Trichinella pseudospiralis secretes a protein related to the Trichinella spiralis 43-kDa glycoprotein

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Molecular and Biochemical Parasitology 78 (1996) 25531

Trichinella pseudospiralis secretes a protein related to the Trichinella spiralis 43-kDa glycoprotein Demetrios K. Vassilatis”*b**, Dickson D. Despommier”, Ramona I. Polvere”, Allen M. Goldd, Lex H.T. Van der Ploeg” “Depturtment of’ Genetics and Molecular Biology, RY-8OY-255, Merck Research Laboratories, RahwaJa. NJ 07065, USA bDepartment of Genetics and Development, Columbia University, 701 W 168 St, New York, NY 10032, USA ‘Division of Environmental Sciences, School of Public Health, and Deptartment of Microbiology, Columbia University, 630 W 168 st, New York, NY 10032, USA ‘Deptarrment of Biochemistry and Molecular Biophysics, Columbia University. 630 W I68 st, New York, NY 10032, USA

Received t 2 October 1995; revised 16 February 1996; accepted 16 February 1996

Abstract A 43-kDa secreted glycoprotein from the intracellular parasitic nematode Trichinellu spirulis has been considered as a factor involved in the formation of the Nurse cell in infected muscle. The closely related intracellular parasitic nematode Trichinellu pseudospiralis that also infects muscle cells, does not form Nurse cells and was thought not to secrete the 43-kDa glycoprotein. This implied a unique role for the 43-kDa glycoprotein in T. spiralis infection and supported the hypothesis of involvement of the 43-kDa glycoprotein in Nurse cell formation. Following cloning of a full length cDNA encoding the 43-kDa protein, antibodies were raised against several domains of the 43-kDa glycoprotein. Here we show that a protein related to the 43-kDa glycoprotein exists in T. pseudospiralis. Immunohistochemical studies reveal important similarities in the distribution of the 43-kDa glycoprotein and the related protein from T. pseudospiralis in muscle infections with either of the two parasites. The 43-kDa glycoprotein may therefore play a common role in the life cycles of these two parasites and probably is not involved in Nurse cell formation. Keywords: T. pseudospirulis; T. spiralis; 43-kDa

glycoprotein;

1. Introduction Trichinella spiralis is a parasitic nematode that invades skeletal muscle cells. Infection with the

* Corresponding author. Abbreviations: SSC, Standard

Saline Citrate; SDS, Sodium Dodecyl Sulfate; PBS, Phosphate Buffered Saline; DAB, Diamino Benzidine.

Excreted

secreted

or ES

nematode alters the pattern of gene expression of the host cell in a process thought to represent muscle cell de-differentiation. The invaded muscle cell is transformed into a Nurse cell over a 20-30 day period post-infection. The resulting Nurse cell is encapsulated along with the parasite and the infected region of the muscle cell in a host derived collagen sheath. A circulatory rete is formed on top of the collagen capsule [l-4]. The natural

0166-6851/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PZI S 166-685 1(96)02596-O

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hosts of T. pseudospiralis, a smaller parasitic nematode closely related to T. spiralis, are avian species but it can also infect mammals. All isolates of Trichinella, except T. pseudospiralis, form Nurse cells upon infection of host muscle [5]. There is no loss of myofibrils in muscle cells infected with T. pseudospiralis and the host cell nuclei appear normal [6]. Muscle stage Ll larvae of T. spiralis and T. pseudospiralis can be isolated by pepsin-HCl digestion of host muscle. In vitro these Ll larvae secrete tens of proteins (Excreted Secreted or ES proteins) from specialized cells called stichocytes [7]. These in vitro derived secreted proteins are thought to represent the secreted products of the nematode while living in the Nurse cell. It has been hypothesized that ES proteins are involved in the mechanisms of muscle cell de-differentiation and in Nurse cell formation [8]. To initiate a study analyzing their role, a few of these proteins have been extensively characterized and some have been purified [9]. We have focused our research on one such protein, the 43-kDa glycoprotein. The T. spiralis 43-kDa secreted glycoprotein has been of particular interest since antibodies raised against the native protein stain the nuclei of developing and mature Nurse cells [lo]. Furthermore, it had been proposed that the 43-kDa protein is absent from T. pseudospiraiis secretions [ 1 1 - 131. We have cloned a cDNA encoding the 43-kDa glycoprotein from T. spiralis and have raised antibodies against domains of the 43-kDa glycoprotein expressed as fusion proteins in E. coli. These include: (i) a fusion protein encoding 183 N-terminal amino acids (N183); (ii) a 313-amino acid fusion protein representing almost the entire 43-kDa polypeptide, except for the signal peptide and the last nine C-terminally located amino acids (313); and (iii) 95 C-terminally located amino acids (C95) of the 43-kDa polypeptide [14,15]. Antibodies against the 3 13 fusion protein recognize a family of proteins in T. spiralis ES and stain the cytoplasm and nuclei of infected muscle cells [ 10,14,16]. However, antibodies which are specific for the 43- kDa glycoprotein (anti-C95 and anti-N183) only stain the cytoplasm of developing Nurse cells and stichocytes of the nematode in the Nurse cell [15].

