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Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida)

Published online by Cambridge University Press:  06 May 2004

A. PARAMÁ ,
R. IGLESIAS ,
M. F. ÁLVAREZ ,
J. LEIRO ,
F. M. UBEIRA  and
M. L. SANMARTÍN
A. PARAMÁ
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
R. IGLESIAS
Affiliation:
Laboratory of Parasitology, Faculty of Sciences, University of Vigo, Lagoas-Marcosende s/n, 36200 Vigo, Spain
M. F. ÁLVAREZ
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
J. LEIRO
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
F. M. UBEIRA
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
M. L. SANMARTÍN
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
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Abstract

This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS–PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30–63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.

Keywords

Philasterides dicentrarchifish pathogenscuticociliatosisciliatecysteine proteasehistolytic activity
Type
Research Article
Information
Parasitology , Volume 128 , Issue 5 , May 2004 , pp. 541 - 548
Copyright
2004 Cambridge University Press

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References

REFERENCES

BANNO, Y., YANO, K. & NOZAWA, Y. (1983). Purification and characterization of a secreted proteinase from Tetrahymena pyriformis. European Journal of Biochemistry 132, 563–568.CrossRefGoogle Scholar
BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle Scholar
BRADY, C. P., DOWD, A. J., BRINDLEY, P. J., RYAN, T., DAY, S. R. & DALTON, J. P. (1999). Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin. Infection and Immunity 67, 368–374.Google Scholar
CAWTHORN, R. J. (1997). Overview of ‘Bumper car disease’ – impact on the North American lobster fishery. International Journal for Parasitology 27, 167–172.CrossRefGoogle Scholar
DRAGESCO, A., DRAGESCO, J., COSTE, F., GASC, C., ROMESTAND, B., RAYMOND, J. & BOUIX, G. (1995). Philasterides dicentrarchi, n. sp. (Ciliophora, Scuticociliatida), a histophagous opportunistic parasite of Dicentrarchus labrax (Linnaeus, 1758), a reared marine fish. European Journal of Protistology 31, 327–340.Google Scholar
FRