with the addition of the polyethylene glycol (PEG)-phosphatidylethanolamine, which displayed high stability, protecting the incorporated siRNA from degradation by blood serum, and also showed high transfection efficiency and low non-specific toxicity (Zhang et al., 2006).

Cationic liposomes are known to interact with serum proteins (e.g. heparin, albumin and lipoproteins) and the anionic glycosaminoglycans in the extracellular matrix, leading to aggregation or release of nucleic acids from the complexes even before reaching the surface of the target cells. Cationic liposomal complexes can also activate the complement system, leading their rapid clearance by macrophages in the reticuloendothelial systems (Mahato et al., 1997). To circumvent these effects, many liposomal carriers have been coated with the hydrophilic molecule PEG, which can form a barrier around the complex providing steric stabilization and reducing the immune clearance (Martina et al., 2007). Recently,

Zimmermann et al. used a PEG conjugated nucleic acid lipid particle (SNALPs) which encapsulated siRNAs and enabled knockdown of apolipoprotein B (ApoB) in the liver of cynomolgus monkeys (Zimmermann et al., 2006). Injection of siRNAs encapsulated in this manner resulted in dose-dependent silencing of ApoB mRNA transcript level in the liver 48 hours after administration, with maximal knockdown of >90% (Zimmermann et al., 2006).

Like cationic lipid carriers, cationic polymers spontaneously form complexes with nucleic acids due to electrostatic interactions between the positively charged amine groups of polycations and the negatively charged phosphate groups of the nucleic acids. The interaction between a cationic polymer/nucleic acid complex and negatively charged cell membranes can enhance its uptake by cells and thus increase transfection efficiency (De Paula et al., 2007). Polyethylenimine (PEI) is a broadly investigated polymeric carrier for siRNA delivery applications. PEIs with various molecular weights, degrees of branching, and other modifications have been intensively utilized for transfection of siRNAs in different cell lines and live animals (Grzelinski et al., 2006; Urban-Klein et al., 2005). PEI has shown effectiveness in a subcutaneous mouse tumour model. Intraperitoneal administration (IP injection) of complexed siRNA led to the delivery of the intact siRNA into the tumours and a notable reduction of tumour growth via siRNA-mediated human epidermal growth factor 2 (HER2) downregulation (Urban-Klein et al., 2005). Furthermore, Grzelinski et al. showed that complexing of unmodified siRNAs with PEI leads to the formation of complexes that condense and completely cover siRNAs as determined by using atomic force microscopy (ATM), and then also demonstrated that delivery of siRNAs against the growth factor pleiotrophin (PTN) complexed with PEI was able to generate antitumour effects in vitro and in vivo (Grzelinski et al., 2006). However, the use of PEI is still under development due to significant concerns regarding its toxicity at high doses (Thomas et al., 2005; Werth et al., 2006).

RNAi interacts with several pathways, and exogenous RNA transfection often activates immune defence leading to unintended and off-target effects in vivo. Many exogenous unmodified siRNAs can induce nonspecific activation of the mammalian immune system via Toll-like receptors (TLRs), which detect pathogen-specific molecular patterns including unmethylated cytosine-guanine motifs (CpG motifs) and viral dsRNA (De Paula et al., 2007).

Research has shown that the activation of immune cells by siRNAs is sequence dependent and sense or antisense strands separately can induce cytokine production as efficiently as duplex siRNAs (Judge et al., 2005; Sioud, 2005). The elimination of this immunostimulatory effect can be achieved by modification of siRNA molecules such as the locked nucleic acid (LAN) modification of the sense and the antisense strands of selected nucleotides within the TLR-7-interacting sequence (Hornung et al., 2005). This chemical modification of the siRNA not only eliminated induction of interferon-gamma (IFN-γ), but also enabled RNAi silencing in plasmacytoid dendritic cells (Hornung et al., 2005). In addition, Sioud (2006) have reported that 2’-OH group of uridines is associated with cellular immune recognition of siRNAs. The modification of the 2’-hydroxyl uridine with either 2’-fluoro, 2’-deoxy or 2’- methyl urdines showed to affect the interaction of siRNA with TLR-7 and TLR-8, abrogating immune activation (Sioud, 2006). Therefore, these kind of chemical modifications of siRNA oligonucleotides can be applied to reduce nonspecific effects without affecting the biological activity of siRNAs in cells. Moreover, significant stability against nuclease degradation has been achieved by modifing siRNA. Introducing a phosphorothioate (PS) backbone linkage at the three prime end and 2’ sugar modifications (i.e. 2’-O-methyl, 2’-deoxy-2’-fluoro) led to exonuclease and endonuclease resistance respectively (Bumcrot et al., 2006).

Another strategy to improve the efficiency of siRNA uptake involves the conjugation of small molecules or peptides to the sense strand of siRNA. Soutschek et al. (2004) demonstrated that conjugation of cholesterol to the three prime end of the sense strand of modified siRNA increased binding to serum albumin, leading to improved delivery to certain targets. Cholesterol-modified siRNA was capable of suppression of ApoB mRNA in mouse liver and jejunum, eventually reducing total cholesterol levels. Subsequently, Wolfrum et al. (2007) synthesized a variety of lipophilic siRNAs and elucidated the requirements for siRNA uptake in vivo. They demonstrated that cholesterol-modified siRNA conjugates enabled siRNA uptake via high-density lipoprotein (HDL) and low-density lipoprotein (LDL) receptors to the liver. More importantly, the HDL-bound cholesterol-siRNA was shown to be 8 to 15-fold more efficient at ApoB expression knockdown when compared to equal amounts of cholesterol-siRNA in vivo (Wolfrum et al., 2007).

Chapter 2

Materials and Methods

2.1. Parasites

Infection of rats and recovery of N. brasiliensis adult worms Generally, 6000 infective third stage N. brasiliensis larvae (L3) were injected subcutaneously into male Sprague-Dawley rats (obtained from the Central Biomedical Services, Imperial College London) through two sites in the flank. Rats were culled by cervical dislocation, 5 to 7 days post-infection and their small intestines were removed. The anterior half of each intestine was cut up and placed onto muslin stretched over a Baermann apparatus filled with phosphate buffered saline (PBS) (Gibco) warmed to 37°C. Parasites were collected from the bottom of a 15 ml Falcon tube after 90 minutes. Adult parasites were washed 4 times in sterile PBS and 4 times in sterile parasite medium, which consisted of Roswell Park Memorial Institute (RPMI) 1640 medium (InVitrogen) supplemented with 1% (w/v) glucose, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 20 µg ml-1 gentamycin, 20 U ml-1 nystatin and 2 mM L-glutamine before being used for further in vitro culture.

Development and recovery of larval stages Faeces were collected from infected rats from day 6 to day 8 post-infection, soaked in water, made into a paste and mixed with an equal volume of granular charcoal (BDH Laboratory Supplies). This was plated onto Petri dishes and incubated at 22°C for at least one week.

Hatched infective L3 could be easily observed under the microscope and remained infective at least for one month. For recovery of larvae, a Baermann apparatus was set up with a 15 ml Falcon tube, a double layer of muslin containing two folded sheets of lens cleaning tissue (Whatman) in the middle. The funnel was filled with water at room temperature. Faecal cultures were placed onto the muslin, and larvae were allowed to settle for 90 minutes. Larvae were then washed 4 times in sterile PBS and counted using a McMaster worm egg counting chamber before being used for infection.

In vitro culture of parasites and collection of excretory-secretory (ES) products Recovered parasites were washed 4 times with sterile parasite medium. Parasites were cultured and maintained in sterile worm medium, unless otherwise stated at 37°C in 5% CO2. The culture medium was changed every 2 days. Excretory-secretory (ES) products were collected from worm culture supernatants, filtered through 0.2 µm filters and concentrated at

4°C in an Amicon concentrator (Amicon) using a 10-kDa molecular weight cut-off membrane. Samples were further concentrated in Centricon 10 microconcentrators (Millipore) at 5,000 x g and 4°C, washed in 25 mM HEPES (pH 7.0), before being aliquoted and stored at -20°C.

Preparation of parasite somatic extracts Parasites were collected, frozen in liquid nitrogen and homogenised using a Bessman Tissue Pulverizer (Spectrum Labs). Two to five volumes of lysis buffer (25 mM Tris base pH 7.0, 0.25 mM η-dodecyl-D-B-Maltoside), and protease inhibitors (1 mM ethylene diamine tetracetic acid (EDTA), 1 mM ethylene glycol bis (2-amino ethyl ether)-N,N,N’,N’-tetracetic (EGTA), 1 mM N-ethylmalemide (NEM). 0.1 µM pepstatin, 1 mM phenyl methyl sulphonyl fluoride (PMSF), 0.1 mM N-tosylamide-L-phenylalanine chloro-methyl ketone (TPCK)) were added. The mixture was then sonicated three times for 1 minute using a 1 second pulse at 40 watts with 1 minute intervals on ice. The samples were left on ice for 30 minutes before centrifugation for 20 minutes at 13,000 x g and 4°C. Once the protein concentration of samples was determined the supernatant was aliquoted and stored at -20°C.

2.2. Culture of 293 T cells and J774 macrophages

The 293 T cells or J774 macrophages were cultured in DMEM (Gibco) containing 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 20 µg ml-1 gentamicin and 10% (v/v) foetal calf serum

(FCS) at 37°C in 5% CO2. Cells were counted in a haemocytometer using 0.2% (v/v) trypan blue (Sigma) to measure viability.

2.3. Proteins

Determination of protein concentration Protein concentration was determined using the BCA microplate assay (Pierce) according to manufacturer’s instructions. Bovine serum albumin (BSA) (Sigma) ranging in concentration

56 from 0.025 mg ml-1 to 2.0 mg ml-1 was used as a standard, and absorbance was read at 560 nm.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Proteins were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) under standard conditions as follows: 12% SDS-PAGE-- Resolving gel: 12% (w/v) acrylamide mix, 0.4 M Tris base, pH 8.8, 0.1% ammonium persulphate, 0.1% (v/v) N,N,N',N'-Tetramethylethylenediamine (TEMED) and 0.1% (w/v) SDS. Stacking gel: 5% (w/v) acrylamide mix, Tris pH 6.8, 0.1% ammonium persulphate, 0.04% (v/v) TEMED and 0.1% (w/v) SDS. Protein samples were boiled for 5 minutes in SDS loading buffer (0.1 M sucrose, 3% (w/v) SDS, 62.5 mM Tris base, pH 6.9, 2 mM Na2EDTA, 0.05% (v/v) β- mercaptoethanol, 0.0025% (v/v) bromophenol blue) and loaded into the gel. Samples were separated in SDS-PAGE running buffer (25 mM Tris base, pH 8.3, 192 mM glycine and 0.1% (w/v) SDS) at a constant current of 25 mA and RT using the mini-PROTEAN II electrophoresis system (Bio-Rad), till the leading edge of the dye had reach the bottom of the gel. Following electrophoresis, proteins were either visualized by staining with Coomassie blue or transferred to nitrocellulose membranes.

Coomassie blue staining of polyacrylaminde gels Gels were stained overnight (O/N) at room temperature (RT) in 0.4% (w/v) Coomassie brilliant blue stain (Sigma) in 10% (v/v) acetic acid and 40% (v/v) methanol. The gel was destained with 10% (v/v) acetic acid and 40% (v/v) methanol until the protein samples were clearly visible.

Western blotting Protein samples were separated by SDS-PAGE or 10-20% gradient SDS gel (Bio-Rad) as described above, and then transferred to nitrocellulose membrane (Hybond ECL; Amersham) using an electrophoretic transfer cell (Bio-Rad). Prior to transfer, the membranes, polyacrylamide gels and other blotting components were equilibrated in transfer buffer (25 mM Tris base, 192 mM glycine and 20% (v/v) methanol). Electrophoretic protein transfer was carried out for 2 hours at a constant current of 100 mA in transfer buffer at 4°C. Following transfer, the membranes were incubated in blocking buffer (PBS with 5%

57 skimmed milk) for 1 hour at room temperature. Blots were then probed with primary antibody in PBS with 5% milk powder and 0.1% Tween 20 O/N at 4°C. The monoclonal mouse anti-HisTag antibody (Sigma) was used at a dilution of 1:3000. Following three 10- minute washes with 0.1% Tween 20 in PBS at room temperature, and the blots were subsequently incubated in an appropriate horseradish peroxidase (HRP) conjugated secondary antibody for 1 hour at RT. For mouse derived primary antibody, goat anti-mouse IgG secondary antibody (Bio-Rad) was used at a dilution of 1:3000. The blots were washed three times for 10 minutes in 0.1% Tween 20 in PBS followed by one 10-minute wash in PBS alone. Reacting antibodies were visulised using enhanced chemiluminescence (ECL): the blots were developed with 5 ml luminol (1.25 mM in 0.1 M Tris base, pH 8.5), 50 µl 6.8

µM p-coumaric acid in DMSO and 15 µl 30% H2O2 that had been mixed for 1 minute and applied to the membranes for 1 minute. The membrane was briefly rinsed in PBS before exposure to X-ray film.

2.4. RNA extraction

Isolated parasites (approximately 100 µl settled volume) were washed in sterile PBS and resuspened in 200 µl TRIzol reagent (InVitrogen). Samples were frozen with liquid nitrogen and homogenised using a Bessman Tissue Pulverizer chilled with liquid nitrogen. Following homogenisation, the parasite/TRIzol powder was collected and allowed to thaw on ice. A further 0.2 volumes of chloroform were added to samples, gently mixed, incubated at room temperature for 3 minutes, then centrifuged at 12,000 x g for 15 minutes at 4°C. The upper aqueous phase was transferred to a fresh tube and RNA was precipitated by an additional 0.5 volumes of isopropanol followed by incubation at room temperature for 10 minutes. The mixture was then centrifuged at 12,000 x g for 10 minutes at 4°C. The supernatant was discarded and the RNA pellet was washed with 500 µl of 75% (v/v) ethanol before centrifugation at 7,500 x g for 5 minutes at 4°C. The supernatant was removed and the pellet air-dried. The RNA pellet was suspended in nuclease free distilled water, and then cleaned up using the RNeasy® Mini Kit (Qiagen). Isolated RNA was quantified by spectrophotometry at 260 nm and the quality of the RNA determined by separating on a 1% agarose gel in 1x TAE

(40 mM Tris base, 20 mM acetic acid and 1 mM EDTA pH 8.0) containing ethidium bromide (0.25 µg ml-1) (Sigma).

2.5. Polymerase Chain Reaction (PCR)

PCRs were carried out in a final volume of 25 µl containing 2.5 U of Taq DNA polymerase (BioLabs), 1x ThermoPol PCR Buffer (BioLabs), 0.25 mM each of dATP, dTTP, dCTP and dGTP (Promega) and 10 pmoles of each primers and the indicated amount of template DNA. Amplification reactions were performed as follows: (1) 95°C for 5 minutes (2) 95°C for 30 seconds (3) Annealing temperature as indicated for 30 seconds (4) 72°C for 1 minute Repeat step (2) to (4) for 30 to 40 cycles (5) 72°C for 10 minutes

2.6. Reverse Transcription PCR (RT-PCR)

For reverse transcription reactions, 2 µg of total RNA was incubated with 1 µg of oligo-dT primer (Promega) at 70°C for 5 minutes and then transferred onto ice. The following mixture were then added to a final volume of 50 µl: 10% (v/v) Moloney Murine Leukaemia Virus Reverse Transcriptase (M-MLV RT) buffer (at a final concentration of 10 mM Tris, pH 8.3,

15 mM KCl, 0.6 mM MgCl2 and 2 mM dithiothreitol (DTT, Promega)), 2.5 mM each of dATP, dTTP, dCTP and dGTP (Promega), 25 U of RNasin Ribonuclease Inhibitor (Promega) and 400 U of M-MLV-RT (Promega). The sample was incubated for 1 hour at 42°C. For PCR, 2 µl of cDNA template generated by reverse transcription was used in a standard reaction as described in 2.5.

2.7. Production of firefly luciferase-encoding mRNA

A 1.8 kb poly(A) tailed Photinus pyralis firefly luciferase-encoding mRNA (Appendix. A) was generated from a PCR product amplified from a 5’ primer incorporated with T7 promoter sequence and a 3’ primer incorporated with poly(A) sequence at the ends of the DNA. A pCRE-Luc plasmid DNA (Clontech) was used as template for the PCR, and removed from the amplified fragment using the QIAquick PCR purification kit (Qiagen). The RiboMAX large scale RNA production system (Promega) was used to generate dsRNA using 10 µg of the PCR products in a 100 µl reaction, according to the manufacturer’s instructions.

The RNA was quantified spectrophotometrically at A260 and visually by resolving 1 µg on 1% agarose TAE gel with ethidium bromide.

2.8. Production of double-stranded (ds)RNA

Double-stranded RNAs were generated from PCR products amplified from three different regions corresponding to the N. brasiliensis acetylcholinesterase subunit B (NbAChE-B) (Appendix. B) and ubiquitin (NbUbq) (Appendix. C) transcripts using primers that incorporated T7 promoter sequence at the ends of the DNA fragments. The NbAChE-B pGEM-T construct or N. brasiliensis adult cDNA were used as template for the PCR, and removed from the amplified fragments using the QIAquick PCR purification kit. The RiboMAX large scale RNA production system was applied to generate dsRNA using 10 µg of the PCR products in a 100 µl reaction, according to the manufacture’s instructions. The

RNA was quantified spectrophotometrically at A260 and visually by resolving 1 µg on 1% agarose TAE gel with ethidium bromide.

2.9. Production of small interfering (si)RNAs

Small interfering (si)RNAs were generated from purified dsRNA (60 µg) using the BLOCK- iT Dicer RNAi Kit (InVitrogen) according to manufacturer’s instructions, and which cleaves

60 dsRNA into fragments of around 22 nucleotides. Cleavage of dsRNA was confirmed by electrophoresis on a 4% agarose TAE gel with ethidium bromide.

2.10. Rapid amplification of 5’ and 3’ cDNA ends (5’-RACE and 3’-RACE)

5’-RACE Full-length 5’ ends of N. brasiliensis VAL-3, 6, 7 and 8 (NbVAL-3, 6, 7 and 8) were obtained using the GeneRacer kit (InVitrogen) according to manufacturer’s instructions. Five µg of total RNA of N. brasiliensis adult were treated with 1 µl of calf intestinal phosphatase (CIP) (10 U µl-1), 1 µl of 10x CIP buffer, 1 µl RNaseOUT (40 U µl-1) in DEPC water in a total volume of 10 µl at 50°C for 1 hour to dephosphorylate non-mRNA. After incubation, RNA was precipitated and air-dried. The dephosphorylated RNA pellet was resuspended in 7 µl DEPC water, and 1 µl of tobacco acid pyrophosphatase (TAP) (0.5 U µl-1), 1 µl of 10x TAP buffer, 1 µl RNaseOUT (40 U µl-1) added in a total volume of 10 µl, which was then incubated at 37°C for 1 hour to remove the mRNA cap structure, and the RNA pellet precipitated. The dephosphorylated and decapped RNA pellet was resuspended in 7 µl DEPC water, 0.25 µg lyophilized GeneRacer RNA Oligo was further added for the ligation reaction, which was firstly incubated at 65°C for 5 minutes to relax the RNA secondary structure and then chilled on ice for 2 minutes. Following the reaction, lyophilized GeneRacer RNA Oligo was ligated to RNA at 37°C for an hour, in the presence of 1 µl 10 mM ATP, T4 ligase (5 U µl-1), 10x ligase buffer and RNaseOUT (40 U µl-1). The ligated RNA sample was then precipitated, air-dried and resuspended in 10 µl DEPC for further reverse transcription reactions. Reverse transcription was performed using 1 µl of the Random Primers (100 ng µl- 1, provided in the kit), 4 µl of 5x RT buffer and 1 µl of Cloned AMV RT (15 U µl-1) and RNaseOUT (40 U µl-1) with 10 µl of ligated RNA at 25°C for 10 minutes followed at 45°C for 1 hour. The reaction was inactivated at 85°C for 15 minutes, before being amplified at the 5’ cDNA end. Two µl of 5’RACE-ready cDNA were used as template for the first PCR reaction, containing 10 pmoles of NbVALs gene-specific primer-1 (GSP1) antisense primer (Table 2.1), together with 30 pmoles of the GeneRacer 5’ Primer (provided in the kit). Following amplification, 1 µl of the reaction was used for a second PCR reaction containing

10 pmoles NbVAL GSP2 antisense primer (Table 2.1), located internally to the NbVALs GSP1, together with 30 pmoles of GeneRacer 5’ Nested Primer (provided in the kit). The 5’ end PCR products of NbVAL-3, 6, 7 and 8 were gel purified, cloned into pCR4-TOPO, and sequenced.

3’-RACE Two µl of N. brasiliensis adult cDNA were used as template for the first PCR reaction in 3’ cDNA amplification, containing 10 pmoles of NbVAL-8 GSP1 sense primer (Table 2.1), together with 30 pmoles of the GeneRacer 3’ Primer (provided in the kit). Following amplification, 1 µl of the reaction was used for a second PCR reaction containing 10 pmoles NbVAL-8 GSP2 sense primer (Table 2.1), located internally to the NbVAL-8 GSP1, together with 30 pmoles of the GeneRacer 3’ Nested Primer (provided in the kit). The 3’ end PCR product of NbVAL-8 was gel purified, cloned into pCR4-TOPO, and sequenced.

Description Code Sequence 5'-3' Firefly luciferase LMRF ATCGAAATTAATACGACTCACTATAGGGCATTCCGGTACTGTTG TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT for mRNA synthesis LMRR TTTTTTTTGAATGCAATTGTTGTTGTT AChEB1-T7F TAATACGACTCACTATAGGGGCTAGATTACTCCTGGCAATATG AChEB1-T7R TAATACGACTCACTATAGGGGTACGTTTCGTCTTTGGTTTGG NbAChE-B AChEB2-T7F TAATACGACTCACTATAGGGACAGCGAAGGCACTAGGATG for dsRNA synthesis AChEB2-T7R TAATACGACTCACTATAGGGGCCGTACTTCTCCCTTGAAGTAG AChEB3-T7F TAATACGACTCACTATAGGGCCACTGCTTACAAGGAAGTAGACAA AChEB3-T7R TAATACGACTCACTATAGGGGAATGGAAACTAGTTGCCTTTAGC NbAChE-B AChEBtF TTCGGCACGAGGATGGGACTTCCC RNAi detection primers AChEBtR CTGAAGAAACCTCCTCCATAAATCC NbUbq Ubq1-T7F TAATACGACTCACTATAGGGAGGCATGCAGATTTTCGTCAAAACC for dsRNA synthesis Ubq1-T7R TAATACGACTCACTATAGGGGTCTTCACGAATATCTGCATGCCTC NbUbq UbqtF GAAGGAGTCTACCCTTCACCTC RNAi detection primers UbqtR TCCATCTTCGAGTTGCTTTCCA NbVAL-1 NbVAL1F CTTCTCAGTGTGATGTTCATTGGG RT-PCR primers NbVAL1R GAGTTCAATCGCCTTTGCTTCATC NbVAL-2 NbVAL2F CGGCTGCTTCCTTCTGCAGCTCAA RT-PCR primers NbVAL2R GAATCATCAGTTTCCGCCTTTGCA NbVAL-3 NbVAL3F CACGACAAAATTCGCGAGTTCTGC RT-PCR primers NbVAL3R GGGCCCAATGGTTCGATTGTTGTG NbVAL-4 NbVAL4F GCTCGTGCTAACTGGCCTCGTCGT RT-PCR primers NbVAL4R GCCAACTTGCATTGATTCACATGT NbVAL-5 NbVAL5F TGAAGCCAGCTCCAACGCCAGATT RT-PCR primers NbVAL5R ATTGGGTCGGCCACTAACCGGTGA NbVAL-6 NbVAL6F CGAAAGATATGGAGTCGACAATCG RT-PCR primers NbVAL6R GGATAGCAGAGCTTGTCGTACTCC NbVAL-7 NbVAL7F GTGGTTTGGGGCGATACCGACAGA RT-PCR primers NbVAL7R GACTCATATCTCTTGTTGGACCTC NbVAL-8 NbVAL8F CGCTGTTCCCGCTGGCGATACTTG RT-PCR primers NbVAL8R CACGTTTAGCCAGCTCTTCTATGG NbVAL-3 VAL3-R2 CACAGATAGTCTTCGTCACAAGCC for 5'-RACE VAL3-R3 GGTGCCAATTCCAGTAGTGGTTTG NbVAL-6 VAL6-R2 CTGTTGGGGCAGTTCTCGCAGATG for 5'-RACE VAL6-R3 CGATTGTCGACTCCATATCTTTCG NbVAL-7 VAL7-R2 GCACAGAGGGCGCCGTCTTCACAC for 5'-RACE VAL7-R3 TCTGTCGGTATCGCCCCAAACCAC NbVAL-8 VAL8-F2 GTCCAGTGATGGCAATGTACCAAG for 3'-RACE VAL8-F3 CGAGTAGGCACTTTCACAGGAGCT NbVAL-3 NbVAL3_49-72Nde1 CATATGATCCAGTCGAAATTCAACTGTCCT into pET29b NbVAL3_ 649-672Not1 GCGGCCGCTATGGTTGCTGGGCCCAATGGTTC NbVAL-4 NbVAL4_58-81Nde1 CATATGGGCGCGTGTCCTAAGACGGAGGGC into pET29b NbVAL4_622-645Not1 GCGGCCGCCCTGACAACACACAGACCGTTCTC NbVAL-5 NbVAL5_49-72Nde1 CATATGAACGCCAGATTTGCTCGGCAGATC into pET29b NbVAL5_619-642Xho1 CTCGAGGCTCATATAGCAGAGTGTGCCACC NbVAL-6 NbVAL6_49-72Nde1 CATATGACGCCGATTAGCCCAGTTTCCATG into pET29b NbVAL6_652-675Xho1 CTCGAGTGGGAGTGCCCAATCAGGATAGCA NbVAL-7 NbVAL7_61-84Nde1 CATATGCATGAATACCACTGCAATAAGGAC into pET29b NbVAL7_631-651Xho1 CTCGAGATTATCAGGCGCCTCACAAAGACC NbVAL-8 NbVAL8_91-114Nde1 CATATGACTCAAAATTTCAACTGTCAAAAC into pET29b NbVAL8_706-729Xho1 CTCGAGGTTTTCTCTTTTCTTAATGCACAA NbFTT-2 NbFTT-2F GAAGGTCGAGAAGGAGCTTCGTGA RT-PCR primers NbFTT-2R GTACGTCCACAAGGTGAGGTTGCT N. brasiliensis actin-F CAGAAGGACTCCTATGTAGGAGATGA Actin primers actin-R CATGATCTGGGTCATCTTTTCTCTGT

Table 2.1. List of primers used in this study

2.11. Iodination of bovine serum albumin (BSA)

Proteins were iodinated using Chloramine T which inserts 125I into the phenolic ring of tyrosine residues. A total of 25 µg of BSA was dissolved in 50 µl PBS, and then 500 µCi Na 125I (MP Biomedicals) was added. Subsequently, 5 µl of 1 mg ml-1 Chloramine T (Sigma) was added and incubated for 3 minutes, and another 5 µl of 1 mg ml-1 Chloramine T was added for a further 5 minutes. The reaction was stopped with 10 µl of saturated tyrosine solution, which samples were loaded onto a Sephadex G-25 column and eluted with PBS in order to separate free isotope from labelled BSA. The flow-through was collected in 1 ml fractions, and the percentage of labelled BSA of the first three fractions was determined by trichloroacetic acid (TCA) precipitation.

2.12. Metabolic labelling of parasite proteins in vitro

Secreted products A total of 10,000 isolated larval N. brasiliensis parasites were incubated in 1 ml of worm medium with 1 mCi L-[35S]methionine (MP Biomedicals) and indicated chemicals at 37°C in

5% CO2 or at 20°C for the indicated incubation time. Parasite secreted products were collected from the worm culture supernatants, and samples were further concentrated in a 2 ml Vivaspin column with 3-kDa molecular weight cut-off membrane (Sartorius Stedim Biotech) at 10,000 x g and 4°C, and washed with 1.5 ml of 25 mM HEPES (pH 7.0) for 4 times, before being stored at -20°C.

Parasite somatic extracts A total of 10,000 isolated larval N. brasiliensis parasites were incubated in 1 ml of worm 35 medium with 1 mCi L-[ S]methionine and indicated chemicals at 37°C in 5% CO2 for the indicated incubation time. Parasites were collected from culture and washed with PBS for 3 times. Worms were resuspended in 50 µl of Studier buffer (SDS-PAGE loading buffer), boiled at 95°C for 5 minutes, then used for scintillation counting (Beckman LS 6500 Scintillation Counter).

2.13. Measurement of N. brasiliensis acetylcholinesterase (NbAChE) activity from parasite secreted products

Ten µl of collected parasite ES proteins were incubated in Ellman reagent (Ellman et al., 1961), containing 168 µl of 100 mM sodium phosphate buffer (pH 7.0), 2 µl of 1 mM acetylthiocholine iodide (Sigma) substrate and 20 µl of 1 mM 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma) for 5 minutes at RT. The absorbance of NbAChE activity was monitored at 412 nm.

2.14. Analysis of AChE activity in native polyacrylamide gels

Collected parasite ES products were resolved by 8% native polyacrylamide gel electrophoresis (8% (w/v) acrylamide mix, 0.4 M Tris pH 8.8, 0.1% ammonium persulphate and 0.1% (v/v) TEMED). Samples were suspended in loading buffer (0.1 M sucrose, 62.5 mM Tris base, pH 6.9, 2 mM Na2EDTA, 0.0025% (v/v) bromophenol blue) and loaded onto the gel. Samples were separated in running buffer (25 mM Tris base, pH 8.3, 192 mM glycine) at a constant current of 25 mA and 4°C using the mini-PROTEAN II electrophoresis system, until the leading edge of the dye had reached two-thirds length of the gel.

After electrophoresis, the gel was incubated with 65 ml of 0.1 M sodium phosphate buffer, pH 6.5 with dissolved 50 mg acetylthiocholine iodide powder for 30 minutes at RT, to which the following were added sequentially: 5 ml of 0.1 M sodium citrate (Sigma), 10 ml of 30 mM CuSO4 (Sigma), 10 ml of sterile water and 10 ml of 5 mM potassium ferricyanide (Sigma). The gel was then developed at RT for 30 minutes. NbAChE active subunits resulted in the appearance of brown bands. The reaction was stopped by sterile water and gel fixed in 10% acetic acid.

2.15. Exsheathment of infective larvae

To exsheath third-stage N. brasiliensis, freshly isolated L3 were exposed to 0.5 ml of 0.3% (w/v) sodium hypochlorite (Sigma) in sterile PBS for 20 minutes at room temperature (RT). Following the incubation, samples were washed 3 times with sterile PBS and worm medium respectively.

2.16. Feeding assay

A total of 10,000 isolated N. brasiliensis L3 were incubated in 0.5 ml culture medium with the indicated chemicals, 10% rat serum (Sigma), rat skin or 30 µg of L3 ES products at 20°C or 37°C for the indicated incubation time. Control larvae were incubated in the absence of stimulants. After incubation, samples were washed with sterile PBS for 3 times and then incubated in 0.5 ml sterile PBS with 2.5 mg ml-1 FITC labelled bovine serum albumin (FITC- BSA) (Sigma) for an hour at RT or 37°C. The proportion of the larval population feeding was determined by fluorescence microscope, with a minimum of 200 larvae counted in triplicate per treatment condition.

Chemicals; L-glutathione reduced (GSH), serotonin (5-HT), L-glutamic acid, gamma- aminobutyric acid, octopamine, dopamine, noradrenaline (norepinephrine), resorcinol, 8- bromo-cGMP, oxotremorine-M, arecoline, and ketoconazole were all purchased from Sigma Chemical Company, except LY294002 and Akt inhibitor IV were both from Calbiochem Company.

2.17. Feeding assay using radiolabeled albumin

To quantitate feeding of N. brasiliensis L3 after chemical treatments, freshly isolated 4,000

L3 parasites were first cultured in 0.5 ml parasite culture medium with the indicated concentrations of chemicals at 37°C in 5% CO2 for the indicated incubation time. After incubation, samples were washed with sterile PBS for 3 times, then incubated in 0.5 ml worm

66 medium with 100,000 cpm 125I-Bovine Serum Albumin (BSA) and indicated chemicals for an hour. Subsequently, samples were washed 5 times with sterile PBS, and ingested 125I-BSA was measured from three replicates of each treatment group.

2.18. Assay of viability

Uptake and reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) was used as a measure of parasite viability (Thomas et al., 1997). About 6,000 larval N. brasiliensis were incubated in 5 mg ml-1 MTT in sterile PBS at 37°C for 2 hours. Resulting formazan crystals formed within the parasites were solubilised by transferring parasites to 500 µl of dimethyl sulfoxide (DMSO) (Sigma) and incubation at RT for an hour with shaking. Absorbance of the supernatant was determined at 550 nm with DMSO as blank. Background levels were established by performing the assay on parasites killed by heating to 95°C for 2 minutes.

2.19. Electroporation

Electroporation of 293 T cells with firefly luciferase-encoding mRNA A total of 5 x 106 293 T cells were suspended in 200 µl DEPC treated PBS containing 50 µg ml-1 of luciferase-encoding mRNA, and electroporated using a 4 mm Gene Pulser cuvette (Bio-Rad) at 400 V with 250 µF (~6 ms) (Bio-Rad Gene Pulser XCell). The cells were then incubated in 1.5 ml DMEM culture medium for 3 hours, and luciferase activity measured (2.22).

Electroporation of N. brasiliensis with firefly luciferase-encoding mRNA A total of 20,000 larval parasites or 100 µl adult worms (settled volume) were suspended in 200 µl DEPC treated PBS containing 400 µg ml-1 luciferase-encoding mRNA, and electroporated using a 4 mm Gene Pulser cuvette at 125 V with a single pulse of 20 ms as

67 described (Correnti and Pearce, 2004). Parasites were then incubated in 1.5 ml of worm culture medium for 3 or 24 hours, and the luciferase activity measured (2.22).

Electroporation of N. brasiliensis with dsRNA or siRNA A total of 20,000 larval parasites or 100 µl adult worms (settled volume) were suspended in 200 µl worm medium containing 1 mg ml-1 dsRNA or 100 µg ml-1 siRNA, and 400 U ml-1

RNasin ribonuclease inhibitor (Promega), pre-incubated for 1 hour at 37°C in 5% CO2. Electroporation was performed in a 4 mm Gene Pulser cuvette at 125 V with a single pulse of 20 ms. After electroporation the medium containing dsRNA or siRNA was removed, and parasites cultured in fresh medium at 37°C in 5% CO2 for the indicated incubation time. Post- treatment, parasites were recovered, washed in sterile PBS and RNA purified. Transcript levels of target genes were determined by RT-PCR using 10 fold serially diluted cDNA.

2.20. Attempted delivery of dsRNA or siRNA into N. brasiliensis by soaking

Soaking of N. brasiliensis with lipofectin-encapsulated dsRNA A total of 20,000 larvae or 100 µl adult parasites (settled volume) were suspended in 200 µl parasite culture medium containing 1 mg ml-1 of dsRNA, 5% lipofectin (InVitrogen) and 400 -1 U ml RNasin ribonuclease inhibitor, and incubated at 37°C in 5% CO2 for 24 hours before being cultured in 500 µl fresh worm medium. Post-treatment, worms were recovered, washed with sterile PBS and RNA purified. Transcript levels of target genes were determined by RT- PCR using 10 fold serially diluted cDNA.

Introduction of polyethyleneimine (PEI)-complexed Cy3-labelled siRNA into J774 macrophages For PEI complex formation, PEI/nucleic acid (i.e. siRNA or dsRNA) ratios were calculated based on nitrogen atoms in PEI to phosphate atoms in nucleic acids (1 µg of siRNA or dsRNA is equivalent to 3 nmoles of phosphate) and expressed as PEI/siRNA or PEI/dsRNA equivalents (N/P ratios) (Marschall et al., 1999; Urban-Klein et al., 2005).

Preparation of jetPEI/Cy3-labelled siRNA was performed according to the supplier's instructions. The jetPEITM reagent (Autogenbioclear) was used to complex with 0.1 µM Cy3- labelled siRNA (Ambion) in 5% sterile glucose solution resulting in the desired N/P ratios (1, 3 and 5), and incubated at room temperature for 30 minutes for further use.

The jetPEI/Cy3-labelled siRNA complexes were then transfected into a total of 5 x 106 J774 macrophages with 1 ml DMEM culture medium, which were then incubated for 24 hours at

37°C in 5% CO2. After incubation, cells were harvested and washed with sterile PBS 3 times. Results were determined and visualized by fluorescence microscopy.

Introduction of PEI-complexed Cy3-labelled siRNA into N. brasiliensis L3 Preparation of jetPEI/Cy3-labelled siRNA was performed according to the supplier's instructions. The jetPEI reagent was used to complex with 0.1, 0.2 and 0.3 µM of Cy3- labelled siRNA in 5% sterile glucose solution resulting in the desired N/P ratio of 5, then incubated at room temperature for 30 minutes for further use.

A total of 5,000 temperature (37°C)-activated larval N. brasiliensis were incubated with prepared jetPEI/Cy3-labelled siRNA solution (200 µl) for 6 hours at 37°C in 5% CO2. After incubation, parasites were harvested and washed 3 times with sterile PBS. The results were determined and visualized by fluorescence microscopy.

Soaking of N. brasiliensis with PEI-complexed siRNA Preparation of PEI-complexed siRNA for parasite target genes was performed as previously described. The jetPEI reagent was used to complex with 0.3 and 1.5 µM of NbUbq siRNA in 5% sterile glucose solution resulting in the desired N/P ratio of 5 and incubated at room temperature for 30 minutes for further use.

