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Infection and Immunity, November 2000, p. 6402-6410, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of tgh-2, a Filarial Nematode Homolog
of Caenorhabditis elegans daf-7 and Human Transforming
Growth Factor
, Expressed in Microfilarial and Adult Stages
of Brugia malayi
Natalia
Gomez-Escobar,
William F.
Gregory, and
Rick
M.
Maizels*
Institute of Cell, Animal and Population
Biology, University of Edinburgh, Edinburgh EH9 3JT, United Kingdom
Received 7 March 2000/Returned for modification 8 May 2000/Accepted 9 August 2000
 |
ABSTRACT |
A novel member of the transforming growth factor
(TGF-
)
family has been identified in the filarial nematode parasite
Brugia malayi by searching the recently developed Expressed
Sequence Tag (EST) database produced by the Filarial Genome Project.
Designated tgh-2, this new gene shows most similarity to a
key product regulating dauer larva formation in Caenorhabditis
elegans (DAF-7) and to the human down-modulatory cytokine
TGF-
. Homology to DAF-7 extends throughout the length of the
349-amino-acid (aa) protein, which is divided into an N-terminal 237 aa, including a putative signal sequence, a 4-aa basic cleavage site,
and a 108-aa C-terminal active domain. Similarity to human TGF-
is
restricted to the C-terminal domain, over which there is a 32%
identity between TGH-2 and TGF-
1, including every cysteine residue.
Expression of tgh-2 mRNA has been measured over the
filarial life cycle. It is maximal in the microfilarial stage, with
lower levels of activity around the time of molting within the mammal,
but continues to be expressed by mature adult male and female
parasites. Expression in both the microfilaria, which is in a state of
arrested development, and the adult, which is terminally
differentiated, indicates that tgh-2 may play a role other
than purely developmental. This is consistent with our observation that
TGH-2 is secreted by adult worms in vitro. Recombinant TGH-2 expressed
in baculovirus shows a low level of binding to TGF-
-receptor bearing
mink lung epithelial cells (MELCs), which is partially inhibited (16 to
39%) with human TGF-
, and activates plasminogen activator
inhibitor-1 transcription in MELCs, a marker for TGF-
-mediated
transduction. Further tests will be required to establish whether the
major role of B. malayi TGH-2 (Bm-TGH-2) is to modulate the
host immune response via the TGF-
pathway.
 |
INTRODUCTION |
Transforming growth factor
(TGF-
) is a stable, multifunctional extracellular growth factor with
an extremely wide range of biological activities in metazoan animals.
In vertebrates, nearly all cells have surface receptors for, and are
stimulated or inhibited by, TGF-
. The nature and polarity of the
response depends on the cell lineage, its state of differentiation and proliferation, and its environment, particularly with respect to the
presence of other growth factors (49). TGF-
plays a crucial role in the coordination of morphogenesis and remodeling of
mesenchymal tissues during embryological development. In
Drosophila melanogaster, the decapentaplegic
(dpp) gene is involved in the specification of the
dorsal-ventral axis (16), in gut morphogenesis, and in wing
development and patterning (27, 37). In Xenopus embryos, dpp homologs ventralize cells, while more distantly
related activin proteins induce mesoderm (53). In
vertebrates, TGF-
-related molecules have been found that control
sexual development (Müllerian inhibiting substance
[11]), pituitary hormone production (inhibins
and
[30, 55]), skeletal muscle growth (myostatins
[34]), and the creation of bone and cartilage (bone
morphogenetic proteins [BMPs] [44]).
TGF-
is a particularly important modulator of the growth,
differentiation, and activities of cells of the immune system
(28), and multiple members of the superfamily are now
associated with immune inhibition (10). The most commonly
reported effects of TGF-
on leukocytes are inhibitory, suppressing
lymphocyte proliferation, although in certain contexts TGF-
exerts
stimulatory effects, as in isotype switching in B lymphocytes
(50). In parasitic infections, TGF-
has emerged as one of
the key cytokines (48), together with interleukin 4 (IL-4)
and IL-10, which down-regulate cellular response and compromise
immunity to a spectrum of intracellular infections, including those
caused by Leishmania species (7, 8, 29, 46, 59),
Mycobacterium tuberculosis (23, 24), Plasmodium chabaudi (54), Toxoplasma
gondii (25), and Trypanosoma cruzi
(17). Similar findings have been reported for infections with extracellular helminths, such as Schistosoma mansoni
(39, 57). In keeping with the pleiotropic functions of
TGF-
, there are also reported examples of a protective role for this
cytokine against some pathogens (36, 38), and indeed it is
highly likely to reduce the severity of immunopathogenic reactions
(56). It is intriguing to consider the possible role of
TGF-
in long-lived chronic infections, such as filariasis, caused by
nematodes of the genera Brugia and Wuchereria, in
which immune down-regulation is a prominent feature (4, 31).
