MOLECULAR AND CELLULAR BIOLOGY OF HELMINTH …
MOLECULAR AND CELLULAR BIOLOGY OF HELMINTH PARASITES
BRATSERA HOTEL, HYDRA, GREECE
6 – 11 SEPTEMBER 2005
Conference Organisers :
Kleoniki Gounaris (London)
Rick Maizels (Edinburgh)
Edward Pearce (Philadelphia)
Murray Selkirk (London)
We gratefully acknowledge donations from:
The Ellison Medical Foundation
Novartis Animal Health
New England BioLabs
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SCIENTIFIC PROGRAMME
All timings include minimum of 5 minutes for discussion.
Tuesday 6 September
16:00 – 18:30 Registration, Bratsera Hotel
18:30 Plenary Lecture: D. Colley: Medical Helminthology: From Bases to Faces
20:00 Welcome Reception, Bratsera Hotel
Wednesday 7 September
9:00 – 10:40 Session 1 : Neurobiology, Drug Targets & Resistance
Chair: Richard Martin
9;00 D. Sattelle: Contributions from comparative genomics, forward and reverse genetics and physiology to neural signalling mechanisms, drug target identification and validation in C. elegans
9:40 C. Welz: Focus on new drugs: Putative receptors for Cyclooctadepsipeptides in parasitic nematodes
10;00 G. Samson-Himmelstjerna: Molecular tools for the qualitative and quantitative assessment of antihelminthic resistance associated single nucleotide polymorphisms in parasitic nematodes
10:20 T. Day: The structure of neuropeptides and neuropeptide receptors in flatworms
10:40 Coffee break
11:10 – 12:50 Session 2 : Neurobiology, Drug Targets & Resistance
Chair: Murray Selkirk
11:10 R. Martin: Variety is the spice of a worm's life: subtypes of ACh receptor and levamisole resistance
11:35 R. Greenberg: Are novel schistosome calcium channel subunits targets for praziquantel action?
12:00 R. Prichard: The role of P-glycoproteins in resistance to ivermectin and other macrocyclic lactones in parasitic nematodes
12:25 A. Maule: RNA interference of neuropeptide signalling in plant parasitic nematodes
Lunch/Free Time
16:30 – 18:10 Session 3 : Structure and Function of Helminth Enzymes
Chair: Niki Gounaris
16:30 A. Page: Biosynthesis and enzymology of the C.elegans cuticle
17:10 C. Burmeister/E. Liebau: Oxidative stress in Caenorhabditis elegans: protective effects of the omega-class glutathione S-transferase (GSTO-1)
17:30 L. Mikes: Cathepsin L involvement in host penetration by cercariae of the fish eye fluke Diplostomum pseudopathaceum
17:50 M. Kennedy: Atomic structure and lipid binding site of a polyprotein allergen of nematodes
18:10 – 20:00 Poster Session 1
Thursday 8 September
9:00 – 10:40 Session 4 : Genetics & Genomics
Chair: Chuck Shoemaker
9:00 M. Berriman: Annotation of parasite genomes
9:40 J. Gilleard: Genetic analysis of Trichostrongylid nematodes of veterinary importance
10:00 C. Fernandez: A transcriptomic survey of Echinococcus granulosus
10:20 R. Davis: Contribution of trans-splicing to mRNA translation and decay and characterisation of nematode cap-interacting proteins
10:40 Coffee break
11:10 – 12:50 Session 5 : Bioinformatics & Functional Genomics
Chair: Malcolm Kennedy
11:10 D. Roos: Designing and mining pathogen genome databases: insights into basic parasite biology, and targets for diagnostic, drug and vaccine development
11:50 B. Connolly: Proteomic analysis of excretory-secretory proteins from Trichinella
12:10 J. Van Hellemond: Mass spectrometric analysis of the S. mansoni tegumental proteome: identification of unique and tegument-specific proteins
12:30 A. Saverwyns: Study of the interaction between adult Ostertagia ostertagi excretory-secretory products and bovine abomasal proteins by cDNA phage display
Lunch/Free Time
16:30 – 18:10 Session 6 : Post-Genomics and Genetic Manipulation
Chair: David Roos
16:30 W. Grant: Parastrongyloides trichosuri as a new model nematode parasite
16:55 C. Britton: Using C. elegans techniques for identification and expression of potential parasite target genes
17:20 J. Lok: Transgenesis in Strongyloides stercoralis: administration, transient expression, silencing and inheritance pattern of plasmid-based reporter constructs
17:45 P. Brindley: Transduction of Schistosoma mansoni sporocysts by VSVG pseudotyped Moloney murine leukaemia retrovirus constructs encoding luciferase and other transgenes
19:45 Boats depart from Hydra port for Vlychos
20:00 Dinner, Vlychos Taverna
Friday 9 September
9:00 – 10:40 Session 7 : Parasite Development
Chair: Klaus Brehm
9:00 C. Grevelding: The role of protein tyrosine kinases in female schistosome development
9:25 C. Behm: Functional analysis in Caenorhabditis elegans of gei-16, a nematode developmental gene with multiple splice variants
9:50 D. Smyth: Enzyme expression associated with activation of larval Nippostrongylus brasiliensis
10:15 E. Devaney: Hsp 90 in Brugia and C.elegans
10:40 Coffee break
11:10 – 12:50 Session 8 : Host-Parasite Interactions
Chair: Alex Loukas
11:10 D. Bird: Mechanisms and evolution of symbiosis; parasites and mutualists invade plants via a shared response pathway
11:35 D. Guiliano: Identification of potential mediators of nurse cell transformation from T. spiralis
12:00 T. Freitas: Schistosoma mansoni TGFβ homologues
12;25 K. Brehm: Do evolutionary conserved molecules mediate an interaction between Echinococcus multilocularis and its mammalian host?
Lunch/Free Time
16:30 – 18:10 Session 9 : Immune Regulation and Modulation
Chair: Rick Maizels
16:30 A. Hoerauf: Regulation of innate and adaptive immunity by filarial worms and their symbionts
17:10 S. Specht: Overexpression of IL-10 from T cells or macropahges leads to apparent different outcomes in filariasis
17:30 S. Hartmann: Functional analysis of regulatory T cells in mice infected with the intestinal nematode Heligmosomoides polygyrus
17:50 H. Haas: The IL-4-inducing principle from S.mansoni eggs (α-1/IPSE) activates human basophils via a novel mechanism: IgE receptor engagment without crosslinking
18:10 – 20:00 Poster Session 2
Saturday 10 September
9:00 – 10:40 Session 10 : Induction of Immunity
Chair: Ed Pearce
9:00 A. MacDonald: B cells dominate IL-10 production during Th1, but not Th2, response induction by dendritic cells in vivo
9:25 K. Pfarr: The Toll-like receptor pathway and the chemokine CCL17 are essential for immune-mediated containment of adult worms and microfilariae in filariasis
9:50 M. Tran: Tetraspanins expressed in the tegument of Schistosoma mansoni are protective vaccine antigens in murine schistosomiasis
10:15 M. Siles-Lucas: The 14-3-3 proteins: new vaccine candidates against platyhelminths
10:40 Coffee break
11:10 – 12:50 Session 11 : Regulation of the Response
Chair: Achim Hoerauf
11:10 E. Pearce: Th2 response polarization during infection with the helminth parasite Schistosoma mansoni
11:50 A. Balic: Development and maintenance of type 2 immune responses in a competitive environment
12:10 C. Zaph: Persistence, function and inter-relationship of central and effector memory CD4+ T cells following infection with a gastrointestinal parasite
12:30 M. Pearson: An orphan seven transmembrane receptor from the tegument of Schistosoma mansoni and its vaccine efficacy in a rodent model of infection.
Lunch/Free Time
16:30 – 18:10 Session 12 : Immunity and the parasite
Chair: Eileen Devaney
16:30 A. Loukas: Vaccination with recombinant aspartic haemoglobinase reduces parasite load and blood loss after hookworm infection
16:55 D. Artis: Resistin-like molecules: novel immune effectors that target parasitic nematodes
17:20 R. Maizels: Regulation by helminth infection of immunity and allergy
17:45 M. Viney: The life and times of the parasitic nematode Strongyloides ratti under immune pressure
20:00 Farewell Dinner, Douskos Taverna, Hydra
Poster Session 1: Wednesday September 7, 18:10 – 20:00
Holly Afferson Purinergic regulation of mucosal mast cell degranulation in Trichinella spiralis infection
Andrew Birnie Quiescin-Sulfhydryl Oxidases, and their role in cuticle collagen biosynthesis in C. elegans
Katerina Doleckova Evaluation of cDNA expression library from the mollusc stage of the bird schistosome Trichobilharzia regenti by PCR
Christian Epe Identification, expression and antigen characterisation of paramyosin of Ancylostoma caninum
Jana Hoeppner Multiple transcription initiation sites and alternative splicing processes generate an unexpectedly large diversity of omega class glutathione S-transferases in Onchocerca volvulus
Martin Kasny Comparison of cysteine and serine proteases of Trichobilharzia regenti and Schistosoma mansoni cercariae
Maren Manske Expression of recombinant glycosylated IPSE (IL-4-inducing principle of Schistosoma mansoni eggs) in 293 HEK-cells
Samantha McCavera Characterisation of a Haemonchus contortus channel that is extremely sensitive to ivermectiin
Paul McVeigh On the FLP-side of nematode neuropeptides
Jan Newton-Howes Analysis of differential gene expression in the free-living and parasitic life cycles of Parastrongyloides trichosuri
Dominic Rees-Roberts Nematode carboxypeptidases inhibit C5a-mediated chemotaxis of human granulocytes
Debbie Scarlett A proteomics approach to identification of secreted antigens from infective larvae of Nippostrongylus brasiliensis
Colin Stack Structural and mutagenesis analysis of the Fasciola hepatica cathepsin L1 reveals insights into its biological function
Fiona Thompson Microarray analysis of gene expression in the Strongyloides ratti life-cycle
Alan Winter Nematode collagen biosynthetic enzymes
Poster Session 2: Friday September 9, 18:10 – 20:00
Daniel Beiting Syndecan-1, a heparin sulfate proteoglycan, is produced by Trichinella spiralis-infected musle cells
Mali Camberis Determining the features of N. brasiliensis infection which drives Th2 Immune responses and protective immunity
Tegan Don Pore-forming proteins from blood-feeding helminths
Julie-Anne Fritz analysis of nematode-specific genes using the free-living nematode, Caenorhabditis elegans
Michael Johnston FMRFamide-like peptide genes in Meloidogyne incognita: charaterisation, expression and RNAi-induced disruption
Henry McSorley TGF-β homologues from parasites: inducers of host immune regulation?
Linda Murray Expression of parasitic nematode genes in Caenorhabditis elegans
Erica Packard Characterisation and development of microsatellite markers for Haemonchus contortus and Teladorsagia circumcincta
Mark Robinson Functional aspects of Trichinella spiralis excretory-secretory proteins
Thomas Schnieder Immunisation of cattle with recombinant Major Sperm Protein (MSP) against Dictyocaulus viviparus
Susan Stasiuk The Insulin/IGF signalling transduction pathway in Parastrongyloides trichosuri - does it play a role in parasitism and aging ?
Christina Strube Expression and purification of bovine lungworm vaccine candidates
Alex Sykes Identification of CPN10 in Strongyloides ratti
Tom Walsh Identifying anthelmintic resistance associated alleles in Haemonchus contortus using real time PCR
Debra Woods Chemical genetics: Identification of new antiparasitic targets for veterinary medicine using novel antihelmintic compounds
MOLECULAR AND CELLULAR BIOLOGY OF HELMINTH PARASITES
HYDRA, GREECE
6 – 11 SEPTEMBER 2005
ABSTRACTS FOR ORAL PRESENTATION
Contributions from comparative genomics, forward and reverse genetics
and physiology to neural signalling mechanisms,
drug target identification and validation in C. elegans
David Sattelle
MRC Functional Genetics Unit, Department of Human Anatomy and Genetics
University of Oxford, Oxford OX1 3QX
david.sattelle@human-anatomy.oxford.ac.uk
Abstract not received
Focus on new drugs: putative receptors for
cyclooctadepsipeptides in parasitic nematodes
C. Welz 1, G. von Samson-Himmelstjerna 1, A. Harder 2, T. Schnieder 1
1 Institute for Parasitology, School of Veterinary Medicine, Hannover, Germany
2 Bayer HealthCare AG, Monheim, Germany
claudia.welz@tiho-hannover.de
Cyclooctadepsipeptides are a new class of anthelmintics. The first member of this class was PF 1022 A. It was isolated from the fungus Mycelia sterilia on the leaves of Camellia japonica at the end of the 1980s. Subsequent to this discovery of an anthelminticly active agent, several semi-synthetic derivatives were developed, one of which was Emodepside. Emodepside shows a better bioavailability than the strongly hydrophobic PF 1022 A. The mode of action is not yet known in detail. In Haemonchus contortus, the barber pole worm in sheep, a putative receptor has been identified. It is a member of the G-protein coupled receptors whose endogenous ligand is still unknown. In Caenorhabditis elegans two orthologous receptors called latrophilin-like protein 1 and 2 are known. In the present study, cDNA-sequences of orthologous structures from different parasitic organisms are described. The investigated parasites are trichostrongylids in cattle, Cooperia oncophora and Ostertagia ostertagi. The agent’s binding site is located within the extracellular N-termini of the receptors. For this reason the N-termini are to be expressed in E. coli and their functionality determined. Comparatively, mammalian latrophilines are analysed. These G-protein coupled receptors show high similarities to the parasitic structures. Latrophilin 1 binds latrotoxin, the main component of the black widow spider venom of Latrodectus spp. The endogenous ligands of latrophilins have also not yet been identified. The following experiments are intended to identify similarities and differences in the receptor’s ability to bind specifically Emodepside, as the most important substance within the cyclooctadepsipeptides. The results are expected to contribute to the characterisation of this new class of anthelmintics by examining the binding affinities of ligand and receptor. Furthermore, rescue experiments in C. elegans knockout mutants for latrophilin-like proteins are planned.