A total of 20,000 larval or 100 µl (settled volume) adult N. brasiliensis were incubated with jetPEI/NbUbq siRNA solution (200 µl) for 24 hours at 37°C in 5% CO2. After incubation, samples were washed with pre-warmed worm medium for 3 times before being cultured in fresh worm medium for 48 hours. Post-treatment, parasites were recovered, washed with

69 sterile PBS and RNA purified. Transcript levels of target genes were determined by RT-PCR using 10 fold serially diluted cDNA.

Soaking of J774 macrophages with LipofectamineTM 2000 (LF2000)-encapsulated firefly luciferase-encoding mRNA A total of 5 x 106 J774 macrophages were suspended in 1 ml DMEM culture medium containing 50 µg ml-1 luciferase-encoding mRNA encapsulated in LF2000 (InVitrogen) at a ratio of 1:2.5 (mRNA (µg):LF2000 (µl)) for 6 hours at 37°C in 5% CO2, followed by measurement of luciferase activity.

Soaking of N. brasiliensis with LF2000-encapsulated siRNA A total of 20,000 larval parasites were suspended in 200 µl parasite culture medium containing 0.3 or 1.5 µM siRNA encapsulated with LF2000 at a ratio of 1:2.5 (siRNA (µg):LF2000 (µl)) and 400 U ml-1 RNasin ribonuclease inhibitor for 48 hours at 37°C in 5%

CO2. After incubation, samples were washed with pre-warmed worm medium for 3 times before being cultured in fresh worm medium at 37°C in 5% CO2 for indicated incubation time. Post-treatment, parasites were recovered, washed with sterile PBS and RNA purified. Transcript levels of target genes were determined by RT-PCR using 10 fold serially diluted cDNA.

2.21. Gel retardation assay

Binding of dsRNA with the various PEI derivatives was determined via agarose gel electrophoresis, using 4% (w/v) agarose in 1x TAE buffer. Aliquots of 10 µg dsRNA were complexed with various amounts of PEI solution to obtain N/P ratios of 1, 3 and 5 in a final volume of 25 µl. The dsRNA bands were visualized by ethidium bromide staining on a UV transilluminator.

2.22. Luciferase assay

293 T cells/J774 macrophages Cells were harvested, washed in sterile PBS, lysed in 400 µl 1x lysis reagent (Promega), and transferred to a microcentrifuge tube. After brief (1 min) centrifugation at 13,000 x g, 20 µl of cell lysate was added to 100 µl of Luciferase Assay Reagent (Promega), and luminescence determined in a FLUOstar OPTIMA luminometer (BMG labtechnologies).

N. brasiliensis Parasites were harvested, frozen in liquid nitrogen and homogenised using a Bessman Tissue Pulverizer. Parasites were resuspended and mixed in 40 µl of 5x lysis reagent (Promega) after homogenization. Following lysis, samples were adjusted to 1x lysis reagent using sterile PBS, and transferred to a microcentrifuge tube. After 1 min centrifugation at 13,000 x g, 20 µl of cell lysate was added to 100 µl Luciferase Assay Reagent, and luminescence read in a FLUOstar OPTIMA luminometer.

2.23. In vivo excision of phagemid

The pBluescript phagemid was excised from lambda Uni-ZAP XR library clones using XL-1 blue MRF’ and non-suppressing SOLR E. coli strains with ExAssist helper phage as described by the manufacturer’s instructions (Statagene).

2.24. Cloning of recombinant N. brasiliensis VALs for expression in E. coli

The NbVALs were cloned into the pET29b expression vector in order to produce proteins with a C-terminal Histidine tag (Novagen). DNA was amplified from N. brasiliensis adult cDNA using specific primers (Table 2.1) that introduced 5’ Nde I and 3’ Not I (in NbVAL-3 and 4) or 3’ Xho I (in NbVAL-5, 6, 7 and 8) restriction sites and proofreading DNA polymerase Phusion PCR enzyme mix (BioLabs). Amplified DNAs were separated by

71 agarose gel electrophoresis, purified using the QIAquick gel extraction kit (Qiagen) and ligated into the pCR4-TOPO vector (InVitrogen). Plasmids were transformed into One Shot TOP10 Escherichia coli competent cells (InVitrogen) according to manufacturer’s instructions, and colonies picked from LB agar plates supplemented with 100 µg ml-1 amplicillin after incubation overnight at 37°C. Positive colonies were confirmed by PCR using gene specific primers and isolated plasmid DNA from colonies as template, and the inserts excised by double digestion using 10 units of Nde I and Not I or Xho I in 1x Orange -1 Buffer (50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 0.1 mg ml BSA, pH 7.5) (Fermentas) at 37°C for 4 hours followed by agarose electrophoresis and gel extraction. The expression vector pET29b was also linearised in this way and ligated with the inserts at a molar ratio of 1:1 or 1:3, together with 1x ligation buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8) and 1 unit of T4 DNA ligase (Fermentas) made up to 10 µl with sterile water, and incubated at 4°C overnight. Four µl of the ligation reactions were transformed into One Shot TOP10 Escherichia coli competent cells (InVitrogen) and selected on LB agar plates supplemented with 50 µg ml-1 kanamycin. Colonies containing the correct NbVAL constructs were confirmed by PCR using gene specific primers and isolated plasmid DNA as template, and isolated plasmids from positive clones were analyzed by DNA sequencing to confirm that the inserts were correctly ligated in frame and that no mutations had occurred within the sequences (Cogenic genomics service company, UK). Constructs were finally transformed into chemically competent E. coli expression strain BL21 (DE3) using standard protocols (InVitrogen).

2.25. Recombinant protein expression in E. coli

Fresh E. coli colonies containing the N. brasiliensis VALs pET29b constructs were picked and used to grow 20 ml overnight cultures in LB supplemented with 25 µg ml-1 kanamycin at 37°C with shaking. The overnight cultures were used for seeding small scale (20 ml) test expression cultures. Once the OD600 had reached 0.6, protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After 3 hours induction, 1 ml of the cultures was harvested and both the uninduced and induced pellets stored at -80°C for further examination.

Large scale expression of NbVALs was carried out in conical flasks containing 500 ml LB supplemented with 25 µg ml-1 kanamycin, seeded with 20 ml overnight cultures and allowed to grow to OD600 1.0 at 30°C with shaking before expression was induced by the addition of 1 mM IPTG. After 3 hours expression at 30°C, the cells were harvested by centrifugation at 4,000 x g for 30 minutes at 4°C, and pellets frozen.

2.26. Purification of recombinant proteins

The 6 histidine residues attached to the C terminus of recombinant proteins allow for their purification by Nickel-chelate affinity chromatography.

Preparation of cleared E. coli lysates under native conditions Small scale expression and solubility tests of recombinant NbVAL proteins were performed before proceeding with large-scale preparations.

Small scale expressed cell pellets were collected and frozen at -80°C overnight. The frozen cell pellets were then thawed for 15 minutes on ice and resuspended in lysis buffer (50 mM

NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) at 5 ml per gram wet weight. One mg ml-1 lysozyme was subsequently added and incubated on ice for 30 minutes. Samples were sonicated for 5 minutes on ice using 30 second bursts at 40 W with 30 second cooling periods. Lysates were centrifuged at 10,000 x g for 20 minutes at 4°C to pellet the cellular debris, and supernatants transferred to fresh tubes. The soluble and insoluble fractions were then resolved and analyzed on 12% SDS-polyacrylamide gels, transferred onto nitrocellulose and recombinant proteins detected with mouse anti-HisTag antibody.

Purification of recombinant NbVAL proteins under denaturing conditions The recombinant NbVALs expressed using pET29b were purified under denaturing conditions. Bacterial pellets from 100 ml cultures were suspended in Buffer A (100 mM

NaH2PO4, 10 mM Tris base, 6 M guanidine hydrochloride, pH 8.0) at 5 ml per gram wet weight, supplemented with a protease inhibitor cocktail (50 µl for 1 gram of cell pellets) (Sigma), then incubated at RT for 1 hour with rotation. Insoluble cell debris was removed by

73 centrifugation at 10,000 x g for 15 minutes at RT followed by filtration through a 0.45 µm filter. The supernatant was added to 0.75 ml Ni-NTA resin (Qiagen), pre-equilibrated with Buffer A, incubated at RT for an hour with rotation and poured into a column. The column was washed with 6 ml Buffer B (100 mM NaH2PO4, 10 mM Tris base, 8 M urea, pH 8.0), washed twice with 5 ml Buffer C (100 mM NaH2PO4, 10 mM Tris base, 6 M guanidine hydrochloride, pH 6.3), and washed with 3 ml of Buffer D (100 mM NaH2PO4, 10 mM Tris base, 6 M guanidine hydrochloride, pH 5.9). Recombinant proteins were eluted 3x with 1 ml

Buffer E (100 mM NaH2PO4, 10 mM Tris base, 6 M guanidine hydrochloride, pH 4.5). Purified NbVALs were dialyzed stepwise from 8 M urea, 6 M urea, 4 M urea, 2 M urea and into PBS supplemented with 1 mM EDTA, then stored at -20°C.

2.27. Generation of polyclonal antibodies

An aliquot (0.5 ml) of purified NbVAL proteins at 0.6 mg ml-1 in sterile PBS was emulsified with an equal volume of 9% aluminium potassium sulphate (Sigma). A drop of 1% phenol red indicator dye (Sigma) was added and the solution was neutralised with drop wise addition of 1 M NaOH, until indicator turned pink. The mixture was incubated at RT for 30 minutes, centrifuged at 3,000 x g for 10 minutes, and the precipitate washed with sterile PBS until the indicator dye was no longer visible. Finally the precipitate was resuspended in 1 ml sterile PBS. Groups of 4 BALC/c mice (Central Biomedical Services, Imperial College London) were injected intraperitoneally with 100 µl (30 µg) of protein. After a period of 28 days and 42 days the mice were boosted with 15 µg of antigens prepared with alum adjuvant respectively. One week after the final immunization, mice were bled out by cardiac puncture. The collected blood was allowed to clot at 4°C O/N, and then centrifuged at 14,000 x g at 4°C for 20 minutes. Sera were subsequently collected, aliquotted and stored at -80°C.

2.28. Bioinformatic analysis

Specific clones were sequenced by the Cogenic genomics service company, UK. Query sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/) or the ExPASy

74 proteomics server (http://www.expasy.ch/) and are annotated by accession number. Nematode sequences were identified by searching the nematode EST databases NEMBASE (http://www.nematodes.org/nematodeESTs/nembase.html) and Nematode.net (http://www.nematode.net/), or the C. elegans genome database, WormBase (http://www.wormbase.org/). Sequences were aligned either using the Multalin server (http://bioinfo.genotoul.fr/multalin/multalin.html), or Clustal W from EMBL-EBI (http://www.ebi.ac.uk/Tools/clustalw2/) and illustrated with BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). The percentage identities and similarities between sequences were calculated using the BLAST 2 Sequences server (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&B LAST_PROG_DEF=megaBlast&BLAST_SPEC=blast2seq).

Nucleotide sequences were translated into amino acid sequences using the Translate tool from the ExPASy proteomics server (http://www.expasy.ch/tools/dna.html). Potential signal peptides and N-linked glycosylation sites protein sequences were predicted using the Signal P server (http://www.cbs.dtu.dk/services/SignalP/) and NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/) respectively. Prediction of protein domain families was analyzed using the ProDom database server (http://prodom.prabi.fr/prodom/current/html/home.php).

2.29. Homology modelling

The HHpred interactive server (http://toolkit.tuebingen.mpg.de/hhpred) (Soding et al., 2005) was used to predict the secondary structure and homology of N. brasiliensis VAL (NbVAL) proteins. The 3-dimensional structure of Necator americanus NaASP-2 (PDB: 1U53) was utilized as a template to build the models of NbVAL proteins. Models were created by using the ModWeb server (http://modbase.compbio.ucsf.edu/ModWeb20-html/modweb.html) (Marti-Renom et al., 2000) and the validity of the modelled structures was examined using the ProSA-web server (https://prosa.services.came.sbg.ac.at/prosa.php) (Wiederstein and Sippl, 2007). Models were visualized using MacPyMOL (DeLano Scientific LLC).

2.30. Statistical analysis

Data were analyzed and plotted using GraphPad Prism 4 (GraphPad Prism software) and are displayed as the mean with standard error (S.E.). The statistical significance between results was determined using the Student’s t-test.

Chapter 3

Activation and initiation of feeding in infective larvae of Nippostrongylus brasiliensis

3.1. Introduction

Many nematodes exist in a developmentally arrested stage which has been considered somewhat analogous to the dauer stage of Caenorhabditis elegans (Hotez et al., 1993). These worms have evolved the ability to shift behaviour and metabolism to deal with many forms of stress in the environment for long term survival. In C. elegans, in response to environmental cues of secreted pheromone, reduced food supply, increased population density, or high temperature during the L1 stage, animals enter an alternative developmental pathway and arrest at the dauer stage. Dauer larvae do not feed, but they can survive at least four to eight times the normal life span of C. elegans, and when favourable conditions are encountered, the dauer larvae start to feed and resume development to the adult (Riddle and Albert, 1997). G Protein Coupled Receptors (GPCRs) on chemosensory amphid neurons are present at the frontline between the environment and C. elegans, sensing environmental cues and transducing them into endocrine signals. Several pathways and numerous components have been shown to regulate signal transduction/transcriptional pathways of dauer formation; however transforming growth factor-beta (TGF-β) and the insulin/insulin-like growth factor- 1 (IGF-1) primarily regulate the decisions between reproductive development and diapause downstream of sensory neurons (Fig. 3.1) (Fielenbach and Antebi, 2008).

In C. elegans, the TGF-β pathway which regulates dauer formation contains core components which are dauer formation (DAF)-7 ligand, DAF-1 (type I), DAF-4 (type II) serine/threonine kinase receptors, and three nuclear effectors, SMADs; DAF-8, DAF-14 and DAF-3. The relationship of these proteins is still unclear, but based on genetic/molecular data, it appears that expression of daf-7 can be activated by food or low levels of pheromone in a pair of sensory neurons. In favourable conditions, high levels of DAF-7 bind and activate the DAF- 1/DAF-4 receptor kinases in target tissue, resulting in the phosphorylatation of DAF-8 and DAF-14. Phosphorylated DAF-8 and DAF-14 then appear to antagonise the activity of DAF- 3, which induces dauer formation in the absence of signalling (Fielenbach and Antebi, 2008; Patterson and Padgett, 2000).

Fig. 3.1. Overview of the transforming growth factor-beta (TGB-β) and insulin-like signalling pathways involved in the regulation of dauer recovery in C. elegans. This diagram is adapted from Beall and Pearce (2002).

The Insulin/IGF-1 signalling (IIS) pathway is another parallel pathway playing an important role in the control of dauer formation and the regulation of life span in C. elegans (Braeckman et al., 2001; Wolkow et al., 2000), and it is considered to originate in amphidial, chemosensory neurons that can sense levels of food and pheromone in the environment. The key component of this pathway is encoded by an orthologue of the mammalian insulin/IGF-1 receptor, DAF-2 (Kimura et al., 1997). When growth conditions are favourable, sensory neurons of C. elegans seemingly release acetylcholine onto muscarinic receptors, thereby regulating the synthesis/release of an insulin-like ligand that further binds to DAF-2 receptor kinase and activates the downstream phosphatidylinositol 3-kinase signalling cascade, resulting in the generation of the phosphatidylinositol (3,4,5)-triphophate or PI-P3. This secondary message activates PDK-1 which then phosphorylates serine/threonine kinase (Akt/PKB), and phosphorylated Akt/PKB subsequently enters the nucleus and phophorylates DAF-16, which acts as a repressor of genes that promote growth and reproduction (Beall and Pearce, 2002; Braeckman et al., 2001).

Inhibition of the IIS pathway by mutation in daf-2 or the downstream gene, PI3 kinase (age- 1), results in an increase of life span by more than two-fold compared to wild-type C. elegans (Friedman and Johnson, 1988; Kenyon et al., 1993). Furthermore, strong daf-2 mutations lead worms to enter the dauer diapause instead of developing to adulthood, and also cause late progeny and reduced broods (Gems et al., 1998; Tissenbaum et al., 2000). The IIS pathway seems to be evolutionarily conserved in a wide range of organisms (Kenyon, 2001); mutations in components of the IIS pathway extend longevity and influence stress resistance in flies (Clancy et al., 2001; Tatar et al., 2001) and also mice (Bartke, 2000). In addition, intriguingly, the canine hookworm Ancylostoma caninum appears to have a similar cascade resulting in the resumption of development of the infective third stage larva (L3) (Tissenbaum et al., 2000).

Dauer formation is regulated by several environmental stimuli, and temperature is one of these important factors in C. elegans. As the growth temperature downshifts from 25°C to 15°C, the resumption of feeding and development of dauer stage larval C. elegans is moderately induced (Riddle and Albert, 1997). Although temperature has a modest influence on dauer diapause and morphogenesis at 25°C, higher temperatures can mobilize regulatory

80 signalling for dauer formation (Ailion and Thomas, 2000, 2003). It has been shown that dauer diapause is more strongly induced at 27°C than 25°C, and also the induction of dauer formation is pheromone dependent at 25°C or lower temperatures (Ailion and Thomas, 2000).

One of the major reasons for attempting to develop RNA interference in N. brasiliensis is to have a means of defining genes which encode proteins which are important for infectivity. In order to do this in strongylid nematodes, it is necessary to target larval stages. Previous attempts in the laboratory to induce RNAi by feeding L1 to L2 stages on E. coli expressing dsRNA had failed, and thus I decided to examine the L3 stage. This presents a problem, as L3 present in the environment do not feed until they infect the mammalian host. However, work on Ancylostoma caninum has shown that L3 can be activated to feed in vitro by exposure to conditions which mimic those encountered in the host (Hawdon and Schad, 1990, 1991, 1992, 1993; Hawdon et al., 1992). I therefore decided to study in vitro activation of N. brasiliensis L3 as a means of maximising uptake and delivery of dsRNA for RNAi in this stage.

3.2. A temperature cue stimulates feeding independently of serum and exsheathment

In order to evaluate the signals required for activation of feeding in N. brasiliensis, developmentally arrested and non-feeding third stage larvae were incubated in RPMI worm medium either at 20°C or 37°C in the presence or absence of a variety of additives. Fig. 3.2 shows that 10% rat serum, 25 mM reduced glutathione or a combination of both could not stimulate feeding in larvae maintained for 6 hours (panel A) and 20 hours (panel B) at 20°C. Rat serum and glutathione were used at higher concentrations (20% and 50 mM respectively) without effect.

As nematodes often use small molecules, such as host-specific chemical cues or dauer pheromone to regulate their development (Butcher et al., 2007; Safer et al., 2007), extracts of rat skin were tested for their ability to stimulate feeding. Secreted products from larvae were

81 also tested, as it was considered that once activated, products secreted by might act as a stimulus for collective feeding within a population of worms. However, neither extracts or minced rat skin, or secreted products from third stage larvae stimulated feeding in larvae held at 20oC (Table. 3.1).

In contrast, approximately 20% of the larval population cultured in worm medium alone at 37°C were observed to feed, and this rose to 60% by 20 hours. The presence of rat serum and glutathione had no additive effect on this process (panels A and B). The time course of temperature-induced activation is illustrated in panel C, indicating that the vast majority of the population were feeding after 48 hours in culture at 37°C. Some variability between batches of larvae was observed, but this was routinely between 80% and 90%. Fig. 3.2, panel D shows some representative images of ingestion of FITC-BSA at different time points by larvae.

Third stage larvae of N. brasiliensis retain their cuticle from the second stage and hence are described as 'sheathed'. This sheath is discarded during the process of skin penetration, and can also be removed by brief exposure to sodium hypochlorite. We therefore investigated whether exsheathment provided a stimulus for activation of feeding. Larvae were chemically exsheathed and then cultured at 20°C. Exsheathed larvae maintained at 20°C for 20 hours were not stimulated to feed, although frequently FITC-BSA could be observed in the mouthparts (Fig. 3.3, panels A and B). The absence of flourescence in the intestine indicated that pharyngeal pumping had not been initiated. Fig. 3.3, panel C shows the time course of activation of chemically exsheathed larvae at 37°C, with representative images of larvae at different time points shown in Fig. 3.3, panel D. Initiation of feeding was slightly slower than control larvae allowed to exsheath naturally, and never reached the same levels in the population. Assessment of MTT reduction showed that chemical exsheathment with sodium hypochlorite significantly impaired parasite viability (Fig. 3.3, panel E).

Fig. 3.2. An elevated temperature cue alone stimulates feeding in N. brasiliensis infective larvae.

N. brasiliensis L3 were incubated in RPMI culture medium with or without (-) the following additives: 10% rat serum (RS), 25 mM glutathione (GSH), 10% rat serum + 25 mM glutathione (RS+GSH), at 37°C or 20°C for 6 hours (panel A) and 20 hours (panel B), prior to assessment of feeding. The results are presented as the mean ± S.E. of the proportion of the population which scored positive for feeding by fluorescence, assayed in triplicate. Panel C: Time course of temperature induced activation, with results presented as the mean ± S.E. of assays in triplicate. Panel D: Representative images of larvae at different time points (100X magnification).

Table. 3.1. Neurotransmitters and exogenous cues do not stimulate the feeding of N. brasiliensis infective larvae at 20°C.

N. brasiliensis L3 were incubated with a broad range of neurotransmitters or exogenous cues (L3 secreted products and extracts or minced rat skin) for 20 hours at 20°C. After incubation, larvae were then cultured in 2.5 mg ml-1 FITC-BSA for 1 hour at 20°C, washed and examined.

Fig. 3.3. Chemical exsheathment is unable to initiate feeding.

Panel A: Control sheathed infective larvae (L3) and larvae exsheathed in sodium hypochlorite (exL3) were maintained at 20°C for 20 hours, and then incubated with FITC- BSA for an hour. No fluorescence was observed in the intestines of either group of parasites (panel A, 400X magnification), but was observed in the mouthparts of some exsheathed larvae (B). Panel C: Time course of temperature induced activation of chemically exsheathed larvae, with results presented as the mean ± S.E. of assays in triplicate. Panel D: Representative images of larvae at different time points (100X magnification). Panel E: Hypochlorite-induced exsheathment impairs parasite viability. Control sheathed (L3) and exsheathed (exL3) larvae were incubated at 37°C for 20 hours, and viability determined by MTT assay. The results are presented as the mean ± S.E. of assays in triplicate. * P < 0.05.

3.3. Temperature-induced activation is dependent upon PI3-kinase and Akt kinase but occurs independently of a cGMP-dependent cholinergic pathway

Both 8-bromo-cGMP and muscarinic agonists promote activation of A. caninum infective larvae, as they mimic early steps in the insulin-like signalling pathway which has also been implicated in dauer recovery in C. elegans (Tissenbaum et al., 2000). However, exposure of N. brasiliensis larvae to 8-bromo-cGMP at concentrations up to 20 mM (Fig. 3.4, panel A), or the muscarinic agonists oxotremorine-M and arecoline at concentrations up to 5 mM (Fig. 3.5, panels A and B) did not stimulate feeding at 20oC. A range of neurotransmitters or other compounds implicated in particular C. elegans behaviours (Bargmann and Horvitz, 1991; Rand and Nonet, 1997) was also tested. Thus, serotonin (5-hydroxytryptamine, 5-HT), gamma-aminobutyric acid (GABA), the FMRFamide-like neuropeptide AF1, glutamate, octopamine, noradrenaline and dopamine were tested concentration up to 1 mM, but all failed to induce feeding at 20oC (Table. 3.1). Oxotremorine-M, arecoline (Fig. 3.5, panels C and D) and 8-bromo-cGMP (Fig. 3.4, panel B) also failed to potentiate activation of feeding at 37oC, and were actually inhibitory at the highest concentrations used.

Events downstream of the insulin-like receptor in C. elegans involve the serine/threonine kinase Akt/PKB, which transduces signals from AGE-1 PI3 kinase to the DAF-16 transcription factor (Paradis et al., 1999; Paradis and Ruvkun, 1998), and a role for this pathway in activation of larval A. caninum has been suggested by the use of inhibitors of PI3 kinase (Brand and Hawdon, 2004) and Akt kinase (Datu et al., 2009). Consistent with these data, both the PI3 kinase inhibitor LY294002 and Akt inhibitor IV blocked activation of N. brasiliensis in a dose-dependent manner (Fig. 3.6, panels A and B).

Although none of the neurotransmitters assayed initiated feeding at 20°C, exposure of parasites to 0.5 mM 5-HT appeared to potentiate the onset of activation induced by high temperature. Fig. 3.7, panel A shows that at 6 hours after incubation, approximately 40% of the larval population had ingested FITC-BSA, in comparison with approximately 20% of controls maintained at 37°C in the absence of 5-HT, although this difference progressively diminished over time such that no significant difference between groups was observed by 24 hours. 5-HT is known to stimulate pharyngeal pumping in C. elegans (Avery and Horvitz,

1990) and Ascaris suum (Brownlee et al., 1995), and thus the rate of uptake of radiolabelled BSA was assayed as a measure of pharyngeal pumping in N. brasiliensis larvae. Fig. 3.7 panel B shows a dose-dependent increase in uptake of 125I-BSA up to the maximal concentration of 5 mM exogenous 5-HT.

Resorcinol has also been observed to effectively stimulate pharyngeal uptake of exogenous FITC and dsRNA in the plant parasitic nematode, Meloidogyne incognita (Rosso et al., 2005), and was therefore tested. However, no enhancement of feeding was observed in N. brasiliensis larvae incubated at 37°C with different concentrations of resorcinol for 6 hours, and in contrast it completely blocked uptake of FITC-BSA (Fig. 3.8). The drug completely impaired larval motility, and thus appeared to be extremely toxic at the concentrations tested.

3.4. Cytochrome P450 (DAF-9) is an alternative regulator for activation of infective larvae

Recent studies have shown that steroid hormones named dafachronic acids are synthesized by DAF-9, a cytochrome P450, and can activate the downstream nuclear receptor DAF-12, promoting dauer recovery in C. elegans (Jia et al., 2002; Motola et al., 2006). Intriguingly, it has been demonstrated that an inhibitor of cytochrome P450, ketoconazole, completely restricted the activation of A. caninum L3s induced by serum/GSH. Moreover, inhibition could be rescued by a synthetic dafachronic acid (Δ7-DA), suggesting that the DAF-9 hormone signalling is involved in the activation of larval parasitic nematodes (Wang et al., 2009). Similarly, the activation of N. brasiliensis L3s was also blocked by ketoconazole in a dose-dependent manner (Fig. 3.9). We do not have access to synthetic dafachronic acids to test whether they could initiate activation at 20oC.

Fig. 3.4. The second messenger cGMP does not initiate activation.

N. brasiliensis L3 were incubated either at 20oC (panel A) or 37oC (panel B) with or without (Ctrl) the indicated concentrations of cell membrane permeable 8-bromo-cGMP for 20 hours, then assayed for feeding. The results are presented as the mean ± S.E. of assays in triplicate. * P < 0.05.

Fig. 3.5. Cholinergic agonists cannot initiate activation.

N. brasiliensis L3 were incubated either at 20oC (panels A and B) or 37oC (panels C and D) with or without (Ctrl) the indicated concentrations of oxotremorine-M (Oxo-M) and arecoline for 20 hours, then assayed for feeding. The results are presented as the mean ± S.E. of assays in triplicate. * P < 0.05; *** P < 0.0005.

Fig. 3.6. Inhibitors of PI3 kinase and Akt kinase block activation of infective L3.

o N. brasiliensis L3 were maintained at 37 C in 5% CO2 with the indicated concentrations of LY294002 (panel A) and Akt inhibitor IV (panel B) for 20 hours, then assayed for feeding. The results are presented as the mean ± S.E. of assays in triplicate. ** P < 0.005; *** P < 0.0005.

Fig. 3.7. Serotonin promotes pharyngeal pumping in infective larvae.

Panel A: Parasites were incubated in the absence (Ctrl) or presence of 0.5 mM serotonin (5- HT) at 37°C for the indicated time and then assayed for feeding. Data are presented as the mean ± S.E. of triplicate samples. Panel B: Larvae were maintained for 47 hours at 37°C in

5% CO2, and then incubated in the absence (Ctrl) or presence of the indicated concentrations of 5-HT for 1 hour before quantitative assessment of feeding with 125I-BSA. Data are presented as the mean ± S.E. of assays in triplicate. Values for uptake significantly different to control parasites (no 5-HT) are indicated ** P < 0.005; *** P < 0.0005.

Fig. 3.8. Resorcinol inhibits feeding in infective larvae.

Infective larvae were maintained at 37°C in 5% CO2 for 5 hours, and then in the absent (Ctrl) or presence of indicated concentrations of resorcinol for 1 hour, prior to assessment of feeding. The results are presented as the mean ± S.E. of assays in triplicate.

Fig. 3.9. A Cytochrome P450 inhibitor blocks activation of N. brasiliensis L3.

o N. brasiliensis L3 were maintained at 37 C in 5% CO2 with the indicated concentrations of ketoconazole for 20 hours, then assayed for feeding. The results are presented as the mean ± S.E. of assays in triplicate. ** P < 0.005; *** P < 0.0005.

3.5. Regulation of protein secretion by activated larvae

A. caninum infective larvae release several important secreted proteins during activation and infection (Hawdon et al., 1996; Hawdon et al., 1995; Hawdon et al., 1999). To test if temperature-induced activation of N. brasiliensis infective larvae resulted in protein secretion, parasites were cultured at 37°C or 20°C and metabolically labelled with [35S]-methionine. In addition, the effect of exposure to 10% rat serum was tested at each temperature. In order that serum components did not interfere with resolution of labelled parasite proteins, worms were exposed to serum for 24 hours at either temperature, washed and then cultured in serum-free medium for a further 24 hours. Culture media were passed through a 0.2 µm filter, concentrated and resolved by SDS-PAGE on a 10-20% gradient gel. As shown in Fig. 3.10, maintenance at 37oC potently stimulated protein secretion by L3s, but host serum did not effect the profile of proteins secreted (lanes 3 and 4).

Adult N. brasiliensis are known to secrete 3 variants of acetylcholinesterase (AChE) termed AChE-A, -B and -C (Hussein et al. 2003). A very sensitive enzymatic assay allows for precise quantitation of the dynamics of protein secretion, and the variants can be distinguished by PAGE on non-denaturing gels. I therefore tested whether temperature- induced activation resulted in AChE secretion by infective larvae. Fig. 3.11 shows that this is the case. AChE activity was rapidly detectable in culture media 3 hours after exposure to elevated temperature, and steadily accumulated over time. However, no AChE activity was detectable in culture media of L3s maintained at 20°C, even after 48 hours of culture (Fig. 3.11, panel A). Native PAGE analysis showed that all three isoforms were released by larvae maintained at elevated temperature (Fig. 3.11, panel C), and RT-PCR confirmed that transcription of AChE-B was initiated at 37°C but not 20oC (Fig. 3.11, panel B).

It has been shown that the PI3 kinase inhibitor LY294002 can block activation of feeding in A. caninum, but has no effect on secretion of the pathogenesis related protein (PRP) ASP-1 (Brand and Hawdon, 2004). However, incubation of N. brasiliensis infective larvae maintained at 37°C for 48 hours with 0.5 mM LY294002 or 20 µM Akt kinase inhibitor IV dramatically inhibited protein secretion (Fig. 3.12), and also protein synthesis in general (Fig. 3.13). Therefore, it is clear that the PI3 kinase and Akt kinase inhibitors blocked general

94 protein synthesis, and which subsequently reduced secretion of all proteins newly synthesized by activated larvae.

Previously, investigators have reported that a steroid hormone-nuclear receptor (DAF-12) signalling cascade mediates larval recovery in C. elegans and also parasitic nematodes (Motola et al., 2006; Wang et al., 2009). In addition, steroid hormones named dafachronic acids (DAs) are ligands for DAF-12, and are synthesized by DAF-9 (cytochrome P450). Dafachronic acids have been shown to stimulate the secretion of AcASP-1 and AcASP-2 from A. caninum L3s, indicating that DAF-9/DAF-12 is a crucial regulatory pathway that initiates development of larval parasites (Wang et al., 2009). Examination of protein secretion by N. brasiliensis L3s incubated with 100 µM P450 DAF-9 inhibitor (ketoconazole) at 37°C for 48 hours showed that the level of protein secretion was significantly decreased compared to controls incubated in the absence of inhibitor (Fig. 3.14), indicating that steroid hormone synthesis by P450 DAF-9 is also required for activation and protein secretion in larval N. brasiliensis.

Fig. 3.10. Host serum does not affect the profile of proteins secreted by activated larvae.

Infective larvae (10,000) were cultured at 20°C and 37°C in the presence or absence of 10% rat serum, metabolically labelled with [35S]methionine for 48 hours, secreted products collected and resolved on a 10-20% gradient SDS gel. Parasites were maintained under the following conditions: Lane 1: 20°C - serum; Lane 2: 20°C + serum; Lane 3: 37°C - serum; Lane 4: 37°C + serum. Migration of molecular weight markers is indicated in kilodaltons.

Fig. 3.11. Acetylcholinesterase is secreted only by activated larvae.

Panel A: Infective larvae were incubated at 37°C and 20°C, and the level of secreted acetylcholinesterase (AChE) enzyme activity in culture medium was measured periodically. Results are presented as nmol. ml-1 min-1 units for 10,000 larvae maintained in 5 ml culture for 48 hours, with the mean +S.E. of assays in triplicate. Panel B: Detection of AChE-B mRNA by RT-PCR in non-activated (L3) and activated (L3A) larvae. The bottom panel shows detection of actin transcripts from the same RNA samples. Panel C: visualisation of secreted AChE isoforms in culture medium from larvae maintained at 20°C (lane 1) and 37oC (lane 2) following resolution by native PAGE.

Fig. 3.12. Inhibitors of PI3 kinase and Akt kinase block protein secretion of activated larvae.

Infective larvae (10,000) were cultured in 1 ml worm medium at 37°C in the presence or absence of inhibitors of PI3 kinase and Akt kinase, metabolically labelled with [35S]methionine for 48 hours, secreted products collected, concentrated to 50 µl and 5 µl resolved on a 10-20% gradient SDS gel. Parasites were maintained under the following conditions: Lane 1: without inhibitors; Lane 2: + 0.5 mM LY294002; Lane 3: + 20 µM Akt inhibitor IV. Migration of molecular weight markers is shown in kilodaltons.

Fig. 3.13. Inhibitors of PI3 kinase and Akt kinase also block overall protein synthesis in activated larvae.

Infective larvae (10,000) were cultured in 1 ml worm medium at 37°C in the presence or absence of inhibitors of PI3 kinase and Akt kinase, and were metabolically labelled with [35S]methionine for 48 hours. Somatic extracts of worms were prepared (50 µl) and 5 µl analysed for incorporation of radiolabel. Parasites were maintained under the following conditions: Ctrl: without inhibitors; LY: + 0.5 mM LY294002; AktIV: + 20 µM Akt inhibitor IV. The results are presented as the mean ± S.E. of assays in triplicate. *** P < 0.0005.

Fig. 3.14. Cytochrome P450 inhibitor blocks protein secretion of activated larvae.

Infective larvae (10,000) were cultured in 1 ml worm medium at 37°C in the presence or absence of cytochrome P450 inhibitor, ketoconazole, metabolically labelled with [35S]methionine for 48 hours, secreted products collected, concentrated to 50 µl and 5 µl resolved on a 10-20% gradient SDS gel. Parasites were maintained under the following conditions: Lane 1: without inhibitor; Lane 2: + 100 µM ketoconazole. Migration of molecular weight markers is shown in kilodaltons.

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A * * NbFTT-2 1 MADNKDELVQRAKLAEPDERYDDMAQSMKKVTELGAELSNEERNLLSVAYKNVVGARRSS AcFTT-2 1 MADNKDELVQRAKLAEQAERYDDMAQSMKKVTELGAELSNEERNLLSVAYKNVVGARRSS CeFTT-2 1 MSDGKEELVNRAKLAEQAERYDDMAASMKKVTELGAELSNEERNLLSVAYKNVVGARRSS

NbFTT-2 61 WRVISSIEQKTEGSEKKQQMAKEYREKVEKELRDICQDVLNLLDKFLIPKAGNPESKVFY AcFTT-2 61 WRVISSIEQKTEGPEKKQQMAKEYRGKVEKELRDICQDVLNLLDKFLIPKAGNPESKVFY CeFTT-2 61 WRVISSIEQKTEGSEKKQQMAKEYREKVEKELRDICQDVLNLLDKFLIPKAGAAESKVFY

** NbFTT-2 121 LKMKGDYYRYLAEVASGEDRSSVVDKSQQSYQEAFDIAKDKMQPTHPIRLGLALNFSVFY AcFTT-2 121 LKMKGDYYRYLAEVASGEDRSSVVDKSQQSYQEAFDIAKDKMQPTHPIRLGLALNFSVFY CeFTT-2 121 LKMKGDYYRYLAEVASGDDRNSVVEKSQQSYQEAFDIAKDKMQPTHPIRLGLALNFSVFF

NbFTT-2 181 YEILNAPDKACQLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDAAADDQD AcFTT-2 181 YEILNAPDKACQLAKQAFDDAIAELDTLNEDSHKDSTLIMQLLRDNLTLWTSDAAADDQD CeFTT-2 181 YEILNAPDKACQLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDAATDDTD

NbFTT-2 241 AGEQGEGAN AcFTT-2 241 AGEQGEGAN (97/98) CeFTT-2 241 ANET.EGGN (92/94)

Fig. 3.15. A gene homologous to ftt-2 in N. brasiliensis.

