Molecular & Biochemical Parasitology 115 (2001) 199–208
Secretion of the novel Trichinella protein TSJ5 by T. spiralis and
T. pseudospiralis muscle larvae�
Sabine Kuratli a, Andrew Hemphill a, Johan Lindh b, Deborah F. Smith b,
Bernadette Connolly a,c,*
a Institute of Parasitology, Uni�ersity of Bern, CH-3012 Bern, Switzerland
b Wellcome Trust Laboratories for Molecular Parasitology, Department of Biochemistry, Imperial College of Science,
Technology and Medicine, UK
c Department of Molecular and Cell Biology, Institute of Medical Sciences, Uni�ersity of Aberdeen, UK
Received 30 January 2001; received in revised form 2 April 2001; accepted 4 April 2001
Abstract
The Trichinella tsJ5 gene is preferentially expressed in muscle larvae of Trichinella spiralis and encodes a novel protein. Previous
observations have shown tsJ5 to be expressed at higher levels in encapsulating species than in non-encapsulating species and
down-regulation of gene expression in T. pseudospiralis to be correlated with a lower protein abundance in the muscle larva of
this species. In the present study we have determined the full-length cDNA sequence of the tsJ5 homologue in T. pseudospiralis
(tpJ5). Antigens recognised by an anti-J5 antibody are found on the cuticular surface of both T. spiralis and T. pseudospiralis
muscle larvae, as well as in the body wall muscle. We show that both the TSJ5 and TPJ5 proteins are found in the
excretory/secretory fractions collected from muscle larva cultured in vitro and that despite the absence of a typical N-terminal
signal sequence, secretion of pTSJ5 is mediated through the classical ER/Golgi secretory pathway. © 2001 Elsevier Science B.V.
All rights reserved.
Keywords: Nematode; Secretion; Immuno-localisation; Crude worm extract; Excretory-secretory; ER/Golgi
www.parasitology-online.com.
1. Introduction
The Newborn L1 larva of the nematode Trichinella
spiralis specifically infects mammalian skeletal muscle,
inducing a realignment of host gene expression and
leading eventually to de-differentiation of the host cell
reviewed in [1,2]. Within this intracellular niche the
larva develops from the pre-infective to the infective
stage and forms an intimate host-parasite complex,
known as the Nurse cell [3]. The infective L1 larva of
Trichinella pseudospiralis [4] develops like T. spiralis
within the physical context of skeletal muscle but does
not form a typical nurse cell and does not encapsulate
within muscle cells. It is still a matter of discussion, as
to whether the T. pseudospiralis infective larva is an
intracellular parasite of the muscle cell as is T. spiralis
[5], or whether the larva is extracellular and free to
move in and between the muscle fibres [6]. In either
case, there are clear morphological differences between
the encapsulating and non-encapsulating species with
respect to the muscle larva (ML) [7–9] and these differ-
ences in biological niche must be underpinned by differ-
ences in gene expression in the ML of the two species.
To date few Trichinella genes have been described
that show substantive differences between expression
levels in the ML of encapsulating and non-encapsulat-
ing species. One interesting candidate is the novel
protein encoded by the T. spiralis gene, tsJ5 [10]. Al-
though a function for pTSJ5 has yet to be determined,
we have shown that a recombinant TSJ5 protein can
alter the in vitro DNA binding properties of the mouse
myogenic transcription factor, MyoD [10]. Expression
of tsJ5 is developmentally regulated in T. spiralis [10]
Abbre�iations : ML, muscle larva; CWE, crude worm extract; ES,
excretory/secretory.
� Note : Nucleotide sequence data reported in this paper are avail-
able in the GenBank™ database under the accession number
AF305831.
* Corresponding author. Tel.: +44-1224-273125; fax: +44-1224-
273144.
E-mail address: b.connolly@abdn.ac.uk (B. Connolly).
