From the Department of Microbiology and Immunology,
College of Veterinary Medicine, Cornell University, Ithaca, New York
14853 and the § Department of Immunology and Infectious
Disease, Harvard School of Public Health,
Boston, Massachusetts 02115
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To optimize reproductive success under the
limitations determined by conditions within an individual host,
parasitic helminths have evolved mechanisms that allow them to detect
and respond to host factors such as species, age, sex, reproductive
condition, and immune status. Using the model helminth
Schistosoma mansoni, we have explored the possibility that
parasitic helminths express signal-transducing receptor molecules on
their surfaces. Here, we present the identification of a schistosome
member of the transforming growth factor receptor family of
cell-surface receptors, the first member of this family to be
identified in a platyhelminth. The putative protein kinase domain of
the schistosome receptor displays up to 58% amino acid identity to
kinase domains of other type I receptor serine-threonine kinases, and
contains a potential "GS domain," suggesting it is a divergent
member of the type I receptor subfamily. This receptor is expressed on
the surface of the parasite's syncytial tegument and expression of
receptor messenger RNA and protein is up-regulated following infection of the mammalian host. The receptor protein can be isolated in a
phosphorylated form from adult parasites, which together with its
surface location, suggests that it functions in transducing signals
across the parasite surface membrane.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An estimated 200 million people throughout tropical and temperate regions of the world are infected with parasitic trematodes of the genus Schistosoma (1). These parasites possess complex life cycles, using humans and other mammals as definitive hosts, while various species of aquatic snails serve as intermediate hosts for larval stages. After infection of the definitive host by infectious stage cercariae of Schistosoma mansoni, the immature parasites undergo a complex program of development to become sexually mature adult males and females at approximately 6 weeks post-infection. During development, the parasites migrate from their point of entry in the skin to the circulatory system and then pass via the heart and lungs to their final destination in the mesenteric veins of the hepatic portal circulation. The severe hepatic and intestinal pathology associated with schistosomiasis is caused by the vigorous host immune response to large quantities of schistosome eggs produced by pairs of adult parasites residing in the mesenteric veins.
Previous studies have suggested that successful completion of schistosome development and migration is dependent on the receipt of appropriate host-derived signals by the parasite, and on transmission of signals between male and female parasites (2-5). Since the schistosome surface is composed of a continuous cytoplasmic tegument bound externally by a lipid bilayer, we hypothesized that schistosomes may possess surface-exposed, membrane-bound receptor proteins that allow signals in the surrounding milieu to be detected. To test this hypothesis, we developed a procedure to specifically label surface proteins expressed on the outer membrane of the parasite tegument using a polar, water-soluble form of biotin (6). Parasite surface proteins could then be isolated from solubilized tegument membranes using a streptavidin affinity matrix and analyzed for potential receptor molecules. As an initial screen for receptor-like proteins, we analyzed purified parasite surface molecules for the presence of protein kinase activity, since many cell-surface receptors are known to possess intrinsic kinase activity, e.g. members of the receptor tyrosine kinase family. Using this approach, we observed that a protein kinase activity specific for serine and threonine residues copurified with schistosome surface proteins (6). The susceptibility of the kinase to biotinylation indicated that it must be exposed at the parasite surface, while the presence of a kinase domain suggested that the protein may span the parasite surface membrane, since protein kinase domains are usually intracellular. Subsequent studies using Triton X-114 to phase-separate purified surface molecules into detergent and aqueous phases have found that the surface-associated kinase partitions into the detergent phase,1 adding weight to the conclusion that it is an integral membrane protein. Based on these observations, we hypothesized that the surface-associated kinase may be a schistosome member of the receptor serine-threonine kinase (RSTK)2 family of proteins, since RSTKs are the only transmembrane proteins known to possess intrinsic serine-threonine kinase activity (7).