Molecular tools for the qualitative and quantitative assessment of anthelmintic resistance associated single nucleotide polymorphisms in parasitic nematodes
Georg von Samson-Himmelstjerna, Thomas Schnieder
Institute of Parasitology, University of Veterinary Medicine,
Buenteweg 17, 30559 Hannover, Germany
Single nucleotide polymorphisms have been described to correlate with benzimidazole (BZ) resistance in the beta-tubulin gene of several ruminant and horse strongyle species. According to early investigations in Caenorhabditis elegans and the sheep nematode Haemonchus contortus the TTC/TAC polymorphism in the beta-tubulin isotype 1 codon 200 was regarded as of most importance for the development of BZ resistance. Later studies in other sheep and horse strongyle species indicated that a similar SNP in the beta-tubulin codon 167 is also involved in the mechanism of BZ resistance in these parasites and may even be of higher significance than the codon 200 SNP. Most recently also one SNP in the glutamate-gated chloride chanel, as target of the macrocyclic lactones, was associated with resistance against this class of anthelmintics in cattle nematode species. We investigated SNP frequencies in susceptible and BZ-resistant populations of several cattle and horse strongyles and observed significant inter-species differences in the SNP frequencies. To achieve this we developed conventional and real time PCR assays which were found to be suitable for genotyping of single larvae and adult worms. For the SNP frequency assessment of a given nematode these methods have to be used on a representative number of individual parasites per population. This precludes the field use of these tests since it is labor and cost intensive. Therefore, the quantitative real time SNP sequencing of DNA isolated of pooled larvae based on the pyrosequencing technology was established. Here we report on the feasability of different qualitative and quantitative SNP genotyping procedures for the assessment of anthelmintic resistance in strongyle parasites. Furthermore, the beta-tubulin sequence variations and differences in SNP allele frequencies in populations with different anthelmintic resistance status of various nematode species will be described.
The structure of neuropeptide and neuropeptide receptors in flatworms
Tim A. Day1, Michael J. Kimber1, Paul McVeigh2,
Judith Humphries1 and Hanan Omar1
1Department of Biomedical Sciences, Iowa State University, Ames IA, USA and
2Parasitology Research Group, Queen's University Belfast
day@iastate.edu
Although amidated neuropeptides have a central role in the biology of both platyhelminths and nematodes, there are some dramatic differences in the complement of these peptides in the two phyla. For one example, only one or two FMRFamide-related peptides (FLPs) have been found in the platyhelminth species thus far examined, while in the nematodes an astounding variety of FLPs is present in each species. For another example, the most abundant amidated neuropeptide in every platyhelminth thus far examined is of the neuropeptide F (NPF) family. NPFs are structurally-related to the vertebrate NPY family of neuropeptides, and the presence of related peptides has not yet been established in the nematodes. Although no amidated neuropeptides other than those in these two large classes (that is, the FLPs and the NPFs) have been identified in platyhleminths, the distribution of the amidating enzyme in schistosomes suggests that others could be present and as yet unidentified.
We have identified a number of putative G protein-coupled neuropeptide receptors from flatworms through a combination of bioinformatics and molecular cloning. One of these is from the model flatworm Girardia tigrina, and that receptor is most similar in primary structure to vertebrate NPY receptors of the Y2 class. Somewhat surprisingly, the Girardia receptor is less similar to the known molluscan NPF receptor. When we co-expressed the Girardia receptor in mammalian cells with chimeric G-proteins, we found that the GPCR does not respond to flatworm NPFs despite its structural similarity to NPY receptors. Instead, the receptor responds most sensitively to flatworm FLPs. Specifically, the most potent ligand is GYIRFamide, a peptide known to be present in G. tigrina. The receptor signals through Gαi and Gαo G-proteins. The G. tigrina receptor also responds to a number of nematode-derived amidated neuropeptides. The most potent of these are peptides encoded on the C. elegans flp-1 gene, all of which share a carboxy-terminal PNFLRFamide motif. There is no data supporting the presence of PNFLRFamides in platyhelminths. The response of the platyhelminth receptor to neuropeptide motifs from nematodes demonstrates that receptors from both phyla share pharmacological properties.
Variety is the spice of a worm’s life:
subtypes of ACh receptor and levamisole resistance
Martin, R.J., Robertson, A.P., Clarke, C.L., Levandoski, M.M., and Qian, H.,
Dept of Biomedical Sciences, Iowa State University, Ames, IA, 50011
The continuous use of chemotherapeutic agents, including anthelmintics has lead to the appearance of resistance. The development of novel anthelmintic drugs to overcome this resistance has been limited. To counter this problem, our approach has been to understand the mode of action of existing anthelmintic drugs and to develop approaches that will counter resistance. We have focused in our studies on levamisole, a cholinergic anthelmintic. We use Ascaris muscle strips and classic pharmacological techniques, larval migration assays, along with patch-clamp techniques to record from single levamisole and nicotine activated channel currents. We found that the novel anthelmintics, paraherquamide and desoxy-paraherquamide, behave like subtype selective competitive cholinergic antagonists that separate out: 1) N-subtypes, preferentially activated by nicotine and oxantel; 2) L-subtypes, preferentially activated by levamisole and 3) B-subtypes, preferentially activated by bephenium. We have also observed that nicotine and methyridine remain active in levamisole-resistant O. dentatum larval migration assays, further suggesting pharmacological separation of cholinergic subtypes in nematode parasites. Recent patch-clamp studies were conducted to characterizes the channel properties of the subtypes. At the single channel level nicotine preferentially activates a 26pS channel but levamisole preferentially activates a 39 pS channel. All of these observations show that there are multiple cholinergic receptor subtypes in nematodes and suggest that some types of levamisole resistance are due to the loss of the L-subtype of receptor. These types of levamisole resistance might be overcome by using anthelmintics that are selective for other AChR subtypes (methyridine).
Are novel schistosome calcium channel subunits targets for praziquantel action?
Robert M. Greenberg1, Peter A.V. Anderson2, Andrea B. Kohn2,
Jessica Roberts-Misterly2, Michael C. Jeziorski3, and Joseph Consiglio1.
1Marine Biological Laboratory, Woods Hole, MA, USA, 02543; 2Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL USA, 32080; 3Neurobiology Institute, Universidad Nacional Autonoma de Mexico, Queretaro, Qro., Mexico, 76001
Praziquantel (PZQ) is the current drug of choice against schistosomiasis and other platyhelminth infections. Although PZQ has been known for some time to disrupt Ca2+ homeostasis in adult schistosomes, the molecular target of the drug remains undefined. Voltage-gated Ca2+ channels couple membrane depolarization to entry of extracellular Ca2+ and represent candidate targets for PZQ action. Ca2+ channels consist of a main pore-forming (1 subunit that is modulated by auxiliary subunits, including the ( subunit. When co-expressed with (1 subunits, ( subunits increase the gated current and exert dramatic effects on the biophysical properties of the channel. Although multiple ( subunit genes are present in mammals, only a single ( subunit gene is found in virtually all invertebrates examined to date. However, we have discovered that schistosomes and other platyhelminths express two ( subunits: a conventional subtype functionally similar to other known ( subunits; and a variant subtype with unusual structural features and functional properties. Instead of enhancing Ca2+ currents when co-expressed with (1 subunits in Xenopus oocytes, the variant schistosome ( subunit decreases current amplitude. The variant subtype also differs from conventional ( subunits in conferring PZQ sensitivity to an otherwise PZQ-insensitive (1 subunit, resulting in an increase in current in the presence of PZQ (100 nM). These unusual properties appear to be related to the absence in the variant subtype of two conserved consensus protein kinase C sites in the BID, a highly conserved domain that is crucial for determining the conformation of the ( subunit. Our results suggest that variant Ca2+ channel ( subunits that are unique to platyhelminths play an essential role in PZQ action. Supported by NIH AI-40522 and by the MBL Neal Cornell Research Fund.
The role of P-glycoproteins in resistance to ivermectin
and other macrocyclic lactones in parasitic nematodes
Roger Prichard, Alain Roulet, Bernadette Ardelli
Institute of Parasitology, McGill University, Montreal, Canada
Ivermectin and other members of the macrocyclic lactone class of anthelmintics have been used for many years to control gastrointestinal nematodes in animals and filarial nematode infections in humans, with great success. However, the development of ivermectin resistance has become a significant problem in nematode parasites of ruminant animals and could develop in filarial nematodes of humans. Ivermectin is an excellent substrate for transport by some P-glycoproteins and P-glycoprotein has been implicated in ivermectin resistance in the nematode Haemonchus contortus. In H. contortus at least 6 P-glycoproteins were sequenced and the expression level of each was assessed, using Real Time PCR, in laboratory and field strains selected with ivermectin or moxidectin and known to be ivermectin resistant. Significant levels of over expression were found in some of these P-glycoproteins in the resistant strains. Repeated treatment with ivermectin also imposes selection on P-glycoprotein in the human filarial nematode, Onchocerca volvulus. A detailed comparison of 28 regions of the O. volvulus P-glycoprotein gene was performed on samples obtained from either ivermectin naive patients and communities, or from patients and communities that were repeatedly treated with ivermectin, in Ghana. The P-glycoprotein locus in O. volvulus was characterized by reduced gene diversity, increased heterozygosity, an increase in the number of markers not in Hardy-Weinberg equilibrium and a disruption of linkage disequilibrium. These effects were associated with increasing ivermectin treatment. These results show ivermectin selects on some P-glycoprotein genes in parasitic nematodes, indicating a contributing mechanism for ivermectin resistance and providing information that will be useful for the development of markers to detect ivermectin resistance in nematodes.
RNA interference of neuropeptide signalling in plant parasitic nematodes
Aaron G. Maule1, Michael J. Kimber2, Sue McKinney1, Chris Holland1, Philip Donnelly1, Steven McMaster1, Gerry Brennan1, Nikki Marks1, and Colin C. Fleming3
1Parasitology Research Group, School of Biology and Biochemistry,
Queen’s University Belfast, Belfast BT9 7BL;
2School of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA;
3Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX.
a.maule@qub.ac.uk
Nematode FMRFamide-like peptides (FLPs) represent the most diverse neuropeptide family known. In C. elegans, some 24 flp genes encode 60 distinct FLPs and available expressed sequence tags indicate similar diversity within parasitic nematodes. FLPs appear to play a central role in motor coordination with a variety of actions on pharyngeal, ovijector and somatic muscles as well as the ability to modulate sensory perception and behaviour. Since many of the leading anthelmintics target motor coordination in nematode parasites, we believe that FLPergic signalling could provide a rich seam of targets for parasite control. Plant parasitic nematodes are estimated to cause losses in plant crops in excess of US$100 billion per year. Current control strategies rely heavily on nematicides for which there are serious environmental and toxicological concerns - several are due for withdrawal. In light of this, novel control measures must be developed for these parasites. Recent studies by other workers have reported that RNA interference (RNAi) can be used to inhibit gene expression in plant parasitic nematodes. However, none of these studies have investigated the sensitivity of neuronally-expressed genes to RNAi and numerous studies have reported that neuronal genes in C. elegans are refractory to RNAi. In contrast, we have found that selected flp genes in plant parasitic nematodes are sensitive to RNAi and that exposure to flp gene dsRNA triggers aberrant phenotypes. In this study we further investigate the utility of RNAi in the silencing of various components of FLP-signalling to establish their potential as control targets in plant parasitic nematodes. The primary focus in this work is the potato cyst nematode, Globodera pallida. We have found that exposure to flp gene dsRNA causes abnormal motor activities in G. pallida that are incompatible with parasite survival. Furthermore, these effects are persistent in that they are still evident 6 days after removal of the dsRNA trigger. The effects of dsRNA on the ability of the parasites to migrate through a sand column were found to be concentration dependent and were still apparent at concentrations as low as 1 ng/ml. Although flp genes from different nematodes display homology, the effects are highly specific in that flp gene homologues from distinct nematode species do not induce motor deficiencies. Further studies have compared the effects of flp-gene silencing with those seen when selected unc gene homologues are silenced to reveal if the differences in neuronal gene sensitivity between plant parasitic nematodes and C. elegans are gene-dependent. The sensitivity of FLP signalling processes in plant parasitic nematodes to RNAi highlights this system as a candidate target for future control strategies.
