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
Hydra Meeting 6-11 September 2005 : Scientific Programme
3
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 excretorysecretory 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
Hydra Meeting 6-11 September 2005 : Scientific Programme
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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 TGF 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 (-1/IPSE) activates
human basophils via a novel mechanism: IgE receptor engagment without
crosslinking
18:10 – 20:00 Poster Session 2
Hydra Meeting 6-11 September 2005 : Scientific Programme
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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
Hydra Meeting 6-11 September 2005 : Scientific Programme
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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
Hydra Meeting 6-11 September 2005 : Poster Sessions
7
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- 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
Hydra Meeting 6-11 September 2005 : Poster Sessions
MOLECULAR AND CELLULAR BIOLOGY OF
HELMINTH PARASITES
HYDRA, GREECE
6 – 11 SEPTEMBER 2005
ABSTRACTS FOR ORAL PRESENTATION
9
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 semisynthetic 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.
Hydra Meeting Wednesday 7 September 2005 : Session 1 : Neurobiology 1
10
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.
Hydra Meeting Wednesday 7 September 2005 : Session 1 : Neurobiology 1
11
The structure of neuropeptide and neuropeptide receptors in flatworms
Tim A. Day1, Michael J. Kimber1, Paul McVeigh2,
Judith Humphries1 and Hanan Omar1
1
Department of Biomedical Sciences, Iowa State University, Ames IA, USA and
2
Parasitology 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.
Hydra Meeting Wednesday 7 September 2005 : Session 1 : Neurobiology 1
12
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).
Hydra Meeting Wednesday 7 September 2005 : Session 2 : Neurobiology 2
13
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.
1
Marine 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 Ca 2+ 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.
Hydra Meeting Wednesday 7 September 2005 : Session 2 : Neurobiology 2
14
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.
Hydra Meeting Wednesday 7 September 2005 : Session 2 : Neurobiology 2
15
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
1
Parasitology Research Group, School of Biology and Biochemistry,
Queen’s University Belfast, Belfast BT9 7BL;
2
School of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA;
3
Department 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.
Hydra Meeting Wednesday 7 September 2005 : Session 2 : Neurobiology 2
16
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.
Hydra Meeting Wednesday 7 September 2005 : Session 3 : Helminth Enzymes
17
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
1
Dept. 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.
Hydra Meeting Wednesday 7 September 2005 : Session 3 : Helminth Enzymes
18
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.
Hydra Meeting Wednesday 7 September 2005 : Session 3 : Helminth Enzymes
19
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.
Hydra Meeting Wednesday 7 September 2005 : Session 3 : Helminth Enzymes
20
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?
Hydra Meeting Thursday 8 September 2005 : Session 4 : Genetics and Genomics
21
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.
Hydra Meeting Thursday 8 September 2005 : Session 4 : Genetics and Genomics
22
A transcriptomic survey of Echinococcus granulosus
Cecilia Fernándeza, John Parkinsonb, Gustavo Salinasa, Alvaro Díaza, Henrique Ferreirac,
Matt Berrimand, Mark Blaxtere & Rick M Maizelsf
a
b
Cátedra de Inmunología, Facultad de Química, Universidad de la República, Uruguay
Program in Genetics and Genomic Biology, Hospital for Sick Children, Toronto, Canada
c
Centro 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
f
Institute 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 signalsequence 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 fulllength cDNA is available for a high proportion of those newly described
[http://nema.cap.ed.ac.uk/Lopho/ 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.
Hydra Meeting Thursday 8 September 2005 : Session 4 : Genetics and Genomics
23
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
Dept 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
1
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 TMGcapped 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.
Hydra Meeting Thursday 8 September 2005 : Session 4 : Genetics and Genomics
24
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 nonpathogenic 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
(http://PlasmoDB.org) 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 phylogeneticallyrestricted targets suitable for diagnostic, drug, and vaccine design.
Hydra Meeting Thursday 8 September 2005 : Session 5 : Functional Genomics
25
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.
Hydra Meeting Thursday 8 September 2005 : Session 5 : Functional Genomics
26
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
1
Department of Biomolecular Mass Spectrometry, Utrecht University, The
Netherlands, 2Institute for Veterinary Research, IVW/GSAH, Utrecht, The Netherlands
and
3
Dept. 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.
Hydra Meeting Thursday 8 September 2005 : Session 5 : Functional Genomics
27
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. Excretorysecretory (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 Cterminus 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-2microglobulin (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.
Hydra Meeting Thursday 8 September 2005 : Session 5 : Functional Genomics
28
Parastrongyloides trichosuri as a new model nematode parasite
Warwick Grant1, Susan Stasiuk1, Jan Newton-Howes1 & Chuck Shoemaker2
1
2
Wallaceville Animal Research Centre, AgResearch, Upper Hutt, New Zealand
Department 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.
Hydra Meeting Thursday 8 September 2005 : Session 6 : Gene Manipulation
29
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.
Hydra Meeting Thursday 8 September 2005 : Session 6 : Gene Manipulation
30
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 mec7::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 singlecopy chromosomal integration of transgenes than microinjection, which favors formation episomal
arrays.
Hydra Meeting Thursday 8 September 2005 : Session 6 : Gene Manipulation
31
Transduction of Schistosoma mansoni sporocysts by VSVG pseudotyped Moloney
murine leukemia retrovirus constructs encoding luciferase and other transgenes
1,2
Kristine J. Kines, 1Victoria H. Mann, 1,2Maria E. Morales,
3
Bernd H. Kalinna and 1,2Paul J. Brindley
1
Department of Tropical Medicine, School of Public Health and Tropical Medicine, and
Interdisciplinary Program in Molecular and Cellular Biology, Tulane University, Health
Sciences Center, New Orleans, Louisiana 70112, USA
3
Department of Molecular Parasitology, Institute of Biology, Humboldt University-Berlin,
Berlin, Germany
2
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 collagenasedigested 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 RTPCR 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.
Hydra Meeting Thursday 8 September 2005 : Session 6 : Gene Manipulation
32
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; HeinrichHeine-University, Institute for Genetics, 40225 Düsseldorf, Germany; * Eberhard-KarlsUniversity, 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 Srcspecific 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 gonadpreferential 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 yeastthree-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.
Hydra Meeting Thursday 8 September 2005 : Session 7 : Development
33
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.
Hydra Meeting Friday 9 September 2005 : Session 7 : Development
34
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.
Hydra Meeting Thursday 8 September 2005 : Session 7 : Development
35
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.
Hydra Meeting Friday 9 September 2005 : Session 7 : Development
36
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 NFreceptor/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 symbiontresponse 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.
Hydra Meeting Friday 9 September 2005 : Session 8 : Interactions
37
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.
Hydra Meeting Friday 9 September 2005 : Session 8 : Interactions
38
Schistosoma mansoni TGF- 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- (TGF-) 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-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-

S.
mansoni Genome Project, we describe the identification of two members of the TGF-
superfamily in S. mansoni. From available sequence within the conserved domain, the first ligand,
SmInAct, shows homology to the TGF-/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- was targeted for knockdown in threehour 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 (p<0.001).
This phenotype persists for at least 11 days and was reproducible across experiments. Worms
treated with dsRNA corresponding to SmInAct showed no obvious phenotypic changes. Further
characterization of SmInAct and SmBMD are underway including the effect of knockdown on the
parasite and/or host in vivo.
Hydra Meeting Friday 9 September 2005 : Session 8 : Interactions
39
Do evolutionary conserved molecules mediate an interaction between
Echinococcus multilocularis and its mammalian host?
Klaus Brehm, Markus Spiliotis, Ricardo Zavala-Gongora,
Christian Konrad and Peter Bernthaler
Institute of Hygiene and Microbiology, University of Würzburg
Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany
kbrehm@hygiene.uni-wuerzburg.de
As bilaterian animals, helminth parasites share a large pool of genetic heritage with mammals.
One of our aims is to understand whether, and to what extent, this plays a role in long-term
persistence of helminths within their mammalian hosts. As a model organism for these
investigations, we have chosen the fox-tapeworm Echinococccus multilocularis whose larval stage
causes alveolar echinococcosis. Our experimental approach mainly bases on an axenic in vitro
cultivation system for the Echinococcus metacestode stage which we have developed to
reconstitute the situation at the host liver during the infection. Using this in vitro system, we
showed that two host hormones/growth factors, insulin and bone morphogenetic protein 2
(BMP2), significantly stimulate parasite survival and growth. As possible mediators of these
effects, we have characterized the parasite surface receptors EmIR, a member of the insulin
receptor family of tyrosine kinases, and several members of the transforming growth factor 
receptor family (type I and type II) of serine/threonine kinases. EmIR is located at the surface of
metacestode vesicles, facing host tissue, and is phosphorylated in response to exogenously added
human insulin. At least one of the identified receptors of the TGF- family, EmTR1, a member of
the ALK1 subfamily of TGFphosphorylates the downstream TGF- signaling factor EmSmadB in vitro. EmIR and EmTR1
are, therefore, promising candidate receptors that could mediate the effects of host insulin and
BMP2 on parasite development.