The RSTKs constitute a large family of cell-surface receptors, with
examples now identified in widely divergent taxa of metazoa, including
Caenorhabditis elegans, Drosophila melanogaster,
and various vertebrates (7). Members of the RSTK family can be further
classified as either type I or type II receptors based on structural
and functional characteristics. Mammalian members of this family
include the type I and type II receptors for growth factors such as
transforming growth factor , the activins and inhibins, bone
morphogenetic proteins, and other factors that play prominent roles in
growth and development. To test our hypothesis that a RSTK may be
expressed on the schistosome surface membrane, we attempted to clone
and characterize schistosome members of this protein family. Here we
report the identification of a schistosome RSTK (S.
mansoni Receptor
Kinase-1, or SmRK-1) which is expressed on the
parasite surface and which may be responsible, at least in part, for
the serine-threonine kinase activity associated with the parasite
surface membrane.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Parasites-- Biomphalaria glabrata snails infected with S. mansoni (NMRI strain) sporocysts were obtained from Biomedical Research Institute (Bethesda, MD). Cercariae were obtained by exposing infected snails to illumination in a 30 °C water bath for 1 h. Schistosomula were obtained by in vitro transformation of cercariae as described previously (8). Adult parasites were recovered from female C57BL/6J mice (Taconic Farms) infected with cercariae 6 weeks (or more) previously using the ring method (9).
Isolation of SmRK-1 cDNA and Sequence Analysis--
Total
RNA was isolated from adult S. mansoni parasites using
RNAzol B (Tel-Test, Inc.) and first-strand cDNA synthesis performed using M-MLV reverse transcriptase (Life Technologies, Inc.) and an
oligo(dT)12-18 primer. A pair of degenerate primers
(forward primer: 5'-AAACCAGCTATTGCTCAYMGNGA-3'; reverse primer:
5'-TCTGGAGCCATATAACGTTTTGTNCCNAC-3'; corresponding to conserved
sequences from subdomains VIB and VIII of other RSTK kinase domains,
respectively) was used to amplify schistosome RSTK sequences from
parasite cDNA by PCR. PCR products were cloned into the pCRII
vector (Invitrogen), sequenced, and used to isolate a larger cDNA
fragment from an adult male S. mansoni cDNA library
constructed in gt11 (10). Phage forward and reverse primers were
used in combination with internal primers to amplify the cDNA
insert as two overlapping fragments from the recombinant phage. These
fragments were cloned into pCRII and sequenced. Additional 5' sequence
was isolated using 5'-rapid amplification of cDNA ends. DNA
sequencing was performed using dye terminator chemistry and a 373A
sequencer (Applied Biosystems Inc.). Sequence analysis was performed
using LASERGENE (DNASTAR Inc.). For phylogenetic analyses, the SmRK-1
kinase domain sequence was aligned with those of other RSTK kinase
domains using the Clustal function of the Megalign program (DNASTAR
Inc.). The Protdist program from the PHYLIP package (11) was used to
compute distance measures for the sequences using the Dayhoff PAM
matrix, and an unrooted tree was generated using the Neighbor-Joining
algorithm (Neighbor program, PHYLIP).
Southern Hybridization Analysis of Genomic DNA--
Genomic DNA
was isolated from cercariae using standard procedures. Following
digestion with restriction enzymes and electrophoresis, DNA was
transferred to nylon membrane (Schleicher & Schuell) and hybridized to
a 1.4-kilobase fragment of the SmRK-1 cDNA labeled with
[-32P]dATP (Amersham) by the random primer method.
Bound probe was detected by autoradiography.
Expression of Recombinant SmRK-1 and Production of Antibodies-- A DNA fragment encoding most of the intracellular portion of the SmRK-1 protein (amino acids Met259-His594) was amplified using primers that incorporated a prokaryotic ribosome-binding site (12) and a 6-histidine tag at the N and C termini, respectively. The PCR product was cloned into pCRII and modifications were confirmed by sequencing before subcloning into the prokaryotic expression vector pKK223-3 (Amersham Pharmacia Biotech). The His-tagged cytoplasmic domain of SmRK-1 was then overexpressed in Escherichia coli Top10F' cells (Invitrogen) and purified under denaturing conditions (8 M urea) on a Ni-NTA-agarose column (Qiagen). Recombinant protein was dialyzed extensively against phosphate-buffered saline and used to raise polyclonal anti-SmRK-1 antisera in mice and rabbits.