Biosynthesis and enzymology of the C. elegans cuticle
Antony P. Page
Institute of Comparative Medicine, University of Glasgow, Glasgow G61 1QH.
The nematode cuticle is an extremely resilient exoskeleton that allows growth, permits locomotion and confers environmental protection. This entire extracellular matrix (ECM) is synthesised 5 times, once in the embryo and subsequently through a process termed moulting. This ECM is composed predominantly of highly cross-linked collagens. Collagen biosynthesis involves numerous co- and post-translational modification, processing and cross-linking steps that in turn are catalysed by specific enzymes. The enzymology of nematode cuticle collagen assembly and crosslinking will be discussed focussing on recent advances using the model nematode Caenorhabditis elegans.
Oxidative stress in Caenorhabditis elegans: protective effects of the
omega-class glutathione S-transferase (GSTO-1)
C. Burmeister1, M. Domagalski1, R.D. Walter1 and E. Liebau1
1Dept. of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg
Oxidative stress provokes various responses and many genes participate in the antioxidant defensive system, including the omega class glutathione S-transferases (GSTs). The GSTO-1 from C. elegans is a member of the GST superfamily that utilizes glutathione in reactions and contributes to the biotransformation and disposition of many compounds, including drugs, carcinogens, and the products of oxidative stress.
Detailed biochemical analysis of the substrate specificity and kinetics of the recombinant GSTO-1 expressed in Escherichia coli revealed high thiol transferase and dehydroascorbate reductase activity. By "halo assay" we showed that the survival of bacteria, overexpressing GSTO-1, significantly increased under several stress conditions. Furthermore, we detected that under oxidative stress conditions mRNA levels of GSTO-1 were highly upregulated in C. elegans.
By microinjection of different GSTO-1-promoter green fluorescent protein constructs, the protein localized exclusively in the intestine of all post-embryonic stages of transgenic C. elegans. Furthermore, mutation analysis demonstrated the involvement of a GATA-box in basic expression. Transgenic C. elegans overexpressing GSTO-1 exhibited an increased resistance under paraquat/cumene hydroperoxide-induced oxidative stress and – as expected – the specific silencing of the GSTO-1 by RNA-interference (RNAi) created worms with an increased sensitivity towards several prooxidants.
Cathepsin L involvement in host penetration by cercariae
of the fish eye fluke Diplostomum pseudospathaceum
Mikes L., Dolecková K. and Kasny M.
Department of Parasitology, Charles University, Viničná 7,
12844 Prague 2, Czech Republic
Trematodes of the genus Diplostomum possess trixenous life cycles, with fish being the second intermediate hosts. Among numerous species, D. pseudospathaceum is widely distributed and common in Central Europe. Infective larvae, the cercariae, directly penetrate fish skin and gills, transform to diplostomula and migrate to the site of final localization, which is the eye lens. The infection can result in blindness or even death in fish fingerlings, thus causing economical losses in freshwater fish industry.
The way the hosts become infected by D. pseudospathaceum cercariae is similar to the case of infections caused by the blood flukes of the family Schistosomatidae parasitizing in homoiotherm vertebrates, thus challenging a comparison. As schistosomes are known to employ peptidases in the penetration process, the attention was focused on these enzymes.
A 24 kDa cysteine peptidase was isolated from cercarial extracts of D. pseudospathaceum by means of ion exchange and affinity chromatography. It is a basic protein (pI 10.1) migrating as a double band around 24 kDa in SDS-PAGE. De novo sequenced tryptic peptides showed significant alignments with cysteine proteases of different origin belonging to the CA clan of cysteine proteases. Fluorometric substrate assays showed a strong activity with Z-Phe-Arg-AMC with a maximum under slightly basic conditions; this could be inhibited by irreversible cysteine protease inhibitor E-64. Parasite mRNA was isolated from cercarial germ balls within sporocysts and transcripted by RT-PCR using a forward primer based on a peptide sequence obtained by mass spectrometry and a reverse oligo-dT primer. The whole sequence of the molecule was obtained employing 3‘ and 5‘ RACE methods. The protein showed a high similarity to cathepsin Ls of adult Fasciola gigantica and F. hepatica liver flukes. Immunohistochemistry as well as ligand histochemistry confirmed localization of the peptidase in penetration glands of cercariae. Substrate specificity and biochemical properties of the peptidase were characterized. Its ability to cleave components of host skin/mucus and involvement in cercarial glycocalyx shedding during penetration were also tested.
Atomic structure and lipid binding site of a polyprotein allergen of nematodes
Malcolm W. Kennedy, Nicola A.G. Meenan, Lindsay C. McDermott,
Alan Cooper and Brian O. Smith.
Institute of Biomedical and Life Sciences, Graham Kerr Building, and Department of Chemistry, University of Glasgow, Glasgow G12 8QQ
malcolm.kennedy@bio.gla.ac.uk
The nematode polyproteins allergens (NPAs) are synthesised as large precursors that are cleaved posttranslationally into multiple copies of ~14kDa functional proteins. These are distributed widely within the tissues of nematodes, and are also secreted into parasitised hosts. There is strong MHC class II restriction of immune responsiveness to NPAs, recombinant forms are functional allergens, and IgE antibody responses to them appear to associate with natural resistance to certain nematode infections in humans (best proven for Ascaris). In some species of parasite, the units of the polyprotein are diverse in sequence, and in others they are identical or nearly so. Such tandemly repetitive polyproteins are rare in nature, and the structures of none have been reported so far. Natural or recombinant forms of individual NPA units have been shown to bind saturated and polyunsaturated fatty acids, and retinol (Vitamin A), and fluorescence-based assays indicate that the binding site is unusually apolar. The proteins appear not to interact directly with artificial membranes to offload/exchange their cargo, so may instead interact with receptors on the parasites’ (or the host’s?) cells. In addition to probably being the nematode functional equivalent of our serum albumin, the activity of secreted NPAs may be to corrupt the host’s lipid-based signalling system.
The atomic structure of a single ~14kDa unit of the NPA of Ascaris (ABA-1A; As-NPA-1A) as solved by protein nuclear magnetic resonance (NMR) has now been solved. This reveals a novel type of helix-rich structure, and provides structural and dynamical evidence that NPA units are themselves derived from a duplication event, giving rise to two discrete, but structurally integrated, domains. Genetic manipulation of the protein previously provided evidence that the lipid-binding site is in the C-terminal domain, and NMR chemical shift data in the presence and absence of lipid ligand reveal the precise location of the binding site and the ligand-interacting side chains. The new structure also partly explains the results of site directed mutagenesis that produces ligand binding defective, or destabilised, ABA-1A proteins, and molecular modelling using NPA sequences from other species (e.g. Brugia) using the new structure as template show features consistent with other biochemical findings. A final curiosity is that ABA-1A refolds extremely rapidly after denaturation, which might be of potential importance to the polyproteinism of the NPAs.
Pathogen genome sequencing: from protozoology to helminthology.
Matthew Berriman and the Pathogen Sequencing Unit
Pathogen Sequencing Unit, Wellcome Trust Sanger Institute, Hinxton, UK
Genome sequencing and comparative genomics has been commonplace in microbiology for several years. In protozoology, the genomes of many important pathogens have been sequenced and comparative genomics is becoming an established tool. As sequencing costs continue to fall, molecular helminthology is moving into a genomic era. Recent parasitic protozoan genome projects may provide us with a taste of things to come in the helminth field.
At the Wellcome Trust Sanger Institute, two genomes – those of Haemonchus contortus and Schistosoma mansoni - are serving as pilot projects. The current status of these genomes will be presented along with a review of current gene-finding efforts. Schistosoma mansoni, in particular, is the largest pathogen genome to be sequenced to date and the unusual composition of its genome presents some interesting challenges for gene finding.
By looking at the protozoa, what can we expect to see in helminth genomics? What problems are we facing now that should be borne in mind in the planning of future projects?
Genetic analysis of Trichostrongylid nematodes of Veterinary Importance
John Gilleard*, Erica Packard*, Victoria Grillo*, Justin Pachebat+ and Paul Dear+
*Division of Infection and Immunity, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, U.K
+ MRC-LMB, Cambridge, U.K
Trichostrongylid nematode parasites such as Haemonchus contortus and Teladorsagia (Ostertagia) circumcincta are amongst the most economically important pathogens of livestock worldwide. The emergence of anthelmintic resistance is making the control of these parasites increasingly difficult. In order to understand the role of particular genes in anthelmintic resistance, better genetic and genomic resources are needed as is a better understanding of the population genetics of these organisms. At present, the definition of nematode species is based on morphological criteria and there are few genetic markers available to define and monitor laboratory and field strains. Consequently, we have developed panels of microsatellite markers for both H.contortus and T.circumcincta and are using these in population genetic studies. Our results suggest that, in the case of Teladorsagia, the conventional morphological criteria do not reflect the genetic differences seen in populations. In the case of Haemonchus our analysis supports the definition of H.contortus and H.placei, as separate species. In addition, there are extremely high levels of genetic divergence between some of the different H.contortus isolates. This not only has important implications for the genetics of anthelmintic resistance but also makes it imperative that the laboratory strains used for experimental work are genetically defined. Consequently we have developed a rapid technique to genotype different strains of H.contortus using our marker panels. We are also placing polymorphic markers on a HAPPY map of the H.contortus genome and progress on this mapping work will be presented.
A transcriptomic survey of Echinococcus granulosus
Cecilia Fernándeza, John Parkinsonb, Gustavo Salinasa, Alvaro Díaza, Henrique Ferreirac, Matt Berrimand, Mark Blaxtere & Rick M Maizelsf
aCátedra de Inmunología, Facultad de Química, Universidad de la República, Uruguay bProgram in Genetics and Genomic Biology, Hospital for Sick Children, Toronto, Canada cCentro de Biotecnologia, Universidade Federal de Rio Grande do Sul, Porto Alegre, Brazil dPathogen Sequencing Unit, The Wellcome Trust Sanger Institute, Hinxton, United Kingdom eInstitute of Evolutionary Biology and
fInstitute of Immunology and Infection Research University of Edinburgh, UK
cfernan@fq.edu.uy
A successful parasitism is established and maintained through events of molecular recognition that involve signals and receptors from the parasite and its hosts. The cestode Echinococcus granulosus is an excellent model for the study of such cross-talks events because it is extremely well adapted to its definitive and intermediate hosts. In order to develop tools for this sort of analysis, we have launched an EST-based gene discovery project of E. granulosus larval stages, focusing on the identification of:
i) genes coding for secreted and membrane-bound proteins, on the grounds that these are likely to be the most critical ones in terms of the host-parasite homeostasis;
ii) genes differentially expressed in parasite materials representative of cross-talk with definitive and intermediate hosts.
Following strategies targeted at cloning copies of mRNAs with an intact 5’ end, we constructed two sets of full-length cDNA enriched libraries from larval worms, the hydatid cyst wall and pepsin-activated larval worms; the libraries were found to include high proportions of signal-sequence encoding genes. A multi-center effort has generated about 10000 ESTs from these libraries and the resulting cDNA sequences have been clustered into 2700 potential gene products. The dataset thus created has increased the quality and the quantity of the molecular information on E. granulosus: 95% of known genes from the parasite were identified in this context; and a full-length cDNA is available for a high proportion of those newly described [ LophDB.php].
We present the key features of the survey in the context of the biology of E. granulosus; in particular, we describe molecules that may be involved in parasite adaptation, such as:
i) a family of serine protease inhibitors, whose expression is up-regulated upon treatment of the larval worms with pepsin and that, therefore, could play a role in the establishment of the larvae in the definitive host;
ii) a group of polypeptides predicted to be secreted and O-glycosylated, identified in the tissue of the hydatid cyst wall; these candidate apomucins could be included in the external layer of the tissue-dwelling cyst, a mucin-rich structure which is critical for parasite survival in the intermediate host.
We also describe the E. granulosus dataset from a perspective of cestode biology, through comparisons with the transcriptomes from other parasitic and free-living platyhelminths (the trematodes Schistosoma mansoni and S. japonicum; and the planaria Schmidtea mediterranea, respectively). Finally, because the strategies used to prepare the E. granulosus libraries yielded separate sets of trans-spliced and oligo-capped cDNAs, we present an analysis of parasite transcripts whose expression involves trans-splicing.
