Identification, Characterization, and Expression of Latrophilin-like Proteins in Parasitic Nematodes
A thesis submitted for the degree of Doctor of Philosophy (PhD) in the subject of Parasitology
by
Claudia Annette Felicitas Welz (formerly Ram), Veterinarian
May 2007
International PhD program “Infection Biology”
Institute for Parasitology
University of Veterinary Medicine Hannover
Identification, Characterization, and Expression of Latrophilin-like Proteins in Parasitic Nematodes
A thesis submitted for the degree of Doctor of Philosophy (PhD) in the subject of Parasitology
by
Claudia Annette Felicitas Welz (formerly Ram), Veterinarian
May 2007
International PhD program “Infection Biology”
Institute for Parasitology
University of Veterinary Medicine Hannover
PhD project funded by
the Ministry for Science and Culture of Lower Saxony through the Georg-Christoph-Lichtenberg Scholarship scheme, the University of Veterinary Medicine through a Research Stipend,
the Hannover Biomedical Research School through a stipend, and the BAYER HealthCare AG
Acknowledged by the PhD committee and head of Hannover Medical School
President: Prof. Dr. Dieter Bitter-Suermann
Supervisor: Prof. Dr. Georg von Samson-Himmelstjerna Co-supervisor: Prof. Dr. Dr. Achim Harder
External expert: Prof. Lindy Holden-Dye Internal expert: Prof. Dr. Andreas Klos
Day of final exam/public defense: 06 July 2007
Πάντα ε, οδν ένει.
Everything flows, nothing stands still.
Heraklit (540 – 480 ante Christum)
To my big, great family
Contents 9
1 Contents
1 Contents... 9
2 Summary... 15
3 Literature... 17
3.1 Introduction... 17
3.2 Parasites ... 17
3.2.1 Taxonomy ... 17
3.2.2 Morphology of Trichostrongylids... 18
3.2.3 Biology of Trichostrongylids... 18
3.3 The Model Nematode Caenorhabditis elegans... 21
3.3.1 Taxonomy ... 21
3.3.2 Morphology and Biology of C. elegans... 22
3.4 G-protein Coupled Receptors ... 22
3.5 Tools for Functional Gene Analysis in C. elegans... 23
3.5.1 Inactivation of Genes ... 23
3.5.2 Expression of Heterologous Genes in C. elegans... 25
3.6 Neurobiology of Nematodes... 26
3.6.1 Neurotransmitters ... 26
3.7 Control of Parasites... 31
3.7.1 Benzimidazoles... 31
3.7.2 Nicotinic Agonists ... 32
3.7.3 Macrocyclic Lactones ... 32
3.7.4 Closantel ... 32
3.7.5 Piperazine ... 33
3.8 Anthelmintic Resistance... 33
3.9 Cyclooctadepsipeptides ... 34
3.9.1 PF1022 A ... 34
3.9.2 Emodepside ... 34
3.9.3 Cyclohexadepsipeptides... 36
3.10 Cyclooctadepsipeptides: Mechanism of Action... 36
3.10.1 Involvement of the GABA System ... 36
3.10.2 Influence of Cyclooctadepsipeptides on Effects of Neurotransmitters... 36
3.10.3 Effects of Emodepside on C. elegans... 36
3.10.4 Hc110-R ... 37
3.10.5 Latrophilin-like Protein 1 in C. elegans... 38
3.10.6 Latrophilin-like Protein 2 in C. elegans... 42
3.11 Black Widow Spider Venom and α-Latrotoxin ... 42
3.12 Mammalian Latrophilins ... 43
3.13 Potassium Channels ... 44
10 Contents
3.13.1 BK-type Potassium Channels... 44
3.13.2 The Potassium Channel SLO-1 in C. elegans... 45
3.13.3 The Potassium Channel SLO-2 in C. elegans... 46
3.14 Real-time PCR ... 47
3.14.1 Detection of PCR Products ... 47
3.14.2 Quantification of Template ... 48
4 Objectives ... 51
5 Material and Methods ... 53
5.1 Maintenance and Collection of Parasites... 53
5.1.1 Haemonchus contortus... 53
5.1.2 Cooperia oncophora and Ostertagia ostertagi... 53
5.1.3 Collection of Adult Nematodes ... 53
5.1.4 Collection of Eggs... 54
5.1.5 Collection of Larvae ... 54
5.2 Mammalian Tissue ... 55
5.3 Isolation of RNA ... 55
5.3.1 Trizol® Method ... 55
5.3.2 QuickPrepTM Micro mRNA Purification Kit ... 56
5.3.3 Quantification of RNA ... 57
5.4 cDNA Synthesis ... 57
5.4.1 3’ RACE System for Rapid Amplification of cDNA Ends ... 58
5.4.2 BD SMARTTM RACE cDNA Amplification Kit... 58
5.5 Primer Design ... 60
5.6 Polymerase Chain Reaction (PCR) ... 60
5.6.1 Qiagen Taq DNA Polymerase ... 61
5.6.2 BD Advantage® 2 Polymerase Mix ... 61
5.6.3 Rapid Amplification of cDNA Ends (RACE)... 62
5.6.4 PhusionTM Hot Start DNA High-Fidelity Polymerase ... 64
5.7 Analysis of PCR Products ... 64
5.8 Isolation of DNA Bands ... 65
5.9 Cloning of PCR Products ... 65
5.9.1 Ligation Using the TOPO TA Cloning® Kit for Sequencing... 65
5.9.2 Ligation Using the StrataCloneTM PCR Cloning Kit... 66
5.9.3 Ligation Using the QIAGEN® PCR Cloning Kit ... 66
5.9.4 Ligation using the Zero Blunt® PCR Cloning Kit ... 66
5.10 Transformation ... 67
5.11 Bacterial Cultures for Plasmid Analysis ... 67
5.11.1 Antibiotics ... 67
5.12 Glycerol Stocks ... 67
5.13 Preparation of Plasmid DNA ... 68
5.13.1 MiniPrep ... 68
Contents 11
5.13.2 MidiPrep ... 68
5.14 Quantification of DNA... 69
5.15 Analysis of Plasmids using Restriction Enzymes ... 69
5.16 Sequencing ... 69
5.17 Sequence Analysis... 70
5.17.1 Aligning and Handling of Sequences ... 70
5.17.2 Prediction of Transmembrane Domains and Signal Peptides ... 70
5.17.3 Detection of Conserved Domains... 71
5.17.4 Phylogenetic Analysis ... 71
5.18 Prokaryotic Expression ... 72
5.18.1 Attaching Restriction Sites ... 72
5.18.2 Digestion of Plasmids for Cloning into pENTRTM 3C... 73
5.18.3 Double Digest ... 74
5.18.4 Ligation ... 74
5.18.5 Gateway® LR ClonaseTM Reaction ... 75
5.18.6 Empty-vector Control... 76
5.18.7 TSS (Transforming and Storing Solution) Transforming Procedure ... 76
5.18.8 Regulation of Expression ... 77
5.18.9 Inducing Expression Cultures... 77
5.18.10 Other Bacterial Strains ... 78
5.18.11 Other Expression Vectors ... 78
5.18.12 Coexpression... 79
5.19 Cell Lysis ... 80
5.20 MagneHisTM Protein Purification System ... 80
5.21 Isolation of Inclusion Bodies... 81
5.22 Refolding of Solubilized Proteins ... 81
5.22.1 Dilution and Refolding Buffer... 81
5.22.2 FPLC and HisTrapTM HP Column... 82
5.23 Protein Analysis ... 82
5.23.1 Conventional Methods... 82
5.23.2 NuPage® System... 83
5.23.3 Coomassie Staining... 83
5.23.4 Drying SDS Gels ... 84
5.23.5 Immunoblot... 84
5.23.6 Testing of Sera ... 85
5.23.7 Analysis by MALDI-MS... 85
5.24 Prokaryotic Expression: Functional Assays ... 86
5.24.1 α-LTX... 86