Surface Labeling of Schistosomes with Biotin--
Freshly
recovered adult S. mansoni were surface-labeled with a
polar, water-soluble form of biotin that does not cross lipid bilayers,
as described previously (6). Briefly, parasites were incubated in a 0.5 mg ml1 solution of sulfosuccinimidobiotin
(Sulfo-NHS-Biotin, Pierce), dissolved in Dulbecco's phosphate-buffered
saline supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 (DPBS+), for 30 min at 4 °C. After
removal of the biotin solution, the parasites were washed twice with
Dulbecco's modified Eagle's medium to quench remaining biotin, and
three times with DPBS+ prior to extraction.
Metabolic Radiolabeling of Schistosome Proteins--
Parasites
were metabolically radiolabeled with [35S]methionine and
[35S]cysteine as described previously (6). Briefly,
freshly recovered S. mansoni were incubated for 6 h at
37 °C, 5% CO2 in Dulbecco's modified Eagle's medium
(minus methionine and cystine), 10 mM HEPES, 2 mM L-glutamine, 50 µg ml1
pyruvate, 150 µg ml
1 oxaloacetate, 5% fetal bovine
serum, 100 units ml
1 penicillin, 100 µg
ml
1 streptomycin (worm culture medium), containing 500 µCi ml
1 [35S]methionine and
[35S]cysteine (Tran35S-label, ICN). Labeling
of parasite proteins with [32P]phosphate was achieved by
incubating freshly recovered adult S. mansoni in
phosphate-free worm culture medium containing 667 µCi
ml
1 [32P]orthophosphate (NEN Life Science
Products Inc.) for 3 h at 37 °C, 5% CO2.
Unincorporated radioactivity was removed by extensive washing of
parasites before protein extracts were prepared.
Preparation of Parasite Extracts--
Following surface labeling
or metabolic radiolabeling, parasite surface membranes and the
underlying tegumental cytoplasm were solubilized by gently incubating
whole parasites in lysis buffer (20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 10 µg ml1
aprotinin, 0.5 µg ml
1 leupeptin, 5 µg
ml
1 pepstatin A, 1 mM phenylmethylsulfonyl
fluoride) for 30 min on ice, after which the supernatant was removed as
the "tegument extract." The phosphatase inhibitors sodium fluoride
(10 mM) and Na3VO4 (1 mM) were also included in the lysis buffer when preparing extracts from [32P]orthophosphate-labeled worms. The
remains of the parasites were then homogenized in a further volume of
lysis buffer, incubated on ice for 30 min, and microcentrifuged at
4 °C for 15 min, the resulting supernatant being called the
"carcass extract." The protein concentration of parasite extracts
was determined using the Protein-Gold Assay Reagent (Integrated
Separation Systems).
Immunochemical Techniques--
To perform immunoprecipitations
from parasite extracts, samples were first precleared by incubating
with 25 µl of Protein G-Sepharose CL-4B beads (Amersham Pharmacia
Biotech) and 5 µl of preimmune serum for 1 h at 4 °C.
Precleared supernatants were incubated for 2-12 h with 5 µl of
antiserum at 4 °C. 25 µl of Protein G-Sepharose beads were then
added and incubation continued for a further 1 h to collect immune
complexes. Beads were pelleted in a microcentrifuge and washed 5 times
with lysis buffer. Bound immune complexes were then eluted in SDS-PAGE
sample buffer containing 2.5% 2-mercaptoethanol at 100 °C for 5 min
and analyzed by SDS-PAGE on 7.5% polyacrylamide gels, followed by
immunoblotting, fluorography, or autoradiography. For immunoblotting,
mouse and rabbit anti-SmRK-1 antisera were used at a dilution of 1:1000
and detected with peroxidase-conjugated sheep anti-mouse immunoglobulin
(Ig) (Amersham Pharmacia Biotech) or peroxidase-conjugated donkey
anti-rabbit Ig G (Jackson Immunoresearch Laboratories, Inc.) diluted to
1:5000. For detection of biotinylated proteins on blots, membranes were
incubated in 0.1 µg ml1 peroxidase-labeled streptavidin
(Kirkegaard & Perry), 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.1% Tween 20 for 25 min at room temperature. Antibody- and streptavidin-peroxidase conjugates bound to blots were
detected using ECL Detection Reagents (Amersham Pharmacia Biotech).