Panel A: Alignment of the predicted N. brasiliensis FTT-2 amino acid sequence with A. caninum and C. elegans FTT-2 (AcFTT-2, Genbank: FJ842376; CeFTT-2, Genbank: CAA91474). Identical amino acids are shaded in black, and similar amino acids in grey. Amino acid residues that directly contact phosphate groups of targets are indicated with an asterisk (Bridges and Moorhead, 2005). The percentage amino acid identity/similarity to NbFTT-2 is indicated at the end of the alignment. Panel B: RT-PCR was used to amplify a Nbftt-2 fragment (439 bp) from non-activated L3 (L3), activated L3 (L3A), L4 and adult N. brasiliensis cDNA. The bottom panel shows expression of the internal control actin by RT-PCR.

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3.6. Discussion

The developmentally arrested larval stage of free-living nematodes is a strategy aimed at dealing with survival in unfavourable environmental conditions, whereas for parasitic nematodes the aim is to survive until favourable conditions (ie the presence of a suitable host in which to continue the life cycle) appear (Hotez et al., 1993; Riddle and Albert, 1997). Dauer larvae of C. elegans are a non-conditional stage in the life cycle, whereas infective larvae of strongylid nematode parasites are an essential part of development. In either case, nematodes which progress through these developmentally arrested larvae can continue to develop on exposure to relevant environmental cues or host specific factors encountered during invasion (Hawdon and Schad, 1990, 1991, 1992, 1993; Hawdon et al., 1992; Riddle and Albert, 1997).

Exposure of N. brasiliensis infective larvae to elevated temperature (37°C) was sufficient to activate signalling pathways which resulted in resumption of feeding after a lag period at 37°C, reaching a maximum of 90% of the population feeding by 48 hours. In A. caninum, host serum is an essential factor for larval activation, and it was also shown to act synergistically with reduced glutathione (GSH), which has also been observed to stimulate development of Onchocerca lienalis microfilariae to the late L1 stage (Hawdon and Schad, 1990, 1991, 1992; Pollack et al., 1988). However, intriguingly, neither GSH nor rat serum initiated the activation of feeding in N. brasiliensis L3s maintained at 20°C, nor enhanced induction of feeding in parasites maintained at 37°C. Therefore, the regulation of resumption of N. brasiliensis L3 development differs from A. caninum in this respect.

Third stage larvae of N. brasiliensis are sheathed in a vestigial cuticle from the second stage, and will discard this (exsheath) during penetration of the skin to continue development in the mammalian host (Kassai, 1982). Exsheathment is thus the first visible feature of parasitic development, followed by resumption of feeding. Studies have shown that several nematodes can be stimulated to exsheath by cues from the host or the external environment (Bailey, 1968; Rogers and Sommerville, 1957). Trichostrongylus retortaeformis infective larvae could be stimulated to exsheath by a medium of low pH and temperature 37°C, and also the stomach fluid of rabbit (Bailey, 1968). Similar data appeared in Trichostrongylus axei and

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Haemonchus contortus, temperature cue (>35°C) or rumen fluid of host stimuli could induce L3 ecdysis (Rogers and Sommerville, 1957). Artificial exsheathment did not act as a stimulus for N. brasiliensis to initiate feeding, however, and in addition impaired parasite viability.

C. elegans can utilize a small molecule signal, the dauer pheromone, to monitor its population density and modulate its development accordingly. When worms encounter harsh growth conditions, they progress to the dauer stage, stop to feed and develop for a while (Butcher et al., 2007; Jeong et al., 2005). It is unclear how dauer pheronomes are synthesized and secreted in vivo, but three distinct ascarosides with dauer pheromone activity have been indentified from C. elegans extracts (Butcher et al., 2007; Jeong et al., 2005). It is known that pheromone can suppress the expression of daf-7 (TGF-β) and str-3 receptor genes in the bilateral ASI neurons to promote dauer formation of C. elegans (Peckol et al., 2001; Ren et al., 1996). Also, it has been shown that ascaroside pheromones signal through the G protein- coupled receptors (GPCRs) SRBC-64 and SRBC-66, and Gα protein GPA-3 in the larval

ASK chemosensory neurons to down-regulate guanylyl cyclase DAF-11 activity and promote dauer formation of C. elegans (Kim et al., 2009), suggesting that the regulation of pheromone-induced larval diapause is mediated by transmission through ASI and ASK neurons.

Activation of strongylid larvae occurs coincident or following penetration of host skin. However, the presence of extracts or minced rat skin did not resume feeding of infective larvae of N. brasiliensis maintained at 20°C. Host-derived chemical cues are also involved in skin penetration behaviour of hookworms (Granzer and Haas, 1991; Haas et al., 2005) and Strongyloides stercoralis (Safer et al., 2007). It has been determined that components of a hydrophilic extract, in addition to fatty acids and lipids from host skin, provide a host-finding signal to larval Ancylostoma duodenale and Necator americanus (Haas et al., 2005). Importantly, furthermore, a specific chemical molecule derived from skin extracts, urocanic acid was examined to have significantly chemoattractive effect on host-finding behaviour of S. stercoralis infective larvae (Safer et al., 2007). Intriguingly, similar behaviour was also observed from N. brasiliensis L3s exposed to urocanic acid or hydrophilic extracts of skin (Chan, unpublished data). Hence, it appears that chemosensory cues stimulate attraction of larval N. brasiliensis to host skin.

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Chemosensation and thermosensation are important sensory systems for C. elegans to interact with the environment. It has been shown that among the three major sensory organs (amphid, phasmid and inner labial sensory organs) of C. elegans, amphid neurons are responsible for the regulation of chemosensory and thermosensory behaviours (Fig. 3.16, panel A) (Bargmann, 1997, 2006). The sensory neurons of the amphid are grouped into three classes depending on their morphology (Fig. 3.16, panels B & C) and function (Table. 3.2); ADF, ASG, ASE, ASI, ASJ, ASK, ADL and ASH have long slender cilia directly exposed to the environment, which detects mostly water-soluble attractants; AWA, AWB and AWC have flattened and branched cilia enclosed by the amphid sheath cell, and respond to volatile odorous substances; and the third class of neurons is represented by AFD which has a brush- like dendritic membrane form within a sheath cell, and can monitor thermal cues from the environment (Bargmann, 1997; Inglis et al., 2007). Importantly, a couple of amphid neurons have an essential role in the regulation of the dauer stage in C. elegans (Bargmann and Horvitz, 1991). ADF, ASG and ASI can regulate the initiation of dauer formation, and dauer recovery is mediated through the ASJ neuron. When these neurons are ablated, the animal constitutively enters into the dauer stage, suggesting that these amphid neurons respond to environmental cues, and regulate dauer formation (Bargmann and Horvitz, 1991). In addition, temperature affects C. elegans behaviour, development and proliferation, and can be observed in thermotactic responses (Mori et al., 2007). C. elegans mainly uses the amphid thermosensory neuron AFD to sense environmental temperature, and thermal signals are then transmitted onto the downstream AIY, AIZ and RIA interneurons driving thermophilic or cryophilic movement (Mori et al., 2007).

It is known that in C. elegans, a cGMP signalling pathway involving the transmembrane guanylyl cyclase (mGC) daf-11 is present in the amphidial neurons (Birnby et al., 2000), and it has been shown that the daf-11 mGC appears to function in the ASI (Murakami et al., 2001) and the ASJ neurons (Birnby et al., 2000) to activate the cGMP pathway promoting resumption of development from the dauer stage. Ablation of the ASI and ASJ neurons leads to the formation of dauers in C. elegans (Bargmann and Horvitz, 1991), and ablation of the ASJ-like neuron causes reduction of larval feeding in S. stercoralis, suggesting that signalling through this neuron regulates activation (Ashton et al., 2007). A membrane permeant analogue of the second messenger cGMP, 8-bromo-cyclic GMP (8-bromo-cGMP), can

104 induce recovery from larval diapause in both C. elegans and A. caninum (Birnby et al., 2000; Hawdon and Datu, 2003).

Furthermore, a cholinergic signalling pathway appears to be involved in regulating activation, as the muscarinic agonists arecoline (a lipophilic compound) and oxotremorine-M (a hydrophilic compound) promote resumption of development in C. elegans and A. caninum (Tissenbaum et al., 2000). Acetylcholine (ACh) is the main neurotransmitter synthesized by choline acetyltransferase (ChAT) of motor neurons (localised in the ventral nerve cord), and act through muscarinic receptors and nicotinic receptors on body wall muscle, pharyngeal muscle and motor neurons to regulate nematode behaviours (Duerr et al., 2008; Martin, 1993; Rand and Nonet, 1997). In addition, studies have illustrated the effects of muscarinic receptors on Ascaris suum muscle depolarization and contraction, using muscarinic agonists or antagonists (Colquhoun et al., 1991; Steger and Avery, 2004).

Intriguingly, a muscarinic antagonist, atropine, significantly inhibits 8-bromo-cGMP- stimulated feeding of A. caninum L3, which suggests that cGMP is an upstream mediator of muscarinic receptor activation in the resumption of reproductive growth (Hawdon and Datu, 2003). However, by contrast, 8-bromo-cGMP or muscarinic agonists did not initiate and promote the activation of feeding in N. brasiliensis L3 maintained at 20°C and 37°C respectively. The results reported here suggest that a cGMP-dependent cholinergic pathway is not involved in the activation of N. brasiliensis infective larvae. It is possible that amphidial neurons homologous to ASJ and ASI are not involved in activation, or that different signalling components are involved. High concentrations of cGMP and muscarinic agonists inhibit feeding at 37°C. It is known that nicotinic acetylcholine receptors are required for pharyngeal pumping in C. elegans (McKay et al., 2004; Raizen et al., 1995). However, a muscarinic receptor, GAR-3, is also localised to the pharynx and regulates pharyngeal muscle contraction. High levels of signalling through this receptor, for example via the application of arecoline, inhibit relaxation of pharyngeal muscle and inhibit feeding (Steger and Avery, 2004), and it is possible that a similar effect is being observed by exposure of N. brasiliensis to high concentrations of arecoline or Oxo-M.

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Fig. 3.16. Structure of amphid neurons in C. elegans.

Panel A: There are three types of sensory organs (amphid, phasmid and inner labial organs) present in C. elegans. Amphid organ with 12 associated neurons (ASE, ASH, ASI, ADF, ASG, ASJ, ASK, ADL, AFD, AWA, AWB and AWC) regulate chemosensory and thermosensory responses which are located in the anterior of the pharyngeal region of the animal. Panel A is adapted from Inglis et al. (2007). Panels B and C: Detailed location and structure of the amphid neurons. cu: cuticle, sh: sheath cell, so: socket cell. Panels B & C are adapted from Bargmann (2006).

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Table. 3.2. Neuronal functions defined by laser ablation. The information in the table is based on Bargmann (1997, 2006) and Inglis et al. (2007).

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I tested a wide range of neurotransmitters for their ability to initiate feeding in N. brasiliensis L3. These neurotransmitters have been the focus of diverse studies on nematode behaviour (Komuniecki et al., 2004). FMRFamide-like neuropeptides are distributed through the nervous system of nematodes, and have been best studies in Ascaris suum (Cowden et al., 1993), Haemonchus contortus (Keating et al., 1995) and C. elegans (Nelson et al., 1998), in which thay have been implicated in a wide range of behaviours including feeding and locomotion (Brownlee et al., 1995; Rogers et al., 2001). AF-1, Ascaris FMRFamide-like peptide 1, was the first neuropeptide to be isolated from A. suum (Cowden et al., 1989), and directly regulates the pharyngeal pumping rate in C. elegans (Rogers et al., 2001; Spanier et al., 2005). In addition, serotonin (5-HT) has also been shown to dramatically increase pharyngeal pumping, and appears to modulate feeding behaviour in response to food availability in the environment (Horvitz et al., 1982; Rogers et al., 2001). Octopamine affects pharyngeal pumping in an opposite manner to serotonin, in that it suppresses M3, one of the motor neurons regulating the timing of pharyngeal muscle contraction (Horvitz et al., 1982; Niacaris and Avery, 2003). Glutamate has also been identified in the M3 motor neuron, causing a rapid depolarization with a cessation of action potentials and a period of suppression of pharyngeal activity (Pemberton et al., 2001). Gamma-aminobutyric acid (GABA) is another important neurotransmitter, which is synthesized by decarboxylation of glutamate via the enzyme glutamic acid decarboxylase, and it acts to relax the body muscles during locomotion and foraging and to contract the enteric muscles during defaecation in C. elegans (Jorgensen, 2005), whereas dopamine regulates behavioural plasticity dependent on environmental cues (Sawin et al., 2000).

Despite their proven role in regulation of nematode behaviour, none of these neurotransmitters initiated feeding in N. brasiliensis L3 maintained at 20°C. Serotonin appeared to potentiate the early onset of activation of N. brasiliensis L3 at 37°C (Fig. 3.7A). However, given that it promoted a quantitative increase in the rate of feeding, presumably by accelerated pharyngeal pumping (Fig. 3.7B), the effect could most easily be explained by increasing the number of larvae in the population which scored positive by flourescence. Significantly, once the majority of the larval population was activated (after 24 hours) there was no significant difference in the numbers which scored positive for feeding in the presence of serotonin. Resorcinol has been reported to induce oesophageal gland secretion

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(Jaubert et al., 2002) and also stimulate pharyngeal pumping (Rosso et al., 2005) in Meloidogyne incognita. However, it had no effect on feeding of N. brasiliensis L3 in this study and in fact was toxic, severely impairing larval motility at the concentrations used (Fig. 3.8).

Elevated temperature (37°C) was essential for activation of feeding in N. brasiliensis L3, whereas it acts as a modulator for dauer recovery in C. elegans. In the latter, the route for temperature sensation and output of that signal to motor and endocrine pathways consists of the amphid sensory neuron AFD coupled to the amphidial interneurons AIY and AIZ (Bargmann, 1997; Mori and Ohshima, 1995). Furthermore, Tissenbaum et al. (2000) have suggested that theromosensory cues acting via AIY and AIZ are coupled to insulin-like secretory neurons, which act to induce dauer recovery. A gene termed daf-28 has been shown to encode an insulin-like protein expressed in ASJ and ASI neurons, and its expression is regulated and inhibited during the normal larval dauer, indicating that daf-28 acts in the DAF-2 signalling cascade of C. elegans dauer arrest (Li et al., 2003b). Elevated temperature appears to be the primary, essential trigger for activation of N. brasiliensis L3. This signal is presumably detected and transduced via thermosensory neurons, which may then couple with neurons homologous in function to ASJ and ASI to release insulin-like ligands which activate development.

It is known that the IIS pathway is critical for regulating dauer recovery in C. elegans, but not TGF-β signalling (Tissenbaum et al., 2000). Mutations in the insulin-like receptor gene daf-2 or PI3 kinase gene age-1 promote dauer formation in C. elegans (Kimura et al., 1997; Morris et al., 1996). Also, the PI3 kinase inhibitor, LY294002 also causes a similar phenotype of dauer formation (Babar et al., 1999). A major target of PI3 kinase signalling is the serine/threonine kinase Akt/PKB, and an increased dosage of Akt relieves the block in dauer recovery seen in PI3 kinase mutants, indicating that Akt signalling acts directly downstream (Paradis and Ruvkun, 1998). Brand and Hawdon (2004) showed that LY294002 completely blocks the activation of infective larvae in A. caninum and Ancylostoma ceylanicum. In addition, an Akt kinase inhibitor results in a significant reduction of feeding (up to 70%) in A. caninum L3 (Datu et al., 2009). In N. brasiliensis, both LY294002 and Akt inhibitor IV suppressed the level of feeding of temperature-activated larvae in a dose-dependent manner

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(Fig. 3.6). Therefore, it appears that the downstream regulators of the insulin-like receptor, PI3 kinase and Akt kinase cascades, are not only responsible for the regulation of resumption of developmental arrest in C. elegans and hookworms, but also in N. brasiliensis.

Several studies have shown that genes in A. caninum are transcriptionally activated or suppressed following serum-induced activation of L3s, and many of the activated genes encoded proteins which were predicted to be extracellular with a possible role in infection (Datu et al., 2008; Mitreva et al., 2005b; Moser et al., 2005). In this study, N. brasiliensis L3 were activated by elevated temperature resulting in protein secretion, and the composition of secreted proteins was unaffected by the presence of host serum. Secretory AChEs, which were previously known to be expressed by adult worms and L4s, were shown to be secreted by activated, but not non-activated L3. Both LY294002 and Akt inhibitor IV showed broad spectrum inhibition of protein synthesis and secretion, in contrast to A. caninum, in which LY294002 was reported to have no effect on the release of ASP-1 (Brand and Hawdon, 2004). The latter results suggest that the pathways for activation of feeding and protein secretion in hookworms diverge downstream of the muscarinic receptor, but both processes seem to be connected in N. brasiliensis, and indeed the whole process of activation may be initiated by a centralised pathway.

One of the key downstream molecules of the IIS pathway in C. elegans is the fork head transcription factor, daf-16 (Ogg et al., 1997). During dauer recovery, DAF-16 is phosphorylated by Akt kinase (Paradis and Ruvkun, 1998) and 14-3-3/FTT-2 proteins can subsequently bind to phosphorylated DAF-16, which potentiates DAF-16 nuclear export and expression of genes associated with development (Cahill et al., 2001; Li et al., 2007; Wang et al., 2006). It has been shown that hookworms use a signalling pathway similar to C. elegans during resumption from developmental arrest, and DAF-16 and FTT-2 homologues have been identified in A. caninum. These proteins show 94% and 91% identity to CeDAF-16 and CeFTT-2 respectively (Gao et al., 2009; Kiss et al., 2009). It is therefore likely that the interaction of AcFTT-2 with phosphorylated AcDAF-16 mediates the IIS cascade on activation of hookworm L3s (Kiss et al., 2009).

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Examination of the draft genome and EST database (The Wellcome Trust Sanger Institute, http://www.sanger.ac.uk/sequencing/Nippostrongylus/brasiliensis/) revealed that a gene homologous to C. elegans and A. caninum ftt-2 is present in N. brasiliensis. The gene has an open reading frame coding for a predicted protein of 249 amino acids with a mass of 28.3 kDa, and its expression was detected in different stages of the parasite by RT-PCR (Fig. 3.15). N. brasiliensis FTT-2 lacks a signal peptide and has 97% and 92% identity to AcFTT-2 and CeFTT-2 respectively, so is quite possibly an orthologue. Contigs highly similar to A. caninum and C. elegans daf-16 were found in N. brasiliensis genome database (data not shown). Accordingly, these data suggest that, as in C. elegans and hookworms, downstream molecules of the IIS pathway are probably present and may be important for the resumption of development in N. brasiliensis L3.

Furthermore, an evolutionarily conserved DAF-9-mediated steroid hormone regulatory cascade which acts via the nuclear receptor DAF-12 has been discovered in C. elegans and A. caninum which controls the developmental resumption of dauer and infective stage parasites respectively (Motola et al., 2006; Wang et al., 2009). Investigators exposed A. caninum to a chemically synthesized dafachronic acid (DA), which surprisingly not only promoted the secretion of AcASP-1 and AcASP-2 of the L3s, but also rescued the activation of larval parasites restricted by ketoconazole, a broad-spectrum cytochrome P450 (DAF-9) blocker for steroid hormone synthesis (Wang et al., 2009). Intriguingly, the level of feeding and protein secretion of temperature-activated larval N. brasiliensis was significantly abolished by ketoconazole, suggesting that an analogous hormone regulatory cascade appears to be conserved in N. brasiliensis. The parasite DAF-9 appears to act at a key position in the pathway where it receives signals converted from environmental cues, and synthesizes steroid hormones which promote larval parasite activation. In addition, it has been shown that supplementation of DA cannot restore the suppression of A. caninum L3 activation mediated by PI3 kinase inhibitor, suggesting that other outputs of the IIS pathway such as DAF-16 probably converge on DAF-12 to regulate resumption of development in larval parasitic worms (Schroeder, 2006; Wang et al., 2009).

In summary, this study suggests that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge downstream of

111 the PI3 kinase cascade (Fig. 3.17). Manipulation of this pathway may improve uptake of exogenous dsRNA or siRNA in order to enhance RNAi.

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Fig. 3.17. A model for regulation of larval activation in N. brasiliensis.

Resumption of feeding is initiated by an elevated temperature cue, perceived by thermosensory neuron(s), and transmitted via interneurons to activate a downstream signalling cascade which involves phosphorylation of PI3 kinase and Akt kinase. Phosphorylated Akt can migrate to the nucleus and lead to the phosphorylation of DAF-16, thereby establishing FTT-2 binding sites. Association of FTT-2 and DAF-16 ultimately results in DAF-16 nuclear export, allowing for gene expression and resumption of development. In addition, signal through IIS pathway can activate P450 DAF-9 to generate endogenous steroid hormones, which bind to the hormone/nuclear receptor, DAF-12, promoting the recovery of infective L3.

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Chapter 4

Development of RNA interference in N. brasiliensis

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4.1. Introduction

Exploitation of RNAi has revolutionised work on C. elegans, and has broadly been adapted as a means to identify and understand functional networking of genes. Importantly, investigators are also attempting to develop RNAi as a means to explore a variety of parasitic nematodes, which are infectious agents causing diseases of humans in developing countries commonly, and resulting in economic losses in the agricultural industry globally.

RNAi in plant parasitic nematodes Infestation of plants by nematodes leads to vast economic losses in farming estimated to be about $125 billion annually worldwide (Chitwood, 2003). Therefore, scientists are attempting to utilize RNAi in order to conduct research with the ultimate aim of controlling plant- parasitic nematode infections. RNAi has been applied in plant-parasitic nematodes by the forced ingestion of dsRNA. More importantly, octopamine has been successfully demonstrated to stimulate ingestion and uptake of dsRNA in several plant-parasitic nematodes such as Heterodera glycines, Globodera pallida and Meloidogyne spp. (Bakhetia et al., 2005; Fanelli et al., 2005; Urwin et al., 2002). Rosso et al. (2005) have utilized 1% resorcinol to stimulate pharyngeal pumping, which induced dsRNA uptake in Meloidogyne incognita.

RNAi has resulted in reduced levels of mRNA for cysteine proteinases, a C-type lectin and major sperm protein (MSP) in second-stage juveniles (J2) of Heterodera glycines (Urwin et al., 2002), and phenotypic effects were observed by targeting cysteine proteinases and the C- type lectin. Likewise in H. glycines, suppression of an aminopeptidase mRNA by RNAi in J2 led to a reduction in the number of female nematodes parasitizing soybean roots after infection (Lilley et al., 2005). Furthermore, knockdown of beta-1,4 endoglucanase reduced the ability of nematodes to invade the roots, and a secreted protein, Gr-ams-1, was shown to be essential for host location, identified by RNAi in Globodera rostochiensis J2 (Chen et al., 2005). The expression of cysteine proteinase (Gp-cp-1) and FMRFamide-like peptides (Gp- flp) of Globodera pallida J2 has been individually suppressed from the intestine and nervous system by soaking with dsRNA, which led a change of sex ratio and inhibition of motility of the parasite respectively (Kimber et al., 2007; Urwin et al., 2002). Bakhetia et al. (2005)

115 demonstrated phenotypic effects on development and reduction of egg production in M. incognita by knocking down the expression of a dual oxidase. In addition, suppression of chitin synthetase in egg-shells of M. incognita was detected after soaking developing eggs in dsRNA (Fanelli et al., 2005). In the intestine of young and mature female M. incognita, RNAi not only down-regulated the expression and enzyme activity of a cathepsin L-like cysteine proteinase, but also reduced the number of established females producing eggs (Shingles et al., 2007).

Plants can be engineered to express dsRNA, silencing essential genes in nematodes. Yadav et al. (2006) described almost complete resistance to root-knot nematode infection. They cloned two genes from M. incognita into a plant expression vector that expresses both sense and anti-sense strands such that the resultant transcript forms a hairpin-shaped dsRNA. This method significantly reduced infection in tobacco plants. After this, another group used a similar strategy to introduce dsRNA for the parasitism gene 16D10 into Arabidopsis, which resulted in effective resistance to infection with M. incognita (Huang et al., 2006). Recently, Dalzell et al. (2009) have presented the first application of work using discrete 21 bp small interfering RNA (siRNA) successfully to silence the transcripts of flp in the root knot nematode M. incognita and the potato cyst nematode G. pallida infective stage juveniles (J2).

RNAi in animal parasitic nematodes RNAi has also been extensively demonstrated in a variety of animal-parasitic nematodes from different phylogenetic clades. The GI nematode N. brasiliensis was the first parasitic nematode in which RNAi was successfully performed. This was achieved by soaking adult stage parasites with dsRNA, which knocked down expression of acetylcholinesterase (NbAChE isoforms A, B and C) by over 80% (Hussein et al., 2002b). RNAi effects have also been reported in the filarial parasite Brugia malayi, in which adult parasites were soaked in dsRNA to target β-tubulin (Bm-tub-1), RNA polymerase II (Bm-ama-1) and a microfilarial sheath protein Bm-shp-1. Targeting β-tubulin and RNA polymerase II was lethal, whereas suppression of Bm-shp-1 led to abnormal sheaths in microfilariae (L1) (Aboobaker and Blaxter, 2003). More recently, RNAi treatment with B. malayi cathepsins L- and Z-like cysteine proteases (Bm-cpl-1 and Bm-cpz) dsRNA resulted in a more than 60% reduction of release of microfilariae from female adults. In addition, either dsRNA or siRNA

116 corresponding to Bm-cpl-5 and pro-region of Bm-cpl genes both caused a significant inhibition of microfilariae release, death of microfilariae and also phenotypic changes in embryos (Ford et al., 2009). RNAi in the third-stage larvae of Onchocerca volvulus, another filarial parasite, also resulted in phenotypic effects after soaking dsRNA for cathepsins L- and Z-like cysteine proteases (Ov-cpl and Ov-cpz) and serine protease inhibitors (Ov-spi-1 and Ov-spi-2) respectively (Ford et al., 2005; Lustigman et al., 2004). RNAi was further demonstrated in another filarial rodent parasite Litomosoides sigmodontis (Pfarr et al., 2006). Levels of actin mRNA were knocked down in adults which were soaked with different concentrations of dsRNA, with reduced release of microfilariae. Also, treatment of L3 and adult female Acanthocheilonema viteae, a rodent filarial nematode, by soaking with chitinase (AvChi) dsRNA inhibited moulting of L3 and hatching of microfilariae, and also led to death of female worms (Tachu et al., 2007).

In Ascaris suum, lung-stage larvae which were soaked with dsRNA had levels of pyrophosphatase (AsPPase) mRNA and enzyme activity significantly suppressed, and this also caused an inhibition of moulting of L3 to L4 (Islam et al., 2005). Issa et al. (2005) investigated the efficiency of three different RNAi techniques; soaking, electroporation and feeding in L1 of the sheep parasitic nematode Trichostrongylus colubriformis. Electroporation was the only method of delivery of ubiquitin (Tc ubq-1) dsRNA that dramatically inhibited the development of parasites. Moreover, both soaking and electroporation with ubiquitin short interfering (synthetic) RNA oligonucleotides (siRNA) resulted in significant delayed development to L3.

RNAi in the blood-feeding nematode Haemonchus contortus was first reported in 2006 (Kotze and Bagnall, 2006). The transcript levels of β-tubulin genes were suppressed in exsheathed L3, L4 and adult parasites by culturing with dsRNA. Meanwhile, Geldhof et al. (2006) also investigated the efficiency of RNAi in H. contortus. Soaking or electroporation in β-tubulin dsRNA showed successful knockdown of β-tubulin mRNA in L1. Also, superoxide dismutase transcript levels were suppressed via electroporation with dsRNA. In larvae of the cattle parasite Ostertagia ostertagi, tropomysin (Oo-tropo), β-tubulin (Oo-btub), ATP synthase (Oo-ATPs), superoxide dismutase (Oo-SODc) and a polyprotein allergen were

117 targeted by soaking and electroporation with dsRNA, with variable decreases in mRNA levels observed (Visser et al., 2006).

In spite of these successes in parasitic nematodes of animals, concerns remain about the efficiency and specificity of this technology (Geldhof et al., 2007; Knox et al., 2007). Most studies have evaluated RNAi in parasitic nematodes employing the soaking methods applied to C. elegans (Tabara et al., 1998), with culture medium containing the dsRNA, and liposome preparations usually supplemented to improve dsRNA uptake into cells. However, parasitic nematodes will not survive for a long period of time under these conditions in vitro. Furthermore, the efficiency of RNA delivery could be improved. Electroporation, a technique which has been used extensively to deliver nucleic acids into microbes, plants and animal cells, has recently been successfully utilized to introduce dsRNA to a trematode, and this was first demonstrated with dsRNA for cathepsin B (SmCB1) in larval Schistosoma mansoni (Correnti et al., 2005; Correnti and Pearce, 2004). This led to a greater than 10-fold reduction in mRNA which persisted in vitro and in vivo for over 3 weeks with considerable growth retardation of the parasite. Similarly, Morales et al. (2008) also demonstrated that electroporation-mediated delivery of dsRNA for the aspartic protease cathepsin D (SmCD) eliminated transcript levels, and eventually caused phenotypic changes in S. mansoni including suppression of aspartic protease enzyme activity, disruption of hemoglobin proteolysis and hindrance of growth in vitro. This method therefore seems to be a potential strategy of delivery for parasitic nematodes, although optimising the current pulse is necessary owing to the fact that different lengths or intensities lead to cell death (Geldhof et al., 2007).

4.2. Manipulating conditions for electroporation of N. brasiliensis using firefly luciferase-encoding mRNA

In order to investigate and realize gene function in parasitic nematodes, developing a simple and reliable system for functional knockouts by RNAi is a pressing need. Correnti and Pearce (2004) successfully established introduction of luciferase-coding mRNA into S. mansoni via electroporation. Since this significant report, electroporation has been utilized as a standard

118 method to deliver dsRNA into schistosomes. Krautz-Peterson and coworkers (2007) have optimized this and compared it with other methods of delivery of dsRNA in this trematode parasite, and concluded that squarewave electroporation was more efficient than either soaking the parasites in an equivalent dose of dsRNA or soaking them in an equivalent dose of dsRNA does in the presence of different proprietary liposome preparations.

To determine whether electroporation could deliver macromolecules into N. brasiliensis, expression of electroporation-delivered luciferase in the parasites was performed. Luciferase mRNA from the firefly Photinus pyralis was synthesised using a 5’ T7 promoter and 3’ poly (A) primer extensions, amplified from the pCRE-Luc plasmid DNA (Fig. 4.1, Panel A). In order to determine whether this mRNA could be translated into functional luciferase, I electroporated 293 T cells with 50 µg ml-1 luciferase mRNA using conditions of 400 V/250 µF (~ 6 ms). Luciferase activity was detected within 3 hours of incubation (Fig. 4.1, Panel B).

Following validation of the methodology, N. brasiliensis adult worms or activated L3 (L3A) were electroporated using 125 V, and a single pulse of 20 ms, in the presence of 400 µg ml-1 of luciferase-encoding mRNA. The parasites were collected at 3 and 24 hours post- electroporation and the expression of luciferase was then examined. Luciferase activity was not been detected in either the adult or larval parasites (Fig. 4.1, Panel C). A single pulse of 200 V for 20 ms was further tried with adult or larval stage worms, but no luciferase activity was measured (Fig. 4.1, Panel D). It therefore appears that either the outer physical barrier of the parasite (ie. the cuticle) impairs delivery, or the parasite translational machinery could not recognize and express luciferase mRNA directly.

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Fig. 4.1. Expression of luciferase in 293 T cells and N. brasiliensis electroporated with luciferase-encoding mRNA.

Panel A: Luciferase mRNA (1.8 kb) was produced from the PCR product of the pCRE-Luc plasmid DNA using primers with 5’ T7 promoter and 3’ poly(A) extensions (PCR). Molecular weight markers are given in base pairs. Panel B: 293 T cells (1 × 106 cells) were electroporated using 400 V/250 µF (~ 6 ms) in the presence of 50 µg ml-1 of the luciferase-encoding mRNA or medium alone. Panel C and D: N. brasiliensis adult or activated L3 (L3A) were electroporated using either 125 V or 200 V and a single pulse of 20 ms in the presence of 400 µg ml-1 of luciferase RNA. Parasites were harvested for assay at 3 and 24 hours post-electroporation. Results are presented as the mean +S.E. of assay performed in triplicate.

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4.3. Attempted RNAi in N. brasiliensis with delivery of NbAChE via electroporation

Despite the observation that electroporation failed to result in delivery and translation of luciferase mRNA in N. brasiliensis, three different dsRNA fragments of about 250 bp were produced, corresponding to distinct regions of the N. brasiliensis acetylcholinesterase B (NbAChE-B) coding sequence, with 5’ and 3’ T7 promoter extensions, amplified from the plasmid DNA of NbAChE-B construct. These dsRNA molecules were termed AChE-B1, AChE-B2 and AChE-B3 (Fig. 4.2).

To ascertain whether electroporation could be used to deliver dsRNA and mediate RNAi in N. brasiliensis, adult or activated L3 (L3A) worms were pre-incubated with 1 mg ml-1 dsRNA of AChE-B1, 2 and 3 containing 400 U ml−1 RNAsin for 1 hour before being electroporated at 125 V for a single pulse of 20 ms and transferred to culture. The secreted products of adult or larval parasites were collected 1, 2 and 3 days post-electroporation and AChE enzyme activity was subsequently determined. Compared to controls (Ctrl), all three different dsRNAs showed a high degree of silencing of NbAChE activity in N. brasiliensis adults, corresponding to about 70-75% knockdown (Fig. 4.3, Panel A). On the other hand, electroporation of dsRNA under similar conditions did not result in the same reduction of enzyme activity in third-stage activated larval parasites, with only about 15-30% knockdown of AChE activity (Fig. 4.3, Panel B). Unfortunately, this approach appeared not to show a consistent knockdown of AChE activity in N. brasiliensis. Thus, in another representative experiment, following electroporation of 1 mg ml-1 AChE-B2 dsRNA to adult worms, the levels of secreted NbAChE activity were determined after 24 hours post-electroporation, and showed no reduction compared to the control (Fig. 4.4, Panel A). The levels of NbAChE-B mRNA in the parasites were also examined at 24 hours post-treatment by RT-PCR, the results suggesting that there was no significant difference to controls (Fig. 4.4, Panel B).

In addition, various conditions of electroporation were tested in N. brasiliensis in an attempt to successfully introduce dsRNA into the cell and silence the target. However, none of these efforts could facilitate RNAi in the parasites (Table 4.1).

121

Fig. 4.2. Production of three distinct dsRNA fragments from NbAChE-B.

Panel A: Three different partial sequence fragments of about 250 bp for NbAChE-B; AChE- B1 (coloured in blue), AChE-B2 (coloured in green) and AChE-B3 (coloured in purple) were generated. Panel B: Double-stranded RNA (dsRNA) for AChE-B1 (lane 1), AChE-B2 (lane 2) and AChE-B3 (lane 3) was produced from the PCR products using primers with T7 promoter extensions (PCR). Molecular weight markers are shown in base pairs (bp).

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Fig. 4.3. RNAi-mediated suppression of NbAChE secretion in N. brasiliensis adults and L3A via electroporation of dsRNA.

Panel A: Adult parasites were exposed to 1 mg ml-1 dsRNA of AChE-B1, 2 and 3 corresponding to the three different regions of NbAChE-B cDNA for 1 hour before being electroporated by a single pulse of 125 V for 20 ms. The enzyme activity of NbAChE was determined from the parasite secreted proteins on a daily basis (Day 1, 2 and 3). Panel B: Results of a similar experiment in which N. brasiliensis L3A were also exposed to 1 mg ml-1 dsRNAs, and NbAChE activity was measured in secreted products. Results are presented as the mean +S.E. of assay performed in triplicate.

123

Fig. 4.4. Inconsistent knockdown of NbAChE-specific mRNA via electroporation.

Adult parasites were pre-incubated in the presence (dsRNA) or absence (Control) of 1 mg ml- 1 dsRNA for AChE-B2 for 1 hour before being electroporated. Post-electroporation, the parasites were incubated in fresh medium for 24 hours. Panel A: Parasite secreted proteins were collected and AChE enzyme activity was measured. Results are presented as the mean +S.E. of assay performed in triplicate. Panel B: Parasite samples were harvested and total RNA extracted. The presence of NbAChE-B and actin transcripts was determined by RT- PCR using 10-fold serially diluted cDNA (neat, 1:10).

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Table 4.1. Attempted delivery of dsRNA via various conditions of electroporation in N. brasiliensis.

Adult parasites were exposed to 1 mg ml-1 dsRNA for AChE-B1, 2 and 3 for 1 hour before being electroporated by a single pulse of different voltages (50, 75, 100 and 125 V) for 20 ms. Secretion of AChE and specific mRNA levels were determined after 24 hours post- electroporation, with none of conditions showing consistent reduction of NbAChE.

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4.4. Induction of RNAi in N. brasiliensis via soaking

Hussein et al. (2002b) have performed RNAi to knock down AChE secretion in N. brasiliensis adults by soaking in dsRNA for NbAChE-B in the presence of Lipofectin. In order to investigate the consistency of this phenomenon, these procedures were re-examined.

Initially, J774 macrophages were used as a positive control to demonstrate that soaking of mammalian cells with luciferase encoding-mRNA in the presence of 5% Lipofectin could administer intracellular delivery of mRNA. Luminescence due to translation of luciferase was consequently observed at 6 hours post-soaking (Fig. 4.5, Panel A). N. brasiliensis adults and L3A were then soaked with 50 µg ml-1 luciferase-encoding mRNA with 5% Lipofectin for 6 hours. In contrast to the consequence of luciferase expression in J774 macrophages, no luminescence was detected in either adult or L3A parasites (Fig. 4.5, Panel B).