Apart from mechanisms of host-parasite communication, evolutionary conserved molecules may
also be involved in nutrient uptake by the parasite. In this context, we have characterized a parasite
protein, EmABP, which is secreted by growing metacestode vesicles and which shows
considerable homology to apolipoprotein AI binding proteins of mammals. Like these, EmABP
binds to apolipoprotein AI (ApoAI) in co-immunoprecipitation assays. Since ApoAI is a major
constituent of HDL particles that transport cholesterol in mammals, one of the functions of
EmABP could be the uptake of cholesterol which E. multilocularis is not capable of producing de
novo.
Taken together, there are several clear indications that evolutionary conserved molecules may play
an important role in host-parasite interactions during alveolar echinococcosis. The significance of
these interactions will have to be clarified in further experiments using the axenic in vitro system
and, in particular, in vivo infection models.
Hydra Meeting Friday 9 September 2005 : Session 8 : Interactions
40
Regulation of innate and adaptive immunity by filarial worms and their symbionts
Achim Hoerauf, Michael Saeftel, Judith Satoguina,
Ken Pfarr, Sabine Specht, Alex Debrah and Sabine Mand
Institute for Medical Parasitology, University Clinic Bonn,
Sigmund-Freud-Str.25, 53105 Bonn
Filarial nematodes induce chronic infections in their hosts. This requires a repertoire of
mechanisms by which they subvert and deviate host immune effector responses. The Litomosoides
sigmodontis model of murine filariasis has been very useful to establish that both Th1 and Th2
responses are associated with parasite control, while reg T cells and the cytokine IL-10 determine
permissiveness. These patterns are also observed in human onchocerciasis.
The filariae are unique within the phylum nematoda in that they harbour Wolbachia, essential
endosymbionts. Apart from being essential for larval growth, fertility and survival of adult worms,
the immunostimulatory capacity of Wolbachia has always been of interest with regard to the
question whether they are pro- or anti-inflammatory. Wolbachia do not make LPS, but Wolbachia
surface protein has been shown to be an inducer of TLR 2 and less so, TLR 4, leading to the
secretion of both pro-inflammatory and anti-inflammatory cytokines in vitro. In vivo and ex vivo,
data from murine and human filariasis suggest that the pro-inflammatory stimulus prevails. Thus,
in the murine river blindness model, keratitis develops in a TLR2 and 4 dependent manner and
requires the presence of Wolbachia. The importance of innate signalling via TLRs will be detailed
in another talk (Ken Pfarr, Saturday), which shows that mice deficient for the TLR co-molecule
MyD88 develop a higher worm burden than WT mice. In onchocercomas, neutrophils are
dependent on the presence of Wolbachia, since following Wolbachia depletion by doxycycline
treatment of onchocerciasis patients, nodules do not show neutrophils around the adult filariae. In
bancroftian filariasis, doxycycline treatment depletes Wolbachia and reduces the serum levels of
TNF and other cytokines, a condition, which is associated with lower adverse events induced by
microfilaricidal treatment, and also lower levels of the VEGF group of lymphangiogenetic growth
factors. The implications for doxycycline in the treatment of lymphedema and urogenital pathology
are discussed.
Hydra Meeting Friday 9 September 2005 : Session 9 : Immune Regulations
41
Overexpression of IL-10 from T cells or macrophages
leads to apparent different outcomes in filariasis
Sabine Specht1, Viviane Helfenstein1, Roland Lang2, Achim Hoerauf1
1
Institute for Medical Parasitology, University Clinic Bonn, Sigmund-Freud-Str.25, 53105
Bonn; 2Institute for Mikrobiology, Immunology and Hygiene, Technische University of
Munich, Trogerstr.9, 81675 München
specht@parasit.meb.uni-bonn.de
Infections with parasitic helminth are quite commonly asymptomatic, and most infected
individuals are able to tolerate the presence of parasites normally considered as „pathogenic“ for
considerable time without effects. Significantly, pathology is more closely associated with
increased immunological reactivity, i.e., acute lymphatic inflammation in lymphatic filariasis, or
hyperreactive immunity to skin microfilariae in onchocerciasis resulting in dermatitis. The fact
that in these disease conditions, the immune system is capable of reacting vigorously to the
parasites, but does not in the majority of infected people with high worm loads, is one indicator
that down-regulation of responsiveness is occurring in helminth infection perhaps via IL-10. IL-10
producing regulatory T cells could be recovered from patients infected with the helminth
Onchocerca volvulus.
To investigate the role of IL-10 in chronic infection and during early innate immune responses we
used a helminth infection model. Mice overexpressing IL-10 under the IL-2 promoter or under the
CD68 promoter restricting IL-10 secretion to T cells respectively macrophages were infected with
the rodent filaria Litomosoides sigmodontis. Interestingly, IL-10 transgenic (tg) mice under the IL2 promoter cleared the infection like the wild type controls, whereas the IL-10 transgenic mice
under the CD68 promoter showed increased susceptibility. These mice had a significantly elevated
number of live adult worms on day 80 p.i. Also, microfilariae were found in the thoracic cavity
and blood in one third of IL-10 tg mice, whereas none of the wild type mice had microfilariae.
These findings were associated with reduced inflammatory cytokine production of both the Th1
and Th2 type. Thus, IL-10 not only produced by T cells but also by macrophages is an important
regulatory cytokine leading to enhanced parasite development.
Hydra Meeting Friday 9 September 2005 : Session 9 : Immune Regulations
42
Functional analysis of regulatory T cells in mice infected with the intestinal
nematode Heligmosomoides polygyrus
Sebastian Rausch, Jochen Hühn*, Bettina Sonnenburg, Richard Lucius, Susanne
Hartmann1
Department of Molecular Parasitology (Institute of Biology), Humboldt-University at
Berlin, Philippstr. 13, 10115 Berlin, Germany;
*German-Arthritis-Center, Schumannstr. 21/22, 10117 Berlin, Germany.
susanne.hartmann@rz.hu-berlin.de
One feature of nematode infections is a marked cellular hyporeactivity which might be due to
regulatory T cells (Tregs). Tregs are described as important suppressor cells which produce the
downregulatory cytokines IL-10 and TGF-
n the present study we investigated the effects of
Tregs in mice infected with the intestinal nematode Heligmosomoides polygyrus. Splenocytes and
mesenterial lymph node cells of mice were isolated at day 6, 12 and 21 post infection. Tregs were
identified using antibodies against CD25 and the integrin E7 by FACS-analysis. We revealed
elevated percentages of Tregs at day 6 p.i. in the mesenterial lymph node cells in comparison to
uninfected controls. This effect could not be observed in splenocytes during the early phase of
infection. From day 12 onward in both organs a significant upregulation of Tregs was detected.
These data were confirmed by mRNA analysis of the Treg marker FoxP3. Co-cultivation of
regulatory T cells from infected animals with naive T cells showed strong immunosuppressive
effects of the regulatory T cells on the cellular proliferation of the naive T cells in vitro. Further
studies have to elucidate the effects of the Tregs in vivo as well as their cytokine pattern. So far,
our data indicate a signifant induction of regulatory T cells during the infection with H. polygyrus
and the potential of these cells to be key mediators of cellular suppression during nematode
infections.
Hydra Meeting Friday 9 September 2005 : Session 9 : Immune Regulations
43
The IL-4-inducing principle from S. mansoni eggs (alpha-1/IPSE) activates human
basophils via a novel mechanism: “IgE receptor engagement without crosslinking”
Silke Blindow*, Gabriele Schramm*, Achim Gronow*, Maren Manske*, Jurgen Galle*,
Bernhard F. Gibbs¶, Christoph Grevelding§, Thomas Weimar&, Brian J. Sutton$, Michael
J. Doenhoff# and Helmut Haas*+
*
Cellular Allergology, Research Center Borstel, Germany, ¶Department of Dermatology,
University of Luebeck, Germany, §Institute for Parasitology, Justus-Liebig-University,
Giessen, Germany, &Institute for Chemistry, University of Luebeck, Germany, $The
Randall Division of Cell & Molecular Biophysics, King’s College, London, UK, #School
of Biological Sciences, University of Wales, Bangor, UK,
hhaas@fz-borstel.de
We have previously isolated and characterized a glycoprotein secreted from S. mansoni eggs,
IPSE, which triggers human basophils to release the Th2 cytokines interleukin- (IL-)4 and IL-13.