As we discuss below, the production of TGF-
-family cytokines by
filariae holds out the possibility that these molecules, as well as
related host effectors, may be directly involved in the
immunosuppression which characterizes some filarial infections.
Four TGF-
family members are encoded in the genome of the
free-living nematode Caenorhabditis elegans (41,
47). One homolog, DAF-7, controls entry to and exit from
developmental arrest represented by the dauer larva and acts via a
well-characterized TGF-
-like signaling pathway (15, 18, 41,
45). Expression of DAF-7 is highest in L1 larvae committed to
non-dauer development, is low in L2 larvae, and is almost undetectable
in L3 and pheromone-induced L2d larvae. Another homolog, UNC-129, acts
as a guide for axon growth (12), while DBL-1 (also named
CET-1) affects body size in hermaphrodite and male worms as well as
tail formation in males alone (35, 51). The precise function
of the fourth gene, designated tig-2, remains to be
determined (51). The Brugia malayi life cycle
contains a series of developmental steps and arrest points which may be
governed by TGF-
homologs, and we postulated that the parasitic mode
of life may select variants able to mimic the host cytokine TGF-
. We
have previously described a member of the TGF-
family from B. malayi, designated transforming growth factor homolog-1
(tgh-1), which is expressed during parasite growth and
development within the mammal and which bears closest similarity with
the dpp/DBL-1 family of signaling molecules (19).
We now report on a second gene, tgh-2, which more closely
resembles C. elegans daf-7 and human TGF-
and which is
expressed at high levels in stages which are either in a state of
arrested development (microfilariae) or have completed their
developmental program (adult worms).
 |
MATERIALS AND METHODS |
Parasites.
Male adult jirds (Meriones
unguiculatus) infected intraperitoneally with B. malayi
organisms were purchased from TRS Labs (Athens, Ga.) and used as a
source of adult parasites and microfilariae. Vector stage parasites
(infective third-stage larvae) were obtained from Aedes
agypti mosquitoes infected with B. malayi parasites via
membrane feeding with infective blood containing 16,000 microfilariae/ml.
Isolation of the tgh-2 cDNA.
A B. malayi expressed sequence tag (EST), MBAFCE6E01, was found to bear
homology to the 3' end of C. elegans daf-7. The full-length protein sequence of DAF-7 was then used to search the database of
approximately 11,000 ESTs from B. malayi deposited in the
EST database (dbEST) in February 1997 by the Filarial Genome Project (58). Two additional ESTs were identified with TGF-
-like
sequences, one which represented the 3' terminus of a novel gene and
one (SWAFCA78) which corresponded to a partially truncated N-terminal sequence. Similarity searching of the same data set with SWAFCA78 identified three further ESTs, two of which appeared to code for the
full-length transcript. These ESTs were obtained from microfilarial, adult female, and adult male conventionally constructed cDNA libraries in the laboratories of M. Blaxter and R. Ramzy (University of Cairo,
Cairo, Egypt) and S. Williams (Smith College, Northampton, Mass.). On
the supposition, later verified, that the two termini represented a
single gene, primers were made corresponding to the 5' and 3' coding
termini as follows: 5'-ATGACGTTCATTGCGGTGTCG-3' (sense) and 5'-AGCACAGGCACACCGCCG-3'
(antisense). These primers were used to amplify full-length
cDNA from a B. malayi microfilarial library constructed in
the laboratory of S. Williams and provided by the Filarial Genome
Project. The PCR was cycled between 94, 55, and 72°C (1 min
each) for 35 rounds, followed by 1 round of 72°C for 10 min. The
reaction product was purified and cloned into the pMOSblue T-vector
(Amersham). Two clones were picked and sequenced in both directions,
with identical results, using ABI PRISM Dye Terminator cycle sequencing
kits (Perkin-Elmer).
RNA extraction and reverse transcription-PCR (RT-PCR).