5.24.2 On-Blot Binding of α-LTX ... 86
5.24.3 Dynabeads® M-270 Carboxylic Acid ... 87
5.25 Eukaryotic Expression... 89
12 Contents
5.25.1 Expression Vectors... 89
5.25.2 Maintenance of Eukaryotic Cells ... 89
5.25.3 Transient Transfection with LipofectamineTM... 90
5.25.4 Transient Transfection with FuGENE 6 Transfection Reagent ... 90
5.26 Eukaryotic Expression: Functional Assays ... 91
5.26.1 Ca2+ Influx Measurement after Transfection with LipofectamineTM... 91
5.26.2 Ca2+ Influx Measurement after Transfection with FuGENE ... 92
5.27 Preparation of Raw Antigen ... 93
5.28 Isolation of Membrane Proteins ... 93
5.29 Protein Quantification with CB-XTM Protein Assay... 94
5.30 Removal of Detergents from Protein Samples... 94
5.31 Specific Anti-Hc110-R Antibodies ... 95
5.32 Construction of Plasmids for Expression in C. elegans... 97
5.33 Real-time PCR ... 100
5.33.1 Design of Primers and Probes for Real-time PCR ... 100
5.33.2 Plasmid DNA Dilution Series... 100
5.33.3 RNA Isolation for Real-time PCR ... 101
5.33.4 DNase Digestion... 101
5.33.5 cDNA Synthesis for Real-time PCR ... 102
5.33.6 Testing for Absence of Genomic DNA ... 102
5.33.7 Real-time PCR Run ... 103
6 Results ... 107
6.1 Depsiphilins (Latrophilin-like Protein 1, lat-1)... 107
6.1.1 Sequences ... 107
6.1.2 Identities between Depsiphilin Sequences ... 108
6.1.3 BLAST Results for Depsiphilin Sequences ... 109
6.1.4 Prediction of Transmembrane Domains and Signal Peptides... 109
6.2 Latrophilin-like Protein 2 (lat-2) ... 110
6.2.1 Identities between lat-2 Sequences... 110
6.2.2 BLAST Results for lat-2 Sequences ... 111
6.2.3 Prediction of Transmembrane Domains and Signal Peptides... 112
6.3 Comparison of Sequences... 112
6.4 Comparison of Predicted Conserved Domains... 115
6.5 The BK-type Potassium Channel SLO-1 (slo-1) ... 116
6.5.1 Identities between slo-1 Sequences... 117
6.5.2 BLAST Results for slo-1 Sequences ... 118
6.5.3 Comparison of SLO-1 Sequences ... 119
6.5.4 Prediction of Transmembrane Domains and Signal Peptides... 120
6.5.5 Prediction of Conserved Domains ... 121
6.6 Bovine and Canine LPH-2... 121
6.7 Prokaryotic Expression ... 122
Contents 13
6.7.1 FPLC... 123
6.7.2 Expression of Canine and Bovine LPH-2 N-termini... 123
6.7.3 MagneHisTM Protein Purification ... 123
6.7.4 Antibodies ... 124
6.7.5 Identification of Protein ... 126
6.7.6 Functional Assays... 127
6.8 Eukaryotic Expression... 131
6.8.1 Ca2+ Influx after Transfection with LipofectamineTM... 131
6.8.2 Ca2+ Influx after Transfection with FuGENE ... 135
6.8.3 Detection of Recombinant Protein in Western Blot ... 139
6.9 Plasmids for Expression of Depsiphilin Genes in C. elegans... 140
6.10 Real-time PCR ... 140
6.10.1 Testing for Absence of Genomic DNA ... 140
6.10.2 Definition of Standards ... 142
6.10.3 Comparison of Amplification Efficiencies ... 143
6.10.4 Real-time PCR Products ... 143
6.10.5 Analysis of Real-time PCR Raw Data ... 145
6.10.6 Analysis of Relative Amounts of Transcripts... 145
6.10.7 Analysis of Copy Numbers ... 149
7 Discussion... 151
7.1 Depsiphilins... 151
7.1.1 Sequences ... 151
7.1.2 Prediction of Conserved Domains in Depsiphilins... 152
7.2 Latrophilin-like Protein 2... 154
7.2.1 Sequences ... 154
7.2.2 Prediction of Conserved Domains in LAT-2 ... 154
7.3 Expression of Isolated N-termini of Depsiphilins ... 155
7.3.1 Antibodies ... 156
7.3.2 Functional Assays on Isolated N-termini ... 157
7.4 Eukaryotic Expression... 159
7.4.1 Western Blot of Membrane Protein... 159
7.4.2 Calcium Influx Measurements ... 162
7.5 Real-time PCR ... 166
7.5.1 Quantification Methods ... 167
7.5.2 Evaluation of Real-time PCR Data ... 168
7.5.3 Transcription Levels of Depsiphilin Normalized to 18 S rRNA ... 169
7.5.4 Significance of Real-time PCR Data... 169
7.6 Heterologous Expression of Depsiphilin in C. elegans... 170
7.7 BK-type Potassium Channel SLO-1 Sequences... 171
7.7.1 Prediction of Transmembrane Regions in SLO-1... 171
7.7.2 Prediction of Conserved Domains in SLO-1... 172
14 Contents
7.8 Conclusions and Perspectives... 173
8 Appendix ... 177
8.1 Additional Data... 177
8.1.1 Predicted Transmembrane Domains... 177
8.1.2 Real-time PCR Raw Data ... 179
8.1.3 Comparison of Real-time PCR Amplification Efficiencies ... 187
8.2 Important Plasmids ... 190
8.2.1 Plasmids for Real-time PCR Standardization... 194
8.3 Published Sequences ... 195
8.3.1 Sequences Used for the Design of Primers ... 195
8.3.2 Sequences Used for Alignments of Genes... 196
8.4 Material... 199
8.4.1 Commercial Primers and Primers as Components of Kits ... 199
8.4.2 Custom Primers ... 199
8.4.3 Oligonucleotides for Real-time PCR... 200
8.4.4 Antibody Concentrations... 203
8.4.5 Escherichia coli Strains... 204
8.4.6 Vectors... 206
8.4.7 Eukaryotic Cell Lines ... 207
8.4.8 Buffers and Solutions... 208
8.4.9 Media ... 212
8.4.10 Reagents and Chemicals ... 212
8.4.11 Enzymes... 214
8.4.12 Commercial Kits ... 215
8.4.13 Disposables ... 216
8.4.14 Technical Equipment... 216
8.4.15 Software ... 219
8.4.16 Databases ... 220
8.4.17 Custom Services ... 220
8.5 Abbreviations ... 221
8.6 IUPAC Code for Nucleotides... 227
8.7 Standard Amino Acid Abbreviations ... 228
8.8 Reference List ... 229
Curriculum Vitae... 250
List of Publications ... 252
Declaration ... 255
Acknowledgements / Danksagung... 256
Summary 15
2 Summary
Claudia Welz (2007): Identification, Characterization, and Expression of Latrophilin-like Proteins in Parasitic Nematodes
Emodepside belongs to the cyclooctadepsipeptides, a new class of anthelmintically active drugs, and is effective against many parasitic nematodes. In the sheep parasite Haemonchus contortus and in the free-living nematode Caenorhabditis elegans putative G-protein coupled receptors (GPCRs) were previously identified as targets for emodepside. In the present work orthologous receptors were identified in the cattle trichostrongylids Cooperia oncophora and Ostertagia ostertagi. These receptors were named depsiphilins. They showed 88 % amino acid sequence identity with their ortholog Hc110-R in H. contortus, 45 – 47 % identity with the orthologous receptor latrophilin-like protein 1 (LAT-1) in C. elegans, and identities of up to 26 % with mammalian latrophilins (LPH). Hc110-R, C. elegans LAT-1, and mammalian LPH are known to bind α-latrotoxin (α-LTX), a component of the black widow spider venom. The newly identified receptors in parasites were examined as recombinant proteins for their binding affinities for α-LTX. Experiments with the isolated N-termini failed to confirm their role as specific binding partners, potentially due to incorrect folding of the truncated receptors. The results of transiently transfected eukaryotic cells expressing full-length receptors were ambiguous; further experiments are planned. Specific polyclonal antibodies against the N-terminus of Hc110-R were developed and successfully tested. Depsiphilins were shown to be transcribed in males, females, eggs, mixed first and second stage larvae, and third stage larvae of H. contortus and O. ostertagi by real-time PCR. An expression plasmid for the heterologous expression of O. ostertagi depsiphilin in C. elegans was prepared.
A further potential target for emodepside was identified in H. contortus and C. oncophora, the putative GPCR latrophilin-like protein 2. It shared structural features with depsiphilins, C. elegans LAT-1, and mammalian LPH. Complete coding sequences for the calcium-gated potassium channel SLO-1 were identified in H. contortus and C. oncophora and, as preliminary sequence, in O. ostertagi. This channel is currently discussed as a participant in the mechanism of action of emodepside. The actual role of the newly described proteins remains to be clarified, but their identification in this work provides a broad basis for future projects.
Literature 17
3 Literature
3.1 Introduction
Infections with parasitic nematodes are responsible for large economic losses in livestock industry. Trichostrongylids can cause severe gastroenteritis, mainly in young ruminants, sometimes even leading to death. The life cycles of gastrointestinal nematodes in cattle and sheep are direct with short generation times. Infection occurs with the ingestion of infectious third-stage larvae on the pasture. Thus the epidemiology of parasitic nematodes is mainly influenced by the access to areas contaminated by infected animals. If the pasture has been contaminated with parasites, elimination of the parasites is difficult. The infectious larvae are viable for months and can survive the winter season on the pasture. Elimination requires interruption of the life cycle, e.g. by alternating pastures between horses and cattle in successive years. Sheep farming in Australia and New Zealand in particular is affected by problems with parasitic nematodes, since large herds are continuously kept on the pasture. Calves for beef production in Germany are in most cases kept in stables, but calves destined for milk production or ecological beef production as well as calves in feedlots, for example in the USA, have access to potentially contaminated environments. Not only the severe and sometimes lethal infections are important for livestock industry, but subclinical effects such as retarded growth and decreased weight gains may result in production losses. Problems are intensified when parasites develop resistances to the main classes of drugs (KAPLAN, 2004). In small ruminants even multidrug resistance has been reported in parasitic nematodes (POMROY, 2006).
3.2 Parasites
3.2.1 Taxonomy
The species Haemonchus contortus, Cooperia oncophora, and Ostertagia ostertagi are parasitic nematodes, affecting the gastrointestinal tract of ruminants. All three species belong to the same subfamily, the Trichostrongylinae:
18 Literature
Kingdom Animalia
Subkingdom Metazoa
Phylum Nematoda
Class Chromadorea
Subclass Rhabditia
Order Rhabditida
Suborder Rhabditina Superfamily Strongyloidea Family Trichostrongylidae Subfamily Trichostrongylinae
Terminology is given according to the Standardized Nomenclature of Animal Parasitic Diseases (SNOAPAD) (TENTER and SCHNIEDER, 2006).
3.2.2 Morphology of Trichostrongylids
Adult trichostrongylids are wire shaped round worms with tapered ends and a length of 5 – 30 mm. They have a filariform pharynx, which means, that the pharynx is long with a terminal bulb. The gut consists of a single layer of epithelial cells with microvilli (TENTER and SCHNIEDER, 2006). Trichostrongylids are dioecious animals, i.e. one individual is having only either male or female reproductive organs. The females have two uteri, two ovaries, and an organ called the ovijector, consisting of the infundibula and the vagina. The vulva is located in the caudal third of the body. The genital system of the males consists of a single testicle, a vas deferens, and a ductus ejaculatorius, which ends in the cloaca. The males also possess two spicula and a bursa copulatrix, which is a lobular modification of the posterior end. The bursa copulatrix surrounds the female vulva during copulation. The morphology of the bursa copulatrix and spicula, as well as surface patterns on the cuticle, can be used for species identification (SCHNIEDER, 2006).
3.2.3 Biology of Trichostrongylids
The life cycles of H. contortus, C. oncophora, and O. ostertagi are monoxenic, i.e. the development cycle does not involve an intermediate host; a schematic life cycle of C. oncophora and O. ostertagi is presented in Figure 1. The adults of all three
Literature 19
species live in the gastrointestinal tract of ruminants and the females lay eggs.
The eggs are 70 – 80 µm in size, oval, and have a thin shell. They are excreted with the host faeces and at that time contain embryonic cells. Detection of the eggs in the faeces of an animal does not allow an exact diagnosis of the species, since the eggs of gastrointestinal nematodes have very similar morphologies. Embryonic development ends with the hatching of the first-stage larvae (L1), which molts to the second-stage larvae (L2). These stages are microbivorous. During development to the infectious third-stage larvae (L3), the cuticle of the L2 is retained as a sheath.