Binding studies with specific antibodies and N-terminal sequencing of the isolated proteins
revealed that IPSE is identical with the S. mansoni egg antigen alpha-1. Immunohistology showed
alpha-1/IPSE to be enriched in and secreted from the sub-shell area of the egg. Furthermore, as
found by RT-PCR and in situ hybridization experiments, IPSE is restricted to the egg stage of the
parasite. Functional tests with natural and recombinant IPSE – the latter in non-glycosylated as
well as in glycosylated form - revealed that IPSE is an immunoglobulin-binding factor with
affinity to both the Fab and the Fc part of the immunoglobulin molecule. IPSE activates human
basophils in an IgE-mediated fashion, however, in contrast to other known IgE-binding factors,
such as antigens, lectins or conventional B cell superantigens, it is not able to crosslink IgE. This
was demonstrated by a variety of assays such as gel precipitation, sandwich blotting and Biacore
analysis. Taken together, IPSE activates human basophils via a novel mechanism to release Th2
cytokines: “IgE receptor engagement without crosslinking”.
Hydra Meeting Friday 9 September 2005 : Session 9 : Immune Regulations
44
B cells dominate IL-10 production during Th1, but not Th2,
response induction by dendritic cells in vivo.
Georgia Perona-Wright1, Stephen Jenkins1, Alison Crawford1,
David Gray1, Edward J. Pearce2 and Andrew S. MacDonald1.
1
Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh,
U.K. and 2Department of Pathobiology, University of Pennsylvania, Philadelphia, U.S.A.
andrew.macdonald@ed.ac.uk
pathology in both autoimmunity and infection. We have investigated the role of IL-10 in the
process of T cell activation and polarisation by dendritic cells (DC) following their exposure to Ag
derived from pathogens that induce either a Th1 or Th2 response (heat-killed Propionebacterium
acnes, Pa, or soluble egg Ag from Schistosoma mansoni, SEA). By comparing wild-type (WT) or
IL-10-deficient (IL-10-/-) murine bone marrow-derived DC transferred into WT or IL-10-/recipients, we have found that DC-derived IL-10 is not required for Th2 induction to SEA,
although it is involved in regulating Th1 development to Pa. Strikingly, IL-10 from a source other
than the initiating DC appears most important for regulating Th1 induction, and promoting Th2
induction, by DC. IL-10-/- mice that were injected with Pa-pulsed WT DC mounted a significantly
greater Pa-
-
production was observed after injection of SEA-pulsed WT DC into IL-10-/- mice, contrasting the
negligible amounts of this cytokine normally measured after transfer of SEA-pulsed DC into WT
mice.To identify the cellular source(s) of IL-10 in recipient mice during immune response
development after DC transfer, we generated bone marrow chimeras in which IL-10 deficiency
was restricted to B cells alone, or both B cells and T cells. Transfer of WT SEA- or Pa-pulsed DC
into these chimeras revealed that B cells dominate IL-10 production during the Th1 response
driven by Pa-conditioned dendritic cells.
This IL-10 inhibited IFN-
effector populations including CD4+, CD8+ and B220+ cells. In contrast, the role of IL-10 during
Th2 development to SEA appeared restricted in both source and cellularity, being made by and
targeting only T cells. These data illustrate the precise regulation of IL-10, the timing of its release
and the specificity of its action during pathogen-focussed, DC-driven immune responses in vivo.
Hydra Meeting Saturday 10 September 2005 : Session 10 : Induction of Immunity
45
The Toll-like receptor pathway and the chemokine CCL17 are essential for the
immune-mediated containment of adult worms and microfilariae in (murine)
filariasis
K. Pfarr*, S. Specht* , A. Hoerauf
Institute for Medical Parasitology, University Clinic Bonn, Bonn, Germany
*Contributed equally to this work
C3H/HeJ mice, deficient in TLR4 signaling, were previously found to have a more robust pattern
of embryogenesis and contained live, fully stretched microfilariae in the pleural cavity, while
control C3H/HeN mice did not. Subsequent experiments have been done to ascertain the cellular
mechanism involving the TLR pathway to control filarial infections on the C3H background. Mice
deficient for MyD88 were included in the study so that the role of other TLRs could be learned.
MyD88-/- mice had significantly more worms in the pleural cavity as their heterozygous litter
mates. MyD88-/- and MyD88+/- mice had similar serum cytokine profiles. At day 56 p.i., a
deficiency in TLR4 signaling lead to significantly fewer NK cells in the pleural cavity. At day 35
p.i., MyD88-/- mice had significantly fewer B, CD4+, CD8+, NK, and NKT cells. These mice also
had significantly fewer granulocytes at the site of infection. As previously shown in our lab, NK
cells and granulocytes are innate immune cells required to control worm number in mice,
suggesting that control of filarial infections is via innate immune cells that are supported by
adaptive immune cells, thus producing a mixed Type 1 and 2 response.
An obvious first innate cell type that could be involved in producing such a response is the
dendritic cell. Examination of draining lymph nodes 24hrs post infection shows that the lymph
nodes are swollen and have more blood vessels than lymph nodes that drain sites receiving just
medium. This is especially apparent when working with mice deficient for the chemokine CCL17,
a molecule secreted by a sub-class of dendritic cells involved in attracting Th2 and Th1 cells to
sites of infection. CCL17-/- mice, have a 5 fold lower worm burden than heterozygous controls at
day 30 p.i. Thus, dendritic cells, especially those secreting CCL17, are innate immune cells
important to directing the adaptive immune response to filarial infections. The exact link between
the TLR pathway and dendritic cells is still to be determined.
Hydra Meeting Saturday 10 September 2005 : Session 10 : Induction of Immunity
46
Tetraspanins expressed in the tegument of Schistosoma mansoni
are protective vaccine antigens in murine schistosomiasis
Mai Tran, Mark Pearson, Danielle Smyth, Don McManus, Malcolm Jones, Alex Loukas
Division of Infectious Diseases and Immunology,
Queensland Institute of Medical Research, Brisbane, Australia.
Tetraspanins (TSP) are a family of glycoproteins containing four conserved transmembrane
regions and two extracellular loops. In association with each other or other types of protein, such
as integrins, MHC and co-stimulatory molecules, tetraspanins have been implicated in the
regulation of cell differentiation, motility, aggregation, signal transduction and metastasis.
We have isolated two tetraspanin cDNAs, termed Sm-tsp-1 and Sm-tsp-2, from Schistosoma
mansoni using signal sequence trap and rapid amplification of cDNA ends.
The extracellular
loop-2 of both TSP-1 and TSP-2 was expressed in E.coli fused to thioredoxin and a C-terminal
hexaHis tag which provided simple purification under non-denaturing conditions by metal affinity
resin. Antiserum was raised against TSP-2 and used to localise its expression to the outer
tegument of adult worms.
Immunoprecipitation of biotinylated whole worm extracts with anti-
TSP-2 serum identified a putative ligand for TSP-2 of approximately 40kDa. Characterisation of
the putative binding partner, as well as the localisation of TSP-1 and its potential binding partner,
is underway.
Vaccination of CBA/J female mice with recombinant TSP-1 or TSP-2 demonstrated high antibody
titres by ELISA and Western blot analysis. Vaccinated animals were challenged with S. mansoni
cercariae, and seven weeks later, displayed significant reductions in worm and liver egg burdens.
We are currently repeating the vaccine trials to confirm our encouraging efficacy data, supporting
the development of tetraspanins as anti-schistosomiasis vaccines.
Hydra Meeting Saturday 10 September 2005 : Session 10 : Induction of Immunity
47
The 14-3-3 proteins: new vaccine candidates against platyhelminths
Mar Siles-Lucas1; Nelson Uribe1; Mike Merli2; Bruno Gottstein3; Antonio Muro1
1
2
Unidad de Parasitología, Facultad Farmacia, Universidad de Salamanca.
Department of Parasitology, University of Hohenheim, Stuttgart, Germany.