Mosquitoes were collected at various time points after feeding with
infected blood. Total RNA was extracted from individual mosquitoes
using either TRIZOLV or RNAZOL (Biotex Inc.) according to the
manufacturer's instructions. RNA from adult worms was extracted as
described previously (19). First-strand cDNA was synthesized from mosquitoes by using the GeneAmp RNA PCR Kit (Perkin-Elmer) using
oligo(dT) as the primer. Infected mosquitoes were detected by
amplifying each first-strand cDNA with primers specific for cystatin
gene, cpi-2, and positive samples were pooled (W. F. Gregory and R. M. Maizels, submitted for publication).
First-strand cDNA from adult worms of the closely related species
Brugia pahangi was synthesized by Emma Lewis (Veterinary
Parasitology, University of Glasgow) as previously described
(19).
To measure the levels of expression of tgh-2 in the mosquito
stages, PCR was performed with gene-specific primers for
tgh-2 and
-tubulin by using 5 µl of pooled cDNA. In
both cases, the presence of introns ensured that contaminating DNA
could not yield false positive results. The PCR was cycled between 94, 55, and 72°C (1 min each) for 35 rounds, followed by 1 round of
72°C for 10 min. The levels of expression of tgh-2 in the
mammalian stages were measured as previously described (19).
The oligonucleotides used were as follows: for tgh-2,
5'-GGTCGCCGCAAACGTAGCTAT-3' (sense) and
5'-AGCACAGGCACACCGCCG-3' (antisense); and for
-tubulin,
5'-AATATGTGCCACGAGCAGTC-3' (sense) and
5'-GCCATACTCCTCACGAATTT-3' (antisense).
Expression of recombinant tgh-2 and production of
antisera.
Primers were designed to amplify the entire coding
region of tgh-2 minus the putative signal peptide from a
B. malayi microfilarial cDNA library. The 5' sequence was
taken from ESTs SWAFCA54 and SWMFCA851 and the 3' untranslated region
(UTR) from MBAFCE6E01. The fragment was digested with
NdeI-BamHI and cloned into the pET-15b vector
(Novagen), which produces proteins containing an N-terminal
six-histidine tag. The oligonucleotides used were
5'-GGCAGCCATATGCCATCGACACACGGAACCACC-3' (sense) and
5'-CGCGGATCCTTAAGCACAGGCACACCGCCG-3' (antisense). Clones
were fully sequenced on both strands. The constructs were transformed
into BL21 (DE3) cells, and protein production was induced by 1 mM IPTG
(isopropyl-
-D-thiogalactopyranoside) for 3 h at
37°C. Recombinant TGH-2 was obtained by purifying the fusion by
following the manufacturer's protocol. Cells were sonicated in the
presence of 6 M urea. After removal of insoluble material by
centrifugation at 12,000 × g for 15 min, the expressed
protein was bound to HIS-Bind resin (Novagen). Bound proteins were
eluted in the presence of 6 M urea, which was removed by overnight
dialysis in phosphate-buffered saline in the presence of 100 mM EDTA to avoid precipitation.
Antisera were produced by subcutaneous immunization of BALB/c and CBA
mice with 20 µg of purified recombinant protein in complete Freund's
adjuvant, followed 1 month later by a booster with 10 µg of protein
in incomplete Freund's adjuvant. A second boosting was carried out a
week later, and sera were collected 1 week after the last boosting.
Western blotting.
Approximately 50 mixed adult worms of
B. malayi were recovered from jirds and cultured overnight
at 37°C in medium supplemented with 25 mM HEPES, 1% glucose, 2 mM
L-glutamine, 100 U of penicillin/ml, and 100 µg of
streptomycin/ml. The culture supernatant containing the
excretory-secretory (ES) products was centrifuged at 2,000 × g for 20 min to remove any microfilariae present in the
culture. The supernatant was then precipitated overnight at
20°C by
adding an equal volume of cold acetone. The following day, the proteins were centrifuged, washed once with cold 50% acetone, and resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (0.4 M Tris-HCl [pH 6.3], 2.3% SDS, 10% glycerol, and
5% 2-mercaptoethanol). For immunoblot analysis, SDS-PAGE-separated proteins were electrophoretically transferred to a nitrocellulose membrane in 39 mM glycine, 48 mM Tris, 0.375% (wt/vol) SDS, and 20%
methanol and then were probed with a 1/200 dilution of anti-TGH-2 serum
or normal mouse serum, followed by an incubation with a horseradish
peroxidase-conjugated rabbit anti-mouse immunoglobulin G diluted
1/2,000. Bound anti-TGH-2 antibodies were visualized using the ECL
detection method (Amersham), followed by autoradiography.