Contribution of Trans-splicing to mRNA Translation and Decay and
Characterization of Nematode Cap-Interacting Proteins
Richard E. Davis1,2, Leah Cohen2, Sabbi Lall2,3, Fabio Piano3,
Cassandra Friedmann2, and Claudette Mihkli2
1Dept of Biochemistry and Molecular Genetics and Pediatrics,
University of Colorado School of Medicine
Depts of Biology, 2CUNY Graduate Center, and 3New York University
richard.davis@uchsc.edu
mRNA metabolism and cap-interacting proteins in nematodes/flatworms must deal with two populations of mRNAs, 1) trans-spliced mRNAs that acquire a 5’ UTR conserved spliced leader and trimethylguanosine cap and 2) non-trans-spliced mRNAs with variable 5’ UTRs and a monomethylguanosine cap. We are investigating the role of trans-splicing on mRNA stability and translation and the adaptation of cap-interacting proteins to two different mRNA caps. We have developed and characterized a cell-free translation and decay system and biolistic methods to examine mRNA translation and decay in vitro and in vivo in Ascaris embryos. The translation system is robust, highly cap-dependent, and exhibits cap and poly(A)-tail synergism. The nematode translation apparatus has evolved to translate TMG-capped mRNAs primarily in the context of the spliced leader sequence. The SL in the context of the TMG-cap synergistically enhances translation in vitro and in vivo. Overall, translation of trans-spliced test mRNAs is typically less efficient in vivo and in vitro than non-trans-spliced test mRNAs. Test mRNAs with the TMG-cap and SL sequence do not have significantly different half-lives than non-trans-spliced mRNAs. Ascaris embryo eIF4E binds both monomethyl- and trimethylguanosine caps and translates both types of mRNAs. eIF4E binding to the TMG cap is enhanced in the context of the spliced leader sequence providing an explanation for the cap-SL synergism. Bioinformatic analyses demonstrate that the SL sequence tends to trans-splice close to the start codon in a diversity of nematode (and flatworm) species. This evolutionary conservation is functionally reflected in the optimal SL to AUG distance for reporter mRNA translation in the Ascaris cell-free system. Overall, the data suggest that SL1 trans-splicing does not have a general or broad effect on translation or mRNA half-life, but may have evolved as a mechanism to provide an optimal mRNA translation initiation context (e.g., heterogeneous transcription initiation?).
The primary general pathway of in vitro mRNA decay in Ascaris embryos is by 3’ to 5’ decay followed by hydrolysis of the resulting cap. This pathway is 15-fold more active than 5’ to 3’ decay initiated by mRNA decapping. C. elegans RNA interference experiments with either decapping enzymes alone or together have not lead to lethal embryonic phenotypes. We have cloned and functionally characterized C. elegans decapping enzymes (DcpS, Dcp1 and Dcp2). DcpS differs from the human enzyme in several substrate requirements including its ability to hydrolyze trimethylguanosine caps. Dcp2 is catalytically active on RNA substrates > 250 nts. Its activity is affected by both 5’ terminal RNA sequences and their context: mRNAs with a spliced leader are decapped 10-fold less efficiently. These data suggest that Dcp2 may be involved in regulated mRNA turnover in nematodes. Dcp2 can also decap TMG-capped RNAs. However, this is not a unique characteristic in nematodes as both human and yeast Dcp2 can function on TMG-capped RNAs. Several nematode cap-interacting proteins exhibit characteristics that are unique from their hosts making them potential and novel helminth drug targets. The in vitro systems provide a tool for testing and evaluating new anthelminthic compounds against mRNA metabolism.
Designing and Mining Pathogen Genome Databases: Insights into Basic Parasite Biology, and Targets for Diagnostic, Drug and Vaccine Development
David Roos
Department of Biology and Penn Genomics Institute
University of Pennsylvania, 415 South University Ave, Philadelphia, USA
droos@sas.upenn.edu
Genomic-scale projects yield vast datasets, from genome and EST sequences, to RNA and protein expression profiles, to interactome and metabolic pathway data, to polymorphisms identified at the population level, and comparative genomics data gleaned from cross-species analysis. Valuable though they may be, however, the emergence of such data -- at ever-increasing rates -- raises an important problem: how to effectively capture, maintain, update, annotate, integrate, and query these resources to advance biomedical research? Genome database development presents challenges for any organism, but certain consistent features apply to taxonomically diverse pathogen species. For example, in contrast to most studies on human metabolic diseases, highly abundant targets are often of greatest interest for drug/vaccine/diagnostic development. Taxonomically-related species permit revealing comparisons between pathogenic and non-pathogenic organisms, facilitating the development of broad-spectrum antibiotics. Correlations between pathogen and host genomes provide additional opportunities for productive exploration.
As new genomic-scale datasets relevant to helminths emerge, it may be useful to consider insights gleaned from the genomics of protozoan parasites. The Plasmodium genome database () provides access to information emerging from various genome sequencing and functional genomics projects for several parasite species, enabling malaria researchers to formulate their own queries. In 2004, PlasmoDB received >6M hits from >45K unique users in >100 countries. Data types available for browsing, downloading, analysis, and dynamic queries include genome and EST sequence for eight Plasmodium species, curated and automated analyses of gene/protein predictions, RNA and protein expression data, data on genetic mutability and population diversity, protein interactome data, ortholog/paralog identification, reagents, publication records, user comments, etc. Particular effort has been invested in extracting information from incomplete datasets (including EST data), comparing expression profiling data across multiple platforms, comparative/phylogenetic approaches, and integrating complex database queries. Combining data from Plasmodium with the related human and veterinary pathogens Toxoplasma and Cryptosporidium provides an integrated apicomplexan parasite database (ApiDB), enabling cross-species comparisons. Comparison with human (and vector) genome(s) has expedited a variety of projects of biological and evolutionary interest, and highlights phylogenetically-restricted targets suitable for diagnostic, drug, and vaccine design.
Proteomic analysis of excretory-secretory proteins from Trichinella
Bernadette Connolly, Mark W. Robinson and Rachel Greig
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, UK
b.connolly@abdn.ac.uk
Excretory-secretory (ES) proteins from helminth parasites are believed to play an important role in host-parasite interactions. For Trichinella spp. these proteins may facilitate the establishment of new infections within the intestine of the host or may play a role in the formation and maintenance of the nurse cell during the muscle stage of the life-cycle. Therefore, the identification of these proteins and the analysis of their expression patterns throughout the life-cycle is important to our understanding of Trichinella infections. Although individual ES proteins from Trichinella spiralis and Trichinella pseudospiralis have previously been identified, little is known about the overall complexity of the ES fractions from different life-cycle stages and how they differ between encapsulating and non-encapsulating species. In the current study, we have used a proteomics approach to address these issues. Protein profiling by 2-dimensional gel electrophoresis (2-DE) has produced information on the complexity of the Trichinella ES proteins. Significant differences between the ES profiles of T. spiralis adults, newborn larvae and muscle larvae are apparent suggesting that ES protein expression is regulated throughout the life-cycle. Furthermore, the use of MALDI-TOF MS and LC-MS/MS has led to the identification of several ES proteins from Trichinella muscle larvae. The initial success of the proteomic analysis has been encouraging given the relatively small database of available Trichinella sequences and does highlight the value of such an approach. In the summer of 2004 the National Human Genome Research Institute announced the inclusion of T. spiralis on the genome sequencing project list and as this project gets underway it is likely that the success rate will continue to increase and currently un-assigned ES proteins will be identified.
Mass spectrometric analysis of the S. mansoni tegumental proteome: identification of unique and tegument-specific proteins.
Bas W.M. van Balkom1,2, Renske A. van Gestel3, Jos F.H.M. Brouwers3, Jeroen Krijgsveld1, Aloysius G.M. Tielens3, Albert J.R. Heck1 and Jaap J. van Hellemond3
1Department of Biomolecular Mass Spectrometry, Utrecht University, The Netherlands, 2Institute for Veterinary Research, IVW/GSAH, Utrecht, The Netherlands and
3Dept. Biochemistry and Cell Biology, Faculty of Veterinary Medicine,
Utrecht University, Utrecht, The Netherlands.
Schistosomiasis, a parasitic disease affecting over 200 million people worldwide, is a major cause of morbidity in (sub-)tropical countries. The blood fluke Schistosoma mansoni, causing the disease, is a long-term inhabitant of the mesenteric veins and employs multiple mechanisms for survival in the mammalian host. The tegument, the highly specialized outer surface of the parasite, forms the site of contact with the host and plays an important role in immune evasion. This layer consists of two closely apposed lipid bilayers that overlay a layer of fused cells, the syncytium. The S. mansoni tegument is a very complex structure and unique in nature, comprising specific lipid components. The S. mansoni genome-sequencing project is now nearing completion, smoothening the path for the analysis of the schistosomal proteome. In this study, we aimed at identifying tegument-specific proteins through isolation of the tegument, followed by mass spectrometric analysis of the protein content, in which the proceedings of the genome sequencing project were used for identification. Thereby we were able to identify a total of 740 proteins, of which 43 were specifically and reproducibly localized in the tegumental fraction, and 207 were specific for the body of the worm. Functional and structural aspects of these tegument-specific proteins will be discussed.
Study of the interaction between adult Ostertagia ostertagi excretory-secretory products and bovine abomasal proteins by cDNA phage display
Saverwyns H., Vercauteren I., Peelaers I., Vercruysse J. and Claerebout E.
Ghent University, Faculty of Veterinary Medicine, Laboratory of Parasitology
Salisburylaan, 133 B-9820 Merelbeke, Belgium
Ostertagia ostertagi, an important abomasal nematode in cattle induces substantial morphological and physiological changes, however many biochemical, immunological and physiological mechanisms are still unknown. Our objective is to obtain a better understanding of the protein-protein interactions which are involved with the pathophysiological and immunological changes in the abomasum caused by an Ostertagia infection. Excretory-secretory (ES) products are known to be released by nematodes and presumably play important roles in host penetration, parasite feeding and escape of host immune-responses but also cause physiological changes in the abomasum during an Ostertagia infection.
A novel phage display system is used to study these protein-protein interactions between parasite (ES) and host (abomasum) during infection. This system allows functional expression of cDNA libraries on the surface of the filamentous phage through the attachment to the C-terminus of the minor coat protein VI. A helminth naïve calf was infected with 100,000 infective O. ostertagi L3 larvae. Upon necropsy (21 days p.i.) abomasal tissue was collected at the fundic region. Bovine abomasal cDNAs were fused in each of the three reading frames to the 3’end of the M13 gene VI expressed by the pG6 phagemid vectors pG6A, B and C. Phages rescued from these three abomasal cDNA expression libraries (each representing a reading frame) were subjected to biopanning against adult O. ostertagi ES products. In total seven different cDNA clones were identified by sequence analysis. They showed homology with bovine proteins such as macrophage lysozyme (lyso), tubulin cofactor A (cofA), beta-2-microglobulin (b2m), guanine nucleotide binding protein G(s) (adenylate cyclase stimulating = GNAS), mitochondrial 12S rRNA and a homo sapiens casein kinase 2 and equus caballus partial 18S rRNA. We have obtained the corresponding full-length cDNAs of lyso, b2m, GNAS and cofA followed by recombinant protein production in Escherichia coli. The recombinant proteins are currently purified for use in a reverse biopanning with adult O. ostertagi phage display cDNA expression libraries followed by identification of the parasite cDNAs of interacting phages.
Parastrongyloides trichosuri as a new model nematode parasite
Warwick Grant1, Susan Stasiuk1, Jan Newton-Howes1 & Chuck Shoemaker2
1Wallaceville Animal Research Centre, AgResearch, Upper Hutt, New Zealand
2Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, MA 01536
Parastrongyloides trichosuri is a nematode parasite of small marsupials with the unusual ability to undergo multiple free-living life cycles in addition to a conventional parasitic life cycle which consists of skin penetrating infective larvae and adults which reside in the small intestine. We have exploited this feature to develop culture conditions for the indefinite maintenance of this species as a free-living nematode and for triggering free-living L1 stage worms to develop into infectious L3. Furthermore, we have developed a variety of genetic tools with which the biology of P. trichosuri can be studied and manipulated. The tools include methods for transgenesis, where the transgenes are expressed and inherited through both the free-living and parasitic life cycles, and the development of single nucleotide and simple sequence repeat polymorphisms and inbred lines (by repeated brother-sister single pair mating) for genetic analysis. To date, we have been unable to reproducibly produce clear-cut RNAi phenotypes in P. trichosuri using methods and gene targets that we have found effective in several strongyle parasites and we will present our experiences to date. From a biological point of view, we are focusing on several aspects that differ significantly between free-living and parasitic life cycles with the aim of discovering key genes required for parasitism in this species. 1) We have shown that the decision between life-cycles is determined by the concentration of an unidentified compound produced by free-living worms in a manner analogous to the Caenorhabditis elegans dauer switch. 2) We have used a collection of expressed sequence tags as a starting point for comparison of gene expression between the life cycles and are characterising the expression of several cloned orthologues of key dauer genes (including daf-7, daf-2, age-1 and daf-16). 3) We have characterised the life span of free-living and parasitic adults and have shown that there is at least a 20-fold extension of life span in parasitic adults compared to free-living adults. 4) We are preparing to test the role these dauer gene orthologues play in regulating this remarkable plasticity of life span in P. trichosuri. In summary, we will introduce P. trichosuri as a powerful new model system in which the key genetic determinants of parasitism and longevity can be discovered and manipulated.
Using C. elegans techniques for identification
and expression of potential parasite target genes.