3
Institute of Parasitology, University of Berne, Switzerland.
marsiles@usal.es
The 14-3-3 proteins represent a family of widely expressed ~30 kDa acididc proteins acting as
spontaneously forming, phosphoserine-threonine binding dimmers. This protein family has a
crucial role in eukaryotic cell signalling events that are involved in controlling progress through
the cell cycle, in transcriptional alterations in response to environmental stimuli and in
programmed cell death. The 14-3-3 proteins have now been isolated and characterized in parasitic
organisms. In Schistosoma and Echinococcus, the biological functions of several 14-3-3 protein
isoforms have been extensively studied. Furthermore, immunoprotective properties against
parasite experimental infections have been revealed upon vaccination with respective recombinant
14-3-3 polypeptides. We have shown that vaccination with a defined 14-3-3 recombinant protein
from E. multilocularis resulted in protection levels ranging from 89 to 95%. Mechanisms of
parasite clearance beyond 14-3-3 vaccination have yet to be completely elucidated, although we
have demonstrated that the induced protection depends on both humoral and cellular specific
responses. In addition, and seeking for the corresponding proteins in Schistosoma, we have
isolated and described two new 14-3-3 proteins in S. haematobium and S. bovis. These new
polypeptides resulted to be very similar to those previously described in S. mansoni. The two new
Schistosoma 14-3-3 proteins have been already produced as recombinant proteins and used for the
first experimental vaccinations against S. mansoni in our lab. These protection experiments have
shown variable results, depending on several variables such as the mouse strain elected for
respective experiments, and the use of different immunomodulators and adjuvants in the vaccine
formulation.
Hydra Meeting Saturday 10 September 2005 : Session 10 : Induction of Immunity
48
Th2 response polarization during infection
with the helminth parasite Schistosoma mansoni
Edward Pearce
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, 19104
Schistosomiasis is a chronic disease caused by infection with helminths of the genus Schistosoma.
Adult S. mansoni parasites live intravascularly, and for transmission of this infection it is
necessary for parasite eggs to traverse the endothelium, and migrate to the intestinal lumen, from
where they can exit the body to continue the lifecycle. This process is dependent on an intact host
CD4 T helper (Th) cell response to egg antigens. Perhaps because of this, eggs have evolved to be
highly immunogenic and capable of inducing potent Th responses. The egg-induced Th response
is unusual in that it is highly Th2-polarized. The selective pressure on the host to mount a Th2
response against eggs is apparent in the fact that Th2 response-defective mice develop acutely
lethal disease when infected with schistosomes. The underlying basis for the Th2 bias in the
immune response to egg antigens will be discussed.
Hydra Meeting Saturday 10 September 2005 : Session 11 : Regulation of Response
49
Development and maintenance of type 2 immune responses
in a competitive environment
Adam Balic, Yvonne M. Harcus and Rick M. Maizels
Institute of Immunology and Infection Research, University of Edinburgh
Gastrointestinal (GI) nematode infections have routinely been used to examine Th2 dependent
host resistance. However, the ability and purpose of the parasites to generate these Th2 responses
in a setting which contains abundant Th1 inducing microbial products has been less well studied.
To investigate these questions we have taken advantage of the ability of
Nippostrongylus
brasiliensis excretory/secretory (NES) antigens to block microbial induced IL-12 production by
dendritic cells and to induce IL-4 and IL-10 production by CD4+ T cells. Indeed, Th2 induction is
seen in peripheral lymph node cells after subcutaneous footpad injection with a single dose of
NES in complete Freund’s adjuvant (CFA). Ex vivo intracellular cytokine staining indicates that
the initial production (72 hours post-injection) of NES-dependent IL-4 and IL-10 in the local
draining lymph node is derived from CD4+ T cells, is independent of IL-4R signalling and is
concurrent with a decrease in the levels of IFN producing CD4+ cells. While initial IL-4
production is independent of IL-4R engagement, production of IL-10 fails to occur in T cells from
IL4Ra-deficient mice, and in this environment the Th2 population does not expand. Inhibition of
Th1 cytokines in the absence of IL-4R signalling restores expansion of the Th2 response. Thus,
IL-4R-dependent IL-10 production during the early, competitive phase of the response to
NES/CFA, is necessary for the expansion and dominance of the classic Th2 outcome to helminth
antigen challenge.
Hydra Meeting Saturday 10 September 2005 : Session 11 : Regulation of Response
50
Persistence, function and inter-relationship of central and effector memory CD4+ T
cells following infection with a gastrointestinal parasite
Colby Zaph1, Kathryn A. Rook1, Markus Mohrs2, Phillip Scott1 and David Artis1*
1
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104
2
Trudeau Institute, Saranac Lake, NY 12983
dartis@vet.upenn.edu
The development and maintenance of memory T cells is a critical component of resistance to
reinfection. Mucosal sites such as the respiratory and gastrointestinal (GI) tracts are primary entry
points for many infectious agents. However little is known about the regulation of CD4 + T cell
memory at mucosal sites. Immunity to primary infection with the parasitic nematode Trichuris
muris is dependent upon CD4+ TH2 cells that develop in the gut-associated lymphoid tissue
(GALT), produce IL-4 and IL-13 and mediate physiological changes in the GI tract (including
goblet cell hyperplasia and expression of RELM) resulting in clearance of the worms and sterile
immunity.
While many of the factors that orchestrate immunity to primary infection with
Trichuris are well defined, those that regulate T cell memory and immunity to rechallenge have
not been analyzed.
Memory T cells are heterogeneous and can be separated into at least two distinct subsets based
upon phenotype, function and migratory pattern. Central memory T (T CM) cells express high
levels of CD62L and can migrate through secondary lymphoid tissues, while effector memory T
(TEM) cells express low levels of CD62L and tend to accumulate at extra-lymphoid sites.
However, the origin, inter-relationship and function of CD4+ TCM and TEM cell subsets have not
been described in vivo.
Here, we demonstrate that memory CD4+ T cells are required for immunity to Trichuris infection
in the GI tract. Both TEM and TCM cells develop, persist following sterile cure of primary infection
with Trichuris and are efficient at conferring resistance to immunity to rechallenge. Further, our
data indicate that the CD4+ TCM cell pool includes both non-polarized cells as previously
demonstrated, and also cytokine-committed CD4+ TCM cells that arise from TEM cells. Finally, we
demonstrate that in addition to expanding the effector T cell pool, CD62Llow TEM cells can also
repopulate the CD62Lhigh TCM cell population, thereby replenishing the pool of pathogen-specific
TCM cells. Taken together, these studies show that distinct, inter-related subsets of memory CD4+
T cells develop after infection, persist in the GALT, and mediate protective immunity to
rechallenge. Furthermore, the data presented here provide a model of mucosal CD4 + T cell
memory and the framework for the design of vaccines against GI-dwelling pathogens.
Hydra Meeting Saturday 10 September 2005 : Session 11 : Regulation of Response
51
An orphan seven transmembrane receptor from the tegument of Schistosoma
mansoni and its vaccine efficacy in a rodent model of infection.
Mark S. Pearson*, Danielle J. Smyth, Mai H. Tran,
Malcolm K. Jones, Donald P. McManus and Alex Loukas
Division of Infectious Diseases and Immunology, Queensland Institute of Medical
Research and Australian Centre for International and Tropical Health, The University of
Queensland, Brisbane, QLD, Australia.
Extracellular domains of molecules on the schistosome surface - are likely to contain epitopes that
are accessible to the host environment, making them attractive candidates for intervention
strategies. A novel cDNA encoding a seven transmembrane (7TM) receptor, termed Sm7TM, was
cloned from Schistosoma mansoni and shared homology with a small group of orphan seven
transmembrane (7TM) receptors of unknown function from vertebrates and invertebrates.
Sm7TM was predicted to possess an extracellular C-terminal tail and this was proven
experimentally by expressing Sm7TM as a 6×His-fusion in COS7 cells and identifying the
extracellular C-terminus on non-permeabilised cells using immunofluorescence. Antisera was
raised against this hydrophilic domain (Sm7TMC - expressed as a soluble fusion in E. coli) and
used to immunolocalise the receptor to the tegument of adult S. mansoni. Mouse vaccination with
Sm7TMC induced a high titre of antibodies which were capable of recognising native Sm7TM in
parasite extracts. Furthermore, vaccinated mice showed a significant mean reduction in worm and
liver egg burdens. Repeat trials are current underway to confirm the protective efficacy of this
molecule.
Hydra Meeting Saturday 10 September 2005 : Session 11 : Regulation of Response
52
Vaccination with recombinant aspartic haemoglobinase reduces parasite load and
blood loss after hookworm infection.
