新发现的旋毛虫分泌物

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