Collette Britton, Peter Geldhof*, Linda Murray,
John Gilleard, Annabelle Couthier and David Knox*
Division of Vet Infection and Immunity, University of Glasgow, UK and
*Moredun Research Institute, Edinburgh, UK.
c.britton@vet.gla.ac.uk
Identification of potential target genes and expression of these in a suitable form are major steps in the development of potential vaccines for parasitic nematodes. We have been using techniques developed in the free-living nematode Caenorhabditis elegans, namely RNA interference (RNAi) and transgene expression, to approach these. RNAi is widely used in C. elegans to identify gene function and has been adapted as a high throughput screening method to identify genes involved in essential processes. We have been examining whether RNAi could be adapted as a screen to identify essential genes in the strongylid parasitic nematode Haemonchus contortus. We targeted ten H. contortus genes which, based on their sequence similarity to essential C. elegans genes, may be predicted to result in larval phenotypes following RNAi. As well as phenotypic examination, enzyme activity assays and RT-PCR were carried out to examine any decrease in protein and mRNA levels following RNAi. For all genes tested we have so far observed no phenotypic differences between RNAi treated and control worms, nor any decrease in specific enzyme activities or mRNA levels of targeted genes. This was found after dsRNA soaking or feeding of the infective L3 larval stage, free-living L2 stage and adult worms. Our findings suggest that the RNAi approaches used in C. elegans may not be effective in gene silencing in strongylid nematodes, which may reflect differences in dsRNA uptake into cells and tissues or in the gene silencing mechanism between nematode species.
To express parasite proteins in a similar form to their native conformation in the parasite, we are carrying out transgene expression in C. elegans. We previously showed that an H. contortus cathepsin L protease gene expressed from an extrachromosal transgenic array could rescue the phenotype of a C. elegans cathepsin L mutant. This showed that the parasite gene was expressed at sufficient levels and in the correct, active conformation in C. elegans. By inserting a His tag at the C-terminal of the H. contortus gene we have expressed and purified H. contortus cathepsin L for testing in vaccination studies. We are also examining expression levels of other Haemonchus genes expressed in C. elegans, using several different C. elegans promoters. For some proteins at least, this approach may be a suitable alternative to expression in bacteria and yeast.
Transgenesis in Strongyloides stercoralis: administration, transient expression, silencing and inheritance patterns of plasmid-based reporter constructs.
James B. Lok1,3, Xinshe Li1, Holman C. Massey, Jr1., Thomas Nolan1, Gerhard A. Schad1, Ariel Junio2, Kelly Kraus2 and Meera Sundaram2.
1 Department of Pathobiology, School of Veterinary Medicine and 2 Department of Genetics, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
jlok@vet.upenn.edu
We are working towards a system for heritable genetic transformation of the parasitic nematode Strongyloides stercoralis. Previously, we reported that constructs fusing the promoters for S. stercoralis act-2 and era-1 to the coding sequence for gfp are expressed in embryonic progeny of free-living female worms microinjected intragonadally with the plasmid. No expression of these constructs was observed in larval progeny. Since then, we have discovered that certain strong C. elegans promoters, myo-2, sur-5 and mec-7, also drive robust gfp expression in embryonic progeny of microinjected female S. stercoralis. The percentage of F1 embryos expressing after microinjection of parental worms with plasmid at 100 ng/µl was 7.8 for myo-2::gfp, 10.3 for mec-7::gfp and 8.2 for sur-5::gfp. Dose titration in the range of 2-200 ng/µl revealed no clear correlation between the percentage of F1 embryos expressing and the concentration of plasmid microinjected. The percentage of expressing embryos was also unaffected by co-injecting reporter constructs with varying concentrations of carrier DNA in the form of either native or enzymatically digested plasmid or S. stercoralis gDNA. Neither approach resulted in construct expression in developing F1 larvae, and development in GFP-expressing embryos was uniformly arrested at the ‘morula’ stage. Despite their lack of expression, approximately 50% of F1 larvae from broods with embryos expressing the sur-5::gfp construct (15 of 30 individuals tested) also contained the gfp coding sequence as determined by single-worm PCR. RT-PCR performed on pools of these individuals failed to detect gfp-specific mRNA. We have derived an F2 generation from these transformants by passage through gerbils and have detected the gfp transgene in F2 larvae by PCR. No GFP expression has been observed in these F2 transformants. Thus, intragonadal microinjection of S. stercoralis with plasmid vector constructs results in heritable DNA transformation of progeny. We hypothesize that in contrast to C. elegans, normally developing S. stercoralis silence these microinjected expression constructs by some mechanism that is not operable in abortively developing embryos. Currently we are investigating modifications of our transformation constructs that may allow expression as well as alternate delivery methods such as biolistic transfer, which, in C. elegans, gives a higher frequency single-copy chromosomal integration of transgenes than microinjection, which favors formation episomal arrays.
Transduction of Schistosoma mansoni sporocysts by VSVG pseudotyped Moloney murine leukemia retrovirus constructs encoding luciferase and other transgenes
1,2Kristine J. Kines, 1Victoria H. Mann, 1,2Maria E. Morales,
3Bernd H. Kalinna and 1,2Paul J. Brindley
1Department of Tropical Medicine, School of Public Health and Tropical Medicine, and 2Interdisciplinary Program in Molecular and Cellular Biology, Tulane University, Health Sciences Center, New Orleans, Louisiana 70112, USA
3Department of Molecular Parasitology, Institute of Biology, Humboldt University-Berlin, Berlin, Germany
Transgenesis techniques offer the promise to determine the importance of schistosome genes that could be targeted in novel interventional strategies. The sporocyst stage of the Schistosoma mansoni is an attractive developmental stage for the introduction of transgenes since it contains germ balls that, if transformed with a transgene, may propagate stable, heritable transgenic lines of schistosomes. We derived in vitro cultures of S. mansoni sporocysts after collecting parasite eggs from livers of experimentally infected mice. Eggs purified on Percoll gradients of collagenase-digested mouse livers were induced to hatch in water to release miracidia, which were transferred to medium MEMSE-j, and the resulting mother sporocysts were cultured under 5% O2, 5% CO2, N2 at 27º C. We modified the murine leukemia virus vector pLNHX (BD Biosciences Clontech) to include luciferse or EGFP reporter genes under control of schistosome gene promoters including the sliced leader RNA gene promoter, and employed the modified pLNHX plasmids along with a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG), and with GP293 producer cells, to generate replication incompetent retrovirus particles pseudotyped with VSVG. These virions were employed to transduce the in vitro derived mother sporocysts cultured in Transwell (Costar) plates in the presence of polybrene. Analysis of RNA isolated from treated sporocysts by RT-PCR demonstrated the presence of transcripts encoding luciferase, which indicated that the sporocysts had been transduced by the VSVG pseudotyped retrovirus. We are employing Southern hybridization and anchored PCR approaches to determine whether proviral forms of the retroviral constructs have integrated into the schistosome genome. This model system represents a route for the introduction of foreign genes into cultured sporocysts and may represent a way forward towards routine transgenesis and insertional mutagenesis of schistosomes.
The role of protein tyrosine kinases in female schistosome development
Christoph G. Grevelding#, Jürgen Knobloch, Katja Kapp*, Svenja Beckmann#, Volker Wippersteg, Stefan Sroka, Reiner Lammers*, Thomas Quack# and Werner Kunz
# Justus-Liebig-University, Institute for Parasitology, 35492 Gießen, Germany; Heinrich-Heine-University, Institute for Genetics, 40225 Düsseldorf, Germany; * Eberhard-Karls-University, Medical Clinic IV, 72076 Tübingen, Germany
Female schistosomes depend on signals from the male to induce mitogenic and differentiation processes in the vitellarium, where vitelline cells are formed for egg production. If the male is separated from the female, mitoses and differentiation of vitelline cells are stopped, and egg production ceases. Upon remating, the stopped processes are reinitiated including egg production. Although this phenomenon has long been known, the molecular basis of this unique male-female interaction is not understood.
Cytoplasmic protein tyrosine kinases (PTKs) of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation. Among other functions, these kinases are involved in regulating the cell architecture. One aim of our study was to biochemically characterize the putative role of PTKs in schistosomes for the control of mitoses and/or differentiation in females. Towards this end, we investigated the influence of chemical inhibitors on mitoses in paired schistosomes kept in culture. The used inhibitors selectively blocked different classes of cellular protein tyrosine kinases (Src or Syk). Evidence was obtained that an Src-specific inhibitor reduced mitogenic activity in paired females but not in males, whereas a Syk-specific inhibitor did not affect mitoses in both genders. Beyond that, the used Src-specific inhibitor also reduced egg production in paired females, a process that was not influenced by the Syk-specific inhibitor. Another aim of our study was to isolate and characterize PTK genes from S. mansoni. Isolation was successfully performed using a degenerate RT-PCR approach. Localization studies demonstrated the tissue-specific activity of a cloned Src-kinase (TK3) or a Syk-kinase (TK4). TK4 showed activity in the parenchyma of both genders, and in the testes of the male or the ovary of the female, but not in the vitellarium. In contrast, TK3 exhibited a gonad-preferential expression including the vitellarium. The Fyn/Src-like kinase TK5 was more ubiquitously expressed including the gonads of both genders. From the inhibitor experiments and the molecular analyses we conclude that Src-kinases are involved in differentiation processes during vitelline-cell development.
Functional assays and yeast-two-hybrid screenings were also been performed to further characterize the isolated schistosome kinases. The Src-kinase activity of TK3 was confirmed by cotransfection experiments in a eukaryotic cell-culture system using the focal adhesion-complex protein p130Cas as a substrate. By yeast-two-hybrid analyses in heterologous (Drosophila melanogaster) and homologous (S. mansoni) systems conserved molecules were identified that are known to be involved in the organization of the cell architecture. This indicates a role that could be assigned to TK3. First experiments to identify potential TK4 binding partners with a yeast-three-hybrid approach resulted in the identification of several clones, which included Src kinases such as TK3, and a novel Src-like kinase from schistosomes designated TK6.
Functional Analysis in Caenorhabditis elegans of gei-16,
a Nematode Developmental Gene with Multiple Splice Variants
Suzannah Hetherington, Flavia Pellerone, Nick Johnson, Alison Knight, Carolyn Behm
School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT, Australia
The gene gei-16 was originally identified as the C. elegans homolog of OvB20, an antigen expressed by the parasitic nematode Onchocerca volvulus. It has since been shown to have a role in embryonic elongation. This gene appears to be nematode-specific, with no clear homolog in humans, mice or flies but clear matches to a range of parasitic nematodes including Ostertagia ostertagi, Strongyloides stercoralis, Parastrongyloides trichosuri and Haemonchus contortus. gei-16 appears to have at least 19 different splice variants in C. elegans. These can be divided into three major types: long variants, short variants and the d variant. Knockdown by RNAi of all variants in C. elegans N2 shows a severe phenotype with a high penetrance of adult lethal, paralysed, uncoordinated, clear and larval arrest additional to the published embryonic lethal, larval arrest, and slow growth phenotypes. RNAi was directed against unique regions of the short and long variants. No obvious phenotype was seen for the short variants, but knockdown of the long variants produced a severe phenotype identical to that found for knockdown of all variants. This may suggest that the long variants are most important for development. Semi-quantitative RT-PCR showed that the long transcript is expressed through all stages of development, but is most highly expressed in the embryonic and early larval stages. Long variant expression levels appeared higher than short variant levels in all stages. A GFP reporter construct is currently being made to elucidate the spatial and temporal expression of gei-16.
Activation of larval Nippostrongylus brasiliensis induces
expression of a calcium-dependent phospholipase A2.
Danielle Smyth, Ayman S. Hussein, Conrad Chan, Fiona Gratrix and Murray E. Selkirk
Division of Cell & Molecular Biology, Imperial College London,
South Kensington Campus, London SW7 2AZ
We are utilising Nippostrongylus brasiliensis as a model system to understand the molecular basis of invasion and survival of gastrointestinal nematode parasites in mammalian hosts. Of interest are the molecules expressed when developmentally arrested, free-living, infective third-stage larvae (L3) resume development after entry into a host. Our investigations into 'in vitro' third stage larvae activation reveal that a simple shift in temperature (from 20ºC to 37ºC) is the only requirement necessary to activate L3s out of their resting state and to begin feeding behaviour, with up to 80% of larvae being activated by 24 hr. Analysis of metabolically labelled parasites shows that this is accompanied by a distinct profile of protein synthesis, notably a set of secreted products of low molecular weight (10 to 20 kDa). In addition, we have found several genes from the N. brasiliensis EST database which are expressed only upon activation of L3s. Acetylcholinesterases secreted by adult worms have been extensively described, and were observed in these experiments to be expressed upon larval activation. Another activation-expressed gene has been identified as a putative phospholipase A2 (PLA2). This shows sequence identity to other PLA2-like genes, with the best match to a PLA2 precursor in C. elegans (65% identity), and numerous homologues in EST databases of other parasitic nematodes. RT-PCR experiments show that the N. brasiliensis PLA2 is not expressed in L1s or unactivated L3, but is clearly induced in activated L3s, and is still highly expressed in gut located adult worms. A recombinant protein was produced in Pichia pastoris, and shows calcium-dependent catalytic activity against a range of substrates, including long chain diacyl phospholipids, which distinguishes the enzyme from the PAF acetylhydrolase already described from N. brasiliensis. Phospholipases have been shown to have roles as virulence factors in bacteria and fungi, however very little is known about nematode enzymes and their possible contribution to infectivity. In order to address this with respect to the PLA2 and a range of other proteins, we are attempting to adapt RNA interference to the early larval stages (L1-L3) of N. brasiliensis, utilising both bacterial feeding and dsRNA soaking approaches. This will allow further functional characterisation of parasite gene products, in particular their contribution to invasion and survival in the mammalian host.