Confocal Microscopy-- Whole-mount adult schistosomes were prepared for confocal microscopy as described by Brownlee et al. (13). Briefly, parasites were fixed in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, for 4 h a 4 °C, and then washed in phosphate-buffered saline, 0.1% bovine albumin, 0.2% Triton X-100, 1 mM NaN3 (PBT) for 24 h at 4 °C. Parasites were then incubated with rabbit anti-SmRK-1 antiserum diluted 1:500 in PBT for 72 h at 4 °C, washed in PBT for 24 h at 4 °C, and incubated with rhodamine-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories, Inc.) diluted 1:250 in PBT for 24 h at 4 °C. After a final 24-h wash in PBT at 4 °C, parasites were mounted in Vectashield (Vector Laboratories, Inc.) on hanging-drop slides and viewed with a MRC-500 confocal scanning laser microscope (Bio-Rad). To visualize surface-bound host Ig, fluorescein-conjugated goat anti-mouse IgG+IgM (Jackson Immunoresearch Laboratories, Inc.) was included at a dilution of 1:250 during the secondary antibody incubation.
Reverse Transcriptase-PCR Assay for SmRK-1
Transcripts--
Total RNA was isolated and cDNA synthesis
performed as described above. To detect SmRK-1 transcripts, the forward
primer 5'-TCTGAAGTCTAGAAACATCTTAG-3' and reverse primer
5'-GATCTGAGTTTGAATTAACTTC-3' (corresponding to nucleotides 1314 to 1336 and 1414 to 1435 of the SmRK-1 cDNA sequence, respectively) were
used to amplify a 121-bp segment of the SmRK-1 cDNA sequence. As a
control, a 230-bp segment of the message for a S. mansoni
calcium-binding protein (CaBP) which is preferentially expressed in
larval stages (14) was amplified using the forward primer
5'-TTTCACTGTATTGCATTAGAATGG-3' and reverse primer
5'-TTATGAACATAAAACATCAAGGAG-3' (corresponding to nucleotides 20 to +4
and +278 to +301 of the CaBP genomic sequence).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and Sequence Analysis of SmRK-1 cDNA-- PCR with degenerate primers based on conserved amino acid sequences from kinase domains of other RSTKs resulted in the amplification of a single 176-bp sequence from adult schistosome cDNA. Cloning and sequence analysis revealed that the fragment contained part of an open reading frame and that the predicted translation product showed considerable amino acid identity to comparable regions of other RSTKs, suggesting that the sequence was indeed derived from a novel schistosome RSTK. After confirming by PCR that the sequence was present in adult male S. mansoni cDNA (data not shown), the fragment was used as a probe to screen an adult male S. mansoni cDNA library. A screen of 1.2 × 105 plaques resulted in the identification of a single positive recombinant phage containing a 3.9-kilobase insert. The cDNA was sequenced and found to contain a single open reading frame of 1782 bases beginning with an ATG codon 19 nucleotides downstream of the 5' end, and a long 3'-untranslated region terminating in a poly(A) tail of 27 residues. Since the initiator ATG codon was not in the context of a Kozak consensus sequence (15) and the 18 nucleotides of sequence upstream remained open, an additional 250 bp of 5' sequence was isolated by 5'-rapid amplification of cDNA ends (data not shown). However, no other potential initiator ATG codons were detected in the additional 5' sequence and an in-frame stop codon occurred 45 nucleotides 5' of the first ATG, leading us to conclude that the entire open reading frame was contained within the initial cDNA isolate. This open reading frame encodes a 594-amino acid protein we call SmRK-1.