Alex Loukas1, Jeffrey M. Bethony2, Susana Mendez2, Ricardo T. Fujiwara2,
Gaddam Narsa Goud2, Najju Ranjit1, Bin Zhan2, Karen Jones2,
Maria Elena Bottazzi2 and Peter J. Hotez2
1
Division of Infectious Diseases and Immunology,
Queensland Institute of Medical Research, Brisbane, Queensland 4006, Australia,
2
Department of Microbiology and Tropical Medicine,
The George Washington University Medical Center, Washington DC 20037, USA
Hookworms infect almost one billion people in developing countries where they are a leading
cause of intestinal blood loss and iron-deficiency anaemia. At the site of host parasite attachment,
adult hookworms ingest blood and lyse the erythrocytes to release haemoglobin. The parasites
subsequently digest haemoglobin in their intestines using a cascade of proteolysis that begins with
the aspartic protease, APR-1. We show that vaccination of dogs with recombinant Ac-APR-1
induced antibody and cellular responses and resulted in significantly reduced hookworm burdens
and faecal egg counts (P = 0.05) in vaccinated dogs compared to control dogs after challenge with
infective larvae of Ancylostoma caninum. Most importantly, vaccinated dogs were protected
against blood loss and did not develop anaemia (P = 0.02), the major pathologic sequelae of
hookworm disease.
IgG from vaccinated animals decreased the catalytic activity of the
recombinant enzyme in vitro, and antibody bound in situ to the intestines of worms from
vaccinated dogs, implying that the vaccine likely interferes with the parasite’s ability to digest
blood. This is the first report of a recombinant vaccine from a hematophagous parasite that
significantly reduces both parasite load and blood loss, and supports the development of APR-1 as
a human hookworm vaccine.
Hydra Meeting Saturday 10 September 2005 : Session 12 : Immunity and the Parasite
53
Resistin-like molecules: novel immune effectors that target parasitic nematodes
David Artis
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, 19104
More than one billion people worldwide are infected with one or more species of gastrointestinal
(GI) nematode parasites and within the livestock industry, reduced productivity and the need for
repeated drug treatment impose a heavy economic burden. Evidence of immunity to nematode
infection in human and livestock populations suggest that long term immunologic intervention
strategies against nematode parasites are an achievable goal.
The importance of CD4+ T helper (Th) cells in regulating immunity to GI nematode infection has
been formally demonstrated in murine model systems. While Th1-associated cytokines, including
IL-12, IL-18 and IFN-, can inhibit protective immunity to infection, Th2 cells that secrete IL-4,
IL-9, and IL-13 mediate expulsion of live worms and host protective immunity. However, the Th2
cytokine-induced immune effector mechanisms that mediate expulsion of nematodes from the GI
tract remain unknown.
Following infection with Trichinella, Nippostrongylus or Trichuris, transcriptome analysis
demonstrated that resistin-like molecule- (RELM/FIZZ2) was one of the most highly expressed
genes in the gut. Therefore, expression of RELM is a conserved mammalian response to
phylogenetically and biologically distinct nematode parasites that inhabit distinct niches in the GI
microenvironment. RELM is a member of the resistin gene family (composed of four members
(RELM, RELM, RELM, and resistin)). This small cysteine-rich protein is expressed by
intestinal goblet cells and secreted into the GI lumen as an 18kDa homodimer. It is highly
conserved in all mammalian species analyzed so far although its functions remain unknown.
We found that expression of RELM was controlled by Th2 cytokines and peak RELM protein
secretion into the gut was coincident with host protective immunity.
Following secretion,
immunofluorescent and ultrastructural analysis demonstrated that RELM bound chemosensory
structures in the nematode cuticle and impaired chemosensory functions. Ongoing studies are
investigating the nematode targets that are bound by RELM and other resistin gene family
members, the biological functions of these targets, and the significance of these interactions in
host protective immunity. Taken together, these results suggest that expression of RELM is a
critical component in the arsenal of type 2-regulated effector mechanisms that precipitate worm
expulsion and host protective immunity following GI nematode infection.
Hydra Meeting Saturday 10 September 2005 : Session 12 : Immunity and the Parasite
54
Regulation by helminth infection of immunity and allergy
R M Maizels, J E Allen, A Balic, C A M Finney,
N Gomez-Escobar, A Harris, M D Taylor, and M S Wilson
Institute of Immunology and Infection Research, University of Edinburgh, UK
We have demonstrated that down-regulatory T cell populations (Tregs) are active in two helminth
infections, the tissue filaria Litomosoides sigmodontis, and the gastrointestinal nematode
Heligmosomoides polygyrus. In both infections, there is expansion of Foxp3+ CD4+CD25+ T cells,
and increased expression of TGF-b and IL-10. In L. sigmodontis, susceptibility of BALB/c mice
can be converted to the resistant phenotype by administration of antibodies to CD25 together with
either anti-GITR or CTLA-4, consistent with the hypothesis that Tregs restrain immunity in
chronic infection. In mice infected with either L. sigmodontis or H. polygyrus, bystander immune
responses are also suppressed; in particular we have demonstrated significant reduction in airway
allergic inflammation in animals harboring nematode infections. Mesenteric lymph node cells
(MLNC) from H. polygyrus infected mice can transfer protection from allergy to uninfected
recpients, with the most potent effect found among the CD4+CD25+ subset. We have also been
studying molecules isolated from nematode parasites (Brugia malayi and Nippostrongylus
brasiliensis) which have been shown to interfere with generation of the inflammatory immune
response, favouring either Th2 or Treg evolution. Both recombinant B. malayi proteins, and
purified N. brasiliensis secreted components, have been shown to block IL12p70 generation by
dendritic cells, which we hypothesise is a key element in driving responsiveness towards the
counter-inflammatory or regulatory phenotype.
Hydra Meeting Saturday 10 September 2005 : Session 12 : Immunity and the Parasite
55
The life and times of the parasitic nematode Strongyloides ratti under immune
pressure.
Mark Viney, Fiona Thompson, Louise Hughes, Gary Barker, Clare Wilkes,
Makedonka Mitreva1 & Jim McCarter1
1
School of Biological Sciences, University of Bristol, Woodland Road, Bristol, UK &
Genome Sequencing Center, Department of Genetics, Washington University School of
Medicine, St. Louis, MO 63108, USA
Mark.Viney@bristol.ac.uk
Parasitic nematodes are stressed by their hosts’ immune responses. For Strongyloides ratti this is
seen both at the individual- and population-level. Individually, parasitic females become
progressively shorter, their per capita fecundity is reduced and they are more posteriorally
positioned in the gut as anti-S. ratti immune responses develop. That these effects are due to an
anti-S. ratti immune response is shown by the fact that these changes either do not occur in
immunocompetent animals or are reversed if animals are immunosuppressed. At the populationlevel, immune-dependent density-dependent processes also act on the survival and per capita
fecundity of parasitic females.
An EST analysis of five life-cycle (free-living and parasitic) stages has discovered c. 20% of S.
ratti genes. A large number of these appear to be nematode- or Strongyloides-specific. There is
extensive stage-specificity of expression (measured as EST abundance), especially between the
parasitic and free-living phases of the life-cycle. Among the most highly represented ESTs are
many with similarity to proteins only predicted from the C. elegans genome sequence: one of
these represents 25% of all ESTs from the S. ratti parasitic females.
These ESTs have been used to construct microarrays. These have been used to compare gene
expression between L2 free-living stages destined for direct (i.e. into infective L3s) or indirect (i.e.
into free-living adult males and females) development. Similarly, we have compared gene
expression between L1s and infective L3s. These analyses have shown that these S. ratti
microarrays work and work well. These arrays are also being used to compare gene expression in
parasitic females under different immune pressures; specifically, to compare gene expression
between worms at 6 and 15 days p.i. The rationale for doing this is to discover individual genes,
metabolic pathways or physiological processes that are particularly important to worms under
immune pressure and, in so doing, to suggest targets for the potential control of nematodes.
Hydra Meeting Saturday 10 September 2005 : Session 12 : Immunity and the Parasite
ABSTRACTS FOR POSTER PRESENTATION
SESSION 1
WEDNESDAY 7 SEPTEMBER
18.10 – 20.00
57
Purinergic regulation of mucosal mast cell degranulation in T. spiralis infection
H. Afferson, M.E. Selkirk and K. Gounaris
Division of Cell and Molecular Biology, Imperial College London, UK.
Corresponding Author: holly.afferson@ic.ac.uk
The parasitic nematode, Trichinella spiralis secretes a variety of extracellular nucleotide metabolising
enzymes. These are postulated to alter the concentrations of extracellular nucleotides and nucleosides
available to interact with host purinergic receptors. Nucleotides and nucleosides can modulate mast
cell activation, including degranulation. As mucosal mast cells secreting mouse mast cell protease-1
(mMCP-1) are crucial for the efficient expulsion of T. spiralis from the murine small intestine, we
sought to investigate whether nucleotide metabolising enzymes can modulate nucleotide induced
mMCP-1-positive mouse bone marrow derived mast cell (mBMMC) degranulation in vitro.