L3 are non-feeding, relying on food resources stored during the earlier stages. L3 are able to endure weeks to months and even the winter period on the pasture and therefore play a key role in epidemiology. Larval development from egg to infectious L3 requires (under optimal conditions) a minimum of 7 – 14 days, usually 21 days, but can take up to three months, depending on environmental conditions.
Prepatency, the time period between infection of the host and appearance of parasite eggs in the faeces, is 17 – 22 days (SCHNIEDER, 2006).
Figure 1: Schematic life cycle of C. oncophora and O. ostertagi. Arrows mark the locations of the adult parasites: C. oncophora (Co) in the small intestine and O. ostertagi (Oo) in the abomasum
20 Literature
The stimuli for L3 exsheathment after entering the host are species-dependent. The larvae appear to receive exsheathment stimuli while passing a part of the alimentary tract of the host proximal to that, where the adult nematodes will reside. H. contortus, which lives in the abomasum of sheep, exsheathes best in slightly acid to neutral conditions like in the rumen, whereas Trichostrongylus colubriformis with the adults staying in the small intestine of sheep, exsheathes in acid conditions, as they are present in the abomasum (LEE, 2002).
3.2.3.1 Haemonchus contortus
The barber pole worm H. contortus is one of the most important gastrointestinal nematodes in sheep (WALLER and CHANDRAWATHANI, 2005). The blood-sucking adults live in the abomasum and lacerate the mucosa of the host with a dorsal tooth to generate hemorrhagic areas (MUNN and MUNN, 2002). The males are 18 – 21 mm, the females 20 – 30 mm in length. Due to the loss of blood heavily infected lambs suffer from anemia, icterus, and edema. Young animals are generally most severely infected and infections can be lethal (SCHNIEDER, 2006).
3.2.3.2 Cooperia oncophora
The adults of C. oncophora are located in the small intestine of cattle. The infection is often observed as a coinfection with O. ostertagi. The main symptoms are diarrhea and lack of appetite resulting in retarded weight gain. The males are 5 – 8 mm, the females 6 – 11 mm (SCHNIEDER, 2006).
3.2.3.3 Ostertagia ostertagi
The males of O. ostertagi are 6 – 8 mm, the females 8 – 12 mm in length. The adults stay in the abomasum of cattle. O. ostertagi is, like some other nematodes including H. contortus, able to arrest its development at the L4 stage and to endure several months without further development. This phenomenon is called hypobiosis and is a strategy to overcome unfavourable environmental conditions. Hypobiotic larvae of O. ostertagi can continue their development after 4 – 6 months and can therefore cause a severe disease called winter ostertagiosis without a new infection of the animals. Hypobiosis is mainly induced by low temperatures (5 – 15° C) or hot, dry
Literature 21
weather for several weeks, but immunological components of the host seem also to play a role. The triggering stimuli for continuing the development are not yet known in detail (SCHNIEDER, 2006). Hormonal stimuli of the host are proposed as triggers, indicating the upcoming reproduction phase of the host. When the offspring larvae produced by the reactivated hypobiotic nematodes are distributed on the pasture, young animals as naive hosts are accessible. The hypobiosis phenomenon is therefore a mechanism to synchronize the life cycles of parasite and host (WHARTON, 2002). Since the L4 stay in the abomasal glands, characteristic nodules are formed in the mucosa. This stage is called the histotropic stage. The main symptoms of an infection with O. ostertagi are diarrhea, lack of appetite, and retarded weight gain.
3.3 The Model Nematode Caenorhabditis elegans
Caenorhabditis elegans is a free-living nematode. It can easily be maintained on agar plates and has a short life cycle; therefore it is a convenient model organism in helminthology (BRENNER, 1974). The genome of C. elegans has been completely sequenced by the C. ELEGANS SEQUENCING CONSORTIUM (1998).
3.3.1 Taxonomy
The non-parasitic nematode C. elegans belongs to a different superfamily than the trichostrongylids (see 3.2.1). The terminology is given according to SNOAPAD (TENTER and SCHNIEDER, 2006):
Kingdom Animalia
Subkingdom Metazoa
Phylum Nematoda
Class Chromadorea
Subclass Rhabditia
Order Rhabditida
Suborder Rhabditina Superfamily Rhabditoidea Family Rhabditidae
22 Literature
3.3.2 Morphology and Biology of C. elegans
The adults of C. elegans are 1 mm in size and are transparent. They have a three-part rhabditoid pharynx and an intestinal wall consisting of a single cell layer.
The adults are mainly hermaphrodites, only a small percentage of a population is represented by males. Hermaphrodites consist of 959 somatic cells. The gonad of hermaphrodites has two lobes, an anterior and a posterior, both consisting of an ovary, an oviduct, and a spermatheca. The spermatheca is the organ where sperm is stored and fertilization takes place. The two lobes of the gonad open into a single uterus, where the fertilized eggs mature. The vulva is located midventrally. The eggs layed by the adult hermaphrodites develop through four larval stages. The fourth-stage larva (L4) and the males produce sperm. Hermaphrodites can mate with males or themselves. The generation time is 3 – 6 days, and the life-span is 2 – 3 weeks (HOPE, 1999). An additional, developmentally arrested stage after the second moult is known as a dauer larva. The dauer larva is a facultative hypobiotic stage in response to unfavourable environmental conditions such as overcrowding or limited food supply (CASSADA and RUSSELL, 1975; GOLDEN and RIDDLE, 1984).
3.4 G-protein Coupled Receptors
G-protein coupled receptors (GPCRs) are membrane-spanning proteins with characteristic structural properties. The membrane-spanning domain consists of seven transmembrane helices. GPCRs play a key role in signal transmission via second messengers. After binding a ligand, the GPCR activates guanine nucleotide-binding proteins (G-proteins). The binding of one ligand molecule to the receptor leads to the activation of many G-protein molecules, resulting in an increased response. G-proteins consist of three subunits, namely α, β, and γ subunit.
Inactive G-proteins bind guanosine-diphosphate (GDP) at the α subunit (Gα).
Activation of the G-protein involves the receptor-catalyzed exchange of GDP to GTP (guanosine-triphosphate) in the binding pocket of the G-protein. The activated α subunit of the G-protein dissociates from the other subunits and catalyzes the activation of a target protein. In some cases the complex of the β and γ subunits is the activating unit for the target protein. The target protein itself activates further regulating proteins (STRYER, 1995b). A well known pathway in a GPCR-mediated
Literature 23
signaling cascade is the phosphoinositide signaling pathway, which involves the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2).
SIMON and coworkers (1991) defined, dependent on the identities of amino acid sequences, four classes of G-proteins, Gαs, Gαi, Gαq, and Gα12. Gαs proteins are stimulatory, whereas Gαi proteins are inhibtory. The class of Gαi proteins also contains the Gαo proteins. The notation of G-proteins is not uniform: some authors note the α as suffix (e.g. Gqα), some put the name of the family as suffix (e.g. Gαq). In this work the latter form is used. The class names are also not uniform, they are found adapted to Gαi/o, Gαq/11, and Gα12/13 (e.g. in HOLLINGER et al., 2001;
MCCUDDEN et al., 2005). In the C. elegans genome genes for 21 Gα subunits are known, with one clear ortholog for each of the four mammalian families of Gα subunits, with C. elegans goa-1, gsa-1, egl-30, and gpa-12 corresponding to mammalian genes for Gαi, Gαs, Gαq, and Gα12, respectively. The remaining 17 Gα subunits, although perhaps more similar to the Gαi/o family, are referred to as the C. elegans specific Gα subunits (BERGAMASCO and BAZZICALUPO, 2006).