To test the capability of lipofectin in introducing dsRNA into the parasite, N. brasiliensis adult worms were soaked with 1 mg ml-1 dsRNA of AChE-B1, 2 and 3 containing 5% Lipofectin and 400 U ml−1 RNAsin for 24 hours. AChE enzyme activity from secreted products of adult parasites was determined on days 1, 2 and 3 post-soaking. The AChE-B2 dsRNA treated parasites appeared to show about 30-40% suppression of enzyme activity compared to the control (Ctrl). Also, AChE secretion was also reduced by about 20% on day 2 and day 3 post-treatment when worms were treated with AChE-B3 dsRNA (Fig. 4.6).

To investigate whether this occurred via RNAi, AChE mRNA levels in dsRNA treated worms were examined. N. brasiliensis adults were soaked under the same conditions of 1 mg ml-1 dsRNA of AChE-B2 or AChE-B3 with 5% Lipofectin, and AChE-B mRNA levels were then determined by RT-PCR at 48 hours or 7 days post-treatment. However, RT-PCR data indicated that mRNA levels of AChE-B were not significantly changed with respect to controls, even after the long period of incubation (7 days) (Fig. 4.7). Additionally, repeat experiments indicated that reduction of AChE secretion was not consistent (data not shown).

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4.5. Attempted knockdown of ubiquitin mRNA

As soaking or electroporation of N. brasiliensis L3/adults did not result in robust RNAi silencing for NbAChE, indicated by either RT-PCR or enzyme activity assay, ubiquitin was then chosen as another target for examination. Ubiquitin had been previously targeted in the GI nematode Trichostrongylus colubriformis and C. elegans via electroporation and soaking with siRNA and dsRNA, which led to a significantly delayed development of larval stage worms (Issa et al., 2005).

N. brasiliensis ubiquitin (NbUbq)-specific dsRNA corresponding to a partial cDNA sequence (GenBank acc.: BM279528) from the EST database was prepared using 5’ and 3’ T7 promoter extensions and amplified from N. brasiliensis larval stage cDNA (Fig. 4.8, Panel A and B). Small interfering RNA (siRNA) for knocking down the expression of NbUbq was generated from the Ubq dsRNA preparation by using the BLOCK-iTTM Dicer Enzyme Kit (InVitrogen). A prominent band representing siRNA of the expected size (20-23 base pairs) was clearly visible on the 4% agarose gel, and a smear of higher mass representing uncleaved NbUbq dsRNA and partially cleaved products could also be seen on the gel (Fig. 4.8, Panel C).

Activated N. brasiliensis L3 (L3A) were incubated with a high concentration of NbUbq dsRNA (1 mg ml-1) to examine whether effective RNAi can be observed following electroporation. Levels of mRNA for ubiquitin in NbUbq dsRNA-treated samples seemed not to be significantly suppressed at 24 or 48 hours following a single pulse of 125 V for 20 ms compared to controls (Fig. 4.9, Panel A).

127

Fig. 4.5. Expression of luciferase in J774 macrophages and N. brasiliensis with Lipofectin-delivered luciferase-encoding mRNA.

6 Panel A: Soaking of J774 macrophages (1 × 10 cells) in the presence (mRNA) or absence ((- -1 )mRNA) of 50 µg ml luciferase-encoding mRNA with 5% Lipofectin for 6 hours before luminescence was determined.

Panel B: Soaking of N. brasiliensis adults or L3A in the presence (mRNA) or absence ((- -1 )mRNA) of 50 µg ml luciferase-encoding mRNA with 5% Lipofectin for 6 hours before luminescence was measured. Results are presented as the mean +S.E. performed in triplicate.

128

Fig. 4.6. RNAi-mediated suppression of NbAChE secretion in N. brasiliensis adults via Lipofectin-mediated delivery of dsRNA

Adult parasites were soaked in 1 mg ml-1 dsRNA of AChE-B1, 2 and 3 with 5% Lipofectin solution for 24 hours respectively. The activity of NbAChE was analyzed from the parasite secreted proteins on Day 1, 2 and 3 post-soaking. Results are presented as the mean +S.E. of assays performed in triplicate.

129

Fig. 4.7. NbAChE-B specific RNAi silencing appeared not to work consistently in N. brasiliensis adults via soaking in dsRNA.

Adult parasites were soaked with the presence (dsRNA) or absence (Control) of 1 mg ml-1 dsRNA of AChE-B2 or AChE-B3 containing 5% Lipofectin for 24 hours before being transferred to fresh worm medium for culture. Panel A: Parasite samples were harvested at 48 hours post-AChE-B2 dsRNA-treatment and total RNA extracted. The presence of NbAChE-B and actin transcripts was determined by RT-PCR using 10-fold serially diluted cDNA (neat, 1:10). Panel B: Parasite samples were collected and total RNA extracted 7 days post-AChE-B3 dsRNA-treatment. The expression of NbAChE-B and actin was determined by RT-PCR using 10-fold serially diluted cDNA (neat, 1:10, 1:100, 1:1000, 1:10000).

130

Fig. 4.8. Production of N. brasiliensis ubiquitin-specific dsRNA and siRNA.

Panel A: The fragment (251 bp) of NbUbq for dsRNA production was prepared from a partial cDNA sequence (GenBank: BM279528) of N. brasiliensis EST database. Panel B: Double-stranded RNA (dsRNA) was produced from the PCR product of NbUbq amplification from N. brasiliensis larval stage cDNA using primers with T7 promoter extensions (PCR). Molecular weight markers are indicated in base pairs. Panel C: Small interfering RNA (siRNA) was generated from the preparation of NbUbq dsRNA using the BLOCK-iTTM Dicer Enzyme Kit. Molecular weight markers are indicated in base pairs (bp).

131

Furthermore, iodinated BSA (125I-BSA) was used to examine and quantify feeding following exposure of L3A to NbUbq dsRNA via electroporation, as it was hypothesized that knockdown of ubiquitin mRNA would either be fatal or would decrease parasite viability and possibly interfere with feeding. However, in contrast to experiments with T. colubriformis, Fig. 4.9, Panels B and C show that electroporated dsRNA did not lead to a significant difference measurable by reduction of iodine-labelled BSA uptake in the treated parasites compared with controls 1 day or 2 days post-electroporation.

Electroporation with a high concentration of siRNA for NbUbq in adult worms was also analyzed. N. brasiliensis adults were pre-incubated with 100 µg ml-1 siRNA for an hour, then electroporated at a single pulse of 125 V for 20 ms. However, there was still no effect on the transcript levels of NbUbq following on days 2, 4, 8 post-electroporation and even after a long-term incubation on day 14 (Fig. 4.10). Similarly, electroporation of NbUbq dsRNA (1 mg ml-1) did not lead to any significant suppression of ubiquitin mRNA levels compared to controls (Fig. 4.10).

Additionally, a similar approach to previous experiments was adopted, using Lipofectin- mediated soaking. However, incubation of larval and adult parasites in 1 mg ml-1 dsRNA for NbUbq with 5% Lipofectin still failed to cause any significant mRNA knockdown when analysed by RT-PCR (Fig. 4.11).

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Fig. 4.9. Attempted suppression of NbUbq in larval N. brasiliensis via electroporation with dsRNA.

Panel A: Activated L3 (L3A) were exposed to the presence (dsRNA) or absence (Control) of 1 mg ml-1 NbUbq dsRNA for 1 hour before being electroporated. Post-electroporation, the parasites were incubated in fresh medium. The parasite samples were harvested and total RNA extracted at 24- and 48-hour post-electroporation. The presence of NbUbq and actin transcripts was determined by RT-PCR using 10-fold serially diluted cDNA (neat, 1:10, 1:100, 1:1000, 1:10000). Panel B and C: Results of a similar experiment in which L3A (10,000) were exposed to 1 mg ml-1 NbUbq dsRNA via electroporation, and placed into fresh culture medium. Worms were collected at 24- and 48-hour post-treatment, and subsequently incubated with 100,000 cpm 125I-BSA in culture medium for 1 hour, washed and counted. Results are presented as the mean +S.E. of assays in triplicate.

133

Fig. 4.10. Electroporation with NbUbq dsRNA or siRNA failed to induce effective RNAi in adult worms.

N. brasiliensis adults were pre-incubated in the presence or absence (Control) of 1 mg ml-1 NbUbq dsRNA (dsRNA) or 100 µg ml-1 NbUbq siRNA (siRNA) for 1 hour before being electroporated then incubated in fresh medium. Parasites were harvested and total RNA extracted on days 2, 4, 8 and 14 post-electroporation. The presence of NbUbq and actin transcripts was determined by RT-PCR using 10-fold serially diluted cDNA (neat, 1:10, 1:100, 1:1000, 1:10000).

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Fig. 4.11. Lipofectin-mediated soaking with NbUbq dsRNA failed to induce RNAi.

N. brasiliensis L3A (Panel A) or adults (Panel B) were pre-incubated in the presence (dsRNA) or absence (Control) of 1 mg ml-1 NbUbq dsRNA with 5% Lipofectin for 24 hours, then incubated in fresh medium. Parasites were collected and total RNA extracted at 48 hours post-treatment. Levels of mRNA for NbUbq and actin were determined by RT-PCR using 10- fold serially diluted cDNA (neat, 1:10, 1:100, 1:1000, 1:10000).

135

4.6. Investigation of polyethyleneimine (PEI)-based delivery

Polyethyleneimine (PEI) is a synthetic polymer with a high cationic charge density potential and a protonable amino nitrogen atom in every third position (Boussif et al., 1995), which has been used to deliver large DNA molecules such as 2.3 Mb yeast artificial chromosomes (YACs) (Marschall et al., 1999) as well as plasmids or small oligonucleotides (Boussif et al., 1995; Urban-Klein et al., 2005; Werth et al., 2006) into mammalian cells in vitro and in vivo. The potency of this transfection reagent depends on the ability of PEI to condense and compact DNA or oligonucleotide molecules into complexes, which form small colloidal particles allowing efficient cellular uptake through endocytosis. In addition, this compact condensation of molecules along with the buffering capacity of PEI in cellular lysosomes or endosomes protects DNA or oligonucleotide molecules from nuclease degradation (Boussif et al., 1995; Urban-Klein et al., 2005).

To evaluate the use of PEI-based delivery for RNAi in N. brasiliensis, PEI-mediated complexation was firstly examined by a gel retardation assay. NbUbq dsRNA (10 µg) was complexed with jetPEITM in 5% glucose at N/P ratios of 0, 1, 3 and 5 (the ratios of the nitrogen residues of PEI to nucleic acid phosphates in the complex), then analyzed on a 3% agarose gel. Gradual differences between the various N/P ratios were observed, and complete binding and neutralization of dsRNA was obtained at a ratio of 5 (Fig. 4.12, Panel A).

To further assess the capacity of PEI for complexation and cellular internalization, fluorescently labelled siRNA was used in polyplex formation. The complexes were composed of 0.1 µM Cy3-labelled siRNA with jetPEITM in 5% glucose at N/P = 0, 1, 3 and 5, which were incubated with J774 macrophages for 24 hours before microscopic analysis. The number of cells which showed red fluorescence due to internalization of polyplexes increased with increasing N/P ratio (Fig. 4.12, Panel B).

136

Fig. 4.12. Examination of jetPEITM complex formation and introduction of dsRNA or siRNA into macrophages.

Panel A: Gel retardation assay of dsRNA-jetPEITM polyplexes prepared in 5% glucose at various N/P ratios (1, 3 and 5). NbUbq dsRNA bands (indicated with arrow) either not complexed to the jetPEITM (N/P = 0 as a control) or dissociated from polyplexes were separated by electrophoresis and visualized by ethidium bromide staining. Molecular weight markers are indicated in base pairs (bp). Panel B: Image of J774 macrophages after 24 hours incubation with polyplexes which were fabricated with 0.1 µM Cy3-labelled siRNA (red) and jetPEITM at different N/P ratios as indicated.

137

Fig. 4.13. Uptake of Cy3-labelled siRNA into the pharynx and intestine of larval N. brasiliensis after jetPEITM complex formation.

Activated N. brasiliensis L3 were incubated in the presence or absence (Control) of 0.1, 0.2 and 0.3 µM Cy3-labelled siRNA complexed to jetPEITM (N/P ratio = 5) for 6 hours at 37°C. No fluorescence was observed in the controls, whereas worms exposed to 0.3 µM Cy3- labelled siRNA showed intense fluorescence in the pharynx and intestine. Scale bar: 0.4 mm; white hollow arrow: pharynx; white solid arrow: intestine.

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PEI-mediated delivery thus appeared to have a high efficiency for complexing and introducing siRNA into mammalian cells. Accordingly, activated larval N. brasiliensis were incubated with Cy3-labelled siRNA to examine how effectively larval worms take up siRNA following jetPEITM-based (N/P ratio = 5) complexation. The intensity of red fluorescence down the length of the intestine was raised by increasing with the concentrations of labelled siRNA from 0.1 µM to 0.3 µM (Fig. 4.13), but was not evident in control worms, which were not exposed to siRNA/PEI. Intense fluorescence was detectable in the pharynx (indicated with a white hollow arrow) and intestine (indicated with a white solid arrow) of the parasites incubated with 0.3 µM complexed siRNA. However, it is not clear whether the RNA was taken up from the intestinal lumen into cells, and spreading into tissues of the parasite.

In order to determine whether PEI-based delivery successfully introduces siRNA into intestinal cells and induces RNAi-mediated knockdown of mRNA, larval parasites were treated for 24 hours with polyplexes, which were fabricated with 0.3 or 1.5 µM NbUbq siRNA and jetPEITM at N/P = 5. In contrast to the Urban-Klein et al. (2005) study showing the efficiency of jetPEITM-complexed siRNA in down-regulation of target mRNAs in rats, no apparent transcriptional knockdown was observed for NbUbq in treated worms (Fig. 4.14, Panel A). Adult parasites were also treated with this transfection reagent complexed to 1.5 µM NbUbq siRNA, which similarly did not result in any reduction of mRNA levels of the target gene compared with controls (Fig. 14.14, Panel B).

4.7. A cationic liposome still could not successfully mediate RNA delivery to induce an RNAi effect in N. brasiliensis

Cationic liposomes have been used to introduce dsRNA or siRNA into in parasitic nematodes and trematodes. For instance, Skelly et al. (2003) and Kotze and Bagnall (2006) have reported the use of dsRNA encapsulated with a commercial liposome, LipofectamineTM 2000 (LF2000), which led to a substantial decrease in cathepsin enzyme activity and reduction in β-tubulin gene expression from S. mansoni and H. contortus L3, L4 and adults respectively. LF2000 is one of several cationic liposome formulations which act by complexing nucleic

139 acid molecules, allowing them to overcome the electrostatic repulsion of phospholipids on the cell membrane and to be taken up by cells, and which have been extensively applied in RNAi (Dalby et al., 2004).

J774 macrophages were used as a positive control to demonstrate delivery of luciferase encoding-mRNA complexed with LF2000 in a final concentration of 50 µg ml-1 in mammalian cells, and luminescence was observed at 6 hours post-treatment. However, luminescence was again not detected in both luciferase mRNA/LF2000-exposed adults and L3A parasites (data not shown).

To examine whether this reagent could effect suppression of NbUbq mRNA in N. brasiliensis, larval worms were soaked with either 0.3 µM or 1.5 µM of NbUbq siRNA encapsulated with LF2000 for 24 hours, but this treatment resulted in no significant reduction of the expression of the target gene compared with the non-treated worms (Fig. 4.15).

140

Fig. 4.14. A small interfering RNA (siRNA)-jetPEITM polyplex failed to induce RNAi in N. brasiliensis.

N. brasiliensis L3A (Panel A) or adults (Panel B) were incubated in the presence (siRNA) or absence (Control) of 0.3 or 1.5 µM NbUbq siRNA complexed with jetPEITM for 24 hours, washed and incubated in fresh medium. Parasites were collected and total RNA extracted at 48 hours post-treatment. The expression of NbUbq and actin was determined by RT-PCR using 10-fold serially diluted cDNA (neat, 1:10, 1:100, 1:1000, 1:10000).

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Fig. 4.15. LipofectamineTM 2000 (LF2000)-encapsulated siRNA also failed to silence the expression of NbUbq in N. brasiliensis.

Activated N. brasiliensis L3 were incubated in the presence (siRNA) or absence (Control) of 0.3 or 1.5 µM NbUbq siRNA encapsulated in LF2000 for 24 hours, washed and incubated in fresh medium. Parasites were collected and total RNA extracted at 48 hours post-treatment. Transcripts of NbUbq and actin were detected by RT-PCR using 10-fold serially diluted cDNA (neat, 1:10, 1:100, 1:1000, 1:10000).

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Fig. 4.16. Expression of AChE-B in different stages of N. brasiliensis.

Panel A: Immunoblot analysis of NbAChE-B in somatic extracts and excretory-secretory (ES) products of N. brasiliensis. Equal amounts (30 µg) of protein prepared from adult (Ad), activated L3 (L3A) and non-activated L3 (L3) parasite extracts and also ES products from adults and activated L3s, were separated via 12% SDS-PAGE, transferred onto a nitrocellulose membrane and probed with anti-AChE-B antibody. The bottom panel shows the same preparations probed with an antibody to the internal control, tubulin, a cytoskeletal protein which is not secreted. Molecular mass is shown in kilodaltons (kDa). Panel B: RT-PCR was used to amplify a 413-bp NbAChE-B partial cDNA fragment from RNA samples of N. brasiliensis L1, L2, non-activated L3 (L3), activated L3 (L3A), L4 and adult (Ad). The bottom panel shows expression of the internal control, actin by RT-PCR. Molecular mass is shown in base pairs (bp).

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Fig. 4.17. Serotonin (5-HT) fails to promote RNAi.

Activated larval N. brasiliensis were exposed to 1 mg ml-1 of NbUbq dsRNA with or without 0.5 mM 5-HT for 1 hour before being electroporated, then incubated in fresh medium. Parasite samples were collected and total RNA extracted at 48 hours post-treatment. Transcripts for NbUbq and actin were detected by RT-PCR using 10-fold serially diluted cDNA (neat, 1:10, 1:100, 1:1000, 1:10000).

144

Table. 4.2. Comparison of core RNAi pathway genes in parasitic nematodes.

Table A: Data for B. malayi and H. contortus were compiled from Geldhof et al. (2007), Ghedin et al. (2007), and Lendner et al. (2008). Table B: The Wellcome Trust Sanger Institute database for N. brasiliensis DNA sequences (http://www.sanger.ac.uk/sequencing/Nippostrongylus/brasiliensis/) was searched for sequences with homology to genes encoding proteins in the RNAi pathway of C. elegans by tBLASTn. Yellow/Pink box and v indicate gene present; -- indicates absence of traces of this gene.

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Fig. 4.18. A putative homologue of C. elegans SID-2 in N. brasiliensis.

Panel A: N. brasiliensis genomic DNA sequences from the Wellcome Trust Sanger Institute database were searched for possible homologues of C. elegans SID-2 (331 amino acids, GenBank: AY466439) by tBLASTn, and two contigs (contig41898 and contig09717) with a high scoring sequence pair (HSP) to SID-2 were found (underlined). Panel B and C: Alignments of translated contigs with CeSID-2. Identical amino acids are shaded in black, and similar amino acids in grey.

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4.8. Discussion

In an attempt to develop RNAi as a method for functional knockouts/knockdowns in N. brasiliensis, diverse approaches were evaluated to deliver dsRNA or siRNA into the parasite. The method of electroporation was firstly adapted as a means to introduce exogenous molecules, and luciferase-encoding mRNA was used as a parameter to elucidate the efficiency of delivery to L3s and adults. Parasites were electroporated with a high concentration (400 µg ml-1) of mRNA using conditions of 125 V/ 20 ms, which had been successfully utilised for Schistosoma mansoni (Correnti and Pearce, 2004). However, given that luciferase could be delivered and translated in mammalian 293 T cells, it was surprising that no activity was observed in either L3s or adults, even at higher voltages.

Luciferase mRNA has also been electroporated into N. brasiliensis egg embryos in this laboratory, but again activity was not observed (Ball, personal communication). It is therefore possible that the luciferase-encoding mRNA is being delivered into nematode cells, but somehow the translational machinery could not recognize and express it. Alternatively, it may be impossible to deliver macromolecules by electroporation into adult and larval stages of N. brasiliensis. The cuticle is likely to present an impenetrable barrier in the absence of cuticular pore canals, which exist in some species (Martin et al., 1987; Watson, 1965a, b). It was considered possible that electroporation could occur via an inside-out process into intestinal epithelial cells, although this would require electrical conductivity from the exterior, and might be disrupted by pharyngeal closure and/or details of the parasite anatomy. This would not be present for electroporation of embryos, but it is unclear whether eggshells, which again present a barrier for delivery, were completely removed.

Davis et al. (1999) have successfully expressed a luciferase reporter gene construct, flanked by a spliced leader (SL) RNA promoter and Ascaris 3’ untranslated region (3’ UTR), in embryos of the parasitic nematode Ascaris suum, delivering constructs by particle bombardment. Investigators also used microinjection and particle bombardment to transfect SL-modified luciferase constructs into embryos, larvae and adults of B. malayi (Higazi et al., 2002). Plasmids with actin or endoplasmic reticulum ATPase promoters have been used to express Green Flourescent Protein (GFP) in free-living female Strongyloides stercoralis

147 following injection into the gonads (Lok and Massey, 2002), and more recently a series of S. stercoralis promoters have been used with a parasite 3' UTR to drive expression of GFP or the red fluorescent protein mRFPmars in a tissue-specific fashion (Junio et al., 2008).

Although it was unclear why luciferase-encoding mRNA was not expressed, we proceeded with using acetylcholinesterase B (NbAChE-B) as a target gene for RNAi. NbAChE-B is one of three secreted variants of NbAChE (-A, -B and -C) in N. brasiliensis, with molecular weights between 69-74 kDa (Hussein et al., 1999a; Hussein et al., 2002a). However, it is still unclear why N. brasiliensis expresses multiple forms of secreted AChE or the purposes in distinct stages of the life cycle. NbAChE-B was most strongly expressed in adult worm extracts or secreted products (Fig. 4.16, Panel A). The expression of NbAChE-B at the RNA level was detectable in activated infective larvae (L3A), L4 and adults (Fig. 4.16, Panel B). It has been suggested that secreted NbAChEs may function to maintain the position of the parasite in the small intestine of the host (Selkirk et al., 2005b). Hence, the secreted AChE-B may play an important role in N. brasiliensis infection, and the most direct approach to investigate the function of this enzyme is through functional knockdown by RNAi.

As in the previous studies on schistosomes, electroporation has been used to effect RNAi in Trichostrongylus colubriformis, Haemonchus contortus and Ostertagia ostertagi (Geldhof et al., 2006; Issa et al., 2005; Visser et al., 2006). Acetylcholinesterase was targeted in N. brasiliensis adult and L3A stages using electroporation with three different dsRNA fragments of NbAChE-B. Although up to 65% reduction of NbAChE activity was observed in adult worms, in comparison to 30% reduction in L3A after treatment (Fig. 4.3), RNAi knockdown via electroporation appears to be inconsistent. Lendner et al. (2008) showed that electroporation of Heligmosomoides polygyrus adults with labelled siRNA for 20 ms at 500 V resulted in focal points of flourescence between the cuticle and the hypodermis. This indicates that electroporation has the capacity to penetrate the cuticle of H. polygyrus adult worms, but that it may occur at defined sites potentially representing pores. Electroporation of N. brasiliensis using conditions of 500 V at 20 ms however, killed both larvae and adult parasites (data not shown). Regarding the inconsistency of RNAi reduction for NbAChE secretion and expression via electroporation, adult parasites were soaked in dsRNA with the transfection reagent Lipofectin, which was used by Hussein et al (2002b) to successfully

148 knock down the mRNA level of NbAChE. However, neither RT-PCR nor activity of NbAChE measurement in culture medium consistently showed observable down-regulation of the target (Fig. 4.6 and 4.7). Ford et al. (2009) have examined the use of Lipofectin mixed with Cy3-labelled Bm-cpl-5 dsRNA in B. malayi adult female worms, and these studies suggested that Lipofectin did not improve the penetration of dsRNA into the parasite. Collectively, these data suggest that Lipofectin seems likely not to have a generalised capacity to facilitate uptake of RNA in parasitic nematodes.

Despite the fact that RNAi is a universal phenomenon in eukaryotic organisms (Hannon, 2002), RNAi in animal parasitic nematodes has been reported to be attempted for 29 genes, with some success achieved for only 14 of these. It is still unknown why some genes are more accessible to RNAi than others. In this study, an attempt to knock down expression of ubiquitin was made. Unfortunately, when NbUbq double-stranded RNA was attempted to be delivered into activated infective larvae (L3A) via electroporation, no significant suppression of ubiquitin mRNA was noted by 24 or 48 hours of post-treatment (Fig. 4.9, Panel A). In addition, this did not lead to a reduction in feeding assayed by uptake of iodine-labelled BSA in L3A compared to controls (Fig. 4.9, panels B and C).

It was shown in Chapter 3 that infective larvae do not feed or ingest exogenous macromolecules, but could be induced to do so by activation at 37oC in a serum-independent manner. Also, serotonergic pathways acted synergistically with elevated temperature to stimulate/enhance uptake of feeding in larval parasites. Even though 5-HT could increase the rate of pharyngeal pumping and enhance uptake of exogenous molecules, when N. brasiliensis were exposed to a high concentration of NbUbq dsRNA with 0.5 mM 5-HT and subsequently electroporated, this still failed to promote RNAi (Fig. 4.17).

In contrast, Urwin et al. (2002) demonstrated RNAi in preparasitic stages of the plant parasitic nematodes Heterodera glycines and Globodera pallida after stimulation of pharyngeal pumping by octopamine. It is possible that the large dsRNA molecules might not be taken up efficiently in the intestinal epithelium of N. brasiliensis. Alternatively, therefore, a possible strategy for improving uptake in the intestine of N. brasiliensis is the use of 21-23 bp small interfering RNAs (siRNA). Issa et al. (2005) have reported that T. colubriformis

149 siRNAs or dsRNAs for ubiquitin introduced by the means of electroporation or soaking successfully inhibited the development of larval parasites, and that siRNA molecules were more effective than dsRNA. Nevertheless, comparatively, neither NbUbq dsRNA nor siRNA could elicit any RNAi inhibition in N. brasiliensis when delivered via electroporation or soaking (Fig. 4.10 and Fig. 4.11).

Polyethylenimine (PEI) is a chemical polymer that contains a high cationic charge density, and has the ability to compact DNA or RNA forming small colloidal particles, which can be taken up into cells through endocytosis (Boussif et al., 1995; Urban-Klein et al., 2005; Werth et al., 2006). PEI has been used to successfully transport siRNA into tumours and displayed full bioactivity at non-toxic concentrations in mice (Urban-Klein et al., 2005). In addition, PEI complexed with influenza siRNA demonstrated antiviral effects in infected mice (Ge et al., 2004), whereas similar targeting of the Ebola L gene led to partial protection against lethal virus infection in guinea pigs (Geisbert et al., 2006). To date, the use of this polymer as a means to optimize RNAi delivery in parasitic nematodes has not been reported. The current study demonstrates that the pharynx and intestinal regions of larval N. brasiliensis worms were filled with intense Cy3-labelled RNA following complexing with PEI (Fig. 4.12, Panel B and Fig. 4.13). However, it is not clear whether the RNA was taken up from the intestinal lumen into cells, and spreading into other tissues or the body cavity was not observed, making systemic RNAi unlikely in either larval or adult parasites. An endocytic pathway mediating internalization of environmental RNA to induce RNAi knockdown has been demonstrated in Drosophila S2 cells (Saleh et al., 2006), which indicates that dsRNA was taken up via an active process involving scavenger receptor-mediated endocytosis. Therefore, it is likely that such an endocytic mechanism does not exist in the intestine of N. brasiliensis, possibly because of the absence of an appropriate receptor for RNA.

Liposomes have been intensively used for formulating drugs to improve pharmacokinetic properties and decrease toxicity (Torchilin, 2006). Similarly, liposomes developed for therapeutic delivery of siRNA are multi-component nanoparticles, typically composed of multiple lipids, including a cationic or fusogenic lipid, and cholesterol (Li and Szoka, 2007; Torchilin, 2006). LipofectamineTM 2000 (LF2000), a cationic lipid carrier, was employed for the administration of siRNA in N. brasiliensis, but did not result in successful RNAi using

150 either low or high concentrations of siRNA. Likewise, siRNA or dsRNA coding for ubiquitin and encapsulated with LF2000 did not elicit any significant RNAi phenotype in T. colubriformis (Issa et al., 2005). So far, the only effectual LF2000-mediated delivery for RNAi was reported for β-tubulin in H. contortus among the animal parasitic nematodes (Kotze and Bagnall, 2006). Interestingly, the investigators found that although a LF2000 formulation was not necessary for RNAi to occur, the effectiveness of gene suppression and the longevity of the result were greater in the presence of liposomes.

Feeding worms with bacteria engineered to express dsRNA is a well-established approach for RNAi in C. elegans (Fraser et al., 2000; Timmons et al., 2001), and this method has been used as a means for high throughput RNAi-based functional genomics screening (Fraser et al., 2000). Issa et al. fed T. colubriformis L1s with E. coli HT115(DE3) expressing tropomyosin (Tctmy-1) dsRNA, and subsequently observed a strong RNAi phenotype in developmental retardation of parasites (Issa et al., 2005). However, in contrast, feeding of dsRNA to H. contortus L1-L2s (Hc-MO3, Hc-mua-6, Hc-unc-87 and Hc-elt-2) and H. polygyrus eggs and L1s (tropomyosin) did not result in any phenotypic effect and RNA silencing (Geldhof et al., 2006; Lendner et al., 2008). In addition, our laboratory has previously examined feeding in N. brasiliensis. GFP-expressing E. coli HT115 were ingested by L1 and L2 and were observable by flourescence in larval intestines. L1 cultured on plates seeded with E. coli developed through to L3s and were infective to rats but consistent knockdown of target mRNAs was not observe in any of 8 different constructs tested (Smyth & Selkirk, unpublished data). Therefore, it is possible that the uptake of bacterial-derived dsRNA from the intestinal lumen into epithelial cells is deficient, and/or core regulators of the RNAi machinery may be missing in parasitic nematodes (Table. 4.2).

Differences in tissue expression of the target mRNA, stability or mRNA turnover could be important factors for RNAi in animal parasitic nematodes (Geldhof et al., 2007). It is still unknown why some genes are more accessible to RNAi than others. In this study, no consistent post-transcriptional silencing was observed for AChE and ubiquitin, and treatment did not result in reduced feeding ability of the parasite, although suppression of secreted AChE has previously been reported in N. brasiliensis, with apparent specificity in knockdown, as somatic AChE appeared unaffected (Hussein et al. 2002). Soaking and

151 electroporation methods have also been attempted in the laboratory using beta-tubulin and an ftt-2 homologue as targets, with no apparent reduction in mRNA observed in larval or adult N. brasiliensis (data not shown).

RNAi is widely and routinely used in C. elegans, and appears to be effective in distinct species of plant parasitic nematodes (Chen et al., 2005; Dalzell et al., 2009; Fanelli et al., 2005; Huang et al., 2006; Kimber et al., 2007; Rosso et al., 2005; Shingles et al., 2007; Urwin et al., 2002), but not in animal parasitic species. There could possibly be two reasons for this circumstance: (i) the RNA is not effectively taken up into tissues, or (ii) later steps in the RNAi mechanism do not function in animal parasitic nematodes. For C. elegans it was found that two transmembrane proteins, SID-1 and SID-2, are responsible for RNA uptake and spreading and mainly expressed in the intestine (Feinberg and Hunter, 2003; Winston et al., 2002; Winston et al., 2007). It is intriguing that SID-2 function is not conserved in Caenorhabditis briggsae, and that expression of C. elegans SID-2 is sufficient to confer environmental dsRNA on C. briggsae (Winston et al., 2007).

There are many other important proteins also participating in the RNAi pathway of C. elegans. RDE-4 and RDE-2 are involved in detecting and retaining exogenous RNA molecules and presenting them for further RNAi processing (Parrish and Fire, 2001; Tabara et al., 2002). In addition, a downstream protein of RNAi, MUT-7, can interact with RDE-2 to mediate the amplification of siRNA (Tops et al., 2005). However, crucially, these genes have not been identified computationally in B. malayi or H. contortus, for which genome sequence and expressed sequence tag (EST) databases are available (Table. 4.2, Table A) (Geldhof et al., 2007; Ghedin et al., 2007). Several publications on filarial nematodes have suggested that a functional RNAi machinery is present (Ford et al., 2005; Ford et al., 2009; Lustigman et al., 2004; Pfarr et al., 2006; Tachu et al., 2007), which may be due to the fact that B. malayi exhibits a gene with similarity to rsd-3 of C. elegans (Table. 4.2, Table A) (Grishok, 2005; Tijsterman et al., 2004). RSD-3 is thought to participate in vesicle trafficking for exogenous dsRNA and contributing to the phenomenon of systemic RNAi of C. elegans (Tijsterman et al., 2004). Hence, RSD-3 might play the same role in filarial nematodes and make them more responsive to RNAi than other species.

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Regarding environmental RNA in parasitic nematodes, electroporation or soaking have been used in a variety of species with limited success (Geldhof et al. 2007) However, electroporation conditions require further optimization owing to the fact that many worms do not survive and importantly it did not work well in some animal parasitic species including N. brasiliensis. Moreover, it is not clear that uptake into cells, spreading into other tissues and thus systemic effects are observed (Lendner et al., 2008). Particle bombardment is an alternative approach for delivering nucleic acids into worms (Davis et al., 1999; Higazi et al., 2002; Jackstadt et al., 1999; Wilm et al., 1999), and interestingly Yuen et al. (2008) have used RNAi to knock down expression of beta-glucuronidase leading to developmental arrest of Drosophila via biolistic bombardment. Experiments in our laboratory have determined that this approach is generally harmful to N. brasiliensis L3s and adults (Ball, personal communication).

Microinjection for RNAi or transformation with DNA constructs is extensively used in C. elegans (Fire et al., 1998; Mello and Fire, 1995), and meanwhile it also has been successfully applied in S. stercoralis (Lok, 2007; Lok and Massey, 2002). Lok has injected free-living female S. stercoralis with dsRNA for unc-54, and demonstrated significant transcriptional knockdown of specific mRNA in the progeny (Lok, 2007). However, microinjection- mediated introduction of RNA was shown to be harmful to H. contortus larvae (Geldhof et al., 2006), probably due to high internal pressure, and this may be a general problem with many parasitic nematodes.

A search for homologues of genes encoding the core RNAi pathway proteins in N. brasiliensis has been made using genomic DNA sequences from the Wellcome Trust Sanger Institute database, which suggests that several essential proteins required for systemic RNAi are missing as in B. malayi and H. contortus (Table. 4.2, Table B). Winston et al. (2007) have shown that C. elegans SID-2 (CeSID-2) functions to transport environmental dsRNA into intestinal epithelial cells, and CeSID-1 subsequently operates to enable uptake of dsRNA into other cells. A mammalian SID-1 homologue, FLJ20174, has been discovered in the plasma membrane of human cells which could improve the uptake of siRNA, resulting in an increase of siRNA-mediated knockdown ability (Duxbury et al., 2005). Likewise, a schistosome SID- 1 homologue (SmSID-1) has recently been identified, which encodes a 1018 amino acid

153 protein having about 19% and 40% similarity with C. elegans and human SID-1 respectively (Krautz-Peterson et al., 2009). In N. brasiliensis, however, a SID-1 homologue has not yet been found, but surprisingly, a SID-2 homologue may exist. Two contigs were found from the N. brasiliensis genome database which, when translated, showed some conservation with the amino acid sequence of CeSID-2 (Fig. 4.18), suggesting that they may represent a homologue. Hence, it is possible that the lack of co-ordinately expressed RNA transport proteins is one reason why RNAi is not effective in most animal parasitic nematodes, including N. brasiliensis. Uptake of environmental RNA into tissues probably requires the transmembrane protein SID-2 or a protein of homologous function to operate in co-operation with another cellular transmembrane protein such as SID-1 to enable systemic RNAi in the parasite.

Intriguingly, some neurons of C. elegans appear to be refractory to RNAi (Kamath et al., 2001), which is likely due to the absence of SID-1 to transport dsRNA into the cells (Winston et al., 2002). Tavernarakis et al. (2000) and Johnson et al. (2005) have transformed C. elegans with constructs designed to produce an endogenous hairpin dsRNA, and successfully knocked down neuronally expressed genes, which were completely refractory to feeding or microinjection-mediated dsRNA interference. This has not been tested in parasitic nematodes, as the inability to complete the life cycle in vitro means that it would be very difficult to select transformants, and as mentioned previously, survival of microinjection procedures tends to be very low.

Genes for components of other processes involved in RNAi such as dsRNA processing, mRNA regulation and amplification seem to be quite well conserved in N. brasiliensis although a homologue of rde-4 appears to be missing not only from N. brasiliensis, but also from H. contortus and B. malayi (Table. 4.2, Table B). RDE-4 is an essential protein required for dsRNA recognition, complex formation with Dicer and generation of siRNAs (Parrish and Fire, 2001). The absence of a homologue of RDE-4 could therefore be a problem associated with inconsistent RNAi in parasitic nematodes, although it is possible that another protein with no clear homology performs a similar function. In general however, the most obvious feature is the lack of genes which encode proteins with known roles in uptake and spreading of dsRNA. This suggests that delivery of dsRNA/siRNA, either via an exogenous

154 route or via endogenous expression, is the primary obstacle to be overcome by workers attempting to develop systems for RNAi in parasitic nematodes.