Hsp90 in Brugia and C. elegans
Eileen Devaney
Division of Veterinary Infection and Immunity,
Faculty of Veterinary Medicine, University of Glasgow
Bearsden Road, Glasgow G61 1QH
Hsp90 is unique amongst the family of HSPs because of the nature of the proteins with which it associates under non-stress conditions. These include a range of signalling and receptor molecules with roles in the cell cycle, cell division and apoptosis. As knockout of hsp90 is lethal in eukaryotes, drugs that inhibit Hsp90 function have been widely used to explore the consequences of inactivating Hsp90. Amongst the best characterised of these compounds is Geldanamycin (GA), a naturally occurring benzoquinone ansamycin that binds in the N-terminal ATP pocket of Hsp90, altering its conformation in such a way as to destabilise client proteins, which are then targeted for degradation via the proteasome. While Hsp90 is clearly essential in C. elegans, as demonstrated by RNAi or analysis of mutants, GA causes no discernible phenotype in the worm. In contrast exposure of Brugia pahangi to GA is lethal for adult worms and irreversibly inhibits the release of Mf, effectively sterilizing adult females. Mf exposed to GA also die after several days in vitro. Similar results were obtained with a second filarial parasite, A. viteae, indicating that the effects of GA reflect inhibition of filarial Hsp90, rather than the Wolbachia Hsp90 homologue, HtpG. These results correlate well with those from a solid phase pull-down assay in which B. pahangi Hsp90 binds GA while C. elegans Hsp90 does not. The differing affinity of C. elegans and Brugia Hsp90 is intriguing given the degree of conservation between the two nematode proteins (92% similar, 87% identical). Hsp90 has been sequenced from a variety of other parasitic nematodes and we are currently investigating the affinity of other nematode Hsp90s for GA. We hope to address whether the ability of Hsp90 to bind GA correlates with phylogenetic position or particular sorts of life cycle (obligate parasite or free-living stages) and to identify specific amino acid residues required for GA binding.
Mechanisms and evolution of symbiosis; parasites and mutualists
invade plants via a shared response pathway.
David Bird and Charles Opperman
Center for the Biology of Nematode Parasitism, NC State University
Raleigh, NC 27695, USA.
Root-knot nematode (Meloidogyne spp: RKN) infects all cultivated crops and is the most economically important plant-parasitic nematode genus worldwide. In contrast, rhizobacteria are beneficial symbionts, responsible for plant assimilation of nitrogen and hence are the origin of much of the world’s protein. Although rhizobia have a restricted host range (legumes), they and RKN induce new organs in the root vasculature, leading to nitrogen nodules and giant cells (GC), respectively.
Because cytokinin is likely involved in root organogenesis, we used a cytokinin-responsive gene promoter driving GUS to spatio-temporally profile this plant hormone’s influence on RKN and rhizobia. Staining was detectible in root hairs of the model legume Lotus japonicus shortly after interaction with rhizobia and was evident at the earliest stages of the nodule primordium. In contrast, a cytokinin response was not detectible during root penetration and migration by RKN, nor in the mature GC. Down-regulation of cytokinin levels in planta via transgenic expression of cytokinin oxidase genes yielded roots with significantly fewer nodules. However, the number of RKN feeding sites also was reduced, consistent with the hypothesis that cytokinin is transiently required for GC initiation, but not for maintenance.
To better understand the symbioses at sub-cellular resolution, we employed confocal microscopy of GFP-tagged microtubules and actin to dynamically profile the cytoskeleton of living Lotus root hairs following exposure to the bacterial elicitor molecules (Nod factors: NF). Remarkably, RKN elicit an identical cytoskeletal response via a signal able to function at a distance. Neither azide-killed RKN nor C. elegans produce this signal. Aspects of the host responses to RKN were altered or abolished by mutations in the NF receptor genes nfr1, nfr5 and symRK, suggesting that RKN produce a molecule with functional equivalence to NF, and which we name Nem factor (NemF). A similar response to NemF was seen in tomato, but not Arabidopsis, which lacks key components of the NF-receptor/response machinery. Because the ability of RKN to establish feeding sites and reproduce was markedly reduced in the mutant lines, we propose that RKN have adapted at least part of the symbiont-response pathway to enhance their parasitic ability.
To obtain a glimpse of RKN gene space, we and our colleagues at the WashU GSC obtained 100,000 ESTs, comparing 14 representative tylenchid species, including 24,000 M. hapla ESTs. Remarkably, ESTs suggested that RKN acquired rhizobial genes (including those associated with NF biosynthesis) via horizontal gene transfer, insinuating a mechanism for evolutionary adaptation to exploit the host the symbiont-response pathway. We currently are building a physical map of the M. hapla genome to produce a minimum tiling path as the basis for a 5-fold redundant draft genome sequence. An initial pilot project has produced more than 8,000 BAC-end sequences, and we are targeting selected RKN loci. M. hapla is a sexually reproducing diploid species with a 62.5Mbp genome and will serve as the reference tylenchid nematode as a platform for comparative genomics. We eagerly await comparison of this plant parasite genome with those of animal parasitic nematodes.
Identification of potential mediators of nurse cell transformation from T. spiralis
D.B. Guiliano1, K. Gounaris1, and M.E. Selkirk1
1 Imperial College London, Division of Cell and Molecular Biology
South Kensington, London, SW7 2AY, U.K.
Transformation of nurse cells by Trichinella spiralis involves the cell cycle re-entry of the host cell nuclei, the loss of markers of terminal differentiation, and a subsequent blockade of nuclear division at G2/M. The molecular mechanisms by which the parasite initiates and maintains this transformation is unknown, however it is believed that parasite secreted (E/S) products interfere with the normal developmental program of the muscle and drive it down a novel pathway. Parasite E/S products have been analyzed in many studies and have been found to contain numerous enzyme activities including proteinases, kinases and nucleases. It has also been shown that the secreted products penetrate the nuclei of the invaded cells, indicating that they could have direct effects on processes such as DNA replication and gene transcription.
The aim of this project is to identify novel proteins that are involved in the myofibre transformation process or nurse cell homeostasis. To this end we are using transcriptomic and proteomic approaches to identify novel secreted proteins. The publicly available T. spiralis expressed sequence tag data has been searched for genes that are expressed during the nurse cell stage of the parasites development and contain secretory leaders. Some of these proteins also contain other motifs such as potential nuclear localization signals that are not normally found on secreted proteins. From our initial screen we identified fourteen candidate molecules that we are currently characterizing. Directed proteomics of fractionated E/S has already allowed us to verify several of these proteins are secreted. Most of these candidates encode novel proteins so it is difficult to predict what their functions might be. However, we are currently generating polyclonal antisera against them for immunolocalization studies. One particularly interesting set of identified proteins contain degenerate nudix domains. Other nudix proteins are characterized bacterial pathogenicity factors that hydrolyze a variety of import signalling and stress induced molecules including adenosine- polyphosphate-adenosine (ApxA) compounds that are involved in controlling cell homeostasis and apoptosis. We are currently testing whether recombinant T.spiralis nudix proteins and parasite E/S can act on these compounds.
Schistosoma mansoni TGF-β homologues
Tori C. Freitas, Jason Correnti, and Edward J. Pearce
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, 19104
ejpearce@mail.med.upenn.edu
Members of the transforming growth factor-β (TGF-β) superfamily of cytokines are a conserved group of signaling proteins that are found in metazoans and regulate a diverse set of cellular processes. Previous work has identified various components of the TGF-β signaling pathway in the blood fluke Schistosoma mansoni including membrane bound receptors and cytoplasmic proteins. However, it was unclear whether or not the parasite itself expressed a TGF-β homolog(s). Here, as a result of a database search of the Wellcome Trust Sanger Institute’s S. mansoni Genome Project, we describe the identification of two members of the TGF-β superfamily in S. mansoni. From available sequence within the conserved domain, the first ligand, SmInAct, shows homology to the TGF-β/Activin/Nodal subfamily with approximately 30% amino acid identity to mammalian activin and inhibin. The second, SmBMD, is a member of the bone morphogenetic protein/decapentaplegic subfamily with approximately 50% amino acid identity to human BMP-6. Using RT-PCR analyses, SmInAct expression was detected in 6 to 27-day-old in vitro cultured schistosomula and adult males and females with weaker expression in two-day-old schistosomula, and no expression in cercariae. Expression of SmBMD was observed across all stages tested suggesting little post-sporocyst stage specificity. To investigate the functional significance of the ligands within the parasite, each TGF-β was targeted for knockdown in three-hour old schistosomula via RNA interference using electroporation for dsRNA delivery. After 7-8 days in culture, SmBMD treated worms were approximately 20% larger than controls (p50 kDa), L3 NES is composed predominantly of low molecular mass proteins in the range 10-50 kDa, perhaps reflecting the higher level of proteolytic activity present in the secreted larval extracts. We are in the process of sequencing the major proteins present in L3 NES using MALDI-TOF mass spectrometry and Edman N-terminal sequence analyses. One interesting component of L3 NES that we have characterised recently is a secreted superoxide dismutase (SOD) activity. During characterisation of the interactions of L3 NES proteins with different innate cell types, we observed that these proteins reproducibly suppressed superoxide production by activated human neutrophils. This was an interesting observation because it has been shown previously by L. Proudfoot and colleagues that L3 NES can also suppress neutrophil recruitment in an LPS-induced model of inflammation. Superoxide suppression was shown to be mediated by an antioxidant activity present in L3 NES rather than by a direct interaction of a component of NES with a neutrophil receptor because similar results were observed in enzyme-free models of superoxide generation. The superoxide suppressing activity was inhibited by potassium cyanide suggesting that it was a member of the Cu/Zn SOD family of enzymes. Examination of the potential involvement of this activity in suppressing neutrophil infiltration is the subject of current investigation. We hypothesise that comprehensive analysis of the proteins secreted by infective N. brasiliensis larvae will provide a basis for understanding the mechanisms used by the parasite to modulate host immunity.
Structural and mutagenesis analysis of the Fasciola hepatica cathepsin L1 reveals insights into its biological function.
C. M. Stack1, S. Donnelly1,2, P. R. Collins1,2, S. Geiger3, R. Marion4,
L. S. Brinen4 and J. P. Dalton1
1Institute for the Biotechnology of Infections Diseases, University of Technology, Sydney, Australia; 2School of Biotechnology, Dublin City University, Dublin, Ireland,3 Department of Chemistry & Pharmacy, Ludwig-Maximilians University, Munich, Germany, 4Department of Cellular & Molecular Pharmacology, University of California, San Francisco, CA USA.
Given the importance of cathepsin L proteases in the virulence of F. hepatica and other helminth pathogens it is important to understand their mechanisms of their synthesis, processing, activation and substrate specificity. These enzymes take part in nutrient acquisition by catabolizing host proteins to absorbable peptides/amino acids, facilitates the migration of the parasite through the host intestine and liver by cleaving interstitial matrix proteins and suppresses host immune responses. Here we present the 1.4 Å X-ray crystallographic structure of procathepsin L1 from F. hepatica. We have also carried out site-directed mutagenesis studies to identify residues in the active site that are important to the substrate specificity of the enzymes and that explain the difference between the activity of cathepsin L1 and a second major F. hepatica protease, cathepsin L2. Moreover, critical residues in the propeptide that are important in regulating auto-activation of the enzymes have been identified. We will discuss these data and their relevance to parasite virulence.
Microarray analysis of gene expression in the Strongyloides ratti life-cycle.
Fiona Thompson, Louise Hughes, Gary Barker, Clare Wilkes & Mark Viney
School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK
F.Thompson@bristol.ac.uk
The establishment, survival and fecundity of Strongyloides ratti is dramatically affected by the host immune response. Furthermore, the alternative developmental pathways of the early larval stages are affected by the immune status of the host. However, the molecular and biochemical nature of these effects have yet to be fully explored. To this end, we have generated a number of cDNA libraries from different stages throughout the S. ratti life-cycle, including those from parasitic female adults which have been subjected to different immune pressures. Microarray chips have been synthesised by amplifying and spotting the available c. 20K cDNA clones: a ‘parasitic chip’ was synthesised from c. 10K cDNA clones obtained from the parasitic libraries; a ‘free-living chip’ contains c. 10K clones from the free-living stage libraries. These microarrays have been differentially probed with cDNA isolated from populations of worms, from various points throughout the life-cycle, using experimental designs that consist of three biological replicates, each with three technical replicates. Specifically, the free-living stage chip has been probed with L2 stage cDNA from lines of worms destined to develop by either the direct or the indirect route. This has shown that some 300 genes have significantly different expression between larvae of different developmental destiny, though the magnitude of this difference is less than a factor of two. We are currently confirming these expression patterns using real-time PCR. Examples of genes that are expressed at a higher level in larvae destined for direct development are those that are predicted to code for heat shock proteins, elongation factors, actin, collagens and glutathione peroxidase. Genes that are expressed at a higher level in larvae destined for the indirect development route include genes with significant alignment to C. elegans hypothetical proteins, ribosomal proteins and thioredoxin peroxidase. Similarly, differences in gene expression between L1s and iL3s have been investigated. The parasitic female chip has been used to compare gene expression in worms under immune pressures; specifically, to compare gene expression in worms from 6 and 15 days post-infection.