Expression of purinergic P1 (A1, A2A, A2B, A3), P2Y2, P2Y4 and P2Y6 receptors in mBMMC were
confirmed by reverse-transcriptase PCR. Initial characterisation studies showed that extracellular
adenosine, NECA, AMP, ADP, ATP, UMP and UTP, in the absence or presence of IgE, did not alter
hexosaminidase or mMCP-1 release from mBMMC. UDP however decreased the basal level of both
hexosaminidase and mMCP-1 release from mBMMC, indicating potential signalling via the P2Y6 or
cysteinyl leukotriene receptors 1 and 2. IgE-sensitised mBMMC exposed to antigen and either
adenosine, NECA, or AMP exhibited potentiation of antigen dependent degranulation. This is
postulated to occur via P1 adenosine receptors, as there is no known receptor for AMP, indicating the
involvement of cell surface 5´-nucleotidase activity able to hydrolyse AMP to adenosine. T. spiralis
secreted proteins and recombinant 5´-nucleotidase, did not directly alter mBMMC degranulation.
Further experiments are underway to determine the effect of these enzymes on the potentiation of
adenosine induced antigen dependent degranulation and UDP-induced decrease in degranulation. RTPCR demonstrated expression of P1 (A1, A2A, A2B, A3), P2Y2, P2Y4 and P2Y6 receptors in epithelial
cells extracted from the small intestine of uninfected mice. Preliminary experiments suggest P2Y4 and
P2Y2 receptor expression in the intestinal epithelium of T. spiralis infected mice is reduced at the
peak of worm expulsion (day 7 to 10). We are also using immunisation experiments to investigate the
role of nucleotide metabolising enzymes on parasite persistence and expulsion in vivo.
Quiescin-sulfhydryl oxidases, and their role in cuticle collagen biosynthesis in C.elegans
Andrew J. Birnie
Institute of Comparative Medicine, University of Glasgow, Glasgow G61 1QH.
Nematodes, both free living and parasitic are dependant upon there Extra Cellular Matrix (ECM) for
multiple aspects of functionality. The cuticle of C.elegans, like other nematodes is composed largely
of collagen-like proteins, with trimeric collagenous proteins forming approximately 80% of the ECM.
The processing of collagen monomers involves many different enzymes and many distinct steps,
namely; chain association, registration, nucleation and propagation. The enzymes that are involved in
the generation of these functional trimers are therefore essential to a number of functions of the
nematode. The generation of a functional triplet collagen chain differs from the nascent folding of
other proteins primarily due to the fact that single monomers cannot fold. Disulphide bond formation
is key in collagen processing at many stages, and is essential in the generation of the mature collagen
chains associated with a cogent ECM. Chain association and registration occur via cysteine-linked
disulphide bond formation at the C-terminal ends of the pro-collagens. This in turn allows for
nucleation of the pro-collagen triple helix and thus the generation of a left handed coil that stretches
from the C- to N- direction in a manner akin to that of a zipper. Of the enzymes involved with the
maturation and processing of cuticular collagens the Quiescin-sulfhydryl oxidases, or QSOX family,
are of particular interest. QSOX enzymes are linked directly to the generation of disulphide bonds and
putatively to the indirect formation of disulphide bonds through an association with protein
disulphide isomerases (PDIs), an enzyme that is also involved in disulphide bond generation. We
generated a deletion mutant in QSOX-1 that resulted in gross morphological defects, synonymous
with a failure to generate a wild type cuticle. The temporal expression of QSOX-1 coincides with
lethargus (cuticle synthesis) and cycles with certain members of the PDI family essential in cuticle
collagen biosynthesis, as well as essential cuticle components.
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
58
Evaluation of cDNA expression library from mollusc-stage of
the bird schistosome Trichobilharzia regenti by PCR
1
Dolecková K., 2Mutapi F., 1Kasny M., and 1Horák P.
1
Department of Parasitology, Charles University, Viničná 7, 12844 Prague 2, Czech Rep.
2
Institute for Infection and Immunology Research, University of Edinburgh, UK.
Bird schistosome Trichobilharzia regenti is recently an intensively studied parasite not only for the
ability of its larvae (cercariae) to cause cercarial dermatitis (swimmer´s itch) in humans, but also for
its unusual way of migration, which involves peripheral nerves, spinal cord, medulla oblongata, brain
and final place of localization – nasal cavity of their definitive bird host (Hrádková and Horák, 2002).
Recent findings in human schistosomes show that proteolytic enzymes play a crucial role in the
process of penetration and migration of the parasite; these have also been recognized as potential
targets for chemotherapy and serodiagnostics (Caffrey et al., 2002). Due to the limitation by
insufficient amount of material for biochemical assays, molecular methods were used to gain some
information about these enzymes in T. regenti cercariae. We performed isolation of poly(A) RNA
from sporocysts with developing cercarie (present in the hepatopancreas of infected snails), RT-PCR
and subsequent semi-nested PCR with degenerate primers designed according to conserved domains
of known cathepsin B from related Schistosoma species. We identified a 1032 bp sequence which
corresponds to a 344 aminoacid chain. The protein contains all conserved domains characteristic for a
cysteine protease, namely for cathepsin B. Due to its 77% similarity to SmCB2 (a tegumental
cathepsin B2 from adult S. mansoni – Caffrey et al., 2002) and 78% similarity to cathepsin B
endopeptidase from S. japonicum, we termed this sequence TrCB2. Furthermore, we constructed a
cDNA expression library from sporocysts with developing cercarie in ZAP Express cDNA system
(Stratagene). This was screened by PCR method using degenerate primers based on the sequences of
cathepsin L (Boophilus microplus; Renard et al., 2000) and B (T. regenti-schistosomula; Dvořák et
al., 2005). We amplified and cloned two relevant sequences (500 and 618 bp) corresponding to
putative cathepsins L and B. The constructed expression library is a good tool for further detection
and characterization of bioactive molecules of T. regenti cercariae and the parasite is a good
comparative model to human schistosomes S. mansoni and S. japonicum.
Identification, Expression and antigen characterization of
paramyosin of Ancylostoma caninum
C. Epe, R. Huesken, N. Wirtherle, T. Schnieder,
Institute of Parasitology, Hannover School of Veterinary Medicine
Hannover 30559 Germany
Earlier, a partial paramyosin (PMY) coding sequence of the canine hookworm A. caninum was
identified by immunoscreening of a cDNA library using affinity-purified dog antisera. Here we
describe the full length coding sequence of this protein and bioinformatic analysis data. The 2634 bp
PMY cDNA sequence corresponds to a 7190 bp preliminary genomic sequence, including 15 exons
of 57-2184bp. The corresponding amino acid sequence is 877 aa long with a molecular weight of 101
kDa. Blast analysis revealed highest identities with PMY genes of C. elegans (93%), O. volvulus
(92%), D. immitis (90%) and B. malayi (89%) and other nematodes. Bioinformatic analysis indicated
that the A. caninum PMY predicted phosphorylation sites are 13 for Serine, 6 for Threonine and 1 for
Tyrosine, whereas 5 O-glycosylation sites were predicted using different calculation pathways.
Furthermore the use of this protein in immunisation studies is discussed. Recent information indicates
that PMY is not only existent in myofibrils but also, among others, in esophagus muscle tissue where
it possibly could be targeted as putative hidden antigen.
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
59
Multiple transcription initiation sites and alternative splicing processes
generate an unexpectedly large diversity of
omega class glutathione S-transferases in Onchocerca volvulus
Höppner, J.1 , Walter, R.D. 1 , Liebau, E. 1
1
Biochemistry; Bernhard Nocht Institut for Tropical Medicine; Hamburg; Germany
Glutathione transferases (GSTs) are a diverse family of enzymes found ubiquitously in aerobic
organisms. They play a vital role in the detoxification of both endogenous and xenobiotic compounds
and are also involved in intracellular transport, biosynthesis of hormones and protection against
oxidative stress. Up to now, thirteen GST classes have been found, one of them being the highly
stress-responsive omega class. The crystallographic structure of the omega class GST shows a GSTfold composed of an N-terminal glutathione-binding domain and a C-terminal domain composed
entirely of alpha-helices. Unlike other GSTs, the omega class GST has an active site cysteine that is
able to form a disulfide bond with glutathione and exhibits glutathione-dependent thiol transferase
and dehydroascorbate reductase activities, reminiscent of thioredoxin and glutaredoxin enzymes.