Indications about the function of the 43-kDa glycoprotein could be obtained by a comparative analysis of its expression pattern in different parasitic nematodes. T. pseudospiralis is particularly important since it fails to form Nurse cells. Here we show that sequences homologous to the 43kDa gene exist in the T. pseudospiralis genome. We also show expression of the 43-kDa homologue in T. pseudospiralis muscle stage larvae. Immunocytolocalizations of mouse muscle infected with T. pseudospiralis, the 43-kDa glycoprotein antibodies reveal a staining pattern similar to that seen in T. spiralis. We conclude that T. pseudospiralis encodes a 43-kDa glycoprotein related protein. These data are consistent with the hypothesis that the 43-kDa glycoprotein from T. spiralis is not a major factor in Nurse cell formation. 2. Materials and methods 2.1. Preparation of genomic DNA from T. spiralis and T. pseudospiralis Ll larvae and Soutrhern blots

Genomic DNA from T. spiralis and T. pseularvae was prepared as described

dospiralis Ll

[141. T. spiralis and T. pseudospiralis genomic DNAs

were digested by restriction endonucleases and the digestion products were fractionated on agarose gels. After transfer to nitrocellulose filters hybridizations with 3’P-labeled probes were performed using the ‘random priming kit’ (Boehringer Mannheim). After hybridization, filters were washed to 0.1 X Standard Saline Citrate (SSC) (1 X SSC: 0.15 M Sodium Chloride, 0.015 M Sodium Citrate), 0.1% Sodium Dodecyl Sulfate (SDS) at 65°C prior to exposure using Kodak XR-5 film. Low stringency Southern hybridizations were done at 50°C in 6 x NET [(l x NET: 150 mM NaCl, 1 mM EDTA, 15 mM Tris-HCl pH 7.5, 1 x Denhardt’s solution, 2% Dextran Sulfate, 0.02% Sodium Pyrophosphate, 0.1% SDS) with tRNA 40 pg mll I (Sigma) added as carrier] overnight, and washed at 55°C in 6 x SSC, 0.1% SDS. The filters were exposed on Kodak XR-5 film.

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2.2. PuriJication of antisera and Western blots

IgG/IgA fractions were isolated using ammonium sulfate precipitation [17] or the Monoclonal Antibody Purification System (MAPS kit from BIORAD). Preparation of T, spiralis and T. pseudospiralis ES products was as described [9]. Samples were subjected to SDS-Polyacrylamide Gel Electrophoresis and electrotransfered to nitrocellulose sheets as described by Towbin [18]. A horseradish peroxidase conjugated goat anti-rabbit secondary antibody was used for detection. The remaining procedure was essentially carried out as described by Tsang [19]. 2.3. hnmunocytolocalizations Slides with paraffin embedded tissue sections of mature infected mouse thigh muscle were used in immunocytolocalizations. The sections were treated as described [14]. Antisera or purified IgG were incubated at 1:lOO- 1:1000 dilutions in 0.1 M Phosphate Buffered Saline (PBS) (NaH*PO, 0.028 M; Na,HPO, 0.028M; NaCl 0.15 M) pH 7.2 at 4°C overnight. A horseradish peroxidase conjusecondary antibody gated goat anti-rabbit (Sigma) was allowed to react at a 1:2000 dilution in 0.1 M PBS pH 7.2 at room temperature for 1 h. The reactions were developed with 0.5 mg ml ~’ Diamino Benzidine (DAB) (Sigma) and 0.01% H,O, (Sigma) in 0.01 M PBS pH 7.6. Sections were examined by light microscopy and photographed on Kodak Kodacolor slide film.

3. Results 3.1. Sequences related to the 43-kDa glycoprotein gene in other species

We investigated whether a gene similar to the one encoding the 43-kDa glycoprotein of T. spiralis exists in free living nematodes (Caenorhabditis elegans), parasitic nematodes (T. spiralis, T. pseudospiralis, Ascaris lumbricoides, Haemoncus contortous, Dirophilaria immitis, Xiphenema digit) and Drosophila melanogaster, Trypanosoma cruzi