Expression of tgh-2 in Sf21 cells.
Sf21 cells
(kindly provided by A. Alcami, Cambridge University) were cultured at
28°C in TC100 medium (Gibco) containing 10% (vol/vol) fetal calf
serum. The full-length tgh-2 cDNA was cloned into pBAC-1
(Novagen), which contains a C-terminal six-histidine tag. Two clones
were fully sequenced on both strands to confirm fidelity. SF21 cells
were cotransfected with tgh-2 and BacPAK6 viral DNA
(Clontech) using Lipofectin (Life Technologies, Inc.). Recombinant
viruses were plaque purified three times on monolayers of Sf21 cells.
The expression of the recombinant viruses was confirmed by metabolic
labeling, and one of these viruses was selected for further experiments.
Recombinant proteins and radiolabeling.
Recombinant
tgh-2 was obtained by purifying the fusion protein by
following the manufacturer's protocol. SF21 cells were infected with
the tgh-2 recombinant virus at a multiplicity of infection of 0.1 PFU/cell. After sonication and removal of the insoluble material, the recombinant protein was then purified as described above.
Recombinant TGH-2 was activated by adding 10 µl of 1 M HCl to
100-µl samples, incubating at room temperature for 10 min, and
neutralizing with 15 µl of 0.72 M NaOH-0.5 M HEPES. To produce recombinant protein for the luciferase assay, 2 × 105
SF21 cells/well seeded in a 24-well plate were infected with the
recombinant virus at a multiplicity of infection of 20 PFU/cell for
2 h at 27°C. The inoculum was removed and replaced by 300 µl
of fresh medium. After a 48-h incubation, the supernatant was removed
and the cells were harvested in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 0.1% bovine serum albumin (BSA), 100 U of
penicillin/ml, and 100 µg of streptomycin/ml. Cells were then
sonicated, and insoluble material was removed by centrifugation at
10,000 × g for 15 min. The supernatant from SF21 cells was diafiltrated in three changes of DMEM that were all supplemented with
0.1% BSA, 100 U of penicillin/ml, and 100 µg of streptomycin/ml according to the manufacturer's instructions (Vivaspin 6 concentrators; Vivascience Ltd.). The final concentration of the
supernatants was 1.2 × 106 cell equivalents per ml.
Recombinant human TGF-
2 (active domain; Boehringer Mannheim) and
B. malayi TGH-2 (Bm-TGH-2) were iodinated by the Iodogen
method (33).
Affinity labeling.
Mv-I-Lu cells (kindly donated by M. Yazdanbakhsh, Leiden University) were maintained in Eagle's minimum
essential medium (Sigma) supplemented with 0.1 M nonessential amino
acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U of
penicillin/ml, 100 mg of streptomycin/ml, and 10% fetal calf serum
(all from Sigma). Cells were detached from the culture flasks by
incubation at 37°C with 1% trypsin-EDTA, washed, and resuspended in
complete medium. Volumes of 1 ml containing 106 cells were
aliquoted into a six-well plate and left overnight to adhere. The
following day, Mv-I-Lu monolayers were incubated on ice for 3 to 4 h with 125I-labeled activated recombinant TGH-2 or TGF-
in binding buffer (33). Different concentrations of
unlabeled inhibitor were added during this incubation period. After
incubation, the cells were washed and lysed by incubation in
solubilization buffer for 40 min on ice. Radioactivity was then counted
in the soluble extracts.
Luciferase assay for TGF-
.
Modified mink lung epithelial
cells (MLECs) (clone 32) were obtained (by kind donation from D. B. Rifkin, Department of Cell Biology, New York University Medical
Center) which were stably transfected with an 800-bp fragment of the 5'
end of the human plasminogen activator inhibitor-1 (PAI-1) gene fused
to the firefly luciferase reporter gene in a p19LUC-based vector
containing the neomycin resistance gene from pMAMneo (1).