GPCRs are grouped into at least six families, showing no sequence homologies to each other. The main families are the families 1, 2, and 3. Family 1 contains most GPCRs, including receptors for odorants. Ligands are small molecules such as catecholamines (family 1a), short peptides (family 1b), or large proteins (family 1c).
Family 2 receptors are also called the secretin-like family, and bind large peptides.
Their relatively long N-termini are involved in ligand binding. Family 3 contains receptors for glutamate and γ-aminobutyric acid (BOCKAERT and PIN, 1999).
3.5 Tools for Functional Gene Analysis in C. elegans
3.5.1 Inactivation of Genes
The completely sequenced genome of C. elegans allows resarchers to produce worms with selectively inactivated genes to investigate the function of a certain gene.
Currently, two main strategies are used: RNA interference (RNAi) and gene deletion (knockout). The term knockdown is used for RNAi-derived reduction of expression, whereas knockout means the total elimination of gene transcription due to deletion or mutation of the gene (BARSTEAD, 1999; LEE et al., 2004).
24 Literature
3.5.1.1 Gene Knockout
Treating C. elegans with chemical mutagens such as ethylmethanesulfonate, diepoxybutane, or trimethylpsoralen combined with UV radiation induces deletions in the DNA. The most appropriate stage for mutagenesis is the L3 / L4 stage, since the germline replicates in these stages. Replicating DNA is most susceptible to chemically induced mutagenesis. The progeny of the treated population can be screened for mutants by polymerase chain reaction (PCR) for mutations in the gene of interest or by their phenotype. Another principle of mutagenesis takes advantage of so-called mutator strains. These strains are relatively active for transpositions of genetic elements. Populations of animals having a transposon-insertion within the gene of interest leading to disruption of the open reading frame, are detected by PCR, and siblings are recovered. Subsequent to the identification of the population containing animals with a mutation or transposition within the respective gene, the mutated animals are selected in several rounds of breeding (BARSTEAD, 1999).
3.5.1.2 RNA Interference (RNAi) in C. elegans
Microinjection of double stranded RNA (dsRNA) into C. elegans L4 or young hermaphrodites leads to a knockdown of the gene with the respective sequence in the progeny of the injected worm (FIRE et al., 1998). The phenomenon is called RNA interference (RNAi). Not only microinjection, but also soaking the worms in dsRNA-containing liquid (TABARA et al., 1998), in vivo production of dsRNA in trangenic C. elegans (TAVERNARAKIS et al., 2000), and feeding the worms with genetically modified Escherichia coli (TIMMONS et al., 2001) leads to this phenomenon. In the classical pathway of RNAi, the dsRNA is bound by an RNA binding protein, RDE-4 (PARKER et al., 2006), and cleaved by a complex called Dicer into small interfering RNAs (siRNAs) (BERNSTEIN et al., 2001). The siRNAs are loaded onto an effector complex, the RNA-induced silencing complex (RISC).
RISC unwinds and separates the siRNA strands, and the resulting single stranded RNA fragments can bind to complementary messenger RNA (mRNA). This mRNA is then cleaved by the RISC complex and can therefore not be translated into protein.
The expression of the respective protein is knocked down (GRISHOK, 2005;
HAMMOND, 2005; ZAWADZKI et al., 2006). Another class of siRNAs is derived from direct amplification performed by the RNA-dependent RNA polymerase RdRP
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(PAK and FIRE, 2007; SIJEN et al., 2001). RNAi screens are widely used in examination of gene functions in C. elegans, and the mechanism also functions in many other organisms (GUNSALUS and PIANO, 2005).
3.5.1.3 RNAi in Parasitic Nematodes
In parasitic nematodes the RNAi technique currently has several drawbacks. Efficacy has been extremely variable and experiments were sometimes not easily reproducible (VISSER et al., 2006). Maintaining parasitic stages under culture conditions is difficult and might influence the phenotype of the parasite; observed changes of the phenotype are therefore not necessarily caused by the knockdown of a certain gene. Furthermore, the developmental stages which can be exposed to RNAi technology are principally the free-living stages rather than the parasitic stages, and the effects of RNAi on subsequent stages are not clear (GELDHOF et al., 2007;
KNOX et al., 2007). Nevertheless, positive outcomes of RNAi experiments have been reported (ISSA et al., 2005; KOTZE and BAGNALL, 2006; LUSTIGMAN et al., 2004).
3.5.2 Expression of Heterologous Genes in C. elegans
The model organism C. elegans can be used as an expression system for heterologous genes or for gene / reporter-gene constructs. Any DNA microinjected into the germline of a hermaphrodite will be replicated (JIN, 1999). To facilitate the handling and maintenance of the DNA, plasmids are usually used. The plasmid contains an ampicillin resistance gene for the maintenance of the plasmid in E. coli.
A large set of vectors was developed in the laboratory of FIRE (1990), containing different promotors and different reporter genes, such as the genes for green fluorescent protein (GFP) or β-galactosidase (MOUNSEY et al., 1999). The microinjection of DNA leads to extrachromosomal insertion, which is heritable. The animals are mosaic mutants, with some cells carrying the DNA and some cells without detectable foreign DNA (STINCHCOMB et al., 1985). Another method for transfection of C. elegans is microbombardment with microcarrier gold beads, leading to integration of DNA into chromosomes. The progeny of the transgenic worms are stably transfected and show no mosaic patterns (PRAITIS et al., 2001).
The expression of genes in C. elegans can be used for studies of expression patterns, for rescue of knockout mutants with mutated genes (e.g. KLOPFENSTEIN
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and VALE, 2004; LEE et al., 2005) or with genes of other species (e.g. COOK et al., 2006; COUTHIER et al., 2004; CULLY et al., 1994; KWA et al., 1995).
3.6 Neurobiology of Nematodes
The chemical complexity of the nematode neurosystem has become apparent only within the last ten years. The nervous system of C. elegans is the most complex organ in the worm. 37 % of the cells in a hermaphrodite belong to the neuronal system (HOBERT, 2007). The most complex neuropil in the animal is a nerve ring encircling the pharynx. Efferent from this nerve ring, a dorsal and a ventral cord extend almost to the tail. Both contain motor neurons; the ventral cord additionally carries sensory neurons and interneurons. The system is completed by several ganglia, mainly in the pharyngeal and tail regions, and by sublateral cords. All nerves are located immediately beneath the hypodermis (THOMAS and LOCKERY, 1999).
