Heat shock proteins HSP70 and HSP60 in Echinococcus granulosus protoscolices.

The heat shock response is a ubiquitous process described in prokaryotic as well as eukaryotic cells (Feige U., Mollenhauer J. 1992: Experientia 48: 621-622). This response is characterized by an induction, due to transcriptional activation, of a specific set of genes (Sorger P.K., Pelham H.R.B. 1987: EMBO J. 6: 993-998); the final products of this activation are the so called heat shock proteins (HSPs). HSPs play different functions in protein synthesis (as molecular chaperones) as well as in protein degradation (Burel C., Mezger V., Pinto M., Rallu M., Trigon S., Morange M. 1992: Experientia 48: 629-634). Furthermore, there are works where a hypothetical role for these proteins in immune response has been described (Kaufmann S.H.E. 1992: Experientia 48: 640643; Mollenhauer J., Schulmeister A. 1992: Experientia: 48: 644-649), and it seems that they are also involved, directly or indirectly, in the development of autoimmune diseases (Cohen I.R. 1991: Rev. Immunol. 9: 567-589; Kaufmann S.H.E. 1991: Curr. Top. Microbiol. Immunol. 167: 1-214). There are a lot of works about the HSP expression in mammals; however, the number of papers on parasite organisms is still low. Whatever the case, it has been previously demonstrated that this protein expression could play a fundamental role in the virulence of parasites (Smejkal R.M., Wolff R., Olenick J.G. 1988: Exp. Parasitol. 65: 1-9), their differentiation (Van der Ploeg L.H.T., Giannini S.H., Cantor C.R. 1985: Science 228: 1443-1446) and in the host adaptation (Maresca B., Carratu L. 1992: Parasitol. Today 8: 260-266). The pattern of these protein expressions has been studied in different parasites such as Trypanosoma, Leishmania, Trichomonas, Trichinella and Haemonchus (Alcina A., Urzainqui A., Carrasco L. 1988: Eur. J. Biochem. 172: 121-127; Mc Farlane J., Blaxter M.L., Bishop R.P., Miles M.A., Kelly J.M. 1990: Eur. J. Biochem. 190: 377-384; Davis S.R., Lushbaugh W.B. 1992: Am. J. Trop. Med. Hyg. 47: 70-77; Van Leewen M.A.W. 1995: Parasitol. Res. 81: 706-709; Ko R.C., Fan L. 1996: Parasitology 112: 89-95); however, the papers where the presence of HSPs in the Excretion-Secretion (ES) products has been described are fewer, as far as we know only in Trichinella (Ko and Fan 1996, op. cit.) and Mesocestoides (Estes D.M., Teale J.M. 1991: J. Immunol. 147: 3926-3934). In this report we study the HSP90, HSP70, HSP60 and HSP25 expression in Echinococcus granulosus protoscolices in control conditions and after heat shock treatment, both in crude somatic extract of protoscolices and in ES products. E. granulosus protoscolices were aseptically collected from liver hydatid cysts in sheep slaughtered at the municipal abattoir in Alcala de Henares, Spain. Viability prior to testing was 95-99% as assessed by the methylene blue exclusion test and microscopic examinations, as previously described (Casado N., Rodriguez-Caabeiro F., Hernandez S. 1986: Z. Parasitenkd. 72: 273-278). The organisms (25,000 protoscolices/Leighton tube) were cultured in 3 ml of medium 199 (Sigma). In vitro incubations were performed at different temperatures, these being: 37°C, 42°C and 45°C for 4 h. At the end of the incubations, the medium was removed, centrifuged (10 min; 12,000 g) and precipitated with 10% TCA (trichloroacetic acid). This solution was centrifuged at 50,000 g for 20 min at 4°C. The pellet was resuspended in phosphate-buffered saline (PBS) and stored at -70°C. Protoscolices were homogenized in 0.5 ml of PBS by sonicator. This homogenate was centrifuged at 100,000 g for 30 min at 4°C. The supernatant was collected and the protein concentration was determined (Bradford, Bio-Rad). Samples (20 μg/well) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Stacking gels containing 4% and separating gels 12% acrylamide were routinely used. Electrophoresis was carried out at a constant current of 40 mA. Electroblot transfer from the polyacrylamide gels was performed as described by Towbin P., Staehelin T. , Gordon J. 1979: Proc. Nat. Acad. Sci. USA 76: 4350-4354). The nitrocellulose blots were washed in PBS containing 0.05% Tween-20, and incubated with 5% non fat powdered milk for 1 h. After the incubations, blots were tested with antisera. Primary monoclonal antibodies (Sigma) were: anti-HSP90 (1/500 dilution), anti-HSP70 (1/5,000 dilution), anti-HSP60 (1/1,000 dilution) and anti-HSP25 (1/500 dilution), diluted in PBS. The peroxidase conjugated secondary antibody (Sigma) was used at 1/1,000 dilution. The peroxidase reaction was carried out of 0.05M Tris buffer containing 0.03% diaminobenzidine (DAB), and 0.001% hydrogen peroxide. For densitometric quantification of blot immunoreactivity a digital analysis image system (Microm, Spain) was used. Immunoblotting revealed that HSP60 and HSP70 were present in the ES products of 42°C and 45°C heated protoFOLIA PARASITOLOGICA 46: 76-78, 1999