Modified MLECs were maintained in DMEM supplemented with 10% fetal
calf serum, 100 U of penicillin/ml, 100 µg of streptomycin/ml, 2 mM
L-glutamine, and 200 µg of G418/ml. Confluent modified
MLECs were trypsinized as described above and resuspended in complete
media at 1.6 × 105 cells/ml. Volumes of 100 µl of
cells/well were seeded in triplicate in an all-white 96-well tissue
culture dish (BMG Biotechnologies). The cells were incubated at 37°C
for 3 h for optimal attachment. Recombinant Bm-TGH-2 from SF21
cell lysates and from SF21 cell supernatants (100 µl) was added
directly to attached cells after aspiratiration of the serum-containing
medium. Similarly, recombinant human TGF-
dilutions (250 to 4 pg/ml;
100 µl/well) made in the same media as the test samples were added to
the cells. Samples were incubated overnight at 37°C. After
incubation, a luciferase assay was performed according to the
manufacturer's instructions (Bright-Glo Luciferase Assay System;
Promega). In brief, 100 µl of Bright-Glo reagent was added to the
100-µl samples in each well. Cells were incubated for 2 min at room
temperature to allow complete lysis. Luciferase activity was assayed
using a Luminometer (BMG Biotechnologies). Luciferase activity was
reported as relative light units. Recombinant baculovirus expressing
the vaccinia protein B15R (2), used as a control in these
experiments, was the kind gift of Antonio Alcami, Department of
Pathology, University of Cambridge, Cambridge, United Kingdom.
Nucleotide sequence accession number.
Sequence analyses were
done with MacVector program Version 6.0. Trees reflecting evolutionary
relationships were generated by phylogenetic analysis using
parsimony (PAUP) (52). The cDNA sequence has been deposited
in GenBank with the accession no. AF104016.
 |
RESULTS |
Identification of a new TGF-
homolog.
The EST data set
generated by the Filarial Genome Project was screened for
the presence of TGF-
-like sequences by searching with the
DAF-7 protein sequence. Three B. malayi ESTs were identified in this way, and from these one nucleotide sequence was then used to
screen the same database, yielding three further clones. From the six
ESTs identified, four represented the 5' end of the cDNA and two the 3'
end (Fig. 1A). The ESTs were present in
cDNA libraries from three stages, microfilaria, adult female, and adult
male. Full-length cDNA was then isolated by PCR from the microfilarial library by using oligonucleotide primers corresponding to the predicted
5' and 3' coding ends of the new gene. This cDNA was named
Bm-tgh-2 (B. malayi TGF-
homolog-2). The
tgh-2 cDNA consists of 1,266 bp encoding a deduced protein
of 349 amino acids (Fig. 1B) with a hydrophobic region at the N
terminus, indicating the presence of a signal peptide.

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FIG. 1.
(A) Map of six EST sequences with similarity to DAF-7.
EST sequences represent single 5' reads from variably truncated clones.
ESTs SWAFCA-54 and SWMFCA-851 include 130 and 68 nucleotides,
respectively, of 5' UTR upstream of the initiation codon (gray bar).
The 22-nucleotide nematode spliced leader is not present in either of
these regions. EST identification codes are given above the black bars
which correspond to sequence data deposited in dbEST. Numbers
correspond to amino acid positions in the C. elegans DAF-7
protein. (B) Nucleotide and deduced amino acid sequence of the B. malayi tgh-2 cDNA. The TGF- homology region, located at the
carboxyl end, is shown in a shaded box. The potential proteolytic
cleavage site is an open box. The potential glycosylation sites (NHS
and NGS) are shown in solid boxes. Nine cysteine residues found in
invariant positions in the active domain are circled. The 3'
polyadenylation site is underlined. The sequences used for the RT-PCR
primers (709 to 729 and 1030 to 1047) are shown with arrows. The full
cDNA sequence has been deposited in GenBank with the accession no.
AF104016.
|
|
The predicted gene product contains the conserved characteristics of
the TGF-
superfamily, namely a large N-terminal preprotein separated
from a C-terminal 110- to 140-amino-acid cysteine-rich active domain by
a tetrabasic cleavage site (6, 26). TGH-2 shows significant
similarity to DAF-7 in both the N-terminal (not shown) and C-terminal
(Fig. 2) segments and contains an RRKR
motif that is thought to serve as a substrate for proteolytic cleavage. Within the C-terminal active domain of all TGF-
superfamily members are seven invariant cysteine residues, six of which form a rigid, heat-stable "cysteine knot" (13). The C-terminal
108-residue span of TGH-2 contains the seven invariant cysteines, as
well as two additional cysteines previously found only in the
vertebrate homologs TGF-
, activin, and myostatin and in the C. elegans homolog daf-7 (Fig. 2).

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FIG. 2.
Alignment of the C-terminal amino acid sequences of
TGH-2 and five other representatives of the TGF- superfamily.