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and rat (Wistar strain). In Southern blots, at low stringency of hybridization, the T. spiralis, T. pseudospiralis and D. immitis lanes revealed restriction fragments hybridizing to the j2P labeled 43-kDa cDNA probe of T. spiralis (data not shown). However, at high stringency (65°C 0.1 X SSC) only the T. spiralis, and T. pseudospiralis displayed hybridization signals, indicating that the T. pseudospiralis genome encodes DNA sequences closely related to the T. spiralis 43-kDa glycoprotein gene. Southern analysis of genomic DNA digested with Rsa I, Hind III and Pst I restriction enzymes showed a single equally strong hybridizing band of size for both parasites (Fig. 1). Southern blots with cDNA probes that correspond to the recombinant regions of the 43-kDa glycoprotein N183 and C95 [14,15] show an identical restriction enzyme pattern as that observed with the entire cDNA probe indicating that homology between the T. spiralis and T. pseudospiralis extends throughout the gene (data not shown). From these experiments we conclude that the sequences detected encoding the 43-kDa glycoprotein in T. spiralis and T. pseudospiralis are closely related. 3.2. T. pseudospiralis synthesizes and secretes a 43-kDa related protein

We investigated whether T. pseudospiralis secretes a protein related to the 43-kDa glycoprotein. Western blots of T. pseudospiralis ES products were reacted with the anti-C95 (Fig. 3, panel A), anti-N183 (Fig. 2, panel B), anti-313 (Fig. 2, panel C) and anti-native 43-kDa glycoprotein (Fig. 2, panel D) antibodies [14,15]. Both T. spiralis 43-kDa glycoprotein specific recombinant antibodies (directed against C95 and N183 fusion proteins) recognize a single protein band of approximately 38-kDa’ (indicated with open arrowheads in Fig. 2). This suggests that T. psezidospiralis secretes a protein similar to the 43-kDa glycoprotein of T. spiralis (Fig. Fig. 2A and B, lanes 3). The T. pseudospiralis protein band appears to have reduced reactivity to these antibodies. The difference in size may be due to a shorter polypeptide backbone for the T. pseudospiralis protein or it may represent an altered glycosyla-

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tion form of the protein, in comparison to the diglycosylated form of the 43-kDa glycoprotein from T. spirulis [9]. The 313 recombinant antibodies recognize a family of proteins in both T. spiralis and T. pseudospiralis ES preparations Fig. 2C). These bands are of a different molecular weight in T. pseudospiralis and T. spiralis. The size differ-

Rsa I InI 1 2

Hind III 1

2

Pst I 1

ences could be due to altered glycosylation patterns or other post-translational modifications. Alternatively, they may reflect differences in the polypeptide backbones. The anti-native T. spiralis 43-kDa glycoprotein antibodies are directed primarily against carbohydrate epitopes and show very strong crossreactivity with the T. pseudospiralis ES secretions recognizing several proteins (Fig. 2D) [10,14]. These experiments indicate that a protein related to the T. spiralis 43-kDa glycoprotein is detected in T. pseudospiralis ES products.

2 3.3. Espression of the 43-kDa honlologue other T. spirulis relcrted proteins in T. pseudospiralis infected muscle tissue

23.1 *

2.3 . 2.0 *

Fig. 1. A T. spiralis homologue of the 43-kDa glycoprotein gene in T. pseudospiralis. DNA (7 mg) from T. spiralis (lanes 1) and T. pseudospiralis (lanes 2) was digested with restriction enzymes as indicated above the lanes and transferred to nitrocellulose filters. The southern blot was hybridized with the 43kDa cDNA from T. spiralis and posthybridizational washes were performed at 65°C and 0.1 X SSC. The molecular size standards in Kb are indicated on the left of the panel.

and

Immunocytolocalizations on muscle sections infected with T. pseudospiralis allowed identification of immunoreacting products with all anti-43-kDa glycoprotein antibodies used. The 43-kDa specific recombinant antibodies antiN183 and anti-C95 (Fig. 3A and Fig. 3B respectively) stain only a region of the stichosome of T. pseudospiralis that is believed to contain, by analogy to T. spiralis, the a-stichocytes [15]. The anti-313 (Fig. 3C) and the anti-native 43-kDa glycoprotein antibodies (Fig. 3E,F) stain the parasite and the cytoplasm of infected muscle cells. The apparently normal nuclei of the infected muscle cell in T. pseudospirulis infections do not stain with the anti313 antibodies but about 25% of these stain with the anti-native 43-kDa glycoprotein antibodies (Fig. 3F arrow) [20]. Pre-immune antibodies did not reveal any staining (Fig. 3D). These results are similar to the staining patterns seen in infected muscle sections of T. spiralis with the exception that the anti-313 antibodies also stain the Nurse cell nuclei in T. spirnlis infections [10,14,15]. It is possible that the lack of nuclear staining with the anti-313 antibodies is due to a reduced reactivity against the T. pseudospiralis proteins. These antibody reagents reveal significant similarities in the distribution patterns of proteins in muscle cells infected by either of the two parasites.