Nematode collagen biosynthetic enzymes
Alan Winter
Institute of Comparative Medicine, University of Glasgow, Glasgow G61 1QH.
We are studying enzymes involved in the biosynthesis of nematode cuticles principally in the free-living species C. elegans. The cuticle is a complex multi-layered extra cellular matrix (ECM) formed predominantly from collagen. Collagen monomers are characterised by repeats of the amino acid sequence Gly-X-Y, where X and Y are any amino acid, most commonly proline and hydroxyproline respectively. Three monomers combine to form a collagen trimer that then associate to form higher order structures. A number of enzymatic steps are required for formation of collagen, 3 of which are examined here. Prolyl 4-hydroxylase (P4H) catalyses the hydroxylation of the Y position prolines in collagen repeat sequences to 4-hydroxyproline (4HyP). 4HyP residues are required for the thermal stability of the triple helix. P4H enzymes are complexes consisting of catalytically active subunits (PHYs) and a subunit required to maintain the complex in an active form within the cells ER. This second subunit is protein disulphide isomerase (PDI). In C. elegans we have shown that 3 P4H subunits, 2 PHYs and 1 PDI, combine in unique ways to form active complexes and that these are essential for cuticle development. We are comparing P4H complexes formed in the closest relative of C. elegans, C. briggsae, and in the distantly related filarial nematode B. malayi. Multi-functional PDI is an ER resident enzyme involved in a number of steps in collagen biosynthesis. It is an essential component of P4H complexes, but also has chaperone activities and catalyses the formation of reducible disulphide bonds in collagen. C. elegans has 3 conserved PDIs, only 1 of which, PDI-2, is involved in P4H. We are studying genetic mutants of all 3 PDIs to determine any overlapping functions as well as the relative importance of their multiple enzymatic activities. Peptidyl prolyl cis-trans isomerase (PPIase) catalyses the interconversion of prolyl imide bonds in peptide substrates. The presence of Gly-Pro-Y and Gly-Pro-4HyP predisposes unfolded collagen chains to form cis peptide bonds. Peptide bound proline residues must be in the trans configuration in the collagen triple helix and the slow cis-trans isomerisation step becomes rate limiting. We are characterising 4 enzymes from 2 different classes of PPIase, 3 FK506-binding proteins (FKBs) and 1 cyclophilin (CYP), using genetic mutants and RNAi to produce combined disruption to determine if these enzymes are required to modify collagens that form the nematode cuticle.
ABSTRACTS FOR POSTER PRESENTATION
SESSION 2
FRIDAY 9 SEPTEMBER
18.10 – 20.00
Syndecan-1, a heparan sulfate proteoglycan, is produced
by Trichinella spiralis-infected muscle cells.
Daniel P. Beiting,* 1 Pyong Woo Park,2 and Judith A. Appleton1
1James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA; and 2Department of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Syndecans are a family of cell surface, transmembrane proteoglycans found on all adherent cells. Members of the syndecan family are comprised of a core protein modified by numerous, highly sulfonated heparan sulfate chains that mediate interactions with extracellular matrix, growth factors, cytokines and chemokines. One member of this family, syndecan-1, is expressed on the surface of epithelial cells, endothelial cells, plasma cells, and immature skeletal muscle cells. In this study we show that mature muscle cells infected with Trichinella spiralis produce syndecan-1. This protein is detected as early as 5 days post-infection and remains detectable into chronic infection (50 days post-infection). Immunohistochemical analysis of nurse cell syndecan-1 demonstrated cytoplasmic and extracellular distribution of the protein, rather than conventional, cell surface localization. Monoclonal antibodies directed at either the carboxy-terminus or amino-terminus of the core protein resulted in the same staining, suggesting that syndecan-1 core protein was not truncated. To examine the role of syndecan-1 in the intracellular habitat of T. spiralis, we infected wild-type and syndecan-1 deficient mice by intravenous injection of T. spiralis newborn larvae. In the absence of nurse cell syndecan-1, parasites developed to maturity, and the inflammatory, cytokine and antibody responses to muscle infection were largely unchanged. In addition to syndecan-1, we also detected perlecan, a related proteoglycan, as well as heparan sulfate associated with the nurse cells in both wild-type and syndecan-1 deficient mice. Our data suggest that syndecan-1 may serve redundant, rather than independent roles in regulation of nurse cell or immune cell activity.
Determining the features of N. brasiliensis infection which drive
Th2 immune responses and protective immunity
M. Camberis, N. Van Panhuys, M. Prout, D. Scarlett and G. Le Gros
Malaghan Institute of Medical Research,
CSB, Victoria University, Wellington, New Zealand
mcamberis@.nz
N. brasiliensis infection is characterised by the invasion, ex-sheathment and secretion of proteolytic enzymes through a 48 hour tissue migration and infection phase. We focus on the importance of each of the phases of worm development to stimulating in vivo Th2 immune responses using mice containing the GFP reporter construct inserted into the IL4 locus. We present results that show the relative importance of IL4 to the generation of Th2 immune responses and the effect IL4 deletion has on worm migration and behaviour through this initial 48-hour phase of infection.
Pore-forming proteins from blood-feeding helminths
Tegan A. Don1,2, Peter O’Donoghue2, Peter J. Hotez3 Najju Ranjit1 and Alex Loukas1.
1Queensland Institute of Medical Research and 2The University of Queensland, Brisbane, Australia;, 3The George Washington University Medical Center, Washington DC, USA.
Tegan.Don@qimr.edu.au
Blood feeding parasites rely on the acquisition of amino acids contained within erythrocytes, namely haemoglobin (Hb), to meet their nutritional requirements for reproduction and survival in the host. While Hb degradation pathways have been at least partially elucidated in hookworms and schistosomes, the molecular mechanisms by which these parasites lyse erythrocytes are still unknown. Extracts from both these helminths exhibit pore-forming haemolytic activity, but the identification of the specific molecules involved has not been determined. A gene-first approach to identify haemolysin-encoding cDNAs was undertaken by scanning the EST datasets for homologues of amoebapores, pore-forming saposin-like proteins of Entamoeba histolytica. Saposin-like proteins with haemolytic capacity were recently identified from the liver flukes Clonorchis sinensis and Fasciola hepatica. We identified mRNAs encoding saposin-like proteins from the dog hookworm, Ancylostoma caninum, and the trematode blood fluke, Schistosoma mansoni. Full length sequences were obtained and for hookworms at least, the mRNAs were identified in the gut of the worms using RNA from intestinal tissue that was extracted by laser capture microdissection. Recombinant proteins were expressed in secreted form using baculovirus and insect cells, and purified on nickel-NTA sepharose. We have shown that the haemoglobin digestion pathway is a suitable target for vaccines against blood-feeding helminths, and therefore reasoned that specific antibodies against haemolytic proteins in the gut might also confer protection to vaccinated animals. The biochemistry and vaccine efficacy of these proteins is currently being explored and will be presented.
Functional analysis of nematode-specific genes using the
free-living nematode, Caenorhabditis elegans
Julie-Anne Fritz and Dr Carolyn Behm
School of Biochemistry & Molecular Biology, Australian National University,
Canberra, A.C.T., Australia, 0200
The free-living nematode, Caenorhabditis elegans, has over 20,000 protein coding genes, a large proportion of which have no clear homologues except in other nematodes. Further, many of these genes encode proteins with no known function. Based on RNA interference (RNAi) data available for C. elegans, a number of these unknown genes clearly have important functions in the biology of the nematode, and may provide good genetic targets for the development of new anthelmintic compounds. One such gene, JAF01, encodes a small transmembrane protein with homologues in several nematode Clades. When the expression of JAF01 is knocked down by RNAi, phenotypes such as locomotory and developmental defects result. Further functional characterisation is underway, using a GFP reporter to analyse the spatial and temporal expression, as well as microarrays, to determine developmental pathways that may be involved. The results of these experiments will be presented.
FMRFamide-Like Peptide Genes in Meloidogyne incognita:
Characterization, Expression and RNAi-Induced Disruption
Michael J.G. Johnston1, Colin C. Fleming2 and Aaron G. Maule1.
1Parasitology Research Group, School of Biology & Biochemistry,
Queen’s University Belfast, Belfast BT9 7BL;
2Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX.
FMRFamide like peptide (flp) genes encode FMRFamide-related peptides (FaRPs) which are the most abundant and diverse neurotransmitters/neuromodulators in nematodes. The complexity and abundance of these neuropeptides in nematodes is evident by the existence of 24 flp genes encoding over 60 distinct FaRPs in C. elegans; 5 flp genes have been characterised from the potato cyst nematode, Globodera pallida and EST data indicate that FaRPs are also abundant in other parasitic nematodes. Since FaRPs are potent modulators of various physiological functions in nematodes, such as locomotion, feeding, reproduction and sensory perception, it is believed that disruption of these neuronally-expressed flp genes would produce aberrant phenotypes and could offer a novel method of control. Although neuronally-based genes in C. elegans are refractory to RNAi-induced disruption, studies have demonstrated that flp genes in the potato cyst nematode, Globodera pallida, are susceptible, such that silencing produces worms with abnormal motility. The present study has fully characterised 5 flp genes from the root knot nematode, Meloidogyne incognita, and also a putative G-protein coupled receptor. The C. elegans sequelogue of this receptor has been shown to be the receptor for flp-18 encoded peptides. The expression of each gene has been investigated using in situ hybridisation techniques and compared to those seen in C. elegans – this was also used to infer function. Also, the sensitivity of both mi-flp-18 and its putative receptor to RNAi-induced silencing has been investigated; aberrant locomotory phenotypes were produced and these effects subsided following removal of the double stranded RNA trigger. The ligand- and receptor-double stranded RNA induced phenotypes are compared.
TGF-( Homologues from Parasites: Inducers of Host Immune Regulation?
Henry J. McSorley, Rick M. Maizels
Institute of Immunology and Infection Research, University of Edinburgh, UK
H.J.McSorley@sms.ed.ac.uk
The most common clinical outcome of lymphatic filiarisis is an asymptomatic state characterised by hyporesponsiveness to parasite antigens. TGH-2, a homologue of human TGF-(, has been identified in the filiarial nematode Brugia malayi, and binds to the TGF-( receptor. TGF-( downregulates immune responses in humans and mice, and has been shown to induce the formation of regulatory T cells (Tr) in vitro. Therefore, if TGH-2 acts in the same way as TGF-(, it could be involved in the induction of hyporesponsiveness to filarial antigens by Brugia. Mice infected with larval Brugia, or implanted with adult worms in the peritoneum are being used to investigate T cell recruitment and differentiation in vivo, to test the hypothesis that TGH-2 induces a Tr population. FACS staining indicates that Brugia infection upregulates markers indicative of Tr such as FoxP3. Further work will investigate the effects of the Brugia malayi TGH-2 on mouse immune responses, as compared to a positive control of mouse TGF-(, and negative controls of putative inactive TGF-( homologues TGH-1 (Brugia malayi) and DAF-7 (C. elegans). The homologues will first be expressed using a baculovirus system in insect cells, cleaving to their active form using co-transfection of human furin transgenic baculovirus. The active proteins will be used in assays of proliferation and cytokine production of stimulated T cells, and their effects on the activation and presentation of antigen presenting cells will be assessed. In vitro and in vivo experiments will be performed to assess whether TGH-2 can induce the differentiation or expansion of Trs, and whether it can induce antigen-specific tolerance.
Expression of parasitic nematode genes in Caenorhabditis elegans.
Linda Murray, Peter Geldhof*, David Knox* and Collette Britton.
Division of Veterinary Infection and Immunity, University of Glasgow, UK and
*Moredun Research Institute, Edinburgh, UK.
l.murray@vet.gla.ac.uk
Vaccination with recombinant parasite proteins expressed in bacteria or yeast has so far produced limited protection. This is in contrast to studies using native proteins purified from parasite extracts. This suggests that expression of protein in an appropriate form may be vital to its ability to induce protection. We are adapting C. elegans transformation technology to express parasite proteins, with the aim of expressing these in a form similar to the native protein. We previously found that a cathepsin L cysteine protease (cpl-1) from Haemonchus contortus was able to rescue a C. elegans cpl-1 mutant, indicating that the protease is expressed in its correct, active form in C. elegans. Addition of a His tag at the C-terminal region of the protease has allowed us to purify the Haemonchus CPL-1 from C. elegans extracts and this will be tested in immunisation studies. We are also testing expression of Hc-cpl-1 under the control of different promoters to try to optimise expression. We have found that transformation of C. elegans with other Haemonchus and Ostertagia genes results in low levels of expression and we are currently examining the effect of different promoters and knockout of related C. elegans genes on expression levels of the parasite genes.
Expression of parasitic nematode genes in Caenorhabditis elegans.