0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S01 6 6 -6851 (01 )00287 -0
S. Kuratli et al. /Molecular & Biochemical Parasitology 115 (2001) 199–208200
and differential gene expression between species has
been observed. While homologues have been identified
in Trichinella brito�i and T. pseudospiralis (tbJ5 and
tpJ5, respectively), expression studies have shown that
transcript levels are higher in the encapsulating species
than in the non-encapsulating species [11]. The down-
regulation of tpJ5 gene expression is correlated with a
lower abundance of the TPJ5 protein in the ML of T.
pseudospiralis. The protein pTSJ5 is, therefore, a possi-
ble candidate for a factor functionally involved in de-
termining or influencing niche choice in T. spiralis.
In this study we show that both the T. spiralis and T.
pseudospiralis J5 proteins are components of the excre-
tory/secretory fraction of ML but that the secreted T.
spiralis protein is modified or processed on secretion
whereas the T. pseudospiralis protein is not. In order to
identify non-conserved domains in the TPJ5 protein
that might explain the observed differences, we have
identified the full-length tpJ5 cDNA sequence. Com-
parison of the predicted amino acid sequence of pTPJ5
with pTSJ5 has revealed regions of conservation and
divergence. Immunolocalisation studies have been per-
formed in which immune sera recognises epitopes in the
body wall muscle of the nematode and on the cuticular
surfaces of both T. spiralis and T. pseudospiralis ML.
Furthermore, despite the absence of a recognisable
N-terminal signal sequence, pTSJ5 secretion has been
found to be mediated through a Golgi/ER dependent
pathway.
2. Materials and methods
2.1. Parasites
The Trichinella isolates T. spiralis (ISS3) and T.
pseudospiralis (ISS13) were maintained in female Swiss
ICR mice that were kept in accordance with Swiss
Government regulations. Infective muscle stage larvae
(ML) were isolated from infected animals as previously
described [12,13].
2.2. Molecular cloning of the tpJ5 cDNA
RNA isolation from T. pseudospiralis ML and re-
verse transcription using oligo(dT)n primer were done
as described earlier [11]. tpJ5 cDNA was amplified by
PCR using the following primer pairs: J5N/J5C [10],
F1415/R1772 [11], F765/R1772 [11], F1415/oligo(dT)n,
F1572 [11] /oligo(dT)n and J5N2 (J. Lindh, PhD thesis,
Imperial College of Science, Technology and Medicine,
London 1996)/R1772. Several overlapping regions were
amplified, cloned into pGEM-T Easy Vector (Promega)
and sequenced using the commercial service provided
by Microsynth (Balgach, Switzerland). Independent
clones of each amplified region were sequenced in order
to verify the cDNA sequences. Sequences were aligned
using CLUSTAL and potential functional domains
were identified using SMART (Simple Modular Archi-
tecture Research Tool) at the EMBL-Heidelberg
(www.smart.embl-heidelberg.de) or ScanProsite Tool
(www.expasy.ch/tools/scnpsite.html).
2.3. Preparation of extracts
Crude worm extract (CWE) was prepared by sus-
pending isolated ML in PBS containing a protease
inhibitor cocktail (Roche Diagnostics, Cat. No. 1836
153). The suspension was sonicated twice for 30 s each
at 4°C using a Sonifier B12 (Branson Sonic Power Co.,
Connecticut) at 50% maximum output, followed by
centrifugation at 10 000 g, for 10 min at 4°C. The
protein concentration in the supernatant was deter-
mined using the BioRad Protein Assay and the CWE
stored in aliquots at −80°C.