Residues that are identical to TGH-2 in any of the other five proteins
are shown in solid boxes; similarities to TGH-2 in any of the other
proteins are outlined and shaded. Gaps introduced to optimize the
alignment are represented by dashes. Two of the conserved cysteines
found only in TGF- , activin, and DAF-7 are shown with asterisks.
Numbers at the start and finish of each line correspond to amino acid
numbers in each respective sequence. Accession numbers for the
sequences shown are as follows: C. elegans DAF-7, U72883;
human TGF- 1, P01137; human activin, X82540; human myostatin,
AF019627; human BMP-2, P12643; and D. melanogaster DPP,
P07713.
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|
TGH-2 is a member of the TGF-
subfamily.
Comparison of the
C-terminal ligand domain of TGH-2 with the entire GenBank protein
database showed that its closest relatives are the other nine-cysteine
proteins C. elegans DAF-7, human TGF-
2, and human
growth/differentiation factor-8 or myostatin (respectively 41, 37, and
32% identical with Bm-TGH-2). Activin, BMP-2 and Dm-DPP show lower
degrees of identity (respectively, 26, 27, and 24%) with Bm-TGH-2. In
general, the N-terminal preprotein domains of TGF-
s are highly
divergent, but the predicted TGH-2 precursor shows 25% identity over
240 residues with the predicted DAF-7 precursor. To investigate further
the relationship between active domains of members of the TGF-
superfamily, their sequences were analyzed by the PAUP program. A
presentation of this tree (Fig. 3) groups
TGH-2 with the DAF-7/TGF-
/activin/myostatin subfamily and shows that
DAF-7 from C. elegans is the closest relative. In contrast,
the previously described TGH-1 groups with the DPP/BMP subfamily.

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FIG. 3.
Phylogenetic representation of the sequence relationship
between active domain of members of the TGF- superfamily. Numbers
represent bootstrap values. The accession numbers are as follows: human
TGF- 1, P01137; human TGF- 2, P08112; C. elegans DAF-7,
U72883; human activin, X82540; human BMP-2, P12643; D. melanogaster DPP, P07713; human inhibin, X72498; human
myostatin, AF019627; C. elegans DBL-1, AF004395;
B. malayi TGH-1, AF010495; D. melanogaster 60A, P27091; C. elegans UNC-129, AF029887;
C. elegans TIG-2 (cosmid F39G3), AF016424.1.
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|
Expression of tgh-2 during the life cycle.
A
full-length tgh-2 cDNA was isolated from the microfilarial
library, and identical sequences were found to be present in the five
ESTs obtained from the adult female and the adult male libraries. Among
the >18,000 B. malayi ESTs now deposited in the NCBI dbEST,
a total of 11 clones have been identified, of which 3 are from
microfilaria, 6 are from adult female, and 5 are from adult male cDNA libraries.
In order to determine further the expression pattern of
tgh-2, first-strand cDNA was synthesized from the closely
related species B. pahangi, taken at 1- to 2-day intervals
over the first 4 weeks postinfection of the jird, and from parasites
taken at 1-day intervals (for 12 days) after mosquitoes were blood fed. These first-strand cDNAs were amplified with primers specific for
tgh-2. To provide a relative quantification of expression levels, each reaction mixture of samples from the mammalian stages contained control primers to simultaneously amplify
-tubulin transcripts (22), as this gene shows similar abundance at
each stage of the life cycle. The abundance of the test transcript was
then expressed as the ratio of the amount of its amplified product to
that of the control transcript. First-strand cDNAs from mosquito stages
were amplified with both tgh-2 and
-tubulin primers, and
the PCR products were analyzed on an agarose gel.
Four separate peaks of mRNA abundance of the tgh-2
transcript were detected in the mammalian stages (Fig.
4A). The first peak corresponds to the
microfilarial stage, the second peak appears just before the first molt
in the host (day 9), the third peak appears before the second
molt of the males in the host (day 18), and the fourth peak
appears just before the second molt of the females in the host (day
23). In the mosquito stages, the tgh-2 transcript started to
be visible 72 h after a blood meal with microfilariae (Fig. 4B).

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FIG. 4.
Expression pattern of the tgh-2 transcript.