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Fig. 2. Comparison of ES products of Trichinella spiralis and Trichinelku pseudospirulis. Four identical panels of mcstcrn blots were reacted with anti 43-kDa glycoprotein antibodies. Lanes 1: 200 ng of purified 43-kDa glycoprotein; Lanes 2: 2 /cg of T. .spirrr/i.u ES products: Lanes 3: 2 /lg of T. pseudospiralis ES products. Panel A was reacted with C-95 antiserum at I:200 dilution. Panel B \\ith N-183 IgCilgA at I:200 dilution. Panel C with 313 IgG/IgA at I:500 dilution and panel D with anti-native 33-kDa plycoprotein IgGiIgA at I:2000 dilution. The position of the 43-kDa glvcoorotein is indicated by a black arrowhead while the T. /,.\c,rrt/r,.,pi~c~/;.\ _. himo!ogue is indicated by a whiie arrowhead.

4. Discussion It had previously been proposed that the 43kDa glycoprotein is unique to T. spiralis and may play a role in the de-diRerentiation of the infected muscle cell and/or the Nurse cell formation [8, IO,1 I .13,14]. We have shown that the genome of the parasitic nematode T. psrudospiralis encodes sequences similar to the 43-kDa glycoprotein gene of T. spirdis. Homologous sequences to the 43-kDa glycoprotein cDNA were not found in the other orders (Strongylata, Ascariduta, Filari&a) of animal parasitic nematodes analyzed. We have also compared the ES products of T. spiralis and T. psatdospiralis and show that a protein closely related to the 43-kDa glycoprotein form T. spirulis is found in the ES products of T. pseudospiralis. As other researchers have shown using monoclonal antibodies, these data indicate

that there is similarity in ES proteins from the two parasites as detected by the 3 13 and the native 43-kDa glycoprotein antibodies [ 12.2 11. The 43kDa glycoprotein related protein from T. psrudospiralis is of a different apparent molecular weight and appears only weakly crossreacting with the 05 and N 183 antibodies. This weak signal may reflect a low abundance of the protein in the ES products or a reduced avidity of the antibodies for the T. p.~WCio.sl)irLlIi.sprotein. Since (i) this is the major protein detected by both of these anti-43-kDa glycoprotein specific antibodies and (ii) the antibodies are raised against nonoverlapping regions of the 43-kDa glycoprotein. it is likely that the antibodies detect a protein closely related to the 43-kDa glycoprotein. We therefore conclude that there are notable antigenic similarities in the ES products of the two parasites and a 43-kDa related protein is expressed by T.

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Fig. 3. mouse anti-313 43 kDa muscle

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Immunolocalizations. Immunohistochemical localizations of the T. pseudospiralis 43-kDa glycoprotein homologue in mature infections. Staining with anti-N183 (A), anti-C95 (B) show reactivity with a subset of the stichocytes of T. pseudospiralis. The (C) antibodies stain the stichosome and infected cell cytoplasm. Preimmune antibodies show no staining (D). Anti-native antibodies stain the parasite and cytoplasm of the infected muscle cell (E,F); in about 25% apparent nuclei of the infected cells also stain (F, arrow).

pseudospiralis. Besides the 43-kDa related protein,

the anti-313 and anti-native 43-kDa antibodies crossreact with several other ES proteins from both parasites (Fig. 2C,D). These crossreacting proteins do not appear to correspond with each other indicating that they may be species specific. However, similarly to the 43-kDa glycoprotein, they may be related proteins of different apparent molecular weight due to different polypeptide length or due to post-translational modifications. There is also similarity in the distribution of

immunologically related proteins in muscle sections infected by either T. pseudospiralis or T. spiralis. The 43-kDa specific antibodies anti-C95 and anti-N183 stain the parasite showing expression of the 43-kDa glycoprotein homologue in the muscle stage of the life cycle. The anti-native 43-kDa glycoprotein antibodies, in addition to staining T. pseudospiralis and the cytoplasm of the infected muscle cells, also appear to stain nuclei in about 25% of the infected muscle fibers. This may indicate that secreted factors from T. pseudospi-

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ralis localize to the nuclei of the infected muscle cell as is observed in T. spiralis infections

[8,10,14]. Our experiments can not address any functional differences that may exist in the two closely related 43-kDa and 38-kDa proteins. The function of the T. spiralis 43-kDa glycoprotein is unknown and data bank searches have not identified any related proteins with significant homology. However, it had been proposed that the 43-kDa glycoprotein may have a role in Nurse cell formation based on its presence in nuclei of infected muscle and its absence from T. pseudospiralis. These arguments no longer seem appropriate since (i) antibodies against recombinant regions of the 43-kDa fail to detect the protein in Nurse cell nuclei [15], and (ii) T. pseudospiralis seems to express a related ES protein product.

Acknowledgements

We thank Dr. George Stewart for providing T. pseudospiralis and tissues infected with T. pseudospiralis.

References

Ul

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