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Chapter 5

Identification and characterization of N. brasiliensis VAH/ASP-Like proteins

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5.1. Introduction

Parasitic nematodes have evolved a range of strategies to invade hosts, while hosts have developed immune and other defence mechanisms against parasites. Secreted protein components of parasitic nematodes are major biologically active mediators involved in immunomodulation of invasion and parasitism in hosts. Recently, a large group of molecules have been identified which are homologous to the vespid venom Antigen 5 (Ag5), termed as vespid venom antigen homologues (VAH), and these proteins are proposed to play an important role in parasite-host interactions. The Ancylostoma caninum secreted protein-1 (AcASP-1) was the first of these proteins to be described, and was characterized as the major component of ES products from serum-activated A. caninum L3 (Hawdon et al., 1996). The nematode VAH/ASP-Like (VAL) molecules show characteristics of a large protein family, the sperm-coating protein (SCP)-like extracellular protein family, also known as SCP/Tpx- 1/Ag5/PR-1/Sc7 (Pfam accession number no. PF00188). Proteins in this family have been identified in a wide variety of organisms throughout the eukaryotic kingdom (Cantacessi et al., 2009a).

AcASP-1 is a 42-kDa molecule, and has been described as a double-domain ASP which has two distinct but related SCP domains with homology to the pathogenesis-related (PR) protein superfamily. PR proteins were initially isolated from plants and are assumed to serve in defence-related signalling (van Loon and Van Strien, 1999). This group of proteins is classified into 17 subfamilies (van Loon et al., 2006), and AcASP-1 belongs to the PR-1 subfamily. PR-1 proteins were first discovered in 1970 and is a dominant group of PR proteins induced by pathogens. Importantly, a direct inhibitory effect of PR-1 members on fungal pathogens, Phytophthora infestans and Uromyces fabae has been shown in plants in vitro and in vivo (Niderman et al., 1995; Rauscher et al., 1999). The nuclear magnetic resonance (NMR) structure of PR-1b (P14a) from tomato was solved and found to represent a unique molecular architecture including four alpha-helices and four beta-strands arranged in an anti-parallel between helices. The tight packing of the alpha-helices on both sides of the central beta-sheet (α-β-α conformation) results in a compact, bipartite molecule core, which is stabilized by hydrophobic interactions and multiple hydrogen bonds (Fernandez et al., 1997). However, the biological function of PR-1 proteins still remains largely unexplored.

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Interestingly, the Sc7 and Sc14 genes from the fungus Schizophyllum commune are specifically expressed during fruiting and with sequence similarities to the PR-1 proteins. The Sc7 appears to be secreted and has been suggested to role in interactions between the dikaryotic hyphae which lead to formation of pseudo-parenchymous tissue (Schuren et al., 1993). Also, the PR-1 related sequences, YJL078c, YJL079c and YKR013w, have been found from the yeast Saccharomyces cerevisiae. YJL078c (Pry3p) is predicted as a glycosylphosphatidylinositol (GPI)-modified protein, representing the major class of cell wall proteins (Yin et al., 2005).

In animals, SCP-domain containing molecules present major components of the venom of a range of insects, such as yellow jackets, paper wasps, hornets and fire ants. The Antigen 5 (Ag5) proteins are the most abundant allergenic molecules within vespid venom and are frequently correlated with eliciting a strong allergenic response in people (Hoffman, 1985a, b). The X-ray crystal structure of Ag5 protein, Ves v 5 from the yellow jacket (Vespula vulgaris) shows hydrogen bonding patterns and hydrophobic interactions which define an α- β-α sandwich structure (Henriksen et al., 2001). Homologues of Ves v 5 are proposed to have the same conformation owing to the high degree of sequence similarity.

Immunogenicity has been shown in that Ag5 proteins result in allergenic reactions typified by increased specific IgE, IgG response and T cell proliferation. Strong immunological cross- reactivity of venom allergens among vespids is also observed (Bohle et al., 2005; King and Lu, 1997; Lu et al., 1993). However, interestingly, the fire ant (Solenopsis invicta) venom allergen, Ag3 (Sol i 3) protein has been identified with no immunological cross-reactivity to the vespid Ag5 and S. invicta Ag3, which share 44 to 50% sequence identity (Hoffman, 1993a, b). The overall structure of Sol i 3 presenting in an α-β-α figuration is very similar to Ves v 5, with major differences in the solvent-exposed loop areas, which may therefore explain the observed lack of relevant cross-reactivity (Padavattan et al., 2008).

Furthermore, two Antigen 5-related genes, named Agr and Agr2 encoding an SCP-domain have been discovered in Drosophila melanogaster respectively (Megraw et al., 1998; Schreiber et al., 1997). Transcriptional levels of Agr and Agr2 are equal in adults of both sexes, and the protein is expressed specifically within the gut. Accordingly, Agr proteins are

158 hypothesized to act as an antipathogenic protein or a novel protease inhibitor (Kovalick and Griffin, 2005). Also, in the tsetse fly (Glossina morsitans morsitans), the vector of trypanosomes, Tsetse Antigen 5 (TAg5), a member of containing SCP-domain proteins has been shown to be expressed primarily in salivary glands and could also be detected in midgut and proventriculus tissue. The sequence has extensive similarities to Drosophila Agr and Agr2, and investigators therefore suggested that TAg5 molecule possibly has a role in mediation of immune responses in the gut of the tsetse fly (Li et al., 2001). More recently, Caljon et al. (2009) observed a significant functional analysis of TAg5 by using TAg5 specific RNAi, which successfully reduced IgE binding in vivo, and also illustrated that TAg5 has the ability to elicit IgE response in mice.

The mammalian cysteine rich secretory proteins (CRISPs) were first identified from rat epididymal fluid, and were characterized as two-domain proteins comprising the structurally conserved SCP domain in the N-terminus, and a cysteine-rich domain (CRD) in the C- terminal sequence. Importantly, CRISPs have been shown to be abundantly expressed in the male reproductive tract of many mammalian species, and have received much attention as potential determinants of male fertility (Gibbs et al., 2008). Crisp-3, one of the mammalian CRISPs, has a broader expression distribution than others. It has been observed in the secretions from several exocrine glands onto mucosal surfaces (Haendler et al., 1993; Kratzschmar et al., 1996), and also within B cells, neutrophils, and eosinophils of the immune system (Udby et al., 2002a; Udby et al., 2002b). Interestingly, within human plasma, presumably as a product of neutrophil and eosinophil secretory granules, CRISP-3 has a high interaction with α-1B-glycoprotein (α1BG). This interaction is not metal ion dependent and has been suggested to inactivate the potentially detrimental effect of CRISP-3 (Gibbs et al., 2008).

Another SCP-domain containing molecule in humans, glioma pathogenesis-related protein (GliPR), also called related to testis specific, vespid and pathogenesis related-1 protein (RTVP-1) has been discovered in the major up-regulated genes of human brain cancer, glioblastoma multiforme/astrocytoma, and glioma cell lines. However, notably, its expression was not detected in any normal tissue and also normal brain tissue of human, which suggests that the function of GliPR/RTVP-1 may be linked to the human immune response to

159 pathogens (Murphy et al., 1995; Rich et al., 1996). A SCP homologue protein was found from cultured human glioblastoma cells as a novel 25-kDa trypsin binding protein (P25TI, also known as PI15) with weak protease inhibitory activity, and is additionally providing indirect support for a role in cancer pathogenesis (Yamakawa et al., 1998). A P25TI homologue, late gestation lung 1 (lgl1) was identified during the screen for glucocorticoide- induced transcripts in foetal rat lungs (Kaplan et al., 1999). Studies of lgl1 knockdown rat and lgl1 knockout mice have demonstrated that secreted LGL1 is important to morphogenesis of lungs and kidneys respectively (Oyewumi et al., 2003; Quinlan et al., 2007).

The C9orf19/Golgi-associated pathogenesis related-1 (GAPR-1) gene has been shown to have a high expression level in human leukocytes, lung, spleen and embryonic tissues (Eberle et al., 2002; Eisenberg et al., 2002). C9orf19/GAPR-1 protein was found to have SCP- domain and structurally homology to the P14a and Ves v 5 proteins (Serrano et al., 2004). Accordingly, with its abundance in immune environments, C9orf19/GAPR-1 may represent an evolutionary association with plant and mammalian immune regulation.

CRISP like proteins have been identified and characterized from snake venom. Helothermine was the first one that has been isolated and identified from the venom of the Mexican beaded lizard Heloderma horridum (Mochca-Morales et al., 1990), and which acts by blocking potassium channel and ryanodine receptors from rodents (Morrissette et al., 1995; Nobile et al., 1996; Olson et al., 2001). Ablomin, triflin and latisemin are toxins from the Japanese mamushi snake, habu snake and erabu sea snake venom respectively, and share about 50% sequence identity with mammalian CRISPs. The biological function of these toxins is to block the depolarization of the smooth muscle contraction induced by high concentrations of K+ (Yamazaki et al., 2002b). In addition, two other venom CRISPs named pseudechetoxin (PsTx) and pseudecin in the venom of the Australian King Brown snake and Red-bellied Black snake respectively, have been asociated with cyclic nucleotide-gated (CNG) ion channel-blocking activity (Brown et al., 1999b; Yamazaki et al., 2002a). Interestingly, a venom CRISP, Tex31 was discovered from the cone snail Conus textile and firstly shown to possess a substrate-specific protease activity (Milne et al., 2003). Also, Shikamoto et al. (2005) have revealed that the triflin tertiary structure has an α-β-α conformation core in the SCP domain, and a similar structure to the voltage-gated potassium channel blocker BgK and

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ShK in the CRD, which may provide useful information for structural and functional studies.

Proteins with SCP domains commonly exist in helminths. The SCP-like extracellular protein- 1 (SCL-1) has been identified from the free-living nematode C. elegans, and is a component of the DAF-2/insulin-like signalling pathway which acts in the regulation of longevity and stress resistance (Ookuma et al., 2003). The other gene, lon-1, encodes a protein with PR-1 homology that is a downstream regulator of TGF-beta (DBL-1) signalling of C. elegans, with roles in body morphogenesis. Knockdown of the gene via RNAi or selected mutants in lon-1 result in a long-bodied phenotype (25% longer) of the worm (Maduzia et al., 2002; Morita et al., 2002). A comprehensive study of SCP-domain containing proteins in the trematode parasite Schistosoma mansoni has been performed by Chalmers et al. (2008), who isolated 13 venom allergen-like proteins (SmVAL-1 to SmVAL-13), of which 12 have a single SCP domain and one (SmVAL-11) a double SCP domain. Transcriptional analysis of SmVALs in different stages of the parasite show a significant variation in profiles, and it has been suggested that several of these proteins may be involved in host invasion and parasitism, although to date there has been no functional analysis.

5.2. Identification of VALs from N. brasiliensis

Amino acid sequence from Ancylostoma caninum secreted protein 1 (AcASP-1) was used as the query sequence to search N. brasiliensis EST databases (NEMBASE and Nematode.net) (Parkinson et al., 2004; Wylie et al., 2004) for possible VALs. Eight distinct EST sequences were found and termed NbVAL-1 (BU493443), NbVAL-2 (BM279311), NbVAL-3 (BU493531), NbVAL-4 (BM278858), NbVAL-5 (BM279479), NbVAL-6 (BM279442), NbVAL-7 (BU493574) and NbVAL-8 (NB00394). Before isolation of full-length sequences of NbVALs, primers were prepared to amplify about a fragment of about 250 bp from each candidate, and transcripts were analyzed from different stages of N. brasiliensis life cycle by RT-PCR. This analysis showed that the NbVALs were all detected in adult stages, but NbVAL-3 and NbVAL-8 in particular were abundantly expressed in activated L3 (L3A) parasites (Fig. 5.1).

161

The phagemid clones (A2H11, O7H03 and O7C07) and bacterial clones (D3H11, D4F03 and aaa36h06/aaa31d09) corresponding to EST BM278858, BM279479, BM279442, BU493531, BU493574, NB00394 were acquired from the Parasitic Nematode EST Project (Prof. Maizels, University of Edinburgh and Dr. Mitreva, Washington University). The NbVAL-4, NbVAL-5 and NbVAL-6 cDNAs were excised from A2H11, O7H03 and O7C07 phagemids, and PCR analysis of these clones showed that the size of inserts was approximately 300 bp (Fig. 5.2). DNA sequencing of the EST clones, and/or the rapid amplification of 5’/3’ cDNA ends (5’ and 3’-RACE) methods were used to obtain the full length cDNA sequence of NbVAL-3, 4, 5, 6, 7 and 8, and the full length cDNA sequence of NbVAL-1 and 2 was completed by the Maizels Lab. The full length NbVAL-1 cDNA was 684 bp and encoded an open reading frame (ORF) of 227 amino acids; NbVAL-2 cDNA was 1335 bp and encoded an ORF of 444 amino acids; NbVAL-3 cDNA was 1365 bp and encoded an ORF of 454 amino acids; NbVAL-4 was 648 bp and encoded an ORF of 215 amino acids; NbVAL-5 cDNA was 645 bp and encoded an ORF of 214 amino acids; NbVAL-6 cDNA was 678 bp and encoded an ORF of 225 amino acids; NbVAL-7 cDNA was 1302 bp and encoded an ORF of 433 amino acids; NbVAL-8 cDNA was 1365 bp and encoded an ORF of 454 amino acids (Fig. 5.3). In addition, the polyadenylation signal, “AATAAA” was identified 10-15 bp upstream of the 3’ poly-A tail in VAL-1, VAL-2, VAL-3, VAL-5, VAL-6 and VAL-7 cDNA (Fig. 5.3).

5.3. Amino acid sequence analysis of N. brasiliensis VALs

Database searches revealed that the deduced amino acid sequences of N. brasiliensis VALs belonged to the PR-1 superfamily. NbVAL-2, NbVAL-3, NbVAL-7 and NbVAL-8 were double SCP domain proteins with predicted molecular masses estimated at 46.7, 51.7, 47.7 and 51.4 kDa, with theoretical isoelectric points (pI) of 8.75, 6.74, 6.16 and 9.67 respectively. NbVAL-1, NbVAL-4, NbVAL-5, and NbVAL-6 were single SCP domain molecules with predicted molecular masses of 25.4, 23.2, 23.4 and 25.4 kDa and pIs of 8.24, 9.01, 8.61 and 5.31 respectively. Whilst both double SCP domain and single SCP domain NbVALs have a consistent number and positioning of cysteine residues, about 20 cysteines were found in the double SCP domain VALs, and 10 in single the SCP domain proteins (Fig. 5.4). A putative hydrophobic signal peptide sequence was identified in all eight NbVAL ORFs (SignalP

162 programme, Fig. 5.4) (Nielsen et al., 1997). Four putative N-linked glycosylation sites (Asn- X-Thr/Ser) were found in VAL-2, between amino acids 16-18, 134-136, 194-196 and 261- 263. Also, three and two putative N-linked glycosylation sites were identified in VAL-8 (between amino acids 11-13, 29-31 and 108-110) and VAL-3 (between amino acids 213-215 and 435-437) respectively (Fig. 5.4, Panel A).

Sequence pair distance analysis indicates that of the double SCP domain NbVALs, VAL-2 shows 28% identity in its amino acid sequence to VAL-3, 25% identity to VAL-7 and only 24% identity to VAL-8; VAL-3 presents about 33% and 25% sequence identities to VAL-7 and VAL-8 respectively, whilst VAL-7 has 24% identity to VAL-8 (Table. 5.1, Panel A). The single SCP domain NbVALs exhibit a similar degree of sequence identity, but VAL-1 is particularly similar to VAL-6 with 59% identity (Table. 5.1, Panel B). This analysis appears to show that the coding segments of either double domain or single domain NbVALs present a family of related sequences which are quite distinct from each other in primary structure.

Alignment of two of the NbVAL protein sequences, NbVAL-1 from the single SCP domain VALs and NbVAL-3 from the double SCP domain VALs, with four SCP domain-containing proteins for which the structure had been determined, human GAPR-1 (Serrano et al., 2004), tomato P14a (Fernandez et al., 1997), snake venom triflin (Shikamoto et al., 2005) and vespid Ves v 5 (Henriksen et al., 2001), revealed areas of similarity within the sequences (Fig. 5.5). NbVAL-1 and NbVAL-3 have conserved cysteine residues within β strand 3 and β strand 4 of P14a, triflin and Ves v 5 which form the central β sheet of the structures. In addition, the sequences contained two conserved histidine residues (H79 and H134 in triflin) and acidic amino acids (normally glutamatic acid E94 and E115 in triflin), with the exception of NbVAL-1 and NbVAL-3 N-terminus (NbVAL-3N, Fig. 5.5). Comparison of the N. brasiliensis VAL sequences with sequences available on the Structural Database of Allergenic Proteins (SDAP) (http://fermi.utmb.edu/SDAP/sdap_fas.html), suggested clustering with allergens of the Pfam A PF00188 family that includes insect SCP domain- containing allergens such as Ves v 5. However, the nematode VALs did not appear to specifically cluster with any SCP domain families (data not shown).

163

Fig. 5.1. Expression of the N. brasiliensis VALs in different stages of the parasite life cycle

Total RNA from N. brasiliensis parasite stages, non-activated L3 (L3), activated L3 (L3A), L4 and adult were used to amplify the NbVAL products by RT-PCR. Primers for actin were used as a positive control to determine relative transcript abundance.

164

Fig. 5.2. Excission of the clones containing the N. brasiliensis VAL cDNAs

Phagemids containing the insert sequences were generated from the EST clones and subjected to PCR analysis using gene specific primers and resolved on a 1% agarose gel. Lane 1: clone A2H11 (BM278858); lane 2: clone O7H03 (BM279479) and lane 3: clone O7C07 (BM279442). Molecular weight markers are indicated in base pairs.

165

NbVAL-1

ATGCGCTCTCTGTATCTTCTCAGTGTGATGTTCATTGGGGCGGCAGCCTCGCCGGCGGTGATTAATAACATGTGCGCCAAGTACCCTGACGCT ACACTAGATGATAATACTAGGACAACATTCACGGAAGGGCACAACCGTTTCAGAACAGCTGTAGCAAACGGCTATGCTTATGTGGGACAGTAC GGTTTCAGTCAGTACGCCGAAGATATGGAAGCTATGGAGTGGTCGTGTCCCGATGAAGCAAAGGCGATTGAACTCGCAAAAGCAAAATGTACT GCTCAACCAACTGCGCCAAAAGGATTCAGAATGAACACCGCTTTGGTGGACAAGGCAGGAAAGACTTCTGATAAAGCCATTGAAGAGGCACTA GGAAAATGGAGAAATGAAATAATGCGTCATGGAATGCCCGAGGATCTGGTCTTCACCGACATTTGGAAGAATAAGATTGGTCATGCCACCAAG ATGATCTGGCACAGCAGCAGCAAACTCGCTTGTGCGTACGCTGATTGCAAGGACAAATACTCGATCGTCTGTCTCTACTCACCAAAAAGTAAC ATGATCGGCAAACCGATCTACAAGAAGGCTGACGAATGGGCGTTTATCTGCACCGGCTGCCCTAAAGACAAGAAATGCGGCGACGAACTTGAC AAGCTCTGCTACCCTCCGTGGGCCCTCCCCTAGACGAACATCGCACAGTAACCGATAGCAAGCGTGAATCATTCGCAAAGGATAGCTTCGAAC TGTTTTTTTAGAAATAAAGATTATCCGTGGCATATCCCTGTTAGGTTGGAAATTAATTCCAGGGATTGCTTTGCAGTCATTATTTTTAGCGAA CACAACTTGGATGAACGTTTACCAATTTC

NbVAL-2

ATGGCAGTTTTGAGGGTTCAACAAGGATCAATACAAAGTTCTAAAAATAGTTCATTGCAGAGCTTATTATTGGCTGTTCGGCTGCTTCCTTCT GCAGCTCAAGATACCCTTTGGGGGTTCTCAAATAATGACGACACAAACAGAAACACATTTTTGACAACGTTGAACGCAGCACGGAATAACACT GCACTTGGATATATCCCGATAGCGAATGGGAGAGGATATCTGCCAACAACATCTAATATGAACCTTTTGGAGTACGACTGCTTCCTCGAAGCG AAGGCGTATCAACAAGTTCAAGGGTGTGCAAAGGCGGAAACTGATGATTCGGTATACTTCGTATTAGACGCTACTCAATCGGGGAAATGTGAT ATACTGGCTGAAATAGCGAGCGTTGTGAACAAGAGCTTTTATGATTCGGGTTTACAAATTGACACCAACCCACCGAGGTTCTGGAAAGCAGAA CAAGCAGATTTTGCAAAGATAGCCAATGCAGACGCCGGTCTGGTGGGATGTTTCCAGAAATACTGCGGCAATGATCTCCATATCTCGTGTTTC TTTGATAAAGGGCTAGCGATAAACAGCACGGATAGGACGAAGGCGACCGTAATTTACGCACCAGGAACAAAAGCGCTCTGTGACGCTACGGAA GGCAGTACTGCCGATTCAACTGGTGGCAACACAGGCTTGTGCATCCGTGGAGCCCCGAAACCCGCCACTACAACATCCTCAACAACAACAACA ACAACGACGACGACGACGACAACAAGATCTTGTCCAAATATAACTGGAAGCTCTAACGACTACATGAGAAATTACTTCCTCAATCAGCACAAC CAATACAGATCCAGCATCGCTCAGGGAAACGTATATAATAAGGTAACAGGAACGAACGTCAGGCAAGCAAGTAGGATGAGCAGAATGACATAC AGCTGTACTTTGGAACAAAATGCGTATGATTGGGCCAGACAGTGTATTAACAAAAAGTCCCCTGGTTCGGATAGATACGAAAACATCTACGCT CACACTGACACTTCACTAGCAAGAAGAGAGGTGGCACGTTTGGCGATGGAACGATGGTGGAGACAGATAACGAACAATGGTCTTCCCGCGAAC CACATATTTGTAGACAAAAAGGGAACCAATATGCTCTCCAAGATTATTTACGACGAAGGACGAAGGGTTGGTTGCTCCGTGGTTAAGTGCTCT ACATTCACCATCGCAGTGTGTCGCTATGATCCAGCTGGGCCAGCCTTCAACACGCAGTACTACTTGCTAGGAGCGGCATGTAACCGTTGCGAC GGAAGGCCTTGTTCGGCGGGACTTTGCGGTTAGGACTGATTCACGACGGAATGTGACAGTGGCGCGAAGAACTTTTGTCCCATTTCATACTGC ATACTGACACAATGTAACTTTCAAAAAGCAATCTTATTCGACGTTTATTCGTTTTTTTTTACTGGATTGTAGAAAGCTATGGGAATAAAACGG CGCTCTGCAAAAAAAAAAAAAAAAAAAAAAAA

NbVAL-3

ATGCGGTGGTTGGTTGTGCTTCTCTACCTAACTCCGCTGGTAGCCGCTATCCAGTCGAAATTCAACTGTCCTAGCGACCTCGGTCAGCAAGAT GACACACGAGAAGAATTCCTCAAGCGGCACAACAACATTCGCCAAAATATCGCTCTAGGAATGCTTGCTGTGAACTACTTCTCGTACTTCTTG GGACCGGCGAAAAACATGTACCAGTTGGAATGGAGCTGCGATCTCGAGAGGATCGCAAAAGCGACCATCAATCAGTGCCAATTCAAAAACGTC AGCACCAAATACGTTTATAACACTAACAAATTCTGGCACTATGGGTTCGAAAACGTTAGAGCAATCGAAGAAGGAGTATACCAATGGGTAGAG CCAATCCACATGAACGATTTTGGGCCAAAATTCGAGTGCCACGACAAAATTCGCGAGTTCTGCAATATGGCGTATCCGACGAACACCAGGCTG GGCTGCGCTTATGAACGATGTGGAAGTGAACTGCTGATATCCTGCGCCTACGAGGAAGGAAAGAAAGTGCCTTTCAAACCACTACTTGAATTG GCACCACACTTTTGTGTGTGCAAGCATTATCCGAACTCGGCTTGTGACGAAGACTATCTGTGTAGGGCGACATCGTTAAACACAACAATCGAA CCATTGGGCCCAGCAACCATAAGAACCACCACAATCACTCGCGCTACTACAACAACCACCGCACCGAAACTGGATGTCCCAAAAGCATCATGC CCCACTTTGAATAACAAGATGACAGATGAGATTAGGCAAATCTTCCTCGACACACACAACAAATGGAGGTCGCTTGTTGCCAGAGGAAAGGCG GAAGATAAGTTATCTCCCGGTGGTTATGCTCCGAAAGCTTCTCGAATGCCTAAACTGTGGTACGACTGCAAACTCGAAGAATCCTCGAAGCAA GTGGCTGAGAAGTGCGCAAATAAACACAGCTATGGGGATTATGGAGAGAATCTATTCGCGGCTTCATTCTCGGGATATGATCAGGATGAGGTG GCAAAAATGGCCGTGGACCAATGGGCTGGTGAGCTGAAGAAGTTTGGAGTGGGCGATAATCTCATATACCCAACACCAGGTCTTACAGGTCAT TACACTCAGGTTGTGTGGCAGAATACCACTAGAGTTGGATGCTATATCAATGACGAGTGCTTCCCAGACAGTGCAGGGTGGAAGACCCAAGTG GTCTGCCAATACAAACCAACAGGAAACTTTATGCGCTGGGAAGTGTACTCGAAAGGAGAACCGTGCAAGAACGACTCCGACTGCAAATGTAAG AATTGCTCCTGTCTCGCTGAAGAGGGACTCTGCTACGATCCCAACTCCATGGCTTACATTTAAATTGTTAATGTTTGACGGAATAAACTTCCA GAACCTTCAAAAAAAAAAAAGGGCGGCCGCTC

NbVAL-4

ATGTTGCTTCCTGCAGTGACACTCGTGCTAACTGGCCTCGTCGTATCCGCCGCTGCTGGCGCGTGTCCTAAGACGGAGGGCTCACAGATGACA GATGCTTTCCGGAAGAAATTCCTAGACGAGCACAATCGTTTGAGATCATTGGTAGCCAACGGCAGGGCGAAGGACAAGGATGGAAGTTACGTT CCTAAGGCGGCTAATATGCTGAAACTGAGGTACGACTGCTCGCTTGAACGATTGGCGGCTGAACATGTGAATCAATGCAAGTTGGCGCCCTCT AAAGCTAGTGGAGCGCGAGCTGCGCTTGGAGAAAGTTTGATTAGGTCCACAGGACGCACTCTTGCTGACCAGGCTGATGTGACGACTGGACTG TGGTTCCTCGATCTCGAGAAGAATGGCATGGGCAGAAACCTGACATTTGGAGCACAGCCGGACGTGTATAAAATCGGCCACTACACTCAGATA GTGTGGGCAACGACAAGAACTATTGGATGCGCGGTTAAGGAGTGTGAAGGTAGTACCATGGCTGTGTGTAATTACCGACCAACAGGAAACATT CTGGGTATGATGGTCTACAAGGAAGGAGAACCGTGCTCAAAATGCCCTAAGGGTGCCATGTGTGAGAACGGTCTGTGTGTTGTCAGGTGAATT GCGGACATGCGAAGGATCGATCTACAATAGTTTGTATAGTCGTGATTTCGCAACGATTTTACAATGCTTCTAAAAATACATCATAGCAAAAAA AAAAAAAAAAAA

166

NbVAL-5

ATGTTCGCCATACTATTGCTAACGCTGCTGAGCCGTGAAGCCAGCTCCAACGCCAGATTTGCTCGGCAGATCGACTCTACTATTGGAGCGCAT GTCGCAGCATGGCACAACTATTTTAGGTCCCAGGTCGCCAGCGGTAATGTTGTCAACGGAAGAACGGGTTCTAATTGCCCTCAGGGCTCGAAT CTGAACCAAATGGTCTACGACGGACTACTGGCTGCTGACGCTCAGAACTACGCCGACACGTGCCCTAACAACGAACAGGGGTCACCGGTTAGT GGCCGACCCAATCAGGGTGAAAATGTGATGATTTACTACACCACCACGCTCAACACCGAATACGCTCTGTTTGGGGCCATACAAAGATGGTGG GACGATATAGCTGTCAACGGTGTCAACCGAAGAATGATTTTCAGACAATTCCTCGCGGATAAAGCACTACCGCCTGTTCGATTCACCCAGATG GCTTGGGCGTTGACGTATCGTGTTGGCTGTGCCGTTGCAGTGTGCCAGGGACGAACGGTCGTCGTCTGCCGCTATTCTCCACGCGGCAACATC GTTGATGAGTCAATATATAGATTAGGCTCTCCGTGCAGCGGTTGTACAACAAGCTGCGTCGGTGGCACACTCTGCTATATGAGCTAATGTTGA GGAGAATATTGACGGATCCAGAGCTGCGTCAGGATTTTTAAAATTTTTAAAAAAATTTCGGGCATATTTTTTAGAATAAACTATTCTATGGGA AAAAAAAAAAAAAAAAAAAAAAAA

NbVAL-6

ATGCGTTCTCTGTATCTTCTCAGTGTGATGTTTATGGGCGCGGCCGCTACGCCGATTAGCCCAGTTTCCATGTGTAAGGAAGCAGATGCCACT CTCTCCGACTTTGACAGAACCCAGTTCTTGGAGCTGCACAACAAATTCAGAGCGGCTGCAGCTAACGGCTTCGCCTACGTAGGCAAATTCGGC TACAGCCAGTATGCCCAAAACATGGAAGCGATGGAGTGGAGGTGCCCGCATGAAGGGAAGGCGATTAAGCTCGCTGAAACGTGCAAGAAGCCC AAAACACCAGCAGGATTCGGAATGAACAGCATCTTTGTGGACAAGGCAGGAAACAAGATCATTAGTGAGGCAATTGAAGAGGCACTGGGAAAA TGGAGAAATGACTTGAAGAAGATGGACCTGCCTGAAGATATGACATTCACGAAAGATATGGAGTCGACAATCGAACGGGCGACTAAGATGCTA TGGCACAACACCGTCCACCTCGCATGTGGTTATGCTGATTGCACGGACAAATACTCGATCGTCTGCCTCTACTCGCCAACTGGTAACAAAGTC GGCGAACAAATTTACGAGCCAACCGACGACTGGAACTTCATCTGCGAGAACTGCCCCAACAGCAAAAACTGTGACCTGGAGTACGACAAGCTC TGCTATCCTGATTGGGCACTCCCATGAACATCATACATCAATTGATACCAGATGTGAATCATTCGCAAGCGATGTCATCAACTTTTCGCAAAT AAAGACTGAATGGCAGTGAAAAAAAAAAAAAAAAAAAAAAAAAAAA

NbVAL-7

ATGGCTCCACCGGGACGTGTTCCGTTGATAGTTGTTATCGTCTTCAACGTTGCTGTTGGTCATGAATACCACTGCAATAAGGACCTCATCATG AACGACGATCTGAGATTTAACATGCTTAACTACCACAACGACATTCGTACCAATATTGCCTGGGGAGCACAGGACTGGCCTACTGGACAAGGA CCGGCGAAAAATATGTACCGTGTGGGATGGGACTGCAAACTCGAAGAGGAAGCCGCCAAATCCCTCCAGCAATGCAGTGAAGCGGAGTACACT GCCGCTCCGAACAACTTTGCAGTGCTGGACGTTATTTCGAAACCGCGCAACAAATGGCCAGCGACGAACTTCGCCCTACACAGAGCCCTTAAT TCATGGTACAAGCCATCAATTGGTTTCGACCAAGGCAGCACACTTGCGGACGAGAAACTTGGCGCTTTTGGCAACATGATCAACTACAAAGCA GTACGACTCGGCTGCTCCTACAAAGTTTGCGGCGACAAGGTCCATGTGCGCTGCATCTACAACGACGGGGCCAACAAAGTCGGTGATACTCTG TACGAGGACGGCTGGGTTTGCTATTCTGACAGTGACTGCACTACCTACGAGAACTCCAGATGCTCTGGAGGTCTTTGTGAGGCGCCTGATAAT CTCGTTCTGAGAACGACGACCACTACAACGACCACAACCGCAAATCCGTCCGCAGGTGGAGATGGTAACTCGCAGTGCGCAAATAATCCTGAA ATGACGGACGAAGCTAGGAAAATGCTCACCGACAAACACAACCAACTAAGAGCACAAACCGCGAAGGGCTTGTCTGTAGACCCGAAATCTGCA ACAGGATTCGCTCCCAAAGGTTCAGCGATGAAGAAAATGAAATACGACTGTGAGATCGAGGCCAGCGCACAGGCGTACACAAATCTGTGCAAA GGACTGCAACACTCTTGGGGACAGTACGGAAAAAATATCTGGATGATCTTCGCAAAAAACTACAACAGAAAAGATGTGGCGGATTGGCCCCCT CATTCATGGTTTGATGAACTGAAGGAGTACGGTGTCGGTGAGAAAAATATATTCAACGCATCGATGATGAACGTGGGTCACTACACTCAGGTG GTTTGGGGCGATACCGACAGATTCGGCTGTGGATTTAAGTCCTGCGCAGGATCAGGATACACCGCTCTCATTTGCCAGTACGCACCACCAGGC AATTGGCTTGATTCGACCATCTATAAAGTAGGAGAGCCATGCTCTGCCTGTCCCGTCGGAACTAAGTGTGAAGACGGCGCCCTCTGTGCCTGA ATATGCTAAGGAGGTCCAACAAGAGATATGAGTCAAAATATAATAAAAACTTCTCATATAAAAAAAAAAAAAAAAAAAAAAAAA

NbVAL-8

ATGCCCCGTCTGTGGTTCGTTTATCAACGAAATTTTTCGCTGTTCTCGCTGGCGATACTTGCGGTTGCCCTCGTAAGTCGAACGAATGGAACT CAAAATTTCAACTGTCAAAACAGCGCGAACAGCGAAATGACCGACGAGCTCAGAGCGTACTACCTCAATTTTCACAACAATCAACGACGGCGC TTGGCAAGAGGCAATTCACCGGCGCGATCCGGAAAATTTAATAAAGCGAAGAATATGTACAAGCTGGAATGGAGCTGCTCCATAGAAGAGCTG GCTAAACGTGCTGTTGCTAACTGCAACGCGGATTTAACGGAAAATCGTTCGTACGGACAAAATCTTGCGGGGTACACCTTTTGGTCCAGTGAT GGCAATGTACCAAGGAGTCACATCAAGATGAAGGAACTAATCAAGAAAACCCTCGAATCGTGGTTCAACCAAGCCAAGGAGCAAGGATGGAAA GACCCGAACAATAAATTCACGAATTCTGGCATTTACGCATTCGCTAATATGGCAAACTCGAAAACGACAGAGATTGGATGTGCTCACAATATT TGTAAAGGCAACACGAGAGTGCAAATTCTTTGCCTGTATAATCGAGTAGGCACTTTCACAGGAGCTAGCATTTATCGAACAGGAAAACCTTGC CAGAAAAACTCTGACTGCACTGTATTTCCTCAATCAACTTGCGATGGCCATGGATTGTGCATTAAGAAAAGAGAAAACCCTGACGATAAAACC AACATCATGTGTCCGCGCAGTTCTTCTACTATGATCGATAAGGTTCGACAAAAATTCCTCAATCTCCACAACCACTACAGATCACTTGTTGCC ACAGGAAGGACTTACGATAAGTTGCTTGGAAAGAACACCCCAAAGGCAGCAAAAATGCAAAAGCTGAAATACTCTTGTGCCCTTGAGGCTACC GCGTGGAGATATTCTCACAATGTCGATTTGCTCATTCCCATACCTGGAAAGAAAAACGCCGGGGAAAATCTTTATAAAAATTTATTCCTGTTC GATAAGTTGAAGGGGCCTCGATGTCGACTGAAGACTGGTTTGAAGAGGTCGAAGATTTTGGTATGGTGGAGCGGCCAATTTCTCCACGAGATT TTCAGATTCCGGCCACTACACGCAGATGTTTGGCAGGCCCCAACAACTTGGATGTGCAATCCAAGACTGCCCCGGTATGCATTAGTAGTCTGC CCTCAAAAAGGCGGGAAACTCATTGACGAGCCAGTTTATACTGCTGGAAGCCCATGTTCTCAATGCGGTAATAGTGAGCTTTGCAATCCTAAC GAAGGACTTTGCTCAGCAGTAGTGACATTTCCAGGAGAGGGGATCAAACCTGGCATGCAATAACCTTTTTTCCCAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAA

Fig. 5.3. Full-length cDNA sequence of the NbVALs.

167

Partial sequences were derived from EST databases, the clones resequenced and completed by rapid amplification of 5’/3’ cDNA ends (5’ and 3’-RACE). The original EST sequences are shaded in grey, start codons shown in blue and stop codons coloured red. Possible polyadenylation signal (AATAAA) sequences are underlined.