The predicted amino acid sequence of SmRK-1 is shown in Fig. 1, panel A. Hydrophobicity analysis using the Kyte-Doolittle method (16) revealed two hydrophobic segments: one at the N terminus and another between residues 137-158. The N-terminal region could constitute a short signal peptide (17) with potential signal peptidase cleavage sites after residues 13 or 16 (18), while the region between residues 137 and 158 may represent a transmembrane domain (Fig. 1, panel A). The predicted molecular mass of the entire SmRK-1 polypeptide is 66.3 kilodaltons (kDa). Removal of the signal peptide would result in a predicted 1.4-1.7 kDa reduction in molecular mass, resulting in a mature SmRK-1 protein of 64.6-64.9 kDa. There is one potential N-linked glycosylation site (Asn12) in the N-terminal part of the protein, which would presumably be removed from the mature receptor with the signal peptide. There are also 12 cysteine residues in the putative extracellular region, 10 of which would be present in the mature protein (Fig. 1, panel A). The three cysteines closest to the transmembrane domain (residues 107, 108, and 113), together with Asn114, constitute a "cysteine box," a motif characteristic of RSTK family members, defined by the consensus sequence C2-3X4-5CN (7). Taken together, these data suggest that the N-terminal part of the protein represents the extracellular ligand-binding domain of SmRK-1.
|
Confirmation of Schistosome Origin of SmRK-1-- To confirm the parasite origin of the SmRK-1 sequence and examine its genomic organization, a 1.4-kilobase fragment of the SmRK-1 cDNA was used as a probe in Southern analysis of S. mansoni genomic DNA isolated from free-living cercarial stages (Fig. 2). Single bands were obtained when probe was hybridized under stringent conditions to cercarial genomic DNA digested with EcoRI and HindIII, confirming that the SmRK-1 cDNA is of schistosome origin and was not inadvertently isolated from contaminating mouse host tissue. These data also suggested that the SmRK-1 gene is represented by a single copy in the S. mansoni genome. Two bands were obtained when probe was hybridized to genomic DNA digested with ClaI, and this is consistent with our observation that a ClaI site is located in the 3'-untranslated region of the SmRK-1 cDNA.
|
|
Localization of SmRK-1 Expression-- To determine if SmRK-1 is expressed on the parasite surface and could therefore contribute to the surface-associated kinase activity, we examined whether native, parasite-expressed SmRK-1 was susceptible to selective surface labeling of parasite proteins. We have previously shown that the water-soluble, membrane-impermeable probe sulfosuccinimidobiotin can be used to selectively and specifically biotinylate the surface proteins of adult schistosomes (6). When freshly isolated adult schistosomes were subjected to surface labeling, SmRK-1 could be immunoprecipitated in a biotinylated form from extracts of these parasites, indicating that the receptor is expressed on the schistosome surface (Fig. 4).
|
|
Phosphorylation of SmRK-1-- To examine the possibility that SmRK-1 may participate in a signaling mechanism within the schistosome tegument, we assessed whether native SmRK-1 is phosphorylated in intact parasites. Freshly recovered adult schistosomes were incubated in medium containing [32P]orthophosphate to label parasite phosphoproteins and SmRK-1 was immunoprecipitated from tegument extracts of these parasites (Fig. 6). SmRK-1 was immunoprecipitated in a phosphorylated form, suggesting that it participates in a protein kinase cascade and may therefore be active in a signaling mechanism. Several other phosphoproteins were also detected in anti-SmRK-1 immunoprecipitates that were absent in the preimmune serum precipitates. The possibility that these species represent specifically co-precipitating molecules that interact with SmRK-1 is currently being addressed.