Due to its large size, having a length of 15 – 30 cm and a diameter of 3 – 6 mm, the pig roundworm Ascaris suum is a convenient system for neurological studies. The neuronal system of A. suum consists of three components: the peripheral, central, and enteric nervous systems. Since the nervous systems of nematodes are highly similar, the derived information may be applied to other nematodes. The nervous system of nematodes is a combined nervous and endocrine system with commonly shared messenger molecules, mainly neuropeptides of 3 – 100 amino acids.
Nematodes lack endocrine glands and a circulatory system (BROWNLEE et al., 2000).
3.6.1 Neurotransmitters
Neurotransmitters are compounds synthesized and stored in neurons where they mediate nerval signals into cellular responses. Their release is dependent on calcium ions (Ca2+) and has inhibitory or stimulatory effects on the postsynaptic cells. The neurotransmitters used by nematodes are mainly classical transmitters also known in mammals. However, some transmitters play a role in nematodes but are uncommon in mammals (THOMAS and LOCKERY, 1999). Which transmitters are used depends on the respective neuron. Neurotransmitters have been studied in C. elegans and A. suum in greater detail.
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Synaptic transmitters are stored in vesicles in the presynaptic terminal. Influx of calcium into the cell causes vesicle release. For termination of action the released transmitters are recycled or degraded. The proteins involved in forming the vesicles are also recovered. An overview of the proteins potentially involved in vesicle formation is given by HARRIS and colleagues (2001).
3.6.1.1 Acetylcholine
A major excitatory transmitter in neuromuscular junctions of C. elegans is acetylcholine (ACh). ACh is used by a third of the cells belonging to the nervous system in C. elegans (RAND, 2007). In C. elegans GPCRs (LEE et al., 1999; LEE et al., 2000; PARK et al., 2003) and ion channels have been identified as receptors for ACh. The ion channels are assumed to consist of five subunits, which are arranged around an ion-pore (MARTIN et al., 2002). Dependent on their affinities, receptors are classified in L-, B-, and N-subtypes. L-subtype receptors have the highest affinity to levamisole, whereas B-type receptors bind bephenium and N-type receptors are sensitive to nicotine (MARTIN et al., 2005). The termination of action is mediated by acetylcholine esterase, which hydrolyzes ACh. The degradation products can be recycled (RAND, 2007). In flatworms, ACh is known to act as an inhibitory transmitter (RIBEIRO et al., 2005).
3.6.1.2 Glutamate
As in vertebrates, the main transmitter for rapid excitatory synaptic signaling in nematodes is glutamate. The excitatory action of glutamate in vertebrates is mediated by ionotropic and metabotropic glutamate receptors (NAKANISHI et al., 1998). In C. elegans at least ten subunits of excitatory ionotropic glutamate receptors have been identified, indicating that a number of different functional types of ionotropic glutamate receptors might be expressed in the worm. The other group of receptors known in vertebrates, the metabotropic glutamate receptors, are GPCRs.
Recently, three genes for metabotropic glutamate receptors have been identified in C. elegans (DILLON et al., 2006). Another target for glutamate in nematodes are glutamate-gated chloride (GluCl) channels, which are unique to invertebrates.
Contrary to the excitatory ionotropic and metabotropic glutamate receptors GluCl channels mediate an inhibitory effect of glutamate by forming channels for
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chloride ions (BROCKIE and MARICQ, 2006). In Xenopus oocysts expressed homomers of GluCl channel subunits show different binding capacities, depending on the subunits expressed: GluClα 1 homomeric channels are sensitive to the anthelmintic drug ivermectin but not to glutamate, whereas GluClβ homomeric channels react to glutamate but not to ivermectin (CULLY et al., 1994; CULLY et al., 1996). DENT and coworkers (1997) identified the alternatively spliced subunits GluClα 2A and GluClα 2B. Homomeric channels of these subunits are ivermectin and glutamate sensitive. Another subunit in two splicing variants, GluClα 3A and GluClα 3B, has been identified later by the same group. Only simultaneous mutations of the three genes for GluClα 1, GluClα 2, and GluClα 3 leads to highly resistant animals, mutations of only two of these genes in C. elegans confers only modest or no resistance (DENT et al., 2000). In C. oncophora and H. contortus orthologous genes for GluClα 3 and GluClβ have been identified (CHEESEMAN et al., 2001;
NJUE and PRICHARD, 2004). In expression studies of these genes amplified from ivermectin-susceptible and ivermectin-resistant C. oncophora in Xenopus oocysts, a single mutation was identified to confer resistance to ivermectin (NJUE et al., 2004).
In nematodes the channels are assumed to consist of five subunits, but the composition is still unknown (MARTIN et al., 2002). Ivermectin-sensitive channels were shown to be expressed in the pharynx of nematodes (DENT et al., 1997;
LAUGHTON et al., 1997).
3.6.1.3 GABA
γ-aminobutyric acid (GABA) is an inhibitory transmitter in mammals. The receptors for GABA are GABAA, a chloride channel, and GABAB, a GPCR. Genes for both are also found in the genomes of nematodes, and GABAA receptors have also been shown to be targets for GABA in nematodes (SCHOFIELD et al., 1987). The activation of GABAA receptors leads, depending on the intracellular chloride concentration, to the influx or efflux of chloride ions and therefore to membrane hyperpolarization or depolarization. In both cases body muscle contraction is inhibited (reviewed in JORGENSEN, 2005). GABAB GPCRs mediate an inhibition of membrane excitability by opening potassium (K+) channels and inhibiting Ca2+ channels (KAUPMANN et al., 1997). Recently an excitatory effect of GABA was discovered in C. elegans. The mechanism involves a cation-selective channel, and the influx of sodium ions causes
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contraction of the enteric muscles (BEG and JORGENSEN, 2003). GABA is cleared from the synaptic cleft by a plasma membrane transporter (SCHUSKE et al., 2004).
3.6.1.4 Dopamine
In mammals the known dopamine receptors are GPCRs. They are classified as D1- and D2-like receptors. D1-like receptors couple positively to adenylate cyclase and therefore increase the level of cyclic adenosine monophosphate (cAMP), whereas D2-like receptors inhibit cAMP formation. Nevertheless, additional second messengers and effector pathways are also recognized (NEVE et al., 2004). In C. elegans four GPCRs for dopamine are currently known, two D1-like and two D2-like receptors (MCDONALD et al., 2006).
3.6.1.5 Serotonin
Serotonin is a modulating transmitter in many physiological mechanisms in C. elegans. According to CARRET-PIERRAT (2006a), C. elegans has 3 – 8 GPCRs for serotonin, which have a predominantly neuronal expression. Another receptor is a serotonin-gated chloride channel (RANGANATHAN et al., 2000).
3.6.1.6 Octopamine and Tyramine
C. elegans was further shown to have physiological processes modulated by biogenic amines other than dopamine and serotonin: octopamine and its biosynthetic precursor tyramine. In some nematode species octopamine is metabolized to noradrenaline, synephrine and epinephrine (FRANDSEN and BONE, 1988). The receptors for octopamine were predicted by database searches, but none have been definitively identified (KOMUNIECKI et al., 2004). Two GPCRs are known for tyramine (REX et al., 2004; REX et al., 2005).