168

NbVAL-2 1 .MAVLRVQQGSIQSSKNSSLQSLLLAVRLLPSAAQDTLWGFSNNDDTNRNTFLTTLNAAR NbVAL-3 1 ...... MRWLVVLLYLTPLVAAIQSKFNCPSDLGQQDDTREEFLKRHNNIR NbVAL-7 1 ...... MAPPGRVPLIVVIVFNVAVGHEYHCNKDLIMNDDLRFNMLNYHNDIR NbVAL-8 1 MPRLWFVYQRNFSLFSLAILAVALVSRTNGTQNFNCQNSANSEMTDELRAYYLNFHNNQR

* * NbVAL-2 60 NNTALGYIPIANGRGYLPTTSNMNLLEYDCFLEAKAYQQVQGC...... AKAETDDSV..Y NbVAL-3 46 QNIALGMLAVNYFSYFLGPAKNMYQLEWSCDLERIAKATINQC...... QFKNVSTK.... NbVAL-7 48 TNIAWG...AQDWPTGQGPAKNMYRVGWDCKLEEEAAKSLQQC...... SEAEYTAAPNNF NbVAL-8 61 RRLARGNSPARSGK..FNKAKNMYKLEWSCSIEELAKRAVANCNADLT.ENRSYGQNLAGY

NbVAL-2 113 FVLDATQSGKCDILAEIASVVN..KSFYDSGLQIDTNPP...RFWKAEQADFAKIANADAG NbVAL-3 97 .YVYNTNKFWHYGFENVRAIEEGVYQWVEPIHMNDFGPK...FECHDKIREFCNMAYPTNT NbVAL-7 100 AVLDVISKPRNKWPATNFALHRALNSWYKPSIGFDQGST...L.ADEKLGAFGNMINYKAV NbVAL-8 119 TFWSSDGNVPRSHIKMKELIKKTLESWFNQAKEQGWKDPNN.KFTNSGIYAFANMANSKTT

* * * * NbVAL-2 169 LVGCFQKYC...GNDLHISCFFDKGLAINSTDRTKAT...VIYAPGTKALCDATEGSTADS NbVAL-3 154 RLGCAYERC...GSELLISCAYEEGKKVPFKPLLELAPHFCVCKHYPNSACDEDYLCRATS NbVAL-7 157 RLGCSYKVC...GDKVHVRCIYNDGANKVGDTLYEDG...WVC..YSDSDCTTYENSRCSG NbVAL-8 179 EIGCAHNICKG.NTRVQILCLYNRVGTFTGASIYRTG.....KPCQKNSDCTVFPQSTCDG

* NbVAL-2 224 TGGNTGLCIRGAPKPATTTSSTTTTTTTTTTTTR.S.CPNITGSSNDYMRNYFLNQHNQYR NbVAL-3 212 LNTTIEPLGPATIRTTTITRATTTTTAPKLDVPKAS.CPTLNNKMTDEIRQIFLDTHNKWR NbVAL-7 210 ..GLCEAPDNLVLRTTTTT..TTTTANPSAGGDGNSQC.ANNPEMTDEARKMLTDKHNQLR NbVAL-8 234 ....HGLCIKKRENPDDKTNIM...... CPRSSSTMIDKVRQKFLNLHNHYR

* NbVAL-2 283 SSIAQGNVYNKVTGTNVR..QASRMSRMTYSCTLEQNAYDWARQCINKKSPGSDRYENIYA NbVAL-3 272 SLVARGKAEDKLSPGGYA.PKASRMPKLWYDCKLEESSKQVAEKCAN.KHSYGDYGENLFA NbVAL-7 266 AQTAKGLSVDPKSATGFA.PKGSAMKKMKYDCEIEASAQAYTNLCKGLQHSWGQYGKNIWM NbVAL-8 276 SLVATGRTYDKLLGKN.T.PKAAKMQKLKYSCALEATAWRYSHNVDLLIPIPGKKNAGENL

* NbVAL-2 342 HTDTSLARREVARLAMERWWRQITNNGLPANHIFVDKKGT..NMLSKIIYDEGRRVGCSVV NbVAL-3 331 ASFSGYDQDEVAKMAVDQWAGELKKFGVGDNLIYPTPGLT..GHYTQVVWQNTTRVGCYIN NbVAL-7 326 IFAKNYNRKDVADWPPHSWFDELKEYGVGEKNIFNASMMNV.GHYTQVVWGDTDRFGCGFK NbVAL-8 335 YKNLFLFDKLKGPRCRLKTGLKRSKILVWWSGQFLHEIFRF.RPLHADVWQAPTTWMCNPR

* * * NbVAL-2 401 K...... CSTFTIAVCRYDPAGPAFNTQYYLLGAACNRCD.GRPCSAGLCG...... NbVAL-3 390 DECFPDSAGWKTQVVCQYKPTGNFMRWEVYSKGEPCKN.DSDCKCKNCSCLAEEGLCYDP. NbVAL-7 386 S.C...AGSGYTALICQYAPPGNWLDSTIYKVGEPCSACPVGTKCEDGALCA...... NbVAL-8 395 ...... LPRYALVVCPQK.GGKLIDEPVYTAGSPCSQCGNSELCNPNEGLCSAVVTFPG.

NbVAL-2 ...... NbVAL-3 449 NSMAYI.. NbVAL-7 ...... NbVAL-8 447 EGIKPGMQ

169

NbVAL-1 61 Q.YGFSQYAEDMEAMEWSCPDEAKAIELAKAKCTAQPTAPKGFRMN...TALVDKAG.KT NbVAL-6 60 K.FGYSQYAQNMEAMEWRCPHEGKAIKLAETC..KKPKTPAGFGMN...SIFVDKAGNKI NbVAL-4 58 D.GSYVPKAANMLKLRYDCSLERLAAEHVNQCKLAPSKASGARAAL...GESLIRSTGRT NbVAL-5 52 RTGSNCPQGSNLNQMVYDGLLAADAQNYADTCPNNEQGSPVSGRPNQGENVMIYYTTTLN

* * NbVAL-1 116 SDKAIEEALGKWRNEIMRHGMPEDLVF.TDIWKNKIG..HATKMIWHSSSKLACAYADCK NbVAL-6 114 ISEAIEEALGKWRNDLKKMDLPEDMTF.TKDMESTIE..RATKMLWHNTVHLACGYADCT NbVAL-4 114 LADQADVTTGLWFLDLEKNGMGRNLTFGAQPDVYKIG..HYTQIVWATTRTIGCAVKECE NbVAL-5 112 TEYALFGAIQRWWDDIAVNGVNRRMIFRQFLADKALPPVRFTQMAWALTYRVGCAVAVCQ

* * * * NbVAL-1 173 DKYSIVCLYSPKSNMIGKPIYKKADEWAFIC.TGCPKDKKCGDELDKLCYPPWALP NbVAL-6 171 DKYSIVCLYSPTGNKVGEQIYEPTDDWNFIC.ENCPNSKNCDLEYDKLCYPDWALP NbVAL-4 172 GSTMAVCNYRPTGNILGMMVYKEGEP....C.SKCPKGAMCENGLCVVR...... NbVAL-5 172 GRTVVVCRYSPRGNIVDESIYRLGSPCSG.CTTSCVGGTLCYMS......

Fig. 5.4. Alignment of NbVALs deduced amino acid sequences

Sequences were aligned using Multalin (Corpet, 1988) and prepared for display using BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). Panel A: Alignment of amino acid sequences of double-SCP-domain NbVALs; Panel B: Alignment of amino acid sequences of single-SCP-domain NbVALs. Identical amino acids are shaded in black, and similar amino acids in grey. Predicted signal peptides are shown in underline-italic, whilst potential N-linked glycosylation sites are boxed in blue. Cysteines are highlighted yellow. The cysteines conserved in NbVALs are indicated with an asterisk.

170

Table 5.1. Amino acid sequence identities of N. brasiliensis VALs

Panel A: The percentage identities/similarities of double SCP domain NbVALs. Panel B: The percentage identities/similarities of single SCP domain NbVALs. The data were obtained using the BLAST 2 Sequences server (Tatusova and Madden, 1999).

171

GAPR-1 1 ------MGKSAS P14a 1 ---MGLFN-ISLLLTCLMVLA------IFHSCEA Vesv5 1 MEISGLVY-LIIIVTIIDLPYGKANNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLT NbVAL-3C 1 ------LDVPKASCPT------LNNKMT Triflin 1 --MIAFIV-LPILAAVLQQSSGNVDFD------SESPRK NbVAL-3N 1 --MRWLVV-LLYLTPLVAAIQSKFNCP------SDLGQQ NbVAL-1 1 --MRSLYL-LSVMFIGAAASPAVINNMCAK------YPDATLD

helix 1 ß-strand 1 helix 2 ======>>>======GAPR-1 7 KQFHNEVLKAHNEYRQKHG------VPPLKLCKNLNR--EAQQYSEALASTRI P14a 25 QNSPQDYLAVHNDARAQVG------VGPMSWDANLAS--RAQNYANSRAGDCN Vesv5 61 KQEKQDILKEHNDFRQKIAR---GLETRGNPGPQPPAKNMKNLVWNDELAYVAQVWANQCQ NbVAL-3C 17 DEIRQIFLDTHNKWRSLVARGK-AEDKLSPGGYAPKASRMPKLWYDCKLEESSKQVAEKCA Triflin 31 PEIQNEIIDLHNSLRRSVN------PTASNMLKMEWYPEAAANAERWAYRCI NbVAL-3N 31 DDTREEFLKRHNNIRQNIALG--MLAVNYFSYFLGPAKNMYQLEWSCDLERIAKATINQCQ NbVAL-1 35 DNTRTTFTEGHNRFRTAVANG---YAYVGQYGFSQYAEDMEAMEWSCPDEAKAIELAKAKC

ß-strand 2 helix 3 >>>>>>> ======GAPR-1 52 LKHSPE----SSRGQCGENLAWA-SYD-Q----TGKEVADRWYSEIKNYN-FQQPGFTSG- P14a 70 LIHSG------AGENLAKG-GGD-F----TGRAAVQLWVSERPSYN-YATNQCVGGK Vesv5 119 YGHDTCRD--VAKYQVGQNVALTGST--AAKYDDPVKLVKMWEDEVKDYNPKKKFSGNDFL NbVAL-3C 77 NKHSYG------DYGENLFAA-SFSGYDQDEVAKMAVDQWAGELKKFGVGDNLIYPTPG Triflin 77 ESHSSRDSRVIGGIKCGENIYMA-TYP-----AKWTDIIHAWHGEYKDFK-YGVGAVPSDA NbVAL-3N 90 FKNVST------KYVYNTNKF-WHYGFENVRAIEEGVYQWVEPIHMNDFGPKFECHDK- NbVAL-1 93 TAQPTAP------KGFRMNTALV-DKAGKTSDKAIEEALGKWRNEIMRHGMPEDLVFTDIW

helix 4 ß-strand 3 ß-strand 4 ======>>>>>>>>>>>> >>>>>>>>> GAPR-1 101 ---TGHFTAMVWKNTKKMGVGK---ASASDG---SSFVVARYFPAGNVVNE--GFFEENVL P14a 114 K--CRHYTQVVWRNSVRLGCGR---ARCNNG---WWFISCNYDPVGNWIGQ--RPY----- Vesv5 176 K--TGHYTQMVWANTKEVGCGS---IKYIQEKWHKHYLVCNYGPSGNFMNE--ELYQTK-- NbVAL-3C 129 L--TGHYTQVVWQNTTRVGCYI-NDECFPDSAGWKTQVVCQYKPTGNFMRW--EVYSKGEP Triflin 131 V--IGHYTQIVWYKSYRAGCAA----AYCPSSKYSYFYVCQYCPAGNIIGKTATPYKSGPP NbVAL-3N 141 ---IREFCNMAYPTNTRLGCAY------ERCGSELLISCAYE-EGKKVPFKPLLELAPHF NbVAL-1 147 KNKIGHATKMIWHSSSKLACAY------ADCKDKYSIVCLYSPKSNMIGK--PIYKKADE

GAPR-1 151 P------PKK------P14a ------Vesv5 ------NbVAL-3C 185 C------KNDSDCKCKNCSCLAEEGLCYDPNSMAYI------Triflin 186 CGDCPSDCDNGLCTNPCTRENEFTNCDSLVQKSSCQDNYMKSKCPASCFCQNKII NbVAL-3N 191 CVCKHYPNSACDEDYLCRATSLNTTIEPLGPATIRTTTITRATTTTTAPK----- NbVAL-1 199 WA------FICTGCPKDKKCG-DELDKLCYPPWALP------

Fig. 5.5. Alignment of NbVALs with different SCP domain-containing proteins

Sequences of NbVAL-1 and NbVAL-3 N-terminus/C-terminus (NbVAL-3N/NbVAL-3C) were aligned with GAPR-1 (GenBank: Q9H4G4), P14a (GenBank: P04284), Ves v 5 (Vesv5;

172

GenBank: Q05110) and Triflin (GenBank: AF384219) using Multalin and prepared for display using BOXSHADE. Amino acids that exactly match are shaded black whilst residues corresponding to conservative substitutions are shaded grey. Residues predicted to form a putative metal biding site are coloured red, and a residue which has been proposed to form part of a putative catalytic triad along with His54 and Glu65 (in GAPR-1 numbering) is marked in green. The major conserved secondary structure elements of GAPR-1, P14a, Ves v 5 and Triflin are indicated above the relevant amino acid sequences.

173

The domain structure of A. caninum ASPs or CRISP family members generally comprises three domains: a signal peptide, a PR-1-like SCP domain and a cysteine-rich hinge (Gibbs et al., 2008; Zhan et al., 2003), which could be observed in the nematode SCP proteins (Fig. 5.6 and 5.7), and the double SCP domain nematode proteins simply consist of two units connected by the cysteine-rich region sequences of the N-terminal domain which is shown in Fig. 5.7. The amino acid sequence alignment of either single domain or double domain N. brasiliensis VALs with nematode VAL homologues indicated that the position of cysteine residues in these sequences was generally conserved (Fig. 5.6 and 5.7). Ten conserved cysteines were found in the primary sequence of nematode single domain SCP proteins, and 20 in the double-domain proteins (Fig. 5.6 and 5.7).

Henriksen et al. (2001) have previously identified seven consensus segments from SCP-like proteins, and the consensus amino acid residues were defined as: alcohol, o (S, T); aliphatic, l (I, L, V); aromatic, a (F, H, W, Y); charged, c (D, E, H, K, R); tiny, u (A, G, S); small, s (A, C, D, G, N, P, S, T, V); turnlike, t (A, C, D, E, G, H, K, N, Q, R, S, T); hydrophobic, h (A, C, F, G, H, I, K, L, M, R, T, V, W, Y); polar, p (C, D, E, H, K, N, Q, R, S, T), while the sequences conserved in all alignment analysis were given in capital letters. The sequence of both single and double domain SCP proteins from different nematode species are mostly consistent with this consensus, with some variability (Fig. 5.6 and 5.7).

The translated cDNA sequences corresponding to the mature NbVAL proteins show about 23%-33% identity and 38%-43% similarity to an A. caninum secretory SCP-related protein, hookworm platelet inhibitor (AcHPI), which is involved in the inhibition of platelet aggregation (Del Valle et al., 2003) (Fig. 5.8). Interestingly, the sequences LDV in NbVAL-3 C-terminus and NbVAL-7 N-terminus, and NGR in NbVAL-2 N-terminus, NbVAL-4 and NbVAL-5, are potential motifs for interaction with integrins (Fig. 5.8) (Ruoslahti, 1996). Furthermore, use of a comprehensive database of protein domain families, ProDom (Bru et al., 2005) showed that the NbVAL-3 N-terminal sequence, NbVAL-2 and NbVAL-8 C- terminal sequence were similar to A. caninum neutrophil inhibitory factor (AcNIF) (Moyle et al., 1994) with 24-25% sequence identity (Fig. 5.9). Importantly, a glutamic acid residue in AcNIF (Glu142) which is thought to be crucial for binding to CR3 (CD11b/CD18) (Rieu et al., 1996) is present in the NbVAL-3 N-terminal sequence (NbVAL-3N) (Glu124) (Fig. 5.9).

174

To explore the tertiary structural characteristics of NbVAL-3N and AcNIF, the ModWeb server (Marti-Renom et al., 2000) was used to create homology models, using the crystal structure of NaASP-2 (Asojo et al., 2005) as a template. These models showed a similar α-β- α sandwich structure as NaASP-2 (Fig. 5.10, Panel A). Also, they revealed that the conserved Glu142 of AcNIF and Glu124 of NbVAL-3 N-terminus are more exposed and aligned along the edge of a longer helix than Glu109 of NaASP-2 (Fig. 5.10, Panel B). This exposure and helical extension has been suggested to facilitate a tighter fit of AcNIF with CR3 integrin (Asojo et al., 2005), and accordingly it suggests that N. brasiliensis VAL-3 might be also an antagonistic ligand of the CR3 receptor or related integrins.

Three-dimensional homology models of the other NbVAL sequences were also constructed using the ModWeb server, and showed well conserved backbone constraints with the determined X-ray structure of NaASP-2 (Asojo et al., 2005), suggesting that the α-β-α sandwich conformation element was common to all N. brasiliensis VALs (data not shown). The residues (His129, Glu106, and Ser70) in NaASP-2 correspond to the putative serine protease catalytic triad in a novel serine protease, Tex31 from the cone snail Conus textile (Asojo et al., 2005; Milne et al., 2003), and which were dimensionally conserved in the NbVAL-3 C-terminus (His319, Glu326 and Ser332) (Fig. 5.11, panel A). However, importantly, comparing to a known serine protease (Fig. 5.11, panel B), the serine residue in either the NbVAL-3 C-terminus (Ser332) or the NaASP-2 (Ser70) is too far away (more than 12 Angstroms) from the histidine (His319 or His129) of catalytic triad to form a hydrogen bond. Furthermore, the human Golgi-associated plant pathogenesis related protein (GAPR-1) forms a dimer in solution, which proposed the histidine, glutamatic acid and serine residues that form the putative serine protease catalytic triad (Serrano et al., 2004) were orientationally conserved in the NbVAL-3 N and C-terminus (His319, Glu326 and Ser164) (Fig. 5.12). In addition, intriguingly, the residues (histidine, glutamatic acid) which make up the putative metal binding site in Trimeresurus flavovirids triflin (Shikamoto et al., 2005) and also Lycopersicon esculentum P14a (Szyperski et al., 1998), were only present on the surface within the cleft of the NbVAL-3 C-terminus (His319, Glu326, Glu352 and His372) (Fig. 5.13).

175

NbVAL-1 1 MRSLYLLSVMFIGAAASPAVINNMCAKYPDATLDDNTRTTFTEGHNRFRTAVANGYAYVG NbVAL-6 1 MRSLYLLSVMFMGAAATPISPVSMC.KEADATLSDFDRTQFLELHNKFRAAAANGFAYVG NbVAL-4 1 ...MLLPAVTLVLTGLVVSAAAGACPKTEGSQMTDAFRKKFLDEHNRLRSLVANGRAKDK AcASP-2 1 ...MLVLVPLLALLAVSVHGNSMRCGN.NG..MTDEARQKFLDVHNSYRSMVAKGQAKDA AdASP-2 1 ...MLVPVALLALLAVAVEGNSMRCGN.NG..MTDEARQEFLDVHNGYRSKVAKGQAKDA AyASP-2 1 .....VLVPLLVLLAVSVDANSVRCGN.NG..MTDEARQKFLDMHNGYRSQVAKGQAKDA NaASP-2 1 ...MMSSITCLVLLSIAAYSKA.GCPD.NG..MSEEARQKFLELHNSLRSSVALGQAKDG Hc24 1 ...MFSLATVAFLTLLSTSGHASMCPDTNG..MSDEVRQTFVNKHNAYRTLVAKGEAKNA OvASP-1 1 ...... MILFIIFPAIVVAVTGYNCPGGK...LTALERKKIVGQNNKYRSDLINGKLKNR BmVAL-1 1 ...... MLFFVIFSITIAVVAGFECPGGR...LTPQQRKDIVRQNNKFRSLLIHGKLKNR WbVAH 1 ...... MLLFVIFSATIVAVAAFECPGGQ...LTPQQRKDIVRQNNKFRSLLIRGKLKNR OvASP-2 1 ...... MILFLIFPAIIVAVTGYDCYRGK...LTPQYREKIVREHNRLRSKLAKGTYKNS NbVAL-5 1 ....MFAILLLTLLSREASSNARFARQ.....IDSTIGAHVAAWHNYFRSQVASGNVVNG SCL-1 1 ...... MYFILCLLAIGVNAQ...... FTSTGQSSIVDAHNKLRSAIAKSTYVAK AcASP-3 1 .MKSYLVISAAILGIAYADADYSKCPQNEI..MNNDMREKVTDMHNAYRSKFARDH.... lpxHNthRtxh Con1

NbVAL-1 61 ..QYGFSQYAEDMEAMEWSCPDEAKAIELAKAKCTAQP...... TAPKGFRMNTALVDKA NbVAL-6 60 ..KFGYSQYAQNMEAMEWRCPHEGKAIKLAETC..KKP...... KTPAGFGMNSIFVDKA NbVAL-4 58 ..DGSYVPKAANMLKLRYDCSLERLAAEHVNQCKLAPS.KASGARAALGESLIR...STG AcASP-2 55 ..ISGNAPKAAKMKKMIYDCNVESTAMQNAKKCVFAHS.H...RKG.VGENIWM...STA AdASP-2 55 ..LGGNAPKAAKMKKMIYDCNVESTAMQDAKKCVFAHS.H...MKG.LGENIYM...STA AyASP-2 53 ..LSGNAPKAAKMKKMVYDCGVESTAMQNAKKCVFTHS.H...MKG.LGENIWM...TTA NaASP-2 54 ..AGGNAPKAAKMKTMAYDCEVEKTAMNNAKQCVFKHS.QPNQRKG.LGENIFM...SSD Hc24 56 KEIGGYAPKAARMLKVTYDCAIEENTMNFAKKCVFAHN.SYSESNN.WGQNLYM...TSI OvASP-1 52 ..NGTYMPRGKNMLELTWDCKLESSAQRWANQCIFGHS.PRQQREG.VGENVYAYWSSVS BmVAL-1 52 ..NGTYMPRGKNMLLLKWSCQLENSAQRWANQCVFGHS.PRNQRQG.IGENVYAYWSSES WbVAH 52 ..NGTYMPRGKNMLQLTWSCQLENSAQRWANQCVFGHS.PRNQRQG.IGENVYAYWSSAS OvASP-2 52 ..AGKWMPKGKNMMEMKWDCELELMAQRWADQCVSGNS.PKDRRGR.IGENVYTQRSDTS NbVAL-5 52 R.TGSNCPQGSNLNQMVYDGLLAADAQNYADTCPNNEQGSPVSGRPNQGENVMI...YYT SCL-1 44 ...GTKKEPATDMRKMVVDSTVAASAQNYANTCPTGHS.....KGTGYGENLYWSWTSAD AcASP-3 54 ...... QASKMRKLVYDCAIEKGIYESDTKCEMKPS...... MEEENVEV....ID hxxhxWsxphttxAtxauppCxhxHs GpNlhh---t Con2 Con3 Con4

NbVAL-1 113 G.KTSDKAIEEALGKWRNEIMR.HGMPEDLVFTDIWKNK....IGHATKMIWHSSSKLAC NbVAL-6 110 GNKIISEAIEEALGKWRNDLKK.MDLPEDMTFTKDMEST....IERATKMLWHNTVHLAC NbVAL-4 112 RTL..ADQADVTTGLWFLDLEK.NGMGRNLTFGAQPDVY...KIGHYTQIVWATTRTIGC AcASP-2 105 RQMDKAQAAQQASDGWFSELAK.YGVGQENKLTTQLWNR.GVMIGHYTQMVWQESYKLGC AdASP-2 105 RQMDKAEAAQQASDGWFAELAK.YGVGQENKLTMQLWNR.GVMIGHYTQMVWQESYKLGC AyASP-2 103 REMDKVKSAEQASQGWFSELAE.YGVGPENKLTMQLWNRPNTQIGHYTQMVWQDTYKLGC NaASP-2 107 SGMDKAKAAEQASKAWFGELAE.KGVGQNLKLTGGLFSR...GVGHYTQMVWQETVKLGC Hc24 111 LNQNKTVAAAESVDLWFDELQQ.NGVPYDNVMTMAVFNR...GVGHYTQVVWQWSNKIGC OvASP-1 108 VEGLKKTAGTDAGKSWWSKLPKLYENNPSNNMTWKVAGQ...GVLHFTQMAWGKTYKIGC BmVAL-1 108 VEKLRNTAGTEAGKSWWSELPKLYKQNPSNNLTDDVARQ...GVLHFTQMAWGKTHKIGC WbVAH 108 VENLRKTAGTEAGKSWWSELPELYKHNPSNNLTDDVSRQ...GVLHFTQMAWGKTHKIGC OvASP-2 108 VAVYGTSGIMIALESWWVELTRSYKNNPSNKYTSIVANR...GVSNFTQLAWGKTYKVGC NbVAL-5 108 TTLNTEYALFGAIQRWWDDIAV.NGVNRRMIFRQFLADKALPPV.RFTQMAWALTYRVGC SCL-1 96 VGSLDSYG.EIAAAAWEKEFQDFGW..KSNAMDTTLFNS...GIGHATQMAWANTSSIGC AcASP-3 94 GNSDDLTVISEAGNSWWSEILDLKGKDVYNSVDNT...... SEIANMAWESHAKLGC thhtxWxsEhtx-a hsHaTQhsWttoxxlGC Con5 Con6

176

cysteine-rich hinge

NbVAL-1 167 AYA...... DCKD..KYSIVCLYSPKSNMIGKPIYKKADEWAFICTGCPKDKKCGDELDK NbVAL-6 165 GYA...... DCTD..KYSIVCLYSPTGNKVGEQIYEPTDDWNFICENCPNSKNCDLEYDK NbVAL-4 166 AVK.....E.CEG..STMAVCNYRPTGNILGMMVYKEGEPCSKCP...... KGAMCE.NG AcASP-2 163 YVE.....W.CSS..MTYGVCQYSPQGNMMNSLIYEKGNPCTKDSDCG.SNASCSAG.EA AdASP-2 163 YVE.....W.CPS..MTYGVCQYSPQGNMMNSIIYEKGNPCTQDSDCG.SNAKCSSG.EA AyASP-2 162 YVE.....W.CSS..MTYGVCQYSPQGNMMNSIIYEKGNPCTQDSDCG.SNARCTAD.KA NaASP-2 163 YVE.....A.CSN..MCYVVCQYGPAGNMMGKDIYEKGEPCSKCENCD.KEKGLCSA... Hc24 167 AVE.....W.CSD..MTFVACEYDSAGNYMGMPIYEVGNPCTNNEDCKCTNCVCSRD.EA OvASP-1 165 GVA.....TQCDGGRTLIVICHYSPGGNMVGEVIYHRGNPCKVDKDC..YTKKCLSK.SG BmVAL-1 165 GIA.....TNCDGGRTLIAICHYSPAGNMLKELIYELGEPCKTDSDC..NTKKCAKK.SG WbVAH 165 GIA.....TNCDGGRTLITICHYSPAGNILKNLIYELGEPCKKDGDC..NTKKCAKK.SG OvASP-2 165 GIA.....THCDGGKAFVAVCQYNPGGNTMGESIYEKGRPCKTDRDC..SSRKCAKE.SG NbVAL-5 166 AVA.....V.CQG..RTVVVCRYSPRGNIVDESIYRLGSPCSGCTTSCVGGTLCYMS... SCL-1 150 GVKNCGRDASMRNMNKIAVVCQYSPPGNTMGRPIYKEGTTCSSCS....GSTKCDTA.SG AcASP-3 145 AVV...... ECSK..KTHVVCRYGPEGKGEGKKIYEKGETCSQCSDYGQGVTCDNDEWEG u hhhhxCpYt Con7

NbVAL-1 219 L.CYPPWALP NbVAL-6 217 L.CYPDWALP NbVAL-4 211 L.CVVR.... AcASP-2 213 L.CVVRG... AdASP-2 213 L.CIVQ.... AyASP-2 212 L.CIVHG... NaASP-2 ...... Hc24 218 L.CIAP.... OvASP-1 217 L.CRK..... BmVAL-1 217 L.CRK..... WbVAH 217 L.CRK..... OvASP-2 217 L.CKLNFFI. NbVAL-5 ...... SCL-1 205 L.CG...... AcASP-3 197 LLCS......

Fig. 5.6. Amino acid sequence alignment of N. brasiliensis single SCP domain VALs with those of other nematodes

Sequences were aligned with Multalin and annotated with BOXSHADE. Amino acids that exactly match are shaded black whilst residues corresponding to conservative substitutions are shaded grey. Signal peptide sequences are underlined, cysteines predicted to form disulphide bridges are highlighted in yellow, and the cysteine-rich hinge region is indicated above the relevant amino acid sequences. Whilst residues thought to form a putative metal binding sites are coloured red, and serine residues of the putative serine protease catalytic

177 triad are marked in green (Serrano et al., 2004; Shikamoto et al., 2005). The seven consensus sequences defined from the vespid venom allergen-5 (Ves v 5) superfamily are shaded purple and labeled Con 1-7 (Henriksen et al., 2001). Abbreviations and GenBank numbers: AcASP- 2, Ancylostoma caninum secreted protein-2, AAC35986; AcASP-3, AY217004; AdASP-2, A. duodenale secreted protein-2, AY288088; AyASP-2, A. ceylanicum secreted protein-2, AAP41593; NaASP-2, Necator americanus secreted protein-2, AAP41952; Hc24, 24 kDa secretory protein of Haemonchus contortus, AAC47714; OvASP-1, Onchocerca volvulus activation-associated secreted protein-1, AAB69625; OvASP-2, AAG40311; BmVAL-1, Brugia malayi Venom allergen-like protein-1, AAK12274; WbVAH, Wuchereria bancrofti vespid allergen antigen homologue, AAD16985; SCL-1, C. elegans SCP-Like extracellular protein, NM_070101.

178

AcASP-6 1 ------MKLFILVLVAILGIAHATDFQCWNF--KST-DTLREHYLKSI AcASP-5 1 ------MPNLLLLLFLSLPGAILST--TCPGN--DLT-DAERTLLTRVH AcASP-1 1 ------MFSP-VVVSVIFTIAICDASPARDSFGCSNS--GIT-DKDRQAFLDFH NaASP-1 1 ------MFSP-VVVSVVFTIAFCNASPARDSFGCSNS--GIT-DSDRQAFLDFH AdASP-1 1 ------MFSSVVVISVISTIAFCDASPARASFGCSNN--GIT-DSDRQAFLDFH AyASP-1 1 ------MFSPVVVISVVLTVAFCDASPVKASFGCSNS--GIT-DSDRQAFLDFH CeVAP-1 1 ------MAVLAVVLLLACLERAVAQT-FGCSNT--KIN-DQARKMFYDAH OoASP-4 1 ------MLATAAAFLLLALVCPYAKAGYGCPRS--LTQSDRTRTIFLDFH NbVAL-3 1 ------MRWLVVLLYLTPLVAAIQSKFNCPSD--LGQQDDTREEFLKRH NbVAL-7 1 ------MAPPGRVPLIVVIVFNVAVGHEYHCNKD--LIMNDDLRFNMLNYH Hc40 1 ------MQIPITIACLVLLAPLWAADKYVICPSD--NGMTNEVRNMFVDTH NbVAL-8 1 ----MPRLWFVYQRNFSLFSLAILAVALVSRTNGTQNFNCQNSANSEMTDELRAYYLNFH NbVAL-2 1 MAVLRVQQGSIQSSKNSSLQSLLLAVRLLPSAAQDTLWGFSNN-----DDTNRNTFLTTL AcASP-4 1 ------MINIHFIALAITSLLPALSEGKPVVFVEPQCKPNG-YLHKNTIDNNVLKPI lpxH

AcASP-6 40 NNLRKKIADG-----SAENKSG-KCPQGKNIYKLSWDCELELKAQQAVD-QCKPNVPEPA AcASP-5 39 NSIRREIAQG-----VANNYHGGKLPAGKNIYRMRYSCELEQAAIDASQTFCSASLEEPQ AcASP-1 45 NNARRRVAKG-----LEDSNSG-KLNPAKNMYKLSWDCAMEQQLQDAIQSCPSGFAGIQG NaASP-1 45 NNARRRVAKG-----LEDSNSG-KLNPAKNMYKLSWDCAMEQQLQDAIQSCPSGFAGIQG AdASP-1 46 NNARRRVAQG-----VEDNKSG-KLNPAKNMYKLEWDCKMEQQLQDAIQSCPGGSAGIQG AyASP-1 46 NNARRRVAQG-----VEDNKSG-KLNPAKNMYKLDWDCEMEQKLQDAIQSCPGGFAGIQG CeVAP-1 41 NDARRSMAKG-----LEPNKCG-LLSGGKNVYELNWDCEMEAKAQEWADGCPSSFQTFDP OoASP-4 43 NEVRRNISLG-----RQPNKQG-FLGPAKNMYELGWDCNLEQKALEQISSCAFS---IKP NbVAL-3 42 NNIRQNIALGM----LAVNYFSYFLGPAKNMYQLEWSCDLERIAKATINQCQFKNVSTK- NbVAL-7 44 NDIRTNIAWG------AQDWPTGQGPAKNMYRVGWDCKLEEEAAKSLQQCSEAEYTAAP Hc40 44 NKLRSQTAQG-----KAKNAFGGFAPKAARMLKVSYDCDMEANMMKWAKQCHFYHPPPAY NbVAL-8 57 NNQRRRLARG------NSPARSGKFNKAKNMYKLEWSCSIEELAKRAVANCNADLTENRS NbVAL-2 56 NAARNNTALGY----IPIANGRGYLPTTSNMNLLEYDCFLEAKAYQQVQGCAKAETDDSV AcASP-4 51 NTRREALAKGTQQNGFDPPNPQTFLPPATDMTKLSWSCDLEQKAIKTINGNCVNPANPTK NthRtxh hxxhxWsxphttxAtxauppCxhxHs Con1 Con2 Con3

AcASP-6 93 G----YSQILKKVKS----TCDPTK-VLKKQIEAWWTKSVKDAGVD------NP-PNN AcASP-5 94 K----YGQNIQAYVTPSIIARPKND-LLEDAVKQWYLPVIYYGQRD------AANKFT AcASP-1 99 -----VAQNTMSWSSSGGFPDPSV--KIEPTLSGWWSGAKKNGVG------PDNKYN NaASP-1 99 -----VAQNTMSWSSSGGYPDPSV--KIEPTLSGWWSGAKKNGVG------PDNKYT AdASP-1 100 -----FSQNVMSWSNSGGFPNSSE--KIESTLSGWWSGAKNNGVG------SDNKYT AyASP-1 100 -----VAQNIISWSGSGGFPNPSE--KINSTLASWWGGAKNNGVA------SDNKYT CeVAP-1 95 T----WGQNYATYMGS--IADPLP--YASMAVNGWWSEIRTVGLTD------PDNKYT OoASP-4 94 G----LAQNIIRMTFRG--YGEEA--ILKFAQKAWVDVVRYGSVT------NRFQ NbVAL-3 97 ------YVYNTNK-FWHYGFENVRAIEEGVYQWVEPIHMNDFGP------KFEC NbVAL-7 97 N----NFAVLDVISK-PRNKWPATNFALHRALNSWYKPSIGFDQG------STLA Hc40 99 R----NYWGQNIYMVGDAYYNFTWPSIAETAVISWWQELQVFGVPEN------NIVVAPD NbVAL-8 111 YGQNLAGYTFWSSDGNVPRSHIKMKELIKKTLESWFNQAKEQGWKD------PNNKFT NbVAL-2 112 Y------FVLDATQSGKCDILAEIASVVNKSFYDSGLQIDTNP------PRFW AcASP-4 111 PN---NGEGLADVLYYGNDYDNTVEGVIQGNLEAWLVKADFNVFPVTTKGTVISYPTYNG GpNlhht thhtxWxsEhtxa Con4 Con5

179

AcASP-6 135 KQGLEDFAKLANGKATKIGCAQKNCN------EQLYVACVINE------PAPAVGMPIYE AcASP-5 141 DPRLYTFANLAYDKNTALGCHYAKCQGP----DRIVISCMYNN------VVPDN-AVIYE AcASP-1 143 GGGLFAFSNMVYSETTKLGCAYKVCG------TKLAVSCIYNG------VGYITNQPMWE NaASP-1 143 GGGLFAFSNMVYSETTKLGCAYKVCG------TKLAVSCIYNG------VGYITNQPMWE AdASP-1 144 GGGLYAFSNMVFSETTKIGCAYKVCG------TKMATSCIYNG------IGYITNAPMWE AyASP-1 144 GGGLYAFSNMVFSETTKLGCAYKVCG------TKLTLSCIYNG------IGYMTGAPMWE CeVAP-1 139 NSAMFRFANMANGKASAFGCAYALCA------GKLSINCIYNK------IGYMTNAIIYE OoASP-4 135 Y-WFETLANIANSKTLKVGCAYKTCGG-----SSMFVSCVYEG------RRLLPNHVLWE NbVAL-3 138 HDKIREFCNMAYPTNTRLGCAYERCGS------ELLISCAYEE------GKKVPFKPLLE NbVAL-7 141 DEKLGAFGNMINYKAVRLGCSYKVCGD------KVHVRCIYND------GANKVGDTLYE Hc40 149 EHKTGHYMQVVWQWTYKIGCAINYCTINK-PWPWTIAGCNYNP------GGDNAYWVVYE NbVAL-8 163 NSGIYAFANMANSKTTEIGCAHNICKGN----TRVQILCLYNR------VGTFTGASIYR NbVAL-2 153 KAEQADFAKIANADAGLVGCFQKYCGN------DLHISCFFDKGLAINSTDRTKATVIYA AcASP-4 168 NTDLLAYSNLVRPTNTEIGCVLERCPATANVPKLVTFYCILNGKNITNGEALYKGTTVNT haHaTQhsWttoxxlGCu hhhhxCpYt Con6 Con7

cysteine-rich hinge

AcASP-6 183 VG---AGCNSKDDCTTYLQSKCSNKVCVAGHPGDATTTTSTPATTAPTTPTIPAGPTTAP AcASP-5 190 PG---TACVKDADCTTYPQSTCKDSLCIIPTP------HPPNPP AcASP-1 191 TG---QACQTGADCSTYKNSGCEDGLCTK------GP NaASP-1 191 TG---QACQTGADCSTYKNSGCEDGLCTK------GP AdASP-1 192 TG---QACKTGADCSTYKNSGCEDSLCTK------GA AyASP-1 192 TG---QACKAGADCTTFKNSGCEDGLCTK------GA CeVAP-1 187 KG---DACTSDAECTTYSDSQCKNGLCYK------AP OoASP-4 183 TG---RACQ----CDAYAASSCSNSLCVTGKP------VGP NbVAL-3 186 LAPHFCVCKHYPNSACDEDYLCRATSLNT------TIEP NbVAL-7 189 DG---WVCYSDSDCTTYENSRCSGG------LCEA Hc40 202 MG---DPCTTDADCKCAG-CVCSQEEALC------IPPE NbVAL-8 213 TG---KPCQKNSDCTVFPQSTCDGHGLCIK------KR NbVAL-2 207 PG--TKALCDATEGSTADSTGGNTGLCIRG------AP AcASP-4 228 GGCKEVTCSAGYACNNATLLCERSATTSSSTSASTSSSTASSTSSS------MAISTSS