|
Stage-specific Expression of SmRK-1-- Immunoblot analysis of extracts from various life cycle stages was performed using anti-SmRK-1 antibodies to determine whether stage-specific regulation of SmRK-1 expression occurred during the parasite life cycle (Fig. 7). SmRK-1 expression was detected from 21 days postinfection of the mammalian host onwards, but not in eggs or larval stages. A monoclonal antibody specific for paramyosin, a protein found in the tegument cytoplasm, was used as a control for protein loading in each lane. These data correlate with those obtained using a reverse transcriptase-PCR assay for SmRK-1 transcripts (Fig. 7). SmRK-1 message was readily detected in adult RNA but not in RNA from 3-h-old schistosomula. In contrast, message for a schistosome CaBP which is expressed in cercariae and early schistosomula (14), was readily detected in RNA from 3-h schistosomula, while only a weak signal could be amplified from adult RNA.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In comparison to pathogens such as viruses, bacteria, and protozoa, a distinguishing feature of helminth infections is the aggregated distribution of infections observed in affected populations, where a minority of individuals are found to harbor the majority of parasites (23). This aspect of helminth infections may partly account for the persistence of parasitic helminths in host populations, since heavily infected, susceptible individuals may act as reservoirs of infection for the rest of the population. This variation in infection intensity within a single population is believed to be the combined result of three interacting factors (24): genetic heterogeneity in the host population, genetic heterogeneity in the parasite population, and phenotypic plasticity on the part of the parasite in response to prevailing intrahost conditions which vary from host to host. Phenotypic plasticity, or parasite responsiveness to host factors such as species, age, sex, reproductive condition, and immune status is advantageous to the parasite in allowing it to maximize reproductive success under the limitations determined by conditions within an individual host. Helminth life history traits influenced by host factors include migration routes taken through host tissues and organs, entry into hypobiotic or dormant states, length of prepatent period, longevity, final body size, and fecundity. Clearly, phenotypic plasticity requires that parasites be able to receive and respond to signals from their hosts.
Phenotypic plasticity by a parasitic helminth in response to a host
factor was recently demonstrated by Amiri et al. (2), who
showed that the host cytokine tumor necrosis factor was a regulator
of fecundity for S. mansoni. Furthermore, transmission of
signals between male and female schistosomes is a well recognized but
poorly defined phenomenon (25). Like other parasitic platyhelminths, the schistosome surface tegument is composed of a layer of cytoplasm, bound externally by a lipid bilayer, which therefore forms a
potentially intimate interface between the parasite and its
environment. These observations led us to hypothesize that parasitic
flatworms may express membrane-bound receptor proteins on their surface
membranes that engage external factors in the surrounding milieu and
transduce signals into the tegumental cytoplasm, thus providing a
mechanism by which signals in the immediate environment might be
detected. Using the S. mansoni model system, we have now
identified a candidate for just such a receptor in that (i) the
predicted amino acid sequence of this candidate protein displays a
considerable level of identity to members of a known family of
signal-transducing receptors, and (ii) the protein is expressed at the
parasite surface, where we would expect interactions between external
ligands and parasite receptors to occur.
With the identification of SmRK-1, platyhelminths now become the most
primitive organisms known to possess RSTKs, an important step toward
elucidating the evolutionary origins of this receptor family. In other
organisms, signaling by TGF-like molecules requires the presence of
both a type I and type II RSTK (26, 27). Ligand is first engaged by the
type II receptor, which is a constitutively active kinase. The
ligand-type II receptor complex then recruits a type I receptor, which
is subsequently phosphorylated by the type II receptor (28).
Phosphorylation of the type I receptor within the GS domain by the type
II receptor results in activation of the type I receptor kinase
activity, which then propagates the signal intracellularly by
phosphorylating cytoplasmic Smad proteins (29). Since the GS domain of
the type I receptor plays a critical role in regulation of type I
receptor kinase activity by the type II receptor (19), it will be of
interest to determine if type I-like receptors such as Daf-1 and
SmRK-1, which possess atypical GS domains (Fig. 1, panel A),
are regulated by type II receptors in the same way as other type I
receptors. To facilitate the examination of this issue, we are
attempting to identify schistosome type II receptors that may act as
partners for SmRK-1. Our observation that SmRK-1 becomes phosphorylated
in adult parasites (Fig. 6) suggests that it may interact with other
protein kinases in the schistosome tegument. Alternatively, SmRK-1
phosphorylation may occur as the result of an autophosphorylation
mechanism.