3.6.1.7 Neuropeptides
Neuropeptides are peptides acting as neuromodulators or neurotransmitters. They are the major class of transmitter compounds in nematodes. 75 % of the nerve cells in A. suum (BROWNLEE et al., 1996), and > 50 % in C. elegans (KIM and Li, 2004)
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were shown to express neuropeptides. Like other neurotransmitters they are highly specific in their action but have a much higher potency than many other transmitters. Their synthesis involves proproteins or precursors. The cleavage and posttranslational modification of the precursor molecules occurs in the endoplasmatic reticulum. The neuropeptides are then bound to a carrier protein and transported in vesicles through the Golgi complex to the nerve terminal. The release is, like the release of classical neurotransmitters, Ca2+-dependent. They are stored in vesicles different than those containing classical transmitters; a differential release is therefore likely possible and would be necessary for the modulating activities of neuropeptides on neurotransmitters. In the simple invertebrate polyp Hydra no transmitters other than neuropeptides have been identified, therefore, neuropeptides are thought to be the original transmitter molecules (BROWNLEE et al., 2000). In C. elegans the neuropeptides can be subdivided into three main classes: insulin-like peptides, neuropeptide-like proteins, and FMRFamide-like peptides. Some neuropeptide-like proteins are antimicrobial and are expressed in the hypodermis rather than in neurons. Their expression is induced upon bacterial or fungal infection (HUSSON et al., 2007).
FMRFamide-like peptides (FaRPs or FLPs) are the most complex group of neuropeptides known from metazoans. In free-living and parasitic nematodes these peptides are proposed to play a fundamental role (MCVEIGH et al., 2005). The name is derived from their similarity to a molluscan neuropeptide called FMRFamide, containing the sequence Phe-Met-Arg-Phe-NH2. FLPs contain the C-terminal tetrapeptide motif X-Xo-Arg-Phe-NH2, where X is any amino acid except cysteine and Xo is any hydrophobic amino acid except cysteine (MCVEIGH et al., 2006). In the snail Lymnaea stagnalis two groups of FMRFamide-like peptides are known, the N-terminally extended peptides and the tetrapeptides. They are derived from the same gene by alternative splicing. Only one tetrapeptide has been identified to date in nematodes: FIRFamide in A. suum. All other currently known FMRFamide-like peptides in nematodes are N-terminally extended peptides (BROWNLEE et al., 2000). FLPs differing in a single amino acid can have different receptors, leading to different actions (BOWMAN et al., 2002). Neuropeptides containing an RFamide motif are also known from mammals. Several FLPs in C. elegans are known to act via GPCRs (reviewed in MCVEIGH et al., 2006). Another mechanism of action
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appears to involve an FMRFamide-gated chloride channel (PURCELL et al., 2002).
Termination of action occurs by enzymatic degradation. The examination of FLPs and their receptors is still ongoing. BROWNLEE and coauthors (2000) emphasize that in addition to the currently known FLPs in C. elegans and A. suum other FLPs might exist, which could be unique to parasitic nematodes. These FLPs need to be studied in the various parasitic species.
3.7 Control of Parasites
For the control of parasites in sheep and cattle several approaches are currently being pursued, e.g. grazing management, optimization of keeping and feeding conditions for the animals as well as breeding approaches to achieve nematode resistant animals. Vaccination strategies against gastrointestinal nematodes in ruminants are still under development. An overview of these control measures was recently given by STEAR (2007). Biological control with nematophagous fungi is also being explored. Nevertheless, the most important strategy is still anthelmintic treatment. Anthelmintic drugs interact with targets in the nematode that are either not present in the host or have lower affinities to the drugs. The major classes of anthelmintic drugs used in ruminants are benzimidazoles, macrocyclic lactones, and nicotinic agonists.
3.7.1 Benzimidazoles
The first member of the class of benzimidazoles was thiabendazole; other commonly used members are fenbendazole, albendazole, and oxfendazole (UNGEMACH, 2003). Thiabendazole was shown to have anthelmintic activity in the early 1960s.
The mechanism of action is the inhibition of polymerization of the nematode’s microtubules by binding to β-tubulin (LACEY, 1988). Microtubules are components of the cytoskeleton and contribute to the intracellular transport of nutrients and substrates. Inhibition of polymerization leads to depletion of ATP resources, exhausting the cells (UNGEMACH, 2003). The worm dies and can be expelled. As precursors of this group, the probenzimidazoles are used. They are metabolized to the active compounds by the host.
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3.7.2 Nicotinic Agonists
The group of nicotinergic agonists contains the imidazothiazoles, such as levamisole, and the tetrahydropyrimidines, such as pyrantel and morantel. Tetrahydropyrimidines and imidazothiazoles are ligands for ACh receptors and lead to membrane depolarization and therefore spastic paralysis of the worm (ECKERT et al., 2005). In higher concentrations the drugs inhibit the acetylcholine esterase (UNGEMACH, 2003). Additionally the drugs cause a flickering open-channel block. The binding site of the drugs is different from the binding site of ACh; the drugs are large cations that might block the cation-selective channel (MARTIN et al., 2002). Morantel induces the greatest block, since the channel possesses two binding sites for morantel (EVANS and MARTIN, 1996).
3.7.3 Macrocyclic Lactones
Avermectins and milbemycins are macrocyclic lactones. The substances belonging to this class are fermentation products of the bacteria Streptomyces spp. or are semi-synthetic derivatives (CONDER, 2002). The first compound available was ivermectin, which was introduced into the market in 1980. The effect of macrocyclic lactones is a rapid paralysis of movement and pharyngeal pumping in the nematode.
The target molecules of avermectins and milbemycins are GABAA receptors and, even more importantly, GluCl channels, which are irreversibly opened. As described above, multiple forms of GluCl channels occur in nematodes, differential sensitivity to the current drugs is likely. Some of these receptors are expressed in the neuromuscular system (WOLSTENHOLME and ROGERS, 2005). In C. elegans channels consisting of different subunits are known, and composition may determine sensitivity to ivermectin.
3.7.4 Closantel
Closantel is a salicylanilide, which is mainly used for the treatment of liver flukes. It is also effective against H. contortus in sheep. Salicylanilides uncouple oxidative phosphorylation in helminths. Due to its plasma-protein binding, closantel is not effective against non-bloodsucking trichostrongylids (CONDER, 2002; UNGEMACH, 2003).
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3.7.5 Piperazine
Piperazine, one of the oldest anthelmintics, acts at GABAA receptors as a GABA agonist, causing flaccid paralysis of the worm by opening chloride-ion channels (DEL CASTILLO et al., 1963; MARTIN, 1982; MARTIN, 1997).