AcASP-6 240 APPPTTAAPTTTS------TIGSIDNTICPQNQVITDSVRLTFLNTHNGLRSQLAQGQ AcASP-5 225 NPPPAMSP------NAEMTDAARKKVLGMHNWRRSQVALGN AcASP-1 219 DVP------ETNQQCPSNTGMTDSVRDTFLSVHNEFRSSVARGL NaASP-1 219 DVP------ETNQQCPSNTGMTDSVRDTFLSVHNEFRSSVARGL AdASP-1 220 DVP------ETNQQCPSNTGMTDSVRDTFLSLHNGFRSSVARGL AyASP-1 220 DVP------ETNQQCPSNTGMTDSVRDTFLSLHNEFRSSVARGL CeVAP-1 215 QAPVV------ETFTMCPSVTDQSDQARQNFLDTHNKLRTSLAKGL OoASP-4 211 KATTTTTTTTTTTRRPAVVKPPAKEPASTTCATNKGMTDELRQIFLTKHNYYRSYVAKGQ NbVAL-3 219 LGPATIRTTTITRATTTTTAPKLDVPKASCPTLNNKMTDEIRQIFLDTHNKWRSLVARGK NbVAL-7 215 PDNLVLRTTTTT--TTTTANPSAGGDGNSQCANNPEMTDEARKMLTDKHNQLRAQTAKGL Hc40 231 YTPLPPTTTSTTTPKPTTTTTVGVPNAGSCPELNNGMTDEARKMFVDKHNEYRSLIAKGQ NbVAL-8 242 ENPDDK------TNIMCPRSSSTMIDKVRQKFLNLHNHYRSLVATGR NbVAL-2 237 KPATTTSSTTTTT------TTTTTTTRSCPNITGSSNDYMRNYFLNQHNQYRSSIAQGN AcASP-4 281 STSASGATTTKAPSPQAQFPTGTSTMCNTRHAYANRMTDNLRNEYVRLHNFRRGLLAKGE lpxHNthRtxh Con1

180

AcASP-6 292 IFMG-NGARARP-ASKMRRMVYNCDAESSARNSAAQCLSS---PGSPSGYTENLHVINNN AcASP-5 260 VQNG-KNAYNCPTATDMYKIEYDCDLENSALAYAKQCSLVGSAEGTRPGEGENVHKG-AL AcASP-1 257 EPD--ALGGNAPKAAKMLKMVYDCEVEASAIRHGNKCVYQHSHGEDRPGLGENIYKTSVL NaASP-1 257 EPD--ALGGNAPKAAKMLKMVYDCEVEASAIRHGNKCVYQHSHGEDRPGLGENIYKTSVL AdASP-1 258 EPD--ALGGNAPKAAKMLKMVYDCEVEASAIRHGNKCVYQHSSGNDRPGLGENIYKTSVQ AyASP-1 258 EPD--ALGGNAPKASKMLKMVYDCEVEASAIRHGNKCVYQHSHGDERPGLGENIYKTSIV CeVAP-1 255 EADGIAAGAFAPMAKQMPKLKYSCTVEANARTWAKGCLYQHSTSAQRPGLGENLYMISIN OoASP-4 271 AKDKLITKGNAPKAARMRKMVYDCRVEASAMRHAKKCKYGHSDGSERPDLGESVWAISYN NbVAL-3 279 AEDKLSPGGYAPKASRMPKLWYDCKLEESSKQVAEKCANKHSYG----DYGENLFAASFS NbVAL-7 273 SVDPKSATGFAPKGSAMKKMKYDCEIEASAQAYTNLCKGLQHSWG---QYGKNIWMIFAK Hc40 291 AKGK--PGQFAPKAARMMKVNYDCDVEANAMEWSKTCTFGLNTAAMLKRWGNNMHMMSSK NbVAL-8 283 TYDK-LLGKNTPKAAKMQKLKYSCALEATAWRYSHNVDLLIPIPGKK-NAGENLYKN-LF NbVAL-2 290 VYNK-VTGTNVRQASRMSRMTYSCTLEQNAYDWARQCINKKSPGSDR---YENIYAHTDT AcASP-4 341 IPQK--GNIYLPKAADMWKISYDCGLEQGAIEHASQCLTGGSGQSSRPGVGENFKVIPAA hxxhxWsxphttxAtxauppCxhxHs GpNlhht Con2 Con3 Con4

AcASP-6 347 FVD-HNSAATQAFNAWWSEIN-TGYMRQAETERNMYSLSV-GIPNFAKMAWETNAHLGCA AcASP-5 318 VTD-PEAAVQTAVQAWWSQISQNGLNAQMKFTAFLKDKPD-APTAFTQMAWAKSVKLGCA AcASP-1 315 KFD-KNKAAKQASQLWWNELKEYGVGPSNVLTTALWNRPNMQIGHYTQMAWDTTYKLGCA NaASP-1 315 KFD-KNKAAKQASQLWWNELKEYGVGPSNVLTTALWNRPNMQIGHYTQMAWDTTYKLGCA AdASP-1 316 KFE-KNKAAKQASELWWNELREFGVGPSNNLTNALWNRPGMQIGHYTQMAWDTTYKLGCA AyASP-1 316 KFE-KNKAAKQASQLWWNELKEFGVGPSNMLTDALWNRPNMQIGHYTQMAWESTYKLGCA CeVAP-1 315 NMP-KIQTAEDSSKAWWSELKDFGVGSDNILTQAVFDRG---VGHYTQMAWEGTTEIGCF OoASP-4 331 RMD-KARSAEMSCDDWFGELAKNGVGRENVLTKKLFYRPNKQIGHYTQMVWQNTYKLGCA NbVAL-3 335 GYD-QDEVAKMAVDQWAGELKKFGVGDNLIYP----TPGL--TGHYTQVVWQNTTRVGCY NbVAL-7 330 NYN-RKDVADWPPHSWFDELKEYGVGEKNIFN----ASMMN-VGHYTQVVWGDTDRFGCG Hc40 349 ANN-KTEAAAEAVAAWFGDLQKYGVPENNVFT----MNVYTTLSKYSQLAWQSSDRIGCV NbVAL-8 340 LFD-KLKGPRCRLKTGLKRSKILVWWSGQFLHEIFRFRPLHADVWQAPTTWMCNPRLPRY NbVAL-2 346 SLA-RREVARLAMERWWRQITNNGLPANHIFVD------KKGTNMLSKIIYDEGRRVGCS AcASP-4 399 RFPTFEDAAKKTVTEWWKPIRNVDYFGNNVNFLPIYDQDP--ISSFTRMAWATTNKVGCS thhtxWxsEhtxa hsHaTQhsWttoxxlGCu Con5 Con6

cysteine-rich hinge

AcASP-6 404 IVR-CGLNT-----NVVCPYSPKS--DGGQIYKMGPFCR---RC-PDYPGTFCNQGLCSF AcASP-5 376 VSN-CQADT-----FTVCRYKAAGNIVGEFIYTKGNVCD---AC---KATCITAEGLCPT AcASP-1 374 VVF-CNDFT-----FGVCQYGPGGNYMGHVIYTMGQPCS---QCSPG-ATCSVTEGLCSA NaASP-1 374 VVF-CNDFT-----FGVCQYGPGGNYMGHVIYTMGQPCS---QCSPG-ATCSVTEGLCSA AdASP-1 375 VVF-CNDFT-----FGVCQYGPGGNYMNHLIYTMGQPCS---QCAAT-ATCSVTEGLCSA AyASP-1 375 VIF-CNDFT-----FGVCQYGPGGNYMNHLIYTIGQPCS---ECEAT-ATCSVTEGLCSA CeVAP-1 371 VEN-CPTFT-----YSVCQYGPAGNYMNQLIYTKGSPCTADADCPGT-QTCSVAEALCVI OoASP-4 390 VEW-CPTMT-----FVVCQYNPPGNYLGQLIYEVGEPCRNDGDCRCPKCKCSRNEALCIV NbVAL-3 388 INDECFPDSAGWKTQVVCQYKPTGNFMRWEVYSKGEPCKNDSDCKCKNCSCLAEEGLCYD NbVAL-7 384 FKS-CAGSG---YTALICQYAPPGNWLDSTIYKVGEPCS---AC--PVGTKCEDGALCA- Hc40 404 VVP-CWSSW----TVVVCEYNPGGDLPGEAIYDVGDPCTKDADCQCPGCTCSRDEALCVA NbVAL-8 399 ALVVCPQK------GGKLIDEPVYTAGSPCS---QC-GNSELCNPNEGLCSA NbVAL-2 399 VVK-CSTFT-----IAVCRYDPAGPAFNTQYYLLGAACN---RC--DGRPC--SAGLCG- AcASP-4 457 IVK-CTTDN---VYVGVCRYSPMGNIVNSNIYQIGNPCS---VRPTQATGCDPVEGLWY- hh-----hhxCpYt Con7

181

AcASP-6 452 ------AcASP-5 424 P------AcASP-1 424 P------NaASP-1 424 P------AdASP-1 425 P------AyASP-1 425 P------CeVAP-1 424 P------OoASP-4 444 P------NbVAL-3 448 PNSMAYI------NbVAL-7 ------Hc40 459 P------NbVAL-8 441 VVTFPGEGIKPGMQ NbVAL-2 ------AcASP-4 ------

Fig. 5.7. Amino acid sequence alignment of N. brasiliensis double SCP domain VALs with those of other nematodes

Sequences were aligned with ClustalW (Corpet, 1988) and annotated with BOXSHADE. Amino acids that exactly match are shaded black whilst residues corresponding to conservative substitutions are shaded grey. Signal peptide sequences are underlined, cysteines predicted to form disulphide bridges are highlighted yellow and cysteine-rich regions are indicated above the relevant amino acid sequences. Residues predicted to form a putative metal binding sites are coloured red, and serine residues of the putative serine protease catalytic triad are marked in green (Serrano et al., 2004; Shikamoto et al., 2005). The seven consensus sequences defined from the vespid venom allergen-5 (Ves v 5) superfamily are shaded purple and marked Con 1-7 (Henriksen et al., 2001). Abbreviations and GenBank numbers: AcASP-6, Ancylostoma caninum secreted protein-1, Q16937; AcASP-4, AY217005; AcASP-5, AY217006; AcASP-6, AY2117007; Hc40, 40 kDa secretory protein of Haemonchus contortus, AAC03562; AdASP-1, A. duodenale secreted protein-1, AAD13339; AyASP-1, A. ceylanicum secreted protein-1, AAN11402; NaASP-1, Necator americanus secreted protein-1, AAD13340; CeVAP-1, C. elegans Venom-Allergen- like Protein-1, AAM54195; OoASP-4, Ostertagia ostertagi activation associated secreted protein-4, AM747039.

182

NbVAL-1 1 ------MRSLYLLSVMFIGAAASPAVINNMCA----KYPDATLDDNTRTTFTE NbVAL-6 1 ------MRSLYLLSVMFMGAAATPISPVSMC-----KEADATLSDFDRTQFLE NbVAL-3C 1 ------LNTTIEPLGPATIRTTTITRATTTTTAPKLDVPKASCPTLNNKMTDEIRQIFLD NbVAL-7C 1 ------GLCEAPDNLVLRTTTTT--TTTTANPSAGGDGNSQCANNPEMTDEARKMLTD NbVAL-4 1 ------MLLPAVTLVLTGLVVSAAAGACPKTEG------SQMTDAFRKKFLD AcHPI 1 ------MSSYLLVLVAILGFAYAEEGDYSLCQ------QREKLDDDMREMFTE NbVAL-2C 1 ------TGGNTGLCIRGAPKPATTTSSTTTTTTTTTTTTRSCPNITGSSNDYMRNYFLN NbVAL-5 1 ------MFAILLLTLLSREASSNAR------FARQIDSTIGAHVAA NbVAL-8C 1 ------HGLCIKKRENPDDKTNIMCPRS------SSTMIDKVRQKFLN NbVAL-8N 1 -----MPRLWFVYQRNFSLFSLAILAVALVSRTNGTQNFN-CQNSANSEMTDELRAYYLN NbVAL-3N 1 ------MRWLVVLLYLTPLVAAIQSKFNCP------SDLGQQDDTREEFLK NbVAL-7N 1 ------MAPPGRVPLIVVIVFNVAVGHEYHCN------KDLIMNDDLRFNMLN NbVAL-2N 1 MAVLRVQQGSIQSSKNSSLQSLLLAVRLLPSAAQDTLWG------FSNNDDTNRNTFLT

* * NbVAL-1 44 GHNRFRTAVANGYAYVG--QYGFSQYAEDMEAMEWSCPDEAKAIELAKAKCTAQ---PTA NbVAL-6 43 LHNKFRAAAANGFAYVG--KFGYSQYAQNMEAMEWRCPHEGKAIKLAET--CKK---PKT NbVAL-3C 55 THNKWRSLVARGKAEDKLSPGGYAPKASRMPKLWYDCKLEESSKQVAEKCAN------KH NbVAL-7C 51 KHNQLRAQTAKGLSVDPKSATGFAPKGSAMKKMKYDCEIEASAQAYTNLCKGL-----QH NbVAL-4 41 EHNRLRSLVANGRAKDK--DGSYVPKAANMLKLRYDCSLERLAAEHVNQCKLAP---SKA AcHPI 42 LHNGYRAAFAR------NYKTSKMRTMVYDCTLEEKAYKSAEKCSEE------NbVAL-2C 54 QHNQYRSSIAQGNVYNK-VTGTNVRQASRMSRMTYSCTLEQNAYDWARQCINK-----KS NbVAL-5 35 WHNYFRSQVASGNVVNG-RTGSNCPQGSNLNQMVYDGLLAADAQNYADTCPNNEQGSPVS NbVAL-8C 37 LHNHYRSLVATGRTYDK-LLGKNTPKAAKMQKLKYSCALEATAWRYSHNVDLLIPIPGKK NbVAL-8N 55 FHNNQRRRLARGNSPAR---SGKFNKAKNMYKLEWSCSIEELAKRAVANCNADLTENRSY NbVAL-3N 40 RHNNIRQNIALGMLAVN-YFSYFLGPAKNMYQLEWSCDLERIAKATINQCQFKN------NbVAL-7N 42 YHNDIRTNIAWGAQDWP----TGQGPAKNMYRVGWDCKLEEEAAKSLQQCSEAEYT--AA NbVAL-2N 54 TLNAARNNTALGYIPIA-NGRGYLPTTSNMNLLEYDCFLEAKAYQQVQGCAKAET----D

NbVAL-1 99 PKGFRMNTAL----VDKAG-KTSDKAIEEALGKWRNEIMRHGMPE-DLVFT---DIWKNK NbVAL-6 96 PAGFGMNSIF----VDKAGNKIISEAIEEALGKWRNDLKKMDLPE-DMTFT---KDMEST NbVAL-3C 109 SYGDYGENLF----AASFSGYDQDEVAKMAVDQWAGELKKFGVGD-NLIY----PTPGL- NbVAL-7C 106 SWGQYGKNIW----MIFAKNYNRKDVADWPPHSWFDELKEYGVGE-KNIF----NASMMN NbVAL-4 96 SGARAALGES----LIRSTGRTLADQADVTTGLWFLDLEKNGMGR-NLTFGA--QPDVYK AcHPI 83 ---PSSEEEN----VDVFSAATLNIPLEAGNSWWSEIFELRGKVY-NKNGK------NbVAL-2C 108 PGSDRYENIY----AHTDTSLARREVARLAMERWWRQITNNGLPA-NHIFV-----DKKG NbVAL-5 94 GRPNQGENVM----IYYTTTLNTEYALFGAIQRWWDDIAVNGVNR-RMIFRQFLADKALP NbVAL-8C 96 NAGENLYKNL----FLFDKLKGPRCRLKTGLKRSKILVWWSGQFL-HEIFR------NbVAL-8N 112 GQNLAGYTFWSSDGNVPRSHIKMKELIKKTLESWFNQAKEQGWKDPNNKFTN------SG NbVAL-3N 93 VSTKYVYNTN----KFWHYGFENVRAIEEGVYQWVEPIHMNDFGPKFECHD------K NbVAL-7N 96 PNNFAVLDVIS---KPRNKWPATNFALHRALNSWYKPSIGFDQGSTLADEK------NbVAL-2N 109 DSVYFVLDAT-----QSGKCDILAEIASVVNKSFYDSGLQIDTNP-PRFWK------AE

183

* * * * NbVAL-1 150 IGHATKMIWHSSSKLACAYADCKDKY------SIVCLYSPKSNMIGKPIYKKADEWA-FI NbVAL-6 148 IERATKMLWHNTVHLACGYADCTDKY------SIVCLYSPTGNKVGEQIYEPTDDWN-FI NbVAL-3C 159 TGHYTQVVWQNTTRVGCYINDECFPDSAGWKTQVVCQYKPTGNFMRWEVYSKGEPCKNDS NbVAL-7C 157 VGHYTQVVWGDTDRFGCGFKSCAGSG----YTALICQYAPPGNWLDSTIYKVGEPCS--- NbVAL-4 149 IGHYTQIVWATTRTIGCAVKECEGST------MAVCNYRPTGNILGMMVYKEGEPCS--- AcHPI 126 TSNIANMVWDSHDKLGCAVVDCSGKT------HVVCQYGPEAKGDGKTIYEEGAPCS--R NbVAL-2C 158 TNMLSKIIYDEGRRVGCSVVKCSTFT------IAVCRYDPAGPAFNTQYYLLGAACN--- NbVAL-5 149 PVRFTQMAWALTYRVGCAVAVCQGRT------VVVCRYSPRGNIVDESIYRLGSPCS--- NbVAL-8C 142 FRPLHADVWQAPTTWMCNPRLPRYAL------VVCPQK-GGKLIDEPVYTAGSPCS--- NbVAL-8N 166 IYAFANMANSKTTEIGCAHNICKGNTR----VQILCLYNRVGTFTGASIYRTGKPCQ--- NbVAL-3N 141 IREFCNMAYPTNTRLGCAYERCGSEL------LISCAYEEGKKVPFKPLLELAPHFC--- NbVAL-7N 144 LGAFGNMINYKAVRLGCSYKVCGDKV------HVRCIYNDGANKVGDTLYEDGWVCY--- NbVAL-2N 156 QADFAKIANADAGLVGCFQKYCGNDL------HISCFFDKGLAINSTDRTKATVIYAP--

* * * NbVAL-1 203 CTGCPKDKKCGDELDKLCYPPWALP------(29/43) NbVAL-6 201 CENCPNSKNCDLEYDKLCYPDWALP------(26/40) NbVAL-3C 219 DCKCKNCSCL--AEEGLCYDPNSMAYI------(28/46) NbVAL-7C 210 --ACPVGTKC--EDGALCA------(29/41) NbVAL-4 200 --KCPKGAMC--ENG-LCVVR------(26/39) AcHPI 178 CSDYGAGVTCDDDWQNLLCIGH------(-/-) NbVAL-2C 209 --RCDGRPCS----AGLCG------(29/46) NbVAL-5 200 --GCTTSCVG----GTLCYMS------(27/38) NbVAL-8C 191 --QCGNSELCN-PNEGLCSAVVTFPGEGIKPGMQ (25/38) NbVAL-8N 219 -----KNSDCTVFPQSTCDG------(25/40) NbVAL-3N 192 --VCKHYPNSACDEDYLCRATS------(23/38) NbVAL-7N 195 -----SDSDCTTYENSRCSG------(28/42) NbVAL-2N 208 ----GTKALCDATEGSTADS------(32/40)

Fig. 5.8. Amino acid sequence alignment of NbVALs with A. caninum platelet inhibitor (AcHPI)

The sequences of AcHPI (GenBank: AAK81732), single domain NbVALs, and N- terminus/C-terminus of double-domain NbVALs were aligned using ClustalW and annotated with BOXSHADE. Amino acids that exactly match are shaded black whilst residues corresponding to conservative substitutions are shaded grey. Cysteines residues predicted to form disulphide bridges are indicated an asterisk. Residues are coloured red (KGD), highlighted pink (LDV) and green (NGR) indicate possible motifs for interaction with integrins (Ruoslahti, 1996). The percentage amino acid identity/similarity to AcHPI is indicated at the end of the alignment.

184

* AcNIF 1 MEAYLVVLIAIAGIAHSNEHNLRCPQNGTEMPGFNDSIRLQFLAMHNGYRSKLAL NbVAL-3N 1 .MRWLVVLLYLTPLVAAIQSKFNCPSD....LGQQDDTREEFLKRHNNIRQNIAL NbVAL-2C 1 ...... RSCPNIT...GSSNDYMRNYFLNQHNQYRSSIAQ NbVAL-8C 1 ...... IMCPRSS...STMIDKVRQKFLNLHNHYRSLVAT

* * AcNIF 56 GHISITEESESDDDDDFGFLPDFAPRASKMRYLEYDCEAEKSAYMSARNCSD... NbVAL-3N 51 GMLAVN...... YFSYFLGPAKNMYQLEWSCDLERIAKATINQCQF... NbVAL-2C 32 GNVYNK...... VTGTNVRQASRMSRMTYSCTLEQNAYDWARQCI.... NbVAL-8C 32 GRTYDK...... LLGKNTPKAAKMQKLKYSCALEATAWRYSHNVDLLIP

§ AcNIF 108 ..SS.SPPEGYDENKYIFENSNNISEAALKAMISWAKEAFNLNKTKEGEGVLYRS NbVAL-3N 91 ..KNVSTKYVYNTNKFWHYGFENV.RAIEEGVYQWV.EPIHMNDF....GPKFEC NbVAL-2C 71 ..NKKSPGSDRYENIYAHTDTSLARREVARLAMERWWRQITNNGLPA..NHIFVD NbVAL-8C 75 IPGKKNAGENLYKNLFLF.DKLKGPRCRLKTGLKRSKILVWWSGQFL..HEIFRF

* * * AcNIF 160 NHDISNFANLAWDAREKFGCAVVNCPLGEIDDETNHDGETYATTIHVVCHYPKIN NbVAL-3N 138 HDKIREFCNMAYPTNTRLGCAYERC...... GSELLISCAYEEGK NbVAL-2C 122 KKGTNMLSKIIYDEGRRVGCSVVKC...... STFTIAVCRYDPAG NbVAL-8C 127 RP....LHADVWQAPTTWMCNP.RL...... PRYALVVCP.QKGG

* * * AcNIF 215 KTEGQPIYKVGTPCDDCSEYTKKADNTTSADPVCIPDDGVCFIGSKADYDSKEFY NbVAL-3N 177 KVPFKPLLELAPHFCVCKHYPNSA...CDEDYLCRATS...... NbVAL-2C 161 PAFNTQYYLLGAACNRCDGRPC...... SAGLCG...... NbVAL-8C 160 KLIDEPVYTAGSPCSQCGNSELC....NPNEGLCSAVVTFPGEGIKPGMQ.....

AcNIF 270 RFREL (-/-) NbVAL-3N ..... (25/40) NbVAL-2C ..... (25/38) NbVAL-8C ..... (24/38)

Fig. 5.9. Amino acid sequence alignment of NbVALs with A. caninum neutrophil inhibitory factor (AcNIF)

The sequences of AcNIF (GenBank: AAA27789), NbVAL-3 N-terminus (NbVAL-3N), NbVAL-2 and -8 C-terminus (NbVAL-2C/-8C) were aligned with Multalin and annotated with BOXSHADE. Amino acids that exactly match are shaded black whilst residues corresponding to conservative substitutions are shaded grey. Cysteine residues predicted to form disulphide bridges are indicated an asterisk. § indicates the conserved glutamic acid residue which is essential for binding to integrin ligands (Rieu et al., 1996). The percentage amino acids identities/similarities to AcNIF is indicated at the end of the alignment.

185

Fig. 5.10. Superposition of the modelled structures of NbVAL-3 and AcNIF with NaASP-2

Panel A: Superposition of the crystal structure of NaASP-2 (PDB: 1U53) (coloured cyan) with the modelled structures of AcNIF (coloured lime) and NbVAL-3 N-terminus (coloured pink). Panel B: NaASP-2 Glu109 (coloured blue) is conserved in NbVAL-3 (Glu124, coloured red) and AcNIF (Glu142, coloured orange). The models of NbVAL-3 N-terminus and AcNIF were constructed using the ModWeb server (Marti-Renom et al., 2000), and structures were visualized using MacPyMOL (DeLano Scientific LLC).

186

Fig. 5.11. Serine is incapable of interacting with the conserved Histidine and Glutamatic acid residues to form the putative active triad in NaASP-2 and NbVAL-3 C-terminus

Panel A: Superposition of the crystal structure of NaASP-2 (PDB: 1U53) (coloured cyan) with the modelled structure of NbVAL-3 C-terminus (NbVAL-3248-445, coloured yellow). Three conserved residues were found in NaASP-2 (Ser70, His129 and Glu106) and NbVAL-

3248-445 (Ser332, His319 and Glu326), and are incapable to form the serine protease catalytic triad. Panel B: The orientation of catalytic triad of a serine protease, trypsin (PDB: 1A0J). The lengths of hydrogen bond between the acidic amino acids and histidine residue are indicated with 3.31 Angstroms and 2.68 Angstroms. The NbVAL-3248-445 model was constructed using the ModWeb server, and structures were visualized using MacPyMOL.

187

Fig. 5.12. Putative active site residues in the GAPR-1 are conserved in NbVAL-3

The two human Golgi-associated plant pathogenesis related protein (GAPR-1) molecules

(PDB: 1SMB), and the modeled NbVAL-3 N/C-terminus (NbVAL-320-209 and NbVAL-3248-

445) possess an α-β-α tertiary structure (Panel A, B and C). Residues (His54, Glu65 and Ser71, coloured white) which have been suggested to form a catalytic triad are shown at a putative active site on the surface of GAPR-1 (Panel D), and conserved residues (His319, Glu326 and Ser164, coloured pink) were found in the NbVAL-3 (Panel E and F). The

NbVAL-3 models were constructed using the ModWeb server, and structures were visualized using MacPyMOL.

188

Fig. 5.13. Putative metal-binding site residues in the snake venom toxin triflin are conserved in NbVAL-3

Four residues (His60, Glu75, Glu96 and His115) which constitute a Cd2+-binding site (can be substituted by Zn2+) in triflin (PDB: 1WVR) (Shikamoto et al., 2005) are shown in a cleft on the protein surface (Panel A & C), and conserved residues (His319, Glu326, Glu352 and

His372) of the NbVAL-3 C-terminus (NbVAL-3248-445) are shown in Panel D. The NbVAL-

3248-445 model was constructed using the ModWeb server, and structures were visualized using MacPyMOL.

189

5.4. Cloning and expression of N. brasiliensis VALs

To identify whether the N. brasiliensis VALs were secreted, recombinant proteins were produced in order to raise polyclonal antibodies. The full-length cDNA minus the signal peptide sequence of the single SCP domain VALs (NbVAL-4, 5 and 6), and cDNAs for the N-terminal domain of NbVAL-3, 7 and 8, again lacking sequence for the signal peptide, were amplified, cloned into the pET29b expression vector and transformed into the E. coli strain TOP10 (Fig. 5.14 and Fig 5.15). DNA sequence analysis indicated that the NbVAL- containing constructs were all in frame followed by six histidine residues at the C-terminal end (data not shown). Plasmids from positive colonies were isolated and transfected into E. coli BL21 (DE3). Bacteria were cultured, and protein expression induced with 10 mM IPTG as described in Materials and Methods. Fusion proteins were identified by SDS-PAGE and western blot analysis using mouse anti-HisTag antibody, which showed that they were predominantly found in the insoluble fractions, with estimated masses of proteins all as expected (Figs. 5.16, 5.17 and 5.18).

The recombinant NbVAL proteins were purified under denaturing conditions by nickel affinity chromatography in the presence of protease inhibitors. Proteins bound to the column were eluted by a pH gradient, and the eluted fractions are shown in Fig. 5.19. From the fractions collected and analyzed on SDS-PAGE gels it was observed that the urea wash buffer of pH 4.5 eluted the majority of the NbVAL His-tagged fusion proteins, and this was further confirmed by western blot analysis using a mouse anti-HisTag antibody (Fig. 5.20, Panel B). The eluted NbVALs were pooled and dialysed stepwise from 8 M urea, to 6, 4, and 2 M urea, then dialysed in PBS. During dialysis, the proteins precipitated out of solution.

Developmental expression of NbVALs was also monitored by western blot analysis using polyclonal antibody raised to the recombinant proteins (Fig. 5.21a and b). The data reveals that NbVALs were predominantly detected in adult ES (Fig. 5.21a and b), and NbVAL-7 and NbVAL-8 were also detected in L3 ES (Fig. 5.21b). Background reactivity made it hard to distinguish specific binding to somatic extracts, although antisera to VAL-1 and VAL-3 bound to non-activated L3 extract, and antisera to VAL-6 and VAL-7 bound to L4 extract (Fig. 5.21a and b).

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Fig. 5.14. Cloning of NbVAL-3, 4 and 5 into the pET29b expression vector

Panels A, B and C: The NbVAL-3 N-terminus, NbVAL-4 and -5 gene products were amplified from N. brasiliensis adult cDNA using primers which introduced 5’ Nde I and 3’ Not I (in NbVAL-3 and -4) or 3’ Xho I (in NbVAL-5) restriction sites. The gel-purified products were subcloned into the pCR4-TOPO vector generating possible subclones (S), which were analysed by PCR with primers specific for the cDNAs. Subclones S1, S2 and S3 for NbVAL-3, S1, S2 and S4 for NbVAL-4, and S1 and S2 for NbVAL-5, contained inserted cDNAs. Panels D, E and F: Plasmid DNA from subclone 3 (S3) of NbVAL-3, S2 of NbVAL-4 and S1 of NbVAL-5 were extracted and digested with the restriction enzymes, and then ligated to pre-digested pET29b. Possible clones (C) were screened by PCR for the presence of inserts. Samples were resolved on 1% agarose gels and detected by ethidium bromide staining. Molecular weight markers are displayed in base pairs (bp).

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Fig. 5.15. Cloning of NbVAL-6, 7 and 8 into the pET29b expression vector

Panels A, B and C: The NbVAL-6, NbVAL-7 N-terminus and NbVAL-8 N-terminus gene products were amplified from N. brasiliensis adult cDNA using primers which introduced 5’ Nde I and 3’ Xho I restriction sites. The gel-purified products were subcloned into the pCR4- TOPO vector generating possible subclones (S), which were analysed by PCR with primers specific for the cDNAs. Subclones S1, S3 and S4 for NbVAL-6, S1 and S2 for NbVAL-7, and S1-S4 for NbVAL-8, contained inserted cDNAs. Panels D, E and F: Plasmid DNA from subclone 4 (S4) of NbVAL-6, S2 of NbVAL-7 and S1 of NbVAL-8 were extracted and digested with the restriction enzymes, and ligated to pre- digested pET29b. Possible clones (C) were screened by PCR for the presence of inserts. Samples were resolved on 1% agarose gels and detected by ethidium bromide staining. Molecular weight markers are displayed in base pairs (bp).

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Fig. 5.16. Test expression of recombinant NbVAL-3 and NbVAL-4 in E. coli

Clone 1 (C1) containing NbVAL-3 (Fig. 5.13, Panel D) and C4 containing NbVAL-4 (Fig. 5.14, Panel E) pET29b constructs were picked for test expression. The soluble (Sol) and insoluble (Insol) protein fractions from uninduced cells grown for 1 hour (Un1) or 3 hours (Un3) were compared with fractions prepared from bacteria that had been induced to express the proteins for 1 or 3 hours. Samples were resolved on 12% SDS-PAGE gels and detected by coomassie blue staining (Pane A: NbVAL-3 & Panel B: NbVAL-4) or western blotting using a mouse anti-HisTag antibody (Panel C: NbVAL-3 & Panel D: NbVAL-4). Molecular weight markers are displayed in kilodaltons.

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Fig. 5.17. Test expression of recombinant NbVAL-5 and NbVAL-6 in E. coli

Clone 1 (C1) containing NbVAL-5 (Fig. 5.13, Panel F) and C1 containing NbVAL-6 (Fig. 5.14, Panel D) pET29b constructs were picked for test expression. The soluble (Sol) and insoluble (Insol) protein fractions from uninduced cells for 1 hour (Un1) or 3 hours (Un3) were compared with fractions prepared from bacteria that had been induced to express the proteins for 1 or 3 hours. Samples were resolved on 12% SDS-PAGE gels and detected by coomassie blue staining (Pane A: NbVAL-5 & Panel B: NbVAL-6) or western blotting using a mouse anti-HisTag antibody (Panel C: NbVAL-5 & Panel D: NbVAL-6). Molecular weight markers are displayed in kilodaltons.

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Fig. 5.18. Test expression of recombinant NbVAL-7 and NbVAL-8 in E. coli

Clone 1 (C1) containing NbVAL-7 (Fig. 5.14, Panel E) and C1 containing NbVAL-8 (Fig. 5.14, Panel F) pET29b constructs were picked for test expression. The soluble (Sol) and insoluble (Insol) protein fractions from uninduced cells for 1 hour (Un1) or 3 hours (Un3) were compared with fractions prepared from bacteria that had been induced to express the proteins for 1 or 3 hours. Samples were resolved on 12% SDS-PAGE gels and detected by coomassie blue staining (Pane A: NbVAL-7 & Panel B: NbVAL-8) or western blotting using a mouse anti-HisTag antibody (Panel C: NbVAL-7 & Panel D: NbVAL-8). Molecular weight markers are displayed in kilodaltons.

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Fig. 5.19. Purification of recombinant NbVALs from E. coli BL21 (DE3) under denaturing conditions

Panels A to Panel F indicate purification of recombinant NbVAL-3 to NbVAL-8 proteins respectively. Histidine tagged N. brasiliensis VAL proteins were purified from cleared cell lysates under denaturing conditions using nickel affinity chromatography. After the flow- through (FT) fractions were collected, columns were washed with 8 M urea in Tris (10 mM)- Phosphate (100 mM) buffer at the respective pH value (pH 8.0, 6.3, 5.9 and 4.5). Molecular weight markers are displayed in kilodaltons.

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Fig. 5.20. Purified NbVAL recombinant proteins

Panel A: Purified NbVAL proteins (3, 4, 5, 6, 7 and 8) resolved by 12% SDS-PAGE and stained with coomassie blue. Molecular weight markers are displayed in kilodaltons. Panel B: A duplicate gel was prepared, purified recombinants were separated by 12% SDS- PAGE, transferred onto nitrocellulose and visualised with a mouse anti-HisTag antibody.

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Fig. 5.21a. Identification of native NbVAL proteins by western blot

Twenty-five µg of N. brasiliensis non-activated L3 (L3), activated L3 (L3A), L4, adult (Ad) somatic extracts, and L3 and adult ES were probed with polyclonal mouse antibody to recombinant NbVAL-1, 3, 4 and 5 (panels A, B, C and D). Molecular weight markers are displayed in kilodaltons.

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Fig. 5.21b. Identification of native NbVAL proteins by western blot

Twenty-five µg of N. brasiliensis non-activated L3 (L3), activated L3 (L3A), L4, adult (Ad) somatic extracts, and L3 and adult ES were probed with polyclonal mouse antibody to recombinant NbVAL-6, 7, 8 and normal mouse serum (pre-bleed) (panels E, F, G and H). Molecular weight markers are displayed in kilodaltons.

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5.5. Discussion

Although a vast number of SCP proteins have been identified which show variation in sequence and structure from a range of plant and animal species, there is very little definitive information on functional roles for members of this protein family. The plant PR-1 proteins, insect venom allergens, mammalian CRISPs and nematode VALs have been classified into SCP protein subfamilies through phylogenetic analysis (Cantacessi et al., 2009a). Tertiary structure alignments of P14a (tomato), Ves v 5 (wasp), triflin (snake), GAPR-1 (human) and NaASP-2 (hookworm), for which protein structural data is available, reveal that all the major secondary structure elements overlap (Fig. 5.5) (Gibbs et al., 2008). Four of the amino acids, two pairs of histidines and glutamic acids, have been proposed to form a divalent metal ion binding site of undefined activity due to the fact that they are evolutionarily conserved/identified within a cleft in SCPs (Szyperski et al., 1998). These residues are also present in most parasite VALs, including N. brasiliensis (Figs. 5.6 and 5.7). However, SCP subfamilies have unique cysteine residues forming individual disulphide bridge distributions (Figs. 5.6 and 5.7) (Gibbs et al., 2008; Shikamoto et al., 2005). Therefore, it is difficult to address an exact function for the parasite VAL proteins via sequence alignments owing to the poorly understood relationship between primary structure and information on biological functions of SCP subfamilies.

The A. caninum secreted protein AcASP-1 was the first SCP-related protein to be characterized across a wide variety of parasitic helminths (Hawdon et al., 1996). As previously described in Chapter 3, many nematode parasites remain in a stage of developmental arrest until they invade the definitive host, in which they receive cues for resumption of development and secretion of a range of proteins. AcASP-1 (42 kDa) is a double SCP domain protein which is abundantly released within half an hour of parasite activation in vitro, and requires the presence of serum for continuous secretion (Hawdon et al., 1996). Subsequently, a single SCP domain ASP, termed AcASP-2, was identified as another major component of the serum-activated A. caninum L3 ES (Hawdon et al., 1999), which therefore would suggest an important role of secreted nematode VALs in transition to parasitism.