We have found that when intact adult schistosomes are specifically surface-labeled with a polar, water-soluble form of biotin, the SmRK-1 protein becomes biotinylated (Fig. 4), indicating that it is expressed on the parasite surface. Experiments using immunofluorescence microscopy further confirmed the localization of the SmRK-1 protein to the parasite surface, where SmRK-1 expression was found to be restricted to the tubercles on the dorsal surfaces of male worms (Fig. 5). We have not been able to detect SmRK-1 expression elsewhere in male tissues or in female parasites using these techniques. However, we have detected SmRK-1 protein in carcass extracts of adult parasites (Fig. 3), suggesting that SmRK-1 may be expressed in other schistosome tissues. Alternatively, this result may represent SmRK-1 protein contained within small amounts of residual tegumental material that was not removed from the parasite carcasses during the preparation of the tegument extract.
Our results indicate that the SmRK-1 protein is expressed at detectable
levels only after the second week of infection of the mammalian host
(Fig. 7). The timing of SmRK-1 expression during the schistosome life
cycle suggests that the protein is either: (i) involved in
host-parasite interactions at the final destination of the parasite
within the mammalian host, since SmRK-1 protein is only expressed after
arrival of the parasite in the hepatic portal circulation, or (ii)
SmRK-1 is involved in male-female interactions, since differentiation
of the separate sexes coincides with initiation of SmRK-1 expression.
The expression of SmRK-1 on the tubercles of males suggests that the
SmRK-1 ligand may be of host origin, since during the normal course of
infection, the tubercles of the adult male are tightly apposed against
the blood vessel endothelial cell membrane (30). A ligand of
endothelial cell origin secreted at the luminal surface would therefore
be in close proximity to the SmRK-1 receptor. Unfortunately, the lack
of homology between SmRK-1 and other RSTKs does not allow us to make
deductions about the nature of the ligand, although it is most likely a
member of the TGF family of growth factors. The hypothesis that a
platyhelminth receptor may bind a vertebrate transforming growth factor
-like molecule is plausible, since it has already been shown that
the Daf-4 RSTK from C. elegans is capable of binding human
bone morphogenetic protein-2 and bone morphogenetic protein-4 when
expressed in mammalian cells (31). While an interaction between Daf-4
and these growth factors would not occur under natural circumstances,
this result emphasizes the highly conserved nature of the RSTK
signaling system, which makes it an ideal candidate system for
transducing signals between parasites and their hosts. It is also of
interest to note that the Daf-1 and -4 RSTKs from C. elegans
are involved in controlling a developmental switch in the nematode life
cycle which occurs in response to conditions in the animal's
environment. When C. elegans larvae are subjected to
starvation or overcrowded conditions, development proceeds to an
arrested third stage dauer larva. Mutations in the daf-1 and
daf-4 genes result in constitutive dauer larva formation
regardless of environmental conditions. Control of developmental responses to environmental signals by RSTK signaling mechanisms might
therefore be a widespread phenomenon among the various invertebrate phyla. The identification of the SmRK-1 ligand will be an important step toward elucidating the function of this receptor and may provide
new insights into the molecular interactions that occur between
parasitic helminths and their hosts.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant RO139085 and the United States Department of Agriculture Animal Health and Disease Research Program (to E. J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031557.
¶ Recipient of a Burroughs Wellcome Fund New Investigator Award in Molecular Parasitology. To whom correspondence should be addressed. Tel.: 607-253-3389; Fax; 607-253-3384.
1 E. Racoosin and E. J. Pearce, unpublished data.
2 The abbreviations used are: RSTK, receptor serine-threonine kinase; SmRK-1, S. mansoni receptor kinase-1; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Ig, immunoglobulin; CaBP, calcium-binding protein; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|