3.8 Anthelmintic Resistance
According to PRICHARD and coworkers (1980) anthelmintic resistance “is present when there is a greater frequency of individuals within a population able to tolerate doses of compound than in a normal population of the same species and [resistance]
is heritable.” It is most common in parasites with a direct life cycle and short generation periods (SANGSTER and DOBSON, 2002). Gastrointestinal nematodes in sheep were repeatedly reported to have developed anthelmintic resistance (GILL et al., 1991; JACKSON and COOP, 2000; KAPLAN, 2004; KOTZE et al., 2002;
WAGHORN et al., 2006). In cattle nematodes resistance is currently less common.
Nevertheless, in Argentina resistance to avermectins has been reported in C. oncophora, O. ostertagi, and Haemonchus placei. In O. ostertagi and H. placei an additional benzimidazole resistance was observed (MEJIA et al., 2003). In New Zealand resistance to ivermectin in cattle nematodes was found to appear in 82 %, resistance to albendazole in 60 % of 59 tested farms (JACKSON et al., 2006).
Another study detected ivermectin resistant C. oncophora in all five farms tested, furthermore evidence for possibly emerging resistances in O. ostertagi and Trichostrongylus spp. (MASON and MCKAY, 2006). The economic impact of resistance is serious; in South Africa some sheep farmers were even forced to abandon sheep production (VAN WYK et al., 1989). No mechanism of resistance is completely understood, but some are partially known. SANGSTER and DOBSON (2002) classify the mechanisms of resistance as pharmacological and genetic.
Pharmacological mechanisms are reduction of anthelmintic concentration and modification of the downstream cascade. Genetic mechanisms include alteration of genes, selective expression of a gene, increased expression of enzymes, and gene deletion. In H. contortus the best studied mechanism of resistance to benzimidazoles is mutation of the β-tubulin gene. Resistance to macrocyclic lactones in H. contortus and in C. oncophora may be related to mutations in the genes coding for glutamate
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and GABAA gated chloride channel subunits. Another mechanism of resistance is the overexpression of P-glycoproteins as molecular transporters to pump the compound out of the cells. The alteration of ACh receptors is thought to be the background for resistance to levamisole, but no molecular evidence yet exists (mechanisms reviewed by WOLSTENHOLME et al., 2004). Alteration of the target may not only occur by mutation but also by alteration in phosphorylation (MARTIN et al., 2002).
Reversion of resistance is partially possible but occurs slowly. If the population is re-exposed to the anthelmintic, resistance reappears within a generation or two, because the resistance alleles are still present in the population at a high frequency (SANGSTER and DOBSON, 2002).
3.9 Cyclooctadepsipeptides
Cyclooctadepsipeptides are a novel group of anthelmintically active compounds with a new mechanism of action. The first member of this class, PF1022 A, was discovered in the late 1980s.
3.9.1 PF1022 A
The compound PF1022 A is a fermentation product of the fungus Mycelia sterilia.
The fungus belongs to the microflora of the flower Camellia japonica (SASAKI et al., 1992). PF1022 A consists of four alternating residues of N-methyl-L-leucine and four residues of D-lactate or D-phenyllactate (see Figure 2). The substance was patented in 1990 by the Japanese company Meiji Seika Kaisha. The anthelmintic activity of PF1022 A was shown for several nematode species, e.g. H. contortus, the canine hookworm Ancylostoma caninum, and the bovine lungworm Dictyocaulus viviparus.
Against Trichostrongylus colubriformis, a gastrointestinal nematode in sheep, the drug is moderately effective when orally applied (SAMSON-HIMMELSTJERNA et al., 2000).
3.9.2 Emodepside
Emodepside is a semi-synthetic derivative of PF1022 A. It was patented in 1993 by Fujisawa Pharmaceutical (Japan), and further examined through a cooperation among Fujisawa Pharmaceutical, Meiji Seika Kaisha (Japan), and Bayer HealthCare
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(Leverkusen, Germany). Compared to PF1022 A, emodepside carries a morpholine ring at each of the two D-phenyllactic acids in para position (see Figure 2). The substance shows a broad anthelmintic spectrum, including the trichostrongylids H. contortus, O. ostertagi, and Cooperia spp., but also D. viviparus, Trichuris spp., A. suum, Toxocara spp., Parascaris equorum and, species-dependent, adult filarial nematodes. In contrast to PF1022 A, emodepside is also effective against Trichinella spiralis larvae in muscles (HARDER et al., 2003). Larval stages of the mouse nematode Heligmosomoides polygyrus and microfilariae of Brugia malayi and Litomosoides sigmoidontis are only partially affected by the drug, whereas other stages of the species are susceptible (HARDER and SAMSON-HIMMELSTJERNA, 2001; ZAHNER et al., 2001). In benzimidazole-, levamisole-, and ivermectin-resistant populations of H. contortus in sheep, as well as an ivermectin-resistant C. oncophora population in cattle, emodepside was shown to be fully active (SAMSON- HIMMELSTJERNA et al., 2005). A so far unknown mechanism of action was therefore proposed. In 2005 emodepside in combination with praziquantel was introduced into the market as a spot-on preparation for cats. The mode of action of cyclooctadepsipeptides in parasitic nematodes is not yet clarified in detail.
Figure 2: Chemical structure of PF1022 A and its derivative emodepside (modified after HARDER et al., 2005)
O N
N O
N
O O
O
O N O
N O
O O
O N
O O
O N
O O
O
O N O N O
O O
O N
O O
O
PF1022 A emodepside
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3.9.3 Cyclohexadepsipeptides
Cyclohexadepsipeptides are an anthelmintically active group related to the cyclooctadepsipeptides. These substances were shown to have a strong anthelmintic activity against H. contortus (JESCHKE et al., 2005; JESCHKE et al., 2006).
3.10 Cyclooctadepsipeptides: Mechanism of Action
3.10.1 Involvement of the GABA System
The activity of emodepside, formerly known as BAY 44-4400, is synergistically enhanced by piperazine (NICOLAY et al., 2000). The involvement of the GABA system in the mechanism of action of emodepside is therefore discussed.
3.10.2 Influence of Cyclooctadepsipeptides on Effects of Neurotransmitters The neuropeptide AF2 induces a biphasic muscle tension and increased cAMP levels in isolated A. suum neuromuscular strips. PF1022 A blocks or reverses the muscle tension, while the increase of cAMP remains unaffected (THOMPSON et al., 2003).
Emodepside irreversibly reduces the contraction of isolated neuromuscular strips induced by ACh or AF2, in contrast to the action of the inhibitory neurotransmitter GABA, the relaxation is slow and incomplete. The inhibitory effect of the neuropeptide PF 2 is similar to the effect of emodepside on the neuromuscular strips.
The electrophysiological response to emodepside is very similar to the response to PF2 (WILLSON et al., 2003).
3.10.3 Effects of Emodepside on C. elegans
In experiments with A. suum emodepside causes muscle relaxation by membrane hyperpolarization. Functional studies by WILLSON and colleagues (2003) indicate that the action is presynaptically mediated. The effect of emodepside on neuromuscular strips is enhanced in low external potassium and abolished by blocking potassium channels. The presence of external Ca2+ ions is required for the action of emodepside. This need for Ca2+ ions might be due to the role of calcium in activating calcium-gated potassium channels or in neurotransmitter release. The