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The biological functions of parasite secreted SCP proteins are largely unknown, with two notable exceptions. A 41-kDa hookworm SCP termed neutrophil inhibitory factor (AcNIF) has been discovered from A. caninum and shown to bind to the β2 integrin, CD11b/CD18 (CR3). CR3 integrin is expressed on the surface of neutrophils and monocytes which binds in a divalent cation-dependent manner to complement, fibrinogen or CD54 (ICAM-1) ligands for mediating immune clearance (Arnaout, 1990); and binding of AcNIF blocks their adhesion to vascular endothelial cells (Lo et al., 1999; Moyle et al., 1994; Rieu et al., 1996). The AcNIF specific binding site composes the region of Pro147-Arg152, Pro201-Lys217 and Asp248-Arg261 within the CD11b I domain, which is a putative metal ion-dependent adhesion site (MIDAS) (Rieu et al., 1996; Zhang and Plow, 1997). Because AcNIF can inhibit neutrophil recruitment, it has anti-inflammatory properties, and it has been shown to inhibit neutrophil recruitment during lung injury in guinea pigs (Barnard et al., 1995), in addition to development of colitis and cerebral ischaemia in rats (Mackay et al., 1996; Meenan et al., 1996). Accordingly, secreted AcNIF probably acts to eliminate the cellular innate responses of host in order to favor the parasite survival during the infection.

A 20-kDa SCP protein, hookworm platelet inhibitor (AcHPI) protein was identified and purified from A. caninum. The protein was demonstrated to bind and antagonise the cell surface integrins GPIIb/IIIa and GPIa/IIa, blocking aggregation and adhesion of platelets to collagen and fibrinogen (Chadderdon and Cappello, 1999; Del Valle et al., 2003). AcHPI was observed in the somatic extracts and secreted products of the adult parasite by western blot and cephalic glands by immunohistochemistry, suggesting that AcHPI was secreted, and could maintain the ability of A. caninum adults to feed on blood from host intestines. Moreover, AcHPI was revealed to have significant homology to AcNIF and AcASP proteins via amino acid sequence alignment (Del Valle et al., 2003).

Three single-SCP domain ASP proteins from the parasite responsible for river blindness, Onchocerca volvulus, have been identified and named OvASP-1, OvASP-2 and OvASP-3 (Lizotte-Waniewski et al., 2000; Tawe et al., 2000). Each OvASP has been shown to have a different expression profile during the development of the parasite: the expression of OvASP- 1 was detected in the L2, L3, moulting L3 and female adult; OvASP-2 transcripts were highly represented in all developmental stages, whereas OvASP-3 was only observed in the L3 stage

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(Tawe et al., 2000). OvASP-1 and OvASP-2 have been demonstrated to induce a minimal inflammatory cell infiltration and also an angiogenic response in the corneas of mice (Tawe et al., 2000). Higazi et al. (2003) showed that the angiogenic activity was exhibited in the N- terminal amphipathic domain of OvASP-2, but this could not be replicated in vitro, suggesting that the protein could not promote angiogenesis directly, but only indirectly. It is known that vascular endothelial cells express a variety of integrins which play a vital role in the process of angiogenesis (Hodivala-Dilke et al., 2003). Thus, OvASPs are unlike the inhibitory effects on neutrophil recruitment and platelet aggregation of AcNIF and AcHPI, which probably agonist with particular integrins to proliferate the formation of blood vessels for supplying an enough blood nutrition to the adults reside in host. However, the mechanism still remains unclear.

The crystal structure of N. americanus ASP-2 has been determined, and is composed of a three-layer α-β-α sandwich structure with a large central cavity flanked by two residues (Glu106 and His192) conserved in all L3 VALs from parasitic nematodes (Asojo et al., 2005). This study showed that NaASP-2 could be a useful homology model, predicting possible binding models of AcNIF to the CR3 receptor, and also highlighting structural and charge similarities of NaASP-2 with CC chemokines, suggest that the protein may act as an agonistic or antagonistic ligand of host chemokine receptors (Asojo et al., 2005).

In this study, eight VAL proteins were identified from N. brasiliensis cDNA sequences. NbVAL-1, 4, 5 and 6 are single-domain VALs with an average predicted mass of 24 kDa, whereas NbVAL-2, 3, 7 and 8 are double-domain VALs with an average predicted molecular mass of 49 kDa. Database searching shows that homologues of either single SCP domain or double SCP domain NbVALs are present in all nematode species. Interestingly, the single domain VAL is more widespread among parasitic nematodes, but only six double-domain types have been reported in the database, from A. caninum, A. duodenale, A. ceylanicum, Necator americanus, Haemonchus contortus and Ostertagia ostertagi. All nematode VALs have conserved cysteine residues known to be involved in the formation of disulfide bonds in other SCPs for which the structure had been determined (Figs. 5.6 and 5.7), and alignment reveals areas of similarity within the sequences (Fig. 5.5) indicative of a common secondary structure. Analysis of NbVALs using the protein databases PROSITE and ProDom showed

202 that NbVALs belong to a group of evolutionarily related secreted proteins, which has been named the SCP/Tpx-1/Ag-5/PR-1/Sc7 family. However, alignments of NbVALs with parasitic nematode VALs showed that they share low identities of about 24%-35% and 24%- 31% with the single and double SCP domain parasite VALs respectively. Although these SCP proteins are common in the different clades of Nematoda, they still predominantly lack clearly defined functions,

A 24-kDa secreted protein (Hc24) expressed by adult Haemonchus contortus has homology to AcASP-1 (Schallig et al., 1997b). A double-domain HcVAL, named Hc40 (40 kDa), was also detected from a cDNA library enriched for membrane and secreted proteins from the gut of the adult (Rehman and Jasmer, 1998). A proteomics study of secreted proteins from adult H. contortus identified multiple distinct spots resolving on 2D gels with sequence similarity to Hc24 (9 spots) and Hc40 (4 spots), suggesting that H. contortus might also have a complex family of VALs (Yatsuda et al., 2003).

In the cattle GI nematode Ostertagia ostertagi, four VAL homologues, OoASP-1, OoASP-2, OoASP-3 (single-domain) and OoASP-4 (double-domain) have been characterized (Geldhof et al., 2003; Visser et al., 2008). Studies showed that OoASP-1 and OoASP-2 were the major antigens observed in the secreted products of adult worms (Geldhof et al., 2003), and these proteins were also localised in the reproductive tract of both male and female adults (Visser et al., 2008). OoASP-3 is also a single domain SCP-containing VAL which has been identified in adult secreted products, and localised to the pharyngeal region of the parasite (Visser et al., 2008). OoASP-4 has an apparent molecular weight of 49.6 kDa and 42% similarity to AcASP-1, and quantitative PCR analysis suggests that it is exclusively expressed in L4s (Visser et al., 2008).

RT-PCR analysis indicated that the mRNA encoding NbVALs were all present in the adult stage. In addition, mRNAs for NbVAL-2, 3, 7 and 8 were observed in activated L3s, with NbVAL-3 and NbVAL-8 showing the strongest expression (Fig. 5.1). However, only NbVAL-7 and NbVAL-8 were detected in L3 secreted products by western blot (Fig. 5.21b). There is a similarity here to AcASP-1 (Hawdon et al., 1996) and AcASP-2 (Hawdon et al., 1999), which are both secreted from infective larvae, suggesting that NbVAL-7 and NbVAL-

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8 might be important in the transition to parasitism. Although a 37oC stimulus is sufficient for activation and protein secretion in N. brasiliensis, it is possible that additional stimuli are required for secretion of specific proteins such as VAL-3.

AcHPI inhibits platelet aggregation by binding to the integrin GPIIb/IIIa (Del Valle et al., 2003). This may occur via the sequence Lys-Gly-Asp (KGD) which is a consensus sequence for interaction with integrins. Intriguingly, some of the NbVALs have potential integrin recognition sites (Fig. 5.8). For example the sequence Leu-Asp-Val (LDV), present in NbVAL-3 and NbVAL-7, is part of a major cell adhesion site (CS1) of the IIICS region of fibronectin, and is an important recognition motif for the integrin α4β1 (Very Late Antigen-4, VLA-4) (Komoriya et al., 1991; Mould et al., 1990; Wayner et al., 1989). The integrin α4β1 is a receptor for vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., 1990), is expressed on human lymphocytes (Hemler et al., 1987) and eosinophils (Bochner et al., 1991), and participates in the recruitment of these cells during inflammation. It is therefore possible that NbVAL-3 and NbVAL-7 may act as antagonists to the integrin α4β1, limiting inflammation during parasite infection.

NaASP-2 has also been suggested to significantly attract leukocytes in vivo by using an air pouch model and an in vitro system of neutrophil chemotaxis (Bower et al., 2008). It seems that NaASP-2 may function to assist/faster parasite migration through the recruitment of a neutrophils rich inflammation infiltrate which will increase tissue swelling and permeability, leading parasite survival and establishment in tissues of the host (Bower et al., 2008).

NbVAL-2, 4 and 5 contain an Asn-Gly-Arg (NGR) motif, which has been identified as part of the cell-binding region of fibronectin, and suggested to contribute to specific recognition by the major platelet receptor for matrix fibronectin, integrin α5β1 (Koivunen et al., 1994). The NGR motif also has been reported to specifically interact with the aminopeptidase N (CD13) showing that CD13 bound to tumor-homing peptide NGR for tumor vasculature, and its interaction was inhibited by anti-CD13 antibody, suggesting CD13 is the receptor for NGR peptides in tumors (Pasqualini et al., 2000). CD13 is a broadly distributed metallopeptidase which acts as a regulator of cytokines, antigen presentation, cell migration, and angiogenesis (Corti et al., 2008). There are therefore multiple potential biological roles

204 for VALs secreted by N. brasiliensis, as for other parasitic nematodes, which may act to enhance their survival in the mammalian host, but appropriate experiments to test these possibilities have not yet been reported.

B. malayi VAL-1 has been expressed in this laboratory as a secreted protein in Pichia pastoris, and, unlike AcNIF or AcHPI, does not inhibit binding of peripheral blood leukocytes to endothelial cells, and has no effect on platelet aggregation induced by agonists or binding of platelets to collagen and fibrinogen. In addition, BmVAL-1 does not appear to bind to any peripheral blood leukocyte when analysed by FACS does not stimulate calcium flux in these cells, and neither stimulates nor inhibits release of myeloperoxidase from granulocytes. It is also not directly chemotactic to granulocytes or mononuclear cells, and does not inhibit chemotaxis of these cells induced by a variety of chemokines such as interleukin-8, C5a or Monocyte Chemoattractant Protein-1. Although these data were obtained for just one parasite VAL, the function of the protein remains unclear (Mullen, unpublished data).

Analysis of protein domains via the ProDom domains server suggests that the NbVAL-3 N- terminal domain and the NbVAL-2 and NbVAL-8 C-terminal domains are similar to AcNIF (Fig. 5.9). It is known that AcNIF binds to the I domain of CR3, inhibiting neutrophil adhesion to endothelial cells and activation (Rieu et al., 1996). Importantly, a key component of the binding motif in integrin ligands is an acidic residue (Haas and Plow, 1994). Asojo et al. (2005) have predicted/modeled the interaction between AcNIF and CR3 showing that Glu142 of AcNIF fits into the MIDAS region. Furthermore, NbVAL-3 has a residue (Glu124) analagous to Glu142 of AcNIF in the primary and predicted tertiary structures (Figs. 5.9 and 5.10), suggesting that NbVAL-3 might be able to interact with the CR3 integrin. It has been reported that secreted products from N. brasiliensis L3 can somehow inhibit neutrophil recruitment in vitro (Keir et al., 2004). In addition, in H. contortus, a 55-kDa secretory glycoprotein (GP55) was purified from the secreted proteins of adult worms and has been suggested to inhibit host neutrophils and reduce H2O2 production via interaction with CR3 (CD11b/CD18) (Anbu and Joshi, 2008). The study demonstrated that GP55 was present in adult secreted products and localised at the parasite surface. As the protein cross-reacted with antibody to AcNIF antibody, the authors suggested that HcGP55 might perform a similar function to AcNIF in order to benefit parasite survival (Anbu and Joshi, 2008).

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One SCP protein, Tex31, from the venom of the cone snail, has been shown to have a serine protease activity (Milne et al., 2003). Sequence alignments and comparisons show that the residues (serine, histidine and glutamatic acid) which constitute the catalytic triad of serine proteases are conserved in many VALs from parasitic nematodes, including N. brasiliensis (Figs. 5.6 and 5.7). The most important point here however is whether these 3 residues are spatially close enough in the tertiary structure of the protein to interact in the catalytic mechanism. In human GAPR-1, the crystal structure has been determined, and it is proposed that a serine protease catalytic triad (Ser71, His54 and Glu65) could be formed at the interface of the dimer, ie one of the residues in the triad could be provided by the other subunit (Serrano et al., 2004; Fig. 5.12, panel D). Interestingly, the orientation of Ser164, His319 and Glu326 in NbVAL-3 were reasonably well conserved to the putative catalytic triad residues in GAPR-1 (Fig. 5.12, panels E and F). However, no experimental evidence has been provided to validate proteolytic activity in GAPR-1 or indeed any nematode VAL, and so this would have to be tested empirically for NbVAL-3.

Vaccination with parasite VALs has shown promise in reduction of parasite burdens in different systems. Immunization of recombinant AcASP-1 successfully led to a reduction of A. caninum third-stage larvae in mice. This effect seemed to be antibody dependent as passive protection was afforded by transfer of sera from vaccinated mice (Ghosh and Hotez, 1999). Subsequently, Sen et al. presented a similar result in BALB/c mice utilizing AcASP-1 as a vaccine, and further demonstrated that immunization of mice with recombinant ASP-1 from A. duodenale or N. americanus conferred protection against A. caninum, reflecting the sequence similarity of these ASPs (Sen et al., 2000). In addition, a study has indicated that the levels of anti-ASP-2 IgE are associated with the protection of heavy hookworm infection in humans (Bethony et al., 2005). Importantly, anti-ASP-2 antibody has been show to lead a significant reduction (60%) of L3 migration of skin invasion, suggesting that VALs may play a crucial role in the early stage of parasite invasion during the infection (Bethony et al., 2005). In the same study, recombinant AcASP-2 formulated with the GlaxoSmithKline Adjuvant (AS03) was found to confer a significant reduction in egg counts and intestinal parasite burden in vaccinated dogs (Bethony et al., 2005).

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Two A. ceylanicum ASPs, AyASP-1 (48 kDa) and AyASP-2 (30 kDa) with identities of 85% and 83% to AcASP-1 and AcASP-2 were cloned from the L3 stage (Goud et al., 2004). Soluble recombinants of AyASP-1 and AyASP-2 expressed in Pichia pastoris have also been examined as vaccine candidates, and these studies indicated that the AyASPs resulted in a significant reduction in the length of the adults, worm numbers and egg output, and inhibition of splenomegaly in hamsters (Goud et al., 2004; Mendez et al., 2005). Importantly, however, reduced antibody responses and lymphocyte proliferation observed during the trial suggested that the immunomodulatory effects of hookworm infection might affect and impair immune responses to the vaccine, indicating difficulties for design of clinical trials of human vaccines (Ghosh et al., 2006).

Expression of NaASP-2 in P. pastoris resulted in a soluble secreted product. Immunisation of rats resulted in antibodies which recognized ASP-2 and inhibited larval migration through skin in vitro (Goud et al., 2005). Recombinant NaASP-2 formulated with the adjuvant Alhydrogel induced a strong antibody response in the Sprague Dawley rat, elevation of IL-6 and a high level of IgG maintained up to three months post-immunization (Fujiwara et al., 2005). More recently, the NaASP-2 vaccine has been successfully tested and is undergoing clinical trials in humans (Bethony et al., 2008b). Phase-1 trials demonstrated that levels of anti-NaASP-2 IgG (primarily IgG1 and IgG4) were dramatically induced in 36 adult participants following a second immunization (112 days), and this antibody elevation lasted about 8 months post-vaccination. In addition, proliferative responses of peripheral blood mononuclear cells (PBMCs) collected from vaccinated participants were detectable at the end of the study. These studies suggested that NaASP-2 is a promising hookworm vaccine candidate, and phase-2 clinical trials are underway undergoing in hookworm-endemic areas (Bethony et al., 2008b). However, a trial of the NaASP-2 vaccine in previously hookworm infected Brazilian adults, it caused mild-to-moderate injection site pain and swelling, and also immediate-type hypersensitivity reactions from the volunteers, which indicates this vaccine is required further development of assay to detect pre-existing anti-NaASP-2 IgE before vaccination (Bethony et al., 2008a).

Vaccination of sheep with the VAL protein, Hc24 from H. contortus with a 15-kDa secreted product resulted in a certain reduction (>70%) of egg output and worm burden. Also, a strong

207 antibody (IgG1) response and increased mastocytosis were observed from the vaccinated animals, suggesting the antigens may mimic the natural response preventing the parasite infection (Schallig et al., 1997a). Unlike Hc24, immunization of cattle with rOoASP-1 derived from the baculovirus expression system did not develop any protection against O. ostertagi infection, and which is likely due to the recombinant protein misfolding (Geldhof et al., 2008).

The filarial nematode B. malayi has cDNA sequences for 3 closely related SCP proteins which are all predicted to be secreted, but only one (BmVAL-1) has been characterised in any details (Murray et al., 2001). This 28 kDa protein was detected by western blot in microfilarial and infective L3 somatic extracts, but only in secreted products from microfilariae (Murray et al., 2001). Studies from our lab have shown that BmVAL-1 is secreted by microfilaria, but not adult worms. It was faintly detected by western blot in adult females but not males, suggesting that this signal was due to expression in microfilariae (Rees-Roberts, 2007). Immunisation of gerbils with recombinant BmVAL-1 resulted in a 64% mean reduction of establishment of adult worms on challenge infection, although because of the inherent variability of parasite recoveries, this did not reach statistical significance and infective L3 somatic extracts, but only in secreted products from microfilariae (Murray et al., 2001).

Another filarial nematode Onchocerca volvulus is also predicted to express 3 secreted SCP proteins, termed OvASP-1-3. Recombinant OvASP-1 expressed in E. coli was reactive with IgG isotypes in sera from parasite-infected or putatively immune individuals, with higher IgG4 but no detectable IgE (MacDonald et al., 2004). Reaction of rOvASP-1 with PBMCs from infected and putatively immune individuals showed a significant production of IFN-γ, with elevated IL-5 only in the infected patients. Also, vaccination of mice with rOvASP-1 formulated with Freund’s complete adjuvant (FCA) or alum, led to partial protection against infection. Although immunization of mice with rOvASP-1 without adjuvant evoked Th1 (IgG2a) and Th2 (IgG1) responses, no striking reduction of L3 infection was observed (MacDonald et al., 2004). When rOvASP-1 was administered with ovalbumin (OVA), it not only effectively induced a high Th1/Th2 mixed response, but also stimulated production of IFN-γ (Th1) but not IL-5 (Th2) from spleen cells in mice (MacDonald et al., 2005).

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Subsequent studies have confirmed that rOvASP-1 exhibits a Th-1 adjuvant effect which appears to be independent of any contaminating bacterial LPS (He et al., 2009; MacDonald et al., 2008). He et al. presented that rOvASP-1 could bind to human PBMCs, mature/activate dendritic cells (DCs) and elicit Th1-biased cytokine production through Toll-like receptor 2 (TLR2) and TLR4 activation (He et al., 2009). However, since the rOvASP-1 was expressed in E. coli, there is no guarantee that the protein is correctly folded, and the adjuvant effect might actually depend upon incorrect folding and fortuitous interaction with an appropriate receptor. Expression of nematode VALs in E. coli generally results in the production of insoluble recombinant proteins (Hotez et al., 2003; Monsalve et al., 1999a, b). This is likely due to the high number of disulphide bonds in VALs, which will either not be formed in the reducing environment of the cell cytosol or improperly formed. NbVALs generally contain 10 and 20 cysteine residues predicted to contribute to 5 and 10 disulphide bonds in the single and the double VALs respectively (Figs 5.6 and 5.7).

As well as the proteins already described, SCP-like sequences have also been identified in many parasitic nematodes from all major clades examined (Cantacessi et al., 2009b; Ding et al., 2000; Gao et al., 2001; Tetteh et al., 1999). In addition to being prime targets for vaccine development, these proteins presumably play important role(s) in the infectious process, and because they are secreted, most probably interact with host molecules in manner(s) which have yet to be determined to promote parasite survival. Development of RNA interference would be an important tool in deciphering these roles.

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Chapter 6

Conclusions

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Gastrointestinal (GI) nematode infections are a major burden on the health and development of humans or livestock. The use of anthemintic chemicals (albendazole or mebendazole) is the main means to control parasitic nematode infections, but the emergence of drug-resistant parasites appears relatively common in several areas of the world (Geary et al., 2010; Kaplan, 2004). Definition of new drug targets and development of anti-nematode vaccines are therefore highly desirable objectives.

Recently, proteomics analysis has been used to identify peptides defining the secretory proteins of nematode parasites (Bennuru et al., 2009; Hewitson et al., 2008; Kiel et al., 2007; Moreno and Geary, 2008; Mulvenna et al., 2009; Nisbet et al., 2008; Robinson et al., 2007; Smith et al., 2009; Yatsuda et al., 2003). In addition, it is believed that a number of secreted products from parasitic nematodes are involved in the modulation/interference of host immune responses during infection in order to promote invasion and aid parasitism (Hewitson et al., 2009). Therefore, analysis of secretome and transcriptome data (Ghedin et al., 2007; Harcus et al., 2004; Li et al., 2009; Maizels et al., 2000; Rabelo et al., 2009) could provide useful information to investigate and reveal potential vaccine candidates for challenging GI nematode infectious diseases.

To develop a means of defining genes which encode proteins that are important for infectivity of parasitic nematodes is desirable. RNA interference (RNAi) is a powerful method for the functional analysis of specific or novel genes, and has been well developed and intensively used in the free-living nematode C. elegans (Fire et al., 1998). Although RNAi has been applied to several animal parasitic nematodes such as N. brasiliensis (Hussein et al., 2002b), B. malayi (Aboobaker and Blaxter, 2003), O. volvulus (Lustigman et al., 2004), A. suum (Islam et al., 2005), T. colubriformis (Issa et al., 2005), H. contortus (Kotze and Bagnall, 2006), L. sigmodontis (Pfarr et al., 2006), Ostertagia ostertagi (Visser et al., 2006), A. viteae (Tachu et al., 2007) and H. polygyrus (Lendner et al., 2008), the results are inconsistent, and solid evidence for reliable knockdown of gene transcripts has not been obtained. Gene silencing by RNAi works well in C. elegans but not in animal parasitic nematodes, which might be due to the lack of genes and their products important in the RNAi pathway, resulting either in defective uptake of processing of dsRNA (Ghedin et al., 2007; Lendner et al., 2008). Both sid-1 and sid-2 have been shown to be critical for uptake,

211 transport and intracellular spreading of exogenous RNA in C. elegans (Feinberg and Hunter, 2003; Winston et al., 2002; Winston et al., 2007); and this mechanism makes C. elegans more amenable that other Caenorhabditis species to distinct delivery approaches such as feeding and soaking.

C. elegans has a simple life cycle, and its development can be completed in vitro. Importantly, RNAi can be easily applied to mediate gene silencing at most stages, and is inheritable so that phenotypic effects can be seen in offspring (Fire et al., 1998; Grishok et al., 2000; Tabara et al., 1998; Timmons and Fire, 1998). In contrast, the life cycle of animal parasitic nematodes is more complicated, notably the infective stage larvae (L3) of strongylids are sheathed with a cuticle, and do not feed and develop in the environment until they receive appropriate stimuli from the mammalian host following infection. Both this and previous studies on A. caninum L3 (Hawdon and Schad, 1990, 1991, 1992, 1993; Hawdon et al., 1992) showed that strongylid nematodes can be activated to feed in vitro by exposure to factors which mimic those encountered in the host. This process was studied in order to improve exposure of infective larvae to exogenous RNA, with the aim of maximising the possibility of successful RNAi.

Activation of third-stage larvae in N. brasiliensis was shown to be initiated solely by elevated temperature (37°C), occurring independently of exposure to host serum or glutathione. Nevertheless, RNAi-mediated gene silencing in activated larvae was still unsuccessful by the methods of electroporation, soaking or polymer (polyethyleneimine, PEI)-mediated introduction of dsRNA or siRNA. Although serotonin (5-HT) could significantly improve the level of uptake of macromolecules, L3 exposed to exogenous dsRNA with 5-HT still failed to show RNAi-mediated gene silencing. Unfortunately, RNAi silencing was also not observed in adult N. brasiliensis using the same delivery methods.

C. elegans SID-2 has been shown to function in the intestine to transport environmental RNA into the worm, and CeSID-1 subsequently plays a role in systemic spreading of RNAi (Winston et al., 2007). Based on the draft genome survey, a SID-2 homologue may exist in N. brasiliensis, but SID-1 seems to be missing. PEI-mediated introduction of RNA into N. brasiliensis L3 indicated that the intestinal and pharyngeal regions were engorged with an

212 intense Cy3-labelled material, but systemic spreading into parasite organs appeared not to occur. It is thus possible that NbSID-2 is not fully expressed in the intestine to facilitate RNA uptake, even though general gene expression of the parasite is up-regulated during activation. Also, transport of exogenous RNA into parasite cells may not be possible without SID-1. On the other hand, key regulatory proteins (e.g. RDE-4 and RDE-2) of the RNAi machinery appear not to be present in N. brasiliensis, and this could possibly explain why RNAi is inefficient.

Furthermore, the data in this thesis showed that a firefly luciferase mRNA was introduced and expressed in human cell lines (293 T cells and J774) by electroporation and soaking, in addition to biolistic bombardment (Ball, personal communication). However, this could not be replicated in either N. brasiliensis adults or temperature-activated L3. It has been suggested that the failure of RNAi in animal parasitic nematodes may be due to inappropriate approaches to introduce RNA into the body of worms (Viney and Thompson, 2008), but this does not explain why RNAi has been relatively successful in plant-parasitic nematodes. The cuticle is likely to present a physical obstruction to block electroporation-mediated delivery, but soaking should promote uptake via the intestine if appropriate RNA transporters are present.

Meanwhile, RNAi in animal parasitic species has been reported to have been attempted for 29 genes, with successful knockdown for only 14 genes. It is difficult to understand why some genes examined appear to be more susceptible to RNAi than others based on the limited available information from publications. Geldhof et al. have proposed that differences in stage specific expression, stability or mRNA turnover of target genes might influence susceptibility to RNAi (Geldhof et al., 2007), although no obvious pattern has emerged so far. A more systematic analysis might be possible, although whole genome sequencing has only been performed for B. malayi and H. contortus thus far (Ghedin et al., 2007).

An approach developed for in vivo RNAi in mammals has been to chemically conjugate siRNA to cholesterol, which can then interact with high-density (HDL) and low-density lipoprotein (LDL) receptors. These particles can then be taken up by cells via receptor- mediated endocytosis, and this approach was shown to enable uptake of siRNA from serum,

213 which led to effective down-regulation of apolipoprotein B (ApoB) expression in liver (Soutschek et al., 2004; Wolfrum et al., 2007). Therefore, a similar approach of receptor- mediated endocytosis could be atempted for N. brasiliensis, although there is little to no information on these processes in parasitic nematodes. Nevertheless, this approach could possibly elucidate whether the import of environmental RNA or components of the RNAi machinery are defective or not in animal parasitic nematodes. In addition, further analysis of the available EST sequences (Harcus et al., 2004) and draft genome data (Wellcome Trust Sanger Institute) of N. brasilensis, integrated with gene expression studies should be performed, and this could be an valuable window for study of RNAi and validation of new therapeutic/vaccine targets in parasitic worms.

VAH/ASP-Like (VAL) molecules have been reported as a major component of secreted proteins from many species of parasitic nematodes (Cantacessi et al., 2009a), and this thesis demonstrates that this is also the case for N. brasiliensis. However, the function of parasite VALs still remains completely undefined. Ancylostoma caninum secreted protein-1 (AcASP- 1) was the first nematode parasite VAL to be described, which with another VAL (AcASP-2) was shown to be abundantly secreted by serum- activated A. caninum L3 in vitro (Hawdon et al., 1996; Hawdon et al., 1999). The crystal structure of a Necator americanus VAL, NaASP- 2, has been determined, and similarities have been highlighted with CC chemokines (Asojo et al., 2005). It has been suggested that NaASP-2 promotes recruitment of cells, predominantly neutrophils and monocytes, but the chemotactic index was extremely low (Bower et al. 2008). Work in this laboratory with B. malayi VAL-1 has demonstrated however that BmVAL-1 is not directly chemotactic to human peripheral blood granulocytes or mononuclear cells, and does not antagonise chemotaxis of these cells to known chemokines. Moreover, B. malayi VAL-1 does not appear to bind to any peripheral blood leukocyte, and so the function of this class of proteins remains unclear (Mullen, unpublished data). Nevertheless, vaccination with NaASP-2 has been shown to reduce parasite burdens in animal models and induce a protective response in vitro and in vivo, and clinical trials of a recombinant vaccine are underway (Bethony et al., 2008b; Fujiwara et al., 2005; Goud et al., 2005).

In this thesis, eight NbVALs were identified, all of which contain predicted signal peptides. Western blot analysis using NbVAL antisera with different stages of the parasite suggested

214 that all NbVALs were present in secreted products of the adult worms, although it is possible that there is some immunological cross-reactivity between the proteins. NbVAL-7 and NbVAL-8 were also detected in secreted products from N. brasiliensis infective stage larvae (L3). The data also showed that N. brasiliensis L3 were activated to secrete these proteins while receiving an elevated temperature (37°C) cue from the environment. Therefore, presumably, among this range of secreted proteins, NbVAL-7 and NbVAL-8 might play an important role in the transition to parasitism. Additionally, RT-PCR showed that NbVAL-3 appeared to be highly expressed in activated L3, but no detectable protein secretion was observed. It is possible that VAL-3 is not secreted by activated L3, or that stimulation of release of NbVAL-3 from secretory granules is influenced by specific host factors, like AcASP-1 and AcASP-2 (Hawdon et al., 1996; Hawdon et al., 1999)

A. caninum neutrophil inhibitory factor (AcNIF) is structurally related to VALs, and can be secreted by the parasite to antagonise the CD11b/CD18 integrin expressed on neutrophils, thus blocking adhesion to endothelium cells and inhibiting recruitment of neutrophils to the site of inflammation (Moyle et al., 1994; Rieu et al., 1994). Protein domain analysis and sequence comparison indicated that the N-terminus of NbVAL-3 or the C-terminus of NbVAL-2 and NbVAL-8 share a 25% identity with AcNIF. NbVAL-3 has a conserved residue (Glu124, which aligns with AcNIF Glu142), which is a key acidic residue suggested to be a critical component of the binding motif for integrin ligands (Haas and Plow, 1994). Thus, NbVALs might serve a similar role in either larval or adult stages of N. brasiliensis, enabling the parasite to interfere with inflammation in order to establish in the host. Furthermore, comparison of NbVALs against the Structural Database of Allergenic Proteins (SDAP) showed that NbVALs are structurally similar to insect venom allergens (i.e. yellow jacket allergen, Ves v 5), suggesting that VAL proteins might also act as an allergen. NaASP- 2 has been observed to be allergenic, as immediate-type hypersensitivity reactions were observed in a phase-1 trial of the hookworm vaccine in previously infected Brazilian adults, and further clinical trials have been suspended (Bethony et al., 2008a). Determination of in vivo biological activities of NbVALs is therefore further required.

Data in this thesis show that protein secretion of N. brasiliensis L3 is dependent solely on exposure to a 37°C temperature cue, and that it can be restricted by inhibitors of PI3 kinase,

215

Akt kinase and cytochrome P450. This differs considerably from A. caninum, in which protein secretion has been reported to be unaffected by the PI3 kinase inhibitor, and therefore it seems that the transcriptional response and protein secretion behaviour in response to activation of parasitic nematodes can be quite distinct. Harcus et al. have shown that a high proportion of sequences from a N. brasiliensis EST dataset (1,234 sequences) represent novel genes, and intriguingly, that a lot of these sequences (32%) code for predicted signal peptides, suggesting that they have evolved for parasite-specific functions presumably related to the host environment (Harcus et al., 2004) Therefore, investigating the variety of profiles and functions of secreted proteins could lead to a better understanding of critical events in host invasion and evasion of the immune system during parasitic nematode infections. The most direct and effective means of attempting to understand the roles of these parasite secreted proteins would be via functional RNAi silencing. Therefore, if RNAi can be successfully developed and applied to animal parasitic nematodes, this could be an extremely beneficial tool which combined with other molecular and cellular approaches, could lead to the discovery of novel targets for drug and vaccine development.

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Appendices

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Appendix. A. The firefly Photinus pyralis luciferase complete coding sequence (Genbank accession number: M15077)

CATTCCGGTACTGTTGGTAAAGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGC GCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGAT ACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTAC GCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATAC AAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCG CGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTC AACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAAT TTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGG ATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAAT GAATACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTC CTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGAT TCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGT GTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATT TCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACA AGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATT GACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGA AGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCA CTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGT AAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGT TAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATC CGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTAC TGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGATTAAGTACAAAGG CTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCAACACCCCAACATCTTCGACG CAGGTGTCGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTG GAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAAC CGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAA AACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCC GTGTAATTCTAGAGTCGGGGCGGCCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGA GTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATG CTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATT C

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Appendix. B. N. brasiliensis acetylcholinesterase subunit B (NbAChE-B) sequence (Genbank accession number: AF052508) ttcggcacgaggatgggacttcccgctagattactcctggcaatatgcgtcttctcgacgtc ggccgctgatgatggtccgacggtggtgctttcctcgggaacaaaaattcatggaatataca tggatgtaaacggacaaacggtgagcgcctacctaggtgttccatttgctacggcggagagg tttgcgatgccgacgttaaccgaaacctacggcggagatattgaagctctacagctttcaaa aacctgtttccaaaccaaagacgaaacgtacccgggatttgatggtgctgaaatgtggaatc caccaacggagctttcagaagattgtctgagtttgaacatctgggtcccggaaaatcccgat ggcaatgtgattgtttggatttatggaggaggtttcttcagtggttccccgtcgcttgcact ttacaatggatccgtgctggccggtaaaacgaacgcagtagttgtgaatgtcaactatcgag ttggaccattcggcttcttctacctcggagccaactcaaaggcacctggaaatgttggtttg ctggatcaaactgcactgaaatggattcataataacatagaatacttcaaaggagatcctag caaggttacgcttttcggtgaatcggctggtggcacctctgtcacgtctcatctgctggccc ccgacagccacagcttgttcagcaaaatcattgttaatagcggatctatacataatgtatgg gcgactagaagtccatgtactatgctacacatctcaatgaaaacagcgaaggcactaggatg tgtcgaaaattacgagatcaaacccatcagagaagcagagggtcgatgcactgtattgggag cagacgctgatacgatttacgagtgcatgaaggagaagacaccggagaagattcagagcgag ggcaactctgacgcgatatatgctgagatgcttccgatggagtggccatttggtccgatcac ctacgacgataactacttcaagggagaagtacggcgaaagctcttcagcggtgagttcaaga cggacgtgtcggcgatcttcggaacggtgaaggacgaaggaacattctggctaccgtactac ctgtctgagagtggcttcagtttctttcctgaccaggattctgacagtgaggcaaacgcagc aaaaatagacgaggccaactatacggcatcaatgcaagctttcgaggggtactttggaaaga gcagcaaagctatagagatcctcaaggaaggattcaaagatttgggtgatgtacaaacaatg tatcgcgacggtgttgctcgattcgttggagacttcttctttacctgcaatttggtggaatt tgtcgatcatgtcacccagaagattagcgaagcctacatgtactacttcaaagcacgatctt ccgcaaacccatggccaaaatggatgggagtcatgcacggatacgagatcgaatatgaattc ggatatcctttcatcaactccactgcttacaaggaagtagacaaggatcgaacaatctccga ggagttcatgcaactaatcaaggaattcgtaaagaacggaaagtttgatgacgaatggccga aatatgagggaggaaaggtaatggtgattgaggacgacgcttcgagaaagatcgaagacaaa gacatccagcagaaatattgcaaaatcattaatgatgccagacaagctctaattgatgaagc taaaggcaactagtttccattcaaatcgggacgacagtatcgaccgaaatgatacagcctgt gataaatctttctatgccgcagcgaacaataaatgattcataaacaaaaaaaaaaaa

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Appendix. C. N. brasiliensis ubiquitin (NbUbq) EST partial sequence (Genbank accession number: BM279528)

ACAGCTCGAAGATGGACGTACTCTTTCCGACTACAACATCCAGAAGGAGTCTACCCTTCACC TCGTCCTTCGTCTCCGAGGAGGCATGCAGATTTTCGTCAAAACCCTCACTGGGAAAACCATC ACTCTCGAAGTTGAAGCGTCGGACACCATCGAAAATGTTAAGGCAAAGATCCAGGACAAGGA AGGAATCCCACCAGATCAGCAGCGATTGATCTTTGCTGGAAAGCAGCTCGAGGATGGACGTA CTCTCTCCGACTACAACATCCAGAAGGAATCTACCCTTCATCTCGTCCTTCGTCTTCGGGGA GGCATGCAGATATTCGTGAAGACTCTCACCGGAAAGACCATCACCCTCGAAGTTGAAGCTTC CGACACAATCGAAAATGTCGAAGCGAATATTCACGACAAGGAAGGAATCCCACCAGATCAGC AACGATTGATCTTTGCTGGAAAGCAACTCGAAGATGGACGCACA

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