2.4. Collection of excretory/secretory (ES) protein and
inhibition by Brefeldin A
Isolated T. spiralis or T. pseudospiralis ML were
washed several times with pre-warmed RPMI 1640
medium (Gibco-BRL) and 20,000 ML were resus-
pended in 3 ml of RPMI containing 100 U ml−1
penicillin, 100 �g ml−1 streptomycin, 2 mM L-glu-
tamine, 0.25% glucose, and protease inhibitors and
maintained at 37°C/5% CO2. After 14 h, the larvae
were allowed to sediment and the culture medium was
collected. Insoluble debris was removed by centrifuga-
tion (14 000 g, 30 m, 4°C). The protein concentration in
the supernatant was measured as described above. Cul-
turing of T. spiralis ML in the presence of brefeldin A
(Sigma) was done according to the procedure described
in [14]. Briefly, freshly isolated ML were pre-incubated
in RPMI 1640 medium supplemented with 10 �g ml−1
brefeldin A and incubated at 37°C/5% CO2 for 3 h. The
parasites were allowed to sediment and the supernatant
discarded and replaced with fresh RPMI 1640 medium
containing 10 �g ml−1 brefeldin A. The incubation was
continued for 14 h at which time ES proteins were
collected as described above. As a control, an equal
number of parasites was treated identically, but without
the addition of brefeldin A. For in vivo metabolic
labelling of proteins, parasites were incubated in me-
thionine-free RPMI medium (Gibco-BRL) for 3 h. The
parasites were allowed to sediment and the supernatant
discarded and replaced with fresh methionine-free
RPMI medium containing 10 �g ml−1 brefeldin A and
supplemented with 0.2 mCi ml−1 [35S]-methionine. The
incubation was continued for 6 h at which time ES
proteins were collected as described above. As a con-
trol, an equal number of parasites was treated identi-
cally, but without the addition of brefeldin A.
S. Kuratli et al. /Molecular & Biochemical Parasitology 115 (2001) 199–208 201
2.5. Immunoblotting
CWE (10 �g) was mixed with an equal volume of
2×SDS-PAGE loading buffer. ES proteins (10 �g)
were concentrated by methanol/chloroform precipita-
tion according to [15], followed by solubilization in
1×SDS-PAGE loading buffer. Both fractions were
heated to 95°C for 5 min prior to fractionation by 10%
SDS-PAGE. Fractionated proteins were blotted onto a
Protran nitrocellulose membrane (Schleicher & Schuell)
and the membrane incubated in blocking solution (20
mM Tris-HCl, 150 mM NaCl, 1% Tween-20, pH 7.6:
TBST) containing 3% bovine serum albumin (BSA) for
3 h at room temperature. Primary antibodies were
diluted in TBST+1% BSA, and were applied overnight
at 4°C. After washing in TBST (4 times, 10 min), the
secondary antibody (anti-rat-IgG) conjugated to alka-
line phosphatase (Promega) was applied according to
the instructions provided by the manufacturer. Proteins
labelled with [35S]-methionine were fractionated by
SDS-PAGE, transferred to nitrocellulose and the mem-
brane exposed to Hyperfilm MP (Amersham) X-ray
film prior to processing for antibody labelling. Unless
otherwise stated, the affinity purified polyclonal rat
anti-TSJ5 antibody, designated Ab-pJA [11] was used
at a dilution of 1:100; immune rabbit anti-pJA serum
[10] was used at a dilution of 1:1000. The monoclonal
anti-tubulin antibody was purchased from Sigma
(Clone No. B-5-1-2, T5168) and was used at a dilution
of 1:2000. The monoclonal antibody 7C2C5 [16] was a
kind gift from H.R. Gamble and was used at a dilution
of 1:10 000.
2.6. Preparation of sections and immunolocalisation
Diaphragms of mice infected with T. spiralis or T.
pseudospiralis were cut into 1–2 mm2 sections. The
tissue was fixed in PBS containing 3% paraformalde-
hyde and 0.05% glutaraldehyde for 30 min at 24°C,
washed 3 times in PBS and incubated in PBS/50mM
glycine at 4°C for 1 h. The sections were then washed
extensively in PBS, dehydrated using a graded series of
ethanol (50-70-90-100%, respectively) for 5 min each at
−20°C and embedded in LR-White resin at −15°C,
with 4 changes of fresh resin over a period of 3 days.
The resin was polymerized at 58°C for 24 h. For
immuno-gold labelling and electron microscopy, ultra-
thin sections were cut using a Reichert & Jung ultrami-
crotome and were picked onto 200 mesh
formvar-carbon-coated nickel grids (PLANO GmbH,
Marburg, Germany). Loaded grids were stored at 4°C
for 48 h maximum. Prior to antibody labeling of sec-
tions, EM grids were incubated overnight in EM-block-
ing buffer (PBS/0.5% BSA/50mM Glycin) at 4°C. The
grids were rinsed in PBS and immunostained with
Ab-pJA serum at a dilution of 1:100 in PBS /0.5% BSA
for 1 h at room temperature. Control sections were
incubated with the corresponding pre-immune serum at
a dilution of 1:100. After washing 5 times in PBS, the
goat anti-rat secondary antibody conjugated to 10 nm
gold particles (Amersham, Zuerich, Switzerland) was
applied at a dilution of 1:5 in PBS/0.5% BSA for 45
min. Grids were washed 6 times in PBS, 5 min each,
rinsed in distilled water and air-dried. Grids were
stained with lead citrate and uranyl acetate [17] and
were subsequently viewed on a Phillips 300 transmis-
sion electron microscope. For indirect immunofloures-
cence sections from LR-White embedded T.