GSTs are considered the most prominent phase II detoxification enzymes in helminths. The omega
class GST from Onchocerca volvulus (OvGST3) is dramatically upregulated in response to
environmental oxidative stress, suggesting an important role in the protection of the parasite against
reactive oxygen species derived from the host´s immune system. The 2.2 kb ovgst3-gene consists of 8
exons, in which exons 1 and 2 code for a potential signal peptide. Alternative splicing of exon 4 and
5, including a splice form that causes early translation termination, results in four different gene
product variants. Furthermore, 5 unique transcripts, each trans-spliced at the 5´-end, were detected.
Taken together, the different initiation start points and the alternative splicing processes of the
OvGST3 gene lead to fourteen transcripts, the resulting proteins possibly having different enzymatic
activities and therefore different functions to perform within the parasite.
Comparison of cysteine and serine proteases of
Trichobilharzia regenti and Schistosoma mansoni cercariae
1
Kasny M., 2Dalton J., 1Mikes L., 1Dolecková K., 2Stack C. and 1Horák P.
1
Department of Parasitology, Faculty of Science, Charles University, Viničná 7, 12844
Prague 2, Czech Republic and 2 Institute for the Biotechnology of Infectious Diseases, PO
Box 123,University of Technology, Broadway, NSW 2007 Sydney, Australia
Invasive larvae (cercariae) of trematodes of the family Schistosomatidae actively penetrate the host
skin. The mechanisms of penetration are partially known in some species, especially those parasiting
in man (e.g. Schistosoma mansoni) (Whitfield et al., 2003; McKerrow and Salter, 2002; Dalton et al.,
1997). The Trichobilharzia regenti species is a bird schistosome with a unique kind of migration
among schistosomes. In contrast to S. mansoni, T. regenti has a strong affinity to nervous tissue and
migrates through peripheral nerves and spinal cord to reach the brain and successively the nasal
cavity of a water bird definitive host of the family Anatidae (Hrádková and Horák, 2002). Proteolytic
enzymes (peptidases) of T. regenti and S. mansoni cercariae probably play a crucial role during the
penetration process. Among these enzymes, the major cysteine and serine peptidases of cercariae of
the bird schistosome T. regenti were biochemically characterized and compared to peptidases of S.
mansoni (human species) cercariae. Some functional similarities of both types of peptidases were
found between cercarial extracts. Molecular techniques were used for screening the cDNA T. regenti
expression library employing PCR with primers based on the sequences of cathepsin L (Boophilus
microplus; Renard et al., 2000) and B (T. regenti-schistosomula; Dvořák et al., 2005). Two fragments
corresponding to putative cathepsin L (500 bp) and B (618 bp) were amplified. The results imply
that similar strategies are probably employed for skin penetration by the two schistosomatids,
although some significant differences have been found in enzymatic equipment of the cercariae.
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
60
Expression of recombinant glycosylated IPSE
(IL-4-inducing principle of Schistosoma mansoni eggs) in 293 HEK-cells
Maren Manske*+, Gabriele Schramm*, Achim Gronow*, Silke Blindow*, Jurgen van der
Bosch*, Daniel Wicklein*, Malcolm W. Kennedy#, Michael J. Doenhoff§ and Helmut Haas*
*Cellular Allergology, Research Center Borstel, Germany, #Environmental & Evolutionary
Biology, University of Glasgow; § University of Wales, Bangor, UK,
mmanske@fz-borstel.de
The eggs of the parasitic trematode Schistosoma mansoni are powerful inducers of a T helper type 2
(Th2) immune response. Recently we have isolated and characterized a glycoprotein from S. mansoni
egg antigen (SmEA) which triggers the release of IL-4 from human basophils (IPSE). It was shown
that IPSE activates human basophils by binding non-antigen-specifically to FcRI-bound IgE. IPSE
was recombinantly expressed in E. coli as un-glycosylated His-tagged fusion protein (His-IPSE). HisIPSE exhibits similar activity as natural IPSE with respect to basophil activation and IgE binding.
However, His-IPSE tends to aggregation, presumably due to the lack of glycosylation and improper
refolding following expression in inclusion bodies. Therefore, His-IPSE is not suitable for structural
analysis like crystallography and NMR. Eukaryotic expression systems, like 293 HEK cells, fold and
process recombinant proteins correctly and are able to add posttranslational modifications such as
glycosylation. Thus, IPSE was expressed as glycosylated His-tagged fusion protein in HEK cells
(HEK-IPSE). HEK-IPSE was secreted into the medium in a fully folded soluble form and was
purified by a two-step chromatography procedure. In a non-reducing SDS-PAGE purified HEK-IPSE
forms 4 bands in the range of 40 to 50 kD which represent different glycoforms. HEK-IPSE shows
the same activity as natural IPSE and His-IPSE concerning basophil activation and IgE binding and is
completely soluble also at high concentrations. These properties make HEK-IPSE a valuable tool for
further structural, binding and mechanistic studies on this factor.
Characterisation of a Haemonchus contortus channel
that is extremely sensitive to ivermectin.
Samantha McCavera*, Adrian T. Rogers, Darran Yates and Adrian Wolstenholme.
Department of Biology & Biochemistry, University of Bath, Bath, BA2 7AY, U.K.
sm267@bath.ac.uk
The avermectins and milbemycins are very effective anthelmintics widely used in animal and human
medicine, but the increase in resistance, especially in animal parasites, is becoming a major concern.
Nematode glutamate-gated chloride channels (GluCl) are a small family of receptors that are the sites
of action of these drugs. There are several genes encoding GluCl subunits and it is not yet clear which
subunit combinations form the most important drug target in vivo. Two of the H. contortus subunits,
GluCl and GluCl3B, form high-affinity ivermectin binding sites when expressed in mammalian
cells and a polymorphism, L256F, in the 3 subunit of Cooperia oncophora has been implicated in
ivermectin resistance. We studied the responses of H. contortus GluCl3B channels to L-glutamate
and ivermectin in the Xenopus oocyte using two-electrode voltage clamp. H. contortus GluCl3B
formed extremely robust chloride channels gated by L-glutamate with an EC50 of 27M, and that
desensitised rapidly. In contrast, the EC50 for L-glutamate of the orthologous C. elegans subunit
(AVR-14B) was 2.2mM. Ivermectin-gated channels opened essentially irreversibly with an EC50 of
~12nM: occasionally responses were observed at concentrations as low as 100pM. The amplitude of
the ivermectin-gated channels did not always vary in a dose-dependent manner, but the rate at which
they opened did. The channels were fully activated by ibotenic acid, but GABA, glycine, L-aspartate,
histamine or 5-HT had no effect. Picrotoxinin and the insecticide, fipronil, were able to fully block
glutamate-gated channels and partially block ivermectin-gated channels. The fipronil block of the
glutamate-gated channels was not reversed by extensive washing. No channels were formed when the
GluCl3A or GluCl subunits were expressed individually in the oocytes and co-expression of
GluCl3B with GluCl3A or GluCl resulted in channels indistinguishable from those formed by
GluCl3B alone. These results, together with the wide distribution of GluCl3B in the nematode
nervous system, are consistent with this subunit being a component of a major ivermectin target in
parasitic nematodes.
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
61
On the FLP-side of nematode neuropeptides
Paul McVeigh1*, Suzanne Leech1, Gunnar R. Mair1, Timothy G. Geary2,
Nikki J. Marks1 & Aaron G. Maule1
1
Parasitology Research Group, Queen’s University Belfast, Belfast, BT9 7BL, UK;
2
Pfizer Animal Health, Kalamazoo, MI, USA
paul.mcveigh@qub.ac.uk
FLPs (FMRFamide-like peptides) represent the largest family of invertebrate neuropeptides. Within
Phylum Nematoda, these neuropeptides display a diversity of structure and function unrivalled by any
neuropeptide family in any other organism. FLPs are related to the seminal FMRFamide, a
cardioactive tetrapeptide isolated from the Venus clam, Macrocallista nimbosa. Prior to this study,
23 flp genes had been reported in C. elegans, encoding 60 distinct FLP neuropeptides, identified
through bioinformatic searches of genome and EST data. This species thus provides the bulk of
current knowledge on nematode FLPs and is a useful benchmark for the FLP complements of
parasitic nematodes. From this background, a series of guided searches of parasitic ESTs using
BLAST software were initiated, utilising all of the known C. elegans FLPs as query sequences. A
total of 968 ESTs representing sequelogs of the 23 known C. elegans flp genes were identified in 30
species, encompassing clades I, III, IV and V, with up to 16 flp genes being identified in any one
parasite species. This suggests that parasitic nematodes have FLP complements approaching that
seen in C. elegans. Numerical analysis of FLP ESTs shows that the three most abundant sequences
represent flp-14, flp-1 and flp-11. The relative abundances of ESTs can be related to their expression
levels, so it appears that the most highly expressed flp gene is flp-14; this tallies with peptide isolation
studies which have frequently cited KHEYLRFamide (AF2), the peptide product of flp-14, as the
most abundant FLP in nematode extracts. Given the explosion in available molecular biological
sequence data since the last reported study of C. elegans FLP diversity in 1999, we hypothesised that
additional unidentified FLPs may await discovery in the EST database. With this in mind, the second
phase of this study was concerned with identifying putative novel FLPs. This involved performing a
speculative BLAST trawl with hexameric search strings derived from theoretical FLP C-termini.