Characterisation and development of microsatellite markers for
Haemonchus contortus and Teladorsagia circumcincta
Erica Packard, Victoria Grillo and John Gilleard
Division of Infection and Immunity, Institute of Comparative Medicine,
Faculty of Veterinary Medicine, University of Glasgow, U.K
We are developing panels of microsatellite markers for the genetic analysis of parasitic nematode populations. Microsatellites of the economically important parasitic nematodes H.contortus and T.circumcincta have been isolated by screening genomic libraries and mining sequence databases. In general, microsatellites from these species are highly polymorphic but only a relative small proportion make good population genetic markers. Many dinucleotide repeats are problematic due to an association with an additional repetitive sequence that appears to be dispersed throughout the genome. Also many markers have a high level of “null” alleles which probably reflects the high level sequence polymorphism in these parasites. Nevertheless useful markers can be found and we have developed panels for use in population genetic analysis. We have studied the inheritance of these microsatellite markers by genotyping broods of progeny from single adult female worms. The results support obligate sexual reproduction but demonstrate that single female worms carry the progeny of multiple males. This presents potential problems for the development of inbred lines. The inheritance studies have also been useful for confirming null alleles for particular markers. We are developing approaches to improve the throughput of worm genotyping by multiplexing multiple markers and examining “bulk” DNA preparations to provide a snapshot of the genetic variation in a parasite isolate.
Functional aspects of Trichinella spiralis excretory-secretory proteins
Mark W. Robinson, Katharina Fischer and Bernadette Connolly
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, UK
m.w.robinson@abdn.ac.uk
Trichinella spiralis excretory-secretory (ES) proteins lie at the host-parasite interface where they may be involved in establishing new infections, in nurse cell formation and/or in modulation of the host immune response. To date, only a few Trichinella ES proteins have been identified and little is known in relation to their putative roles during infection. Consequently, the identification of these proteins and the elucidation of their potential functions will further our understanding of Trichinella infections and of the host-parasite interaction. The application of a global proteomics approach to study the ES proteins from T. spiralis L1 larvae has led to the identification of several novel proteins. A number of the secreted proteins appear on 2-D gels as several isoforms and immuno-analysis suggests that differential glycosylation may be responsible for at least some of these. Furthermore, data from mass spectrometry also suggests that post-translational processing of some precursor proteins also occurs on their secretion from the parasite. We have used a range of molecular and biochemical analyses to investigate the functions of selected ES proteins. These include the ORF9.10 protein, a novel 38kDa protein and a group of small molecular weight T. spiralis-specific proteins. Basic information with regard to the timing of expression (in relation to life-cycle stage) gene copy number and transcript analysis has been obtained as well as data on the biochemistry of these selected proteins. The data presented will be discussed with regard to the potential roles of these ES proteins in Trichinella infections.
Immunization of cattle with recombinant Major Sperm Protein (MSP)
against Dictyocaulus viviparus.
T. Schnieder, G. von Samson-Himmelstjerna, C. Strube and C. von Holtum
Institute of Parasitology, University of Veterinary Medicine, Hannover, Germany
Thomas.schnieder@tiho-hannover.de
Natural infections with the bovine lungworm, Dictyocaulus viviparus, rapidly stimulate a strong protective immunity, which lasts for about six to twelve months without booster infection. An X-ray attenuated live vaccine had been available on the market in several European countries for about forty years. In Europe despite of routine anthelmintic treatments in cattle, D. viviparus is still highly prevalent in about 40 % of cattle herds and continues to cause considerable economical losses in cattle farming. Experience with the live vaccine showed that immunprophylaxis is the most efficient way to control lungworm infections. MSP is the most abundant protein in nematode sperm cells, comprising about 15% of the total cell protein. MSP plays an essential role in nematode sperm motility. Within the sperm's pseudopod, individual MSP molecules form long chains, which further associate with each other to form a dense network of bundles. The constant assembly of this network at the leading edge of the pseudopod and disassembly at the back end is what moves the pseudopod membrane, allowing the cell to crawl forward. Although MSP is abundant in the sperm of all nematode species, it has not been found in any other organism. Immunization with a recombinant MSP could possibly reduce the production of new larvae and thus control the spread of infection. MSP is an immunodominant protein of approximately 17 kDa found as water soluble protein in adult male worms. Recombinant MSP was isolated from a D. viviparus adult worm λ-ZAPII cDNA library by immunoscreening with rabbit anti-lungworm hyperimmune sera, cloned into pGEX-2T and expressed in E. coli as GST fusion protein. Three immunizations with 200 µg recombinant MSP fusion protein three weeks apart using alum or Quil-A as an adjuvant were followed by experimental infection with 3300 infective D. viviparus larvae three weeks after the last immunization. As expected, immunization with MSP did not prevent the establishment of adult worms, however a strong IgG1 response was stimulated from eleven days after the first immunization onwards and lower numbers of larvae were produced.
The Insulin/IGF signalling transduction pathway in Parastrongyloides trichosuri-
does it play a role in parasitism and aging?
Susan Stasiuk1, Chuck Shoemaker2 and Warwick Grant1.
1Wallaceville Animal Research Centre, AgResearch, Upper Hutt, New Zealand
2Department of Biomedical Sciences, Tufts University School of Veterinary Medicine,
North Grafton, MA 01536
Early L1 stage larvae of Parastrongyloides trichosuri, a nematode parasite of small marsupials, make a developmental choice to become either a short lived free-living nematode or a long lived parasite. This developmental choice appears to be triggered by a compound produced by the worms which acts as a developmental signal, reminiscent of the free-living nematode Caenorhabditis elegans dauer pheromone (daumone). The evidence for the existence of this signal comes from experiments in which a conditioned medium was prepared by sequentially culturing several generations of P. trichosuri in the same liquid culture. The medium derived from this culture regime induced pronounced infective larval development (i.e. entry into the parasitic life cycle) under conditions in which only free-living worms develop in control medium.
In C. elegans, daumone is believed to influence the signalling state of the insulin/IGF signalling pathway, the end result of which is to initiate either normal adult development or diapause entry which culminates in the production of a dauer larva. The insulin/IGF pathway has also been linked to variation in lifespan in C. elegans. The key genes involved in the insulin/IGF pathway are the putative IGF-receptor, daf-2; the phosphatidylinositol 3-OH kinase, age-1 and the forkhead transcription factor, daf-16. We have cloned the putative orthologues of these components of the insulin/IGF signalling pathway from P. trichosuri. We have a putative full-length age-1 gene orthologue which shares 43% amino acid identity with C. elegans, a putative full-length daf-16a gene orthologue which shares 45% amino acid identity with C. elegans and 58% identity with Strongyloides stercoralis and we have a 620bp internal fragment of a daf-2 gene homologue that shares 61% amino acid homology with C. elegans.. We will present data on the expression of these genes and their likely role in the free-living/parasitic life cycle switch in P. trichosuri.
Expression and purification of bovine lungworm vaccine candidates
Christina Strube*, Georg von Samson-Himmelstjerna and Thomas Schnieder
Institute of Parasitology, Hannover School of Veterinary Medicine, Germany
christina.strube@tiho-hannover.de
The bovine lungworm Dictyocaulus viviparus is a very important parasite in cattle farming with a prevalence of about 40 % in European countries. In calves and adult animals infections with the bovine lungworm induce a protective immunity lasting for six to twelve months. This has been explored by the development of a lungworm vaccine in the early sixties of the last century. The live attenuated vaccine currently available in a few countries, has certain drawbacks since it has only a short shelf life and is costly to produce. A vaccine based on a recombinant protein can overcome these limitations. Candidates for such a vaccine are the D. viviparus extracellular superoxide dismutase (SOD), paramyosin, and phenyl-ethanolamine N-methyltransferase (PNMT). The latter one is predominantly expressed in lungworm inhibited larvae. Thus, the immunization with these proteins could also protect against hypobiotic stages. The SOD, paramyosin and PNMT were cloned into pGEX vectors to express them as fusion proteins with GST, which is assumed to enhance immune response. The expressed GST-fused vaccine candidates were isolated by GST-affinity purification followed by FPLC and confirmed by MALDI-TOF analysis. The immunoreactivity and the protective potential of each of the above mentioned recombinant proteins will be analysed in challenge trials.
Identification of CPN10 in Strongyloides ratti
Alex Sykes1*, Michelle Jennens3, Narelle Villa2, Malcolm Jones1,
Carolyn Jones3, Andrew Thompson3 and James McCarthy1
1Queensland Institute for Medical Research, Herston, QLD. 2Cellabs Pty Ltd, Brookevale, NSW. 3Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, WA.
Relatively little is known about the biochemistry and immunobiology of the parasitic nematodeStrongyloides. To develop tools to study this parasite, we immunised mice with somatic worm extractof adult S. rattiand generated a panel of 3 monoclonal antibodies (MABs). One MAB we have studied that recognized a 10 kDa protein on immunoblot in parasitic adults. Immunohistochemistry was subsequently performed on both frozen and formalin fixed parasite sections. In fixed tissue, positive immunoreactivity was observed in 4 distinct regions immediately subjacent to the cuticle and corresponds to hypodermal cells. In unfixed frozen adult sections immunoreactivity with this MAB occurred as cuticular localization and as a slight halo of immuno positive material around each parasite section. These data suggest that this antigen occurs at a hypodermal location, is water soluble and does not lose antigenicty after cross-linking with formalin. EM localization with colloidal gold and subsequent proteomic analysis will help identify this unknown antigen. Characterisation of this somatic antigen will lead to a better understanding of Strongyloides sp. and due to the suspected nature of the antigen recognized by 8C3, potentially aid in diagnosis of human infection.
Identifying anthelmintic resistance associated alleles in
Haemonchus contortus using real time PCR.
T. K. Walsh, and A. J. Wolstenholme
Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY
bsstkw@bath.ac.uk
Haemonchus contortus is a parasitic nematode affecting ruminants and is a severe problem in livestock farming around the world. As with other gastrointestinal parasites, control is mainly by treatment with anthelminthics. However, resistance is becoming widespread and represents a serious threat to agricultural incomes. We are developing real-time PCR assays for the detection of resistance alleles, in the belief that such assays will be useful for monitoring the spread of resistance and for permitting an informed choice of treatment regimes. Resistance is best understood for the benzimidazoles, where it is mediated by a single nucleotide polymorphism (SNP) in the β tubulin gene (TTC to TAC). This results in the substitution of phenylalanine for tyrosine at amino acid 200 preventing the binding of benzimidazoles to the β tubulin subunit. Resistance is recessive, therefore the frequency of resistance alleles in a population is crucial in understanding the potential development of the resistant phenotype in the field. Real-time PCR enables the rapid identification of SNPs and has been used to detect resistance alleles. Taqman probes are an established real time PCR technology and have been used extensively for SNP determination. Scorpion® probes are a relatively new real time PCR technology that relies on an intermolecular mode of action. Both techniques have been applied to the SNP at amino acid 200 to determine the prevalence of resistant and wild type alleles of the β tubulin in Haemonchus contortus. The sensitivity and detection limits of the two techniques have been defined and the two techniques have been compared and their accuracy confirmed using established allele specific PCR techniques. Preliminary results suggest that Scorpion probes are able to detect concentrations of 2.25 x10 –9 μg of plasmids containing the SNP. Taqman probes are slightly less sensitive, detecting down to 2.10 x10 –8 μg . A SNP in the GluCl(3 gene of Cooperia oncophora has been identified in an ivermectin-resistant isolate. We are developing an assay for the detection of this SNP in H. contortus and C. oncophora, and this can be used to test its importance in field isolates. Similar tests can be applied to any other candidate polymorphism associated with resistance.
Chemical Genetics: Identification of New Antiparasitic Targets for
Veterinary Medicine using Novel Anthelmintic Compounds
Glyn Ball+, Timothy G. Geary*, Debra J. Woods*,
Karen G. Greenwood* and John S. + Gilleard
+Faculty of Veterinary Medicine, University of Glasgow, UK;
*Veterinary Medicine Discovery Biology, Pfizer Animal Health
With widespread drug resistance in goat and sheep nematodes and emerging resistance in cattle nematodes, the need for new veterinary anthelmintics, not cross resistant with known resistance mechanisms, is high. With few mechanisms targeted by antiparasitic molecules, recent advances in genomics technologies offer new opportunities for novel target identification. The free living nematode C. elegans is a useful model for parasitic nematode species and has previously been used to define the target and mode of action of several anthelmintic drugs. By performing mutagenesis to produce drug resistant mutant strains, followed by classical genetic mapping, it was possible to identify the mutation responsible for resistance. We are using a similar approach to identify the molecular targets of several novel classes of compound with anthelmintic activity. (-ketoamides are a class of compound structurally related to the anthelmintic closantel. Closantel itself has not been amenable to genetic analysis since it has limited activity against C.elegans, however some of the (-ketoamides have much more potent activity. The activity of a number of (-ketoamides has been characterised against C.elegans and we are currently performing mutagenesis experiments to isolate resistant mutants. We plan to map mutations by snip-SNP mapping, which is much more rapid than conventional mapping approaches. We will present our data characterising the effects of the novel anthelmintic compounds on C. elegans motility and development, along with preliminary results of mutant screens and the snip-SNP mapping.
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