(a) Expression data from semiquantitative RT-PCR for the
tgh-2 gene performed on a time course of RNA samples taken
from B. pahangi microfilariae, infective L3, at 1- to 2-day
intervals over the first 4 weeks postinfection, and adults. The life
cycle stage and timing of the molt relating to this time course are
indicated on the x axis. The y axis represents
the ratio of detected test mRNA over control -tubulin mRNA,
determined as previously described (19). (b) First-strand
cDNAs from microfilariae, uninfected mosquitoes (U), and infected
mosquitoes taken every day for 12 days after blood feeding were used as
templates in PCRs using the tgh-2-specific primer pair
indicated in Fig. 1B. The positive control for -tubulin used primers
described in Materials and Methods. The time points associated with
each molt are indicated.
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Secretion of TGH-2 by adult parasites in culture.
Polyclonal
antibodies raised against recombinant TGH-2 were used to identify
proteins secreted by adult worms in culture by Western blotting.
Antisera to TGH-2 reacted against a 12-kDa protein released from adult
parasites cultured in serum-free medium. This corresponds to the
expected molecular mass of the TGH-2 active domain. No reactivity
was observed on Western blot analysis of ES products probed with
normal mouse serum (Fig. 5).

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FIG. 5.
Secretion of TGH-2 by parasites in culture. Shown is a
Western blotting of adult parasite ES products probed with normal mouse
serum (lane 1) or mouse anti-recombinant TGH-2 antibodies (lane 2)
followed by ECL detection and autoradiography. The positions of the
molecular mass markers (in kilodaltons) are shown on the left.
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Expression of the TGH-2 polypeptide in insect cells.
For
functional characterization of the 349-amino acid TGH-2
polypeptide, the entire protein-coding region, including a His tag
at the C-terminal end, was expressed in Sf21 insect cells under the
control of the polyhedrin promoter. As shown by SDS-PAGE analysis of
35S-labeled cells, TGH-2 protein was secreted to the medium
by insect cells as a 38.5-kDa polypeptide. The 40.6-kDa protein in
TGH-2-infected insect cell extracts corresponds to the polypeptide with
a signal sequence still bound due to the inability of insect cells to
process properly the high amount of TGH-2 protein expressed. This
reconciles with the expected molecular mass of the TGH-2 polypeptide
(39.4 kDa) with a 1.2-kDa peptide tag. Time course experiments revealed that the expression levels of the recombinant protein reached a maximum
48 h after infection (data not shown).
TGH-2 shows low level of binding to mammalian receptors.
To
measure whether Bm-TGH-2 binds directly to mammalian TGF-
receptors, we assayed the binding of iodine-labeled recombinant protein
to mink lung cells. As shown in Fig.
6, baculovirus-expressed B. malayi TGH-2 (Bm-TGH-2) does bind to these cells, and binding can
be inhibited by nanogram per-milliliter-concentrations of homologous
ligand or mammalian TGF-
administered at concentrations of nanograms
per milliliter. In four separate experiments, maximal inhibition of
TGH-2 binding by TGF-
averaged 29% (range, 16 to 38%).

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|
FIG. 6.
TGH-2 binding to mammalian cells.
125I-labeled TGF- and TGH-2 were incubated with mink
lung cells and washed. Incubations were performed in the presence of up
to 4 ng of inhibitor/ml as indicated; 1 ng of inhibitor/ml represents a
33-fold molar excess over 125I-labeled ligand, and all
assays were performed in the presence of 2 mg of BSA/ml. Shown are
binding of TGF- inhibited by various concentrations of unlabeled
TGF- ( ), binding of TGH-2 inhibited by unlabeled TGF- ( ),
and binding of TGH-2 inhibited by unlabeled TGH-2 ( ). CPM, counts
per minute. (B) Bioactivity of TGH-2 produced by baculovirus-infected
insect cells. SF21 cells were infected with TGH-2 virus, B15R virus, or
wild-type virus added at a multiplicity of infection of 20 PFU/cell. At
48 h postinfection, cells and supernatants were collected and
assayed for their ability to induce PAI-1 expression by using the MLEC
luciferase assay (1). *, responses to TGH-2 are
significantly higher than those of medium alone (P values of
<0.025 for both cell lysate and supernatant samples) and significantly
higher than responses to the control recombinant B15R (P
value of <0.025 for cell lysate, P value of <0.05 for
supernatant) using Student's t test for unpaired samples.