spiralis-infected or T. pseudospiralis-infected mouse di-
aphragms were applied to poly-L-lysine-coated (100
�g/ml) glass coverslips and were stored for 48 h maxi-
mum prior to use. The coverslips were rinsed 3 times in
PBS and incubated in blocking buffer (PBS/2%BSA/
50mM glycine) for 1 h. Rat Ab-pJA serum was applied
at a dilution of 1:100 in PBS/0.5% BSA/50mM glycine
for 1 h, followed by 5 washes in PBS. The FITC-conju-
gated goat anti-rat secondary antibody was used at a
dilution of 1:40 in PBS/0.5% BSA/50mM glycine. Sec-
tions were then washed 5 times, 5 min each in PBS and
the sections examined on a Leitz Laborlux S fluores-
cence microscope.
3. Results
3.1. Secretion of pTSJ5 and pTPJ5 in �itro
Preliminary observations suggested that the TSJ5
protein localised on or near the surface of the ML and
might thus be secreted or excreted from the parasite
during this stage. In order to address this question,
Western analysis of the ML crude worm extract (CWE)
and excreted/secreted (ES) protein fractions was per-
formed. Given the difference observed in both gene and
protein expression between T. spiralis and T. pseudospi-
ralis with regard to TSJ5, extracts were collected and
examined for both species. Isolated T. spiralis and T.
pseudospiralis ML were cultured in vitro as described in
materials and methods. Excreted/secreted products re-
leased during this incubation time were collected, con-
centrated and analyzed by SDS-PAGE and
immunoblotting.
A single protein was detected in the CWE of both T.
spiralis and T. pseudospiralis ML by the affinity purified
Ab-pJA (Fig. 1a, lanes 1 and 2). As previously ob-
served, pTPJ5 migrated as a protein of slightly smaller
molecular mass than pTSJ5 and was less abundant [11].
The affinity purified Ab-pJA also detected a single
protein band in ES extracts from both T. spiralis and T.
pseudospiralis (Fig. 1b, lanes 1 and 2). A positive con-
trol monoclonal antibody, 7C2C5, directed against the
major ES products of Trichinella [16] readily reacted
S. Kuratli et al. /Molecular & Biochemical Parasitology 115 (2001) 199–208202
with secretory components of the expected molecular
mass range for T. spiralis and T. pseudospiralis (Fig. 1b,
lanes 3 and 4). In contrast, the anti-�-tubulin mono-
clonal antibody showed no reactivity with either ES
fraction but readily labeled the CWE (Fig. 1a, lanes 3
and 4) indicating that parasites had largely retained
their structural integrity during in vitro maintenance.
Thus, detection of pTSJ5 and pTPJ5 in the ES fraction
was not due to leakage from dead or damaged larvae
but these proteins were secreted or excreted by the ML
in vitro and were components of the respective ES
fractions of both Trichinella species. However, the T.
spiralis ES protein migrated as a 95 kDa protein com-
pared with 110 kDa for the CWE protein, suggesting
that processing or modification of CWE TSJ5 protein
had occurred on in vitro incubation/secretion. Protein
instability is unlikely to account for this, as the size of
the CWE protein does not change even after prolonged
incubation of CWE at room temperature (data not
shown). In contrast, the T. pseudospiralis protein mi-
grated as a 105 kDa protein in both the CWE and ES
fractions. Interestingly, while the TPJ5 protein is less
abundant in the CWE fractions the relative abundance
of the two proteins in the ES fractions appeared to be
reversed. Protein bands co-migrating with the ES forms
of pTSJ5 and pTPJ5, as identified by immunoblotting,
were detectable by Coomassie Blue staining of corre-
sponding SDS-PAGE gels and constitute approxi-
mately 1% of total ES protein. The possibility that the
smaller protein detected by Ab-pJA in the T. spiralis ES
extracts was a protein sharing antigenic epitopes with
pTSJ5 was considered unlikely for the following rea-
sons. First, the 95 kDa protein was detected only in the
ES and not in the crude worm extract fractions pre-
pared from identically treated T. spiralis ML and,
secondly, the antibody detected the same sized protein
in both the ES and the crude worm extract fractions
prepared from T. pseudospiralis ML.