These searches yielded 10 multi-species sequelogs encoding peptides with the characteristic FLP
signature. Six are present in C. elegans, bringing the present benchmark flp gene total in a single
nematode species is 29. However, additional non-C. elegans FLP ESTs were identified, such that the
current putative nematode flp gene total is 33, encoding a battery of almost 90 distinct neuropeptides.
The secreted peptide status of these sequences was further confirmed by SignalP analysis, which
identified putative signal peptides on at least one member of each group of ESTs. The implications of
such a bewildering array of neuropeptides in an organism with only 300 neurones will be discussed in
the context of flp gene expression data generated by our laboratory.
Analysis of differential gene expression in the free-living
and parasitic life cycles of Parastrongyloides trichosuri
Jan Newton-Howes1, Alan McCulloch1, Chuck Shoemaker2 and Warwick Grant1
1
Wallaceville Animal Research Centre, AgResearch, Upper Hutt, New Zealand
2
Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North
Grafton, MA 01536
The nematode parasite Parastrongyloides trichosuri is extremely well suited for studies which aim to
discover the genetic basis of parasitism because of ease of access to pure populations of both freeliving and parasitic life cycles. P. trichosuri is also one of more than 30 nematode species included in
the Parasitic Nematode Genome Sequencing Project based at Washington University, Mo, USA. As
a part of that project, 7964 ESTs have been sequenced from libraries derived from 3 distinct life cycle
stages: (1) a mixed stage free-living [FL] library, (2) an infective larval [iL3] library and (3) a
parasitic adult [PA] library, and the sequences deposited in public databases. Raw sequence data
from these ESTs has been aggregated into 3490 contigs on the basis of sequence overlap. Of these,
2023 contigs comprise ESTs wholly from parasitic life-cycle stages (iL3 + PA), 188 are represented
in all libraries (iL3, PA and FL)and 1278 are from free-living stages alone (FL) with singleton ESTs
being heavily represented in all categories. By analogy with C. elegans we estimate that the 3490
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
62
contigs represent about 15% of the P. trichosuri transcriptome. We ranked the stage-specific
transcripts according to expression level in a “digital northern” analysis and selected, on the basis of
this in silico analysis, a group of genes which appear highly expressed only in the parasitic life cycle.
We then tested this prediction by quantitative real-time PCR analysis (Q-PCR) of cDNA from
different life cycle stages. A total of 46 contigs were selected using these criteria from which we
currently have real-time PCR data from 39. The Q-PCR data showed that there was greater than fourfold difference in expression between free-living and parasitic stages in 27/39 (69%) of cases. In
addition, we analysed expression patterns for a number of constitutively expressed genes (act-1 and
gap3dh)and genes selected for particular biological interest based on predicted function. In no cases
did the real-time data contradict the in silico data by showing that selected contigs had little or no
expression in parasitic cDNA but the level of expression in vitro often did not closely parallel
expression in silico. Overall, the greatest difference in expression seen so far has been a 2 14-fold
difference between parasitic and free-living stages in a gene (give an accession number here?)
showing moderate [contig depth =3] expression in the IL library. By comparison one contig with
high expression [124 ESTs, all in the IL library only], showed only a 25 fold increase in parasitic
compared with the free-living stages. The approach described above has helped us to define candidate
parasitic stage specific genes for functional manipulation (attempted knock down by RNAi and/or
overexpression or mis-expression by transgenesis), with the aim of defining genes that are specific to
and essential for parasitism i.e. “parasitism genes” We will also present data on a more general survey
for "parasitism genes" by comparison of life-cycle stage expression of all ESTs by microarray.
Nematode carboxypeptidases inhibit C5a-mediated chemotaxis of human granulocytes.
D. Rees-Roberts and M. E. Selkirk
Division of Cell & Molecular Biology, Biochemistry Building, Faculty of Life Sciences, South
Kensington Campus, Imperial College London, London SW7 2AZ
dominic.rees-roberts@imperial.ac.uk
The onset of inflammation during helminth infection can be detrimental to both the host and the
parasite. We are therefore investigating whether parasitic nematodes employ mechanisms to block the
action of inflammatory mediators and thus prolong worm survival in the host. Secreted products from
Trichinella spiralis, T. pseudospiralis and Nippostrongylus brasiliensis all inhibit the chemotaxis of
human granulocytes in vitro towards Complement C5a. In addition, T. spiralis and N. brasiliensis
secreted products reduce chemotaxis of granulocytes to Platelet Activating Factor, whereas those
from T. pseudospiralis inhibit the response to Interleukin 8. The anaphylatoxins C3a and C5a are
inactivated by mammalian zinc metallocarboxypeptidases via specific cleavage of a C-terminal lysine
residue. We therefore hypothesised that a similar activity might be secreted by parasitic nematodes.
Carboxypeptidase activity was detected in secreted products of T. spiralis. A search of EST databases
for zinc carboxypeptidases produced good candidates from T. spiralis and Brugia malayi, which were
subsequently cloned and sequenced. Both possess N-terminal signal peptides, complete active sites
and homology to mammalian zinc carboxypeptidases (48% and 52% respectively). These possible
modulators of inflammation are currently being expressed in Pichia pastoris for functional
characterisation in vitro and in vivo.
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
63
A proteomics approach to identification of secreted antigens
from infective larvae of Nippostrongylus brasiliensis
Debbie Scarlett, Mali Camberis, Petra van der Linden-Ross and Graham Le Gros
Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, New Zealand
dscarlett@malaghan.org.nz
Infection of mice with the parasitic nematode N. brasiliensis is associated with a polarised Th2-type
immune response characterised by eosinophilia, IgE, IL-4, IL-5 and IL-13 production. These
responses appear to be induced by products secreted by the nematode (termed NES) since
supernatants harvested from in vitro cultures of either the infective L3 larvae, lung emerging L4, or
gut L5 adults induce similar effects. Heat inactivation or protease treatment abolishes the Th2polarising activity of L5 NES, suggesting that the key allergens secreted by these worms are proteins.
The objectives of this work are to identify proteins secreted by infective L3 larvae of N. brasiliensis
using a combination of indirect activity assays and direct protein sequencing techniques, and to
characterise the mechanisms by which these proteins interact with the innate immune system to drive
a polarised Th2 immune response. Despite inducing similar Th2-polarising effects in vivo, the
molecular composition of L3 NES differs quite considerably from that secreted by adult N.
brasiliensis worms. While the latter contains several high molecular mass glycoproteins (>50 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
1
Institute 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.
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
64
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 freeliving 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 cistrans 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.
Hydra Meeting : Wednesday 7 September 2005 : Poster Session 1
ABSTRACTS FOR POSTER PRESENTATION
SESSION 2
FRIDAY 9 SEPTEMBER
18.10 – 20.00
66
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
1
James 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 postinfection). 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@malaghan.org.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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
67
Pore-forming proteins from blood-feeding helminths
Tegan A. Don1,2, Peter O’Donoghue2, Peter J. Hotez3 Najju Ranjit1 and Alex Loukas1.
1
Queensland 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 nickelNTA 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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
68
FMRFamide-Like Peptide Genes in Meloidogyne incognita:
Characterization, Expression and RNAi-Induced Disruption
Michael J.G. Johnston1, Colin C. Fleming2 and Aaron G. Maule1.
1
Parasitology Research Group, School of Biology & Biochemistry,
Queen’s University Belfast, Belfast BT9 7BL;
2
Department 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 Gprotein 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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
69
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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
70
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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
71
The Insulin/IGF signalling transduction pathway in Parastrongyloides trichosuridoes it play a role in parasitism and aging?
Susan Stasiuk1, Chuck Shoemaker2 and Warwick Grant1.
1
Wallaceville Animal Research Centre, AgResearch, Upper Hutt, New Zealand
2
Department 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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
72
Identification of CPN10 in Strongyloides ratti
Alex Sykes1*, Michelle Jennens3, Narelle Villa2, Malcolm Jones1,
Carolyn Jones3, Andrew Thompson3 and James McCarthy1
1
Queensland 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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
73
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.
Hydra Meeting : Friday 9 September 2005 : Poster Session 2
NOTES