This experiment was performed three times, with positive stimulation by
TGH-2 on each occasion. WT, wild type.
|
|
In order to further show that TGH-2 has a biological effect similar to
that of TGF-
, we measured the responsiveness of an MLEC line which
had been transfected with a luciferase reporter construct linked to the
promoter for a gene up-regulated by TGF-
, plasminogen activator
inhibitor-1 (1). Modified MLECs cultured in the presence of
cell lysates and cell supernatants from insect cells infected with the
wild-type virus and B15R virus (a 40- to 44-kDa vaccinia virus
secretory glycoprotein that functions as a soluble interleukin-1
receptor [2]) did not generate significant levels of
luciferase activity. However, modified MLECs cultured with either cell
lysate or cell supernatant from insect cells infected with the TGH-2
virus both showed significant increases in luciferase activity. The
relative luminescence generated by recombinant Bm-TGH-2 was similar to
the signal observed by stimulation with 8 pg of recombinant human
TGF-
/ml.
 |
DISCUSSION |
Host defenses against pathogens rely on a network of cytokines for
activation and coordination of the immune response. It is not
surprising, therefore, that infectious organisms encode cytokine-like
molecules and other products which interfere with cytokine-mediated
communication (3, 43). Most known examples come from
viruses, which are likely to have evolved most of their cytokine mimics
by horizontal gene capture from their hosts. Such a process would be
less common among eukaryotic parasites, and for metazoan helminths in
particular it seems more likely that convergent evolution between
members of ancient gene families may give rise to functional cytokine
homologs in parasites. The TGF-
superfamily emerged early in the
evolution of metazoa, and helminths would have had the opportunity to
adapt these molecules for the purposes of subversion of host responses.
We therefore investigated homologs of TGF-
in B. malayi,
a prominent tissue-dwelling filarial nematode. We have isolated and characterized two related genes. One, designated
tgh-1, belongs to the differentiation-inducing subfamily of
DPP and BMP and is expressed at a relatively low level
during growth points of the parasite (19). The second
which we describe here is more abundant, more closely related to DAF-7
and to human TGF-
itself, is secreted by the parasite in culture,
shows some binding for host TGF-
receptors, and seems to have a
similar biological effect as human TGF-
on the MLECs. Perhaps the
most striking feature of this new gene is that it is maximally
expressed in the blood stage microfilarial parasites, which are exposed
to the full force of the host immune system and are at the same time in
a state of arrested development. Moreover, the appearance of
microfilariae during natural infection is most closely associated with
suppression of the host immune response. These considerations suggest
that TGH-2 may have an immunomodulatory function, rather than inducing growth and differentiation. Thus, on the basis of sequence
similarities, expression patterns, and receptor binding, TGH-2 is a
candidate for an immune evasion molecule. Further studies are now
necessary to test this supposition.
TGH-2 is one of a growing set of helminth homologs of host immune
system molecules. For example, B. malayi homologs of
macrophage migration inhibitory factor have recently been cloned
(40), while migration inhibition factor-like biological
activity has been demonstrated in a range of nematode species
(42) and a gamma interferon-like protein reported in the
nematode Trichuris muris (21). It is also likely
that parasites will express cytokine-binding proteins and receptors.
Thus, receptors for TGF-
-like ligands have been cloned from B. malayi (20) and S. mansoni (14), while the existence of tumor necrosis factor alpha receptors in S. mansoni is implied by the dependence of egg-laying on the
presence of this cytokine (5). As parasite genome projects
expand apace (9), it is likely that many more cytokine
mimics will be discovered to play major roles in the successful immune
evasion by helminth organisms.
 |
ACKNOWLEDGMENTS |
We thank the Leverhulme Trust and the Medical Research Council
for support.
We thank Steve Williams of the Filarial Genome Project for provision of
B. malayi cDNA libraries. We thank Don Riddle (Columbia, Mo.) for sharing the DAF-7 sequence prior to publication and for general advice and Mark Blaxter (ICAPB, Edinburgh, United Kingdom) for
advice on phylogenetic analysis. We thank D. B. Rifkind (New York
University Medical Center) and Antonio Alcami (University of Cambridge)
for their kind gifts of a transfected MLEC line and B15R-containing
baculovirus, respectively.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Cell, Animal and Population Biology, University of Edinburgh, West
Mains Road, Edinburgh EH9 3JT, United Kingdom. Phone: (44) 131 650 5511. Fax: (44) 131 650 5450. E-mail:
rick.maizels{at}ed.ac.uk.
Editor:
J. M. Mansfield
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Infection and Immunity, November 2000, p. 6402-6410, Vol. 68, No. 11
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