3.2. Comparison of the T. spiralis J5 protein with its
T. pseudospiralis homologue
A homologue of the tsJ5 gene was previously iden-
tified in T. pseudospiralis and approximately 928 bp of
the genomic sequence determined [11]. Comparison of
the predicted amino acid sequence encoded by this
fragment of the T. pseudospiralis gene with the corre-
sponding region of pTSJ5, indicates that the two
proteins are very similar over this region [11]. In order
to determine if differences in protein composition out-
side of this region could explain the differences between
pTSJ5 and pTPJ5 observed by Western analysis, the
entire coding sequence of the tpJ5 gene was determined.
The full-length tpJ5 cDNA sequence was amplified by
RT-PCR using primers designed against the tsJ5 gene
(materials and methods). Overlapping fragments were
sequenced in order to eliminate errors due to mis-incor-
poration and to identify sequence polymorphisms in the
primers. Together the amplified regions represented a
cDNA of 2098 nucleotides excluding the poly(dA)n tail;
the comparable tsJ5 cDNA is 2180 nucleotides in
length. In comparison, the two cDNAs showed a high
degree of conservation, with overall sequence identity
of 86% at the nucleotide level (data not shown); exon
sequences in the previously cloned 928 bp genomic tpJ5
fragment were �93% identical with the corresponding
region of tsJ5 [11]. The decrease in overall nucleotide
identity was due primarily to the presence of sequence
gaps in the tpJ5 cDNA; one in a region of GAG/GAA
repeats found in tsJ5 (nucleotides 1221–1266) and the
second at the 3� end of the coding region (nucleotides
1850–1863).
A single open reading frame of 1998 nucleotides was
identified within the tpJ5 cDNA, conceptual translation
of which, from the first in-frame Met (at nucleotide 39)
to a stop codon at nucleotide 2034, yielded a putative
Fig. 1. Western analysis of T. spiralis and T. pseudospiralis CWE and ES protein fractions. (a) 10 �g of total crude worm extract of T. spiralis
(lanes 1 and 3) and T. pseudospiralis (lanes 2 and 4) were fractionated by 10% SDS-PAGE and Western blotted. Strips were reacted with
affinity-purified Ab-pJA (lanes 1and 2) or with the monoclonal anti-�-tubulin antibody (lanes 3 and 4). (b) 10 �g of ES protein of T. spiralis (lanes
1, 3 and 5) and T. pseudospiralis (lanes 2, 4 and 6) were fractionated by 10% SDS-PAGE and Western blotted. Strips were reacted with
affinity-purified Ab-pJA (lanes 1 and 2), mAb 7C5C2 (lanes 3 and 4) or monoclonal anti-�-tubulin antibody (lanes 5 and 6). Primary antibodies
were diluted as described in Materials and Methods and antibody binding was detected using alkaline-phosphatase conjugated anti-rat IgG.
Molecular weight markers are shown in kDa.
S. Kuratli et al. /Molecular & Biochemical Parasitology 115 (2001) 199–208 203
Fig. 2. Alignment of the predicted amino acid sequence of pTSJ5 and pTPJ5. Putative N-glycosylation sites are underlined and putative bipartite
nuclear localization signals are shown in bold.
protein (pTPJ5) of 665 amino acids (Fig. 2). The calcu-
lated molecular mass of 75 kDa for pTPJ5 is slightly
smaller than the 76 kDa calculated molecular mass for
the T. spiralis protein, pTSJ5. The native pTSJ5 and
pTPJ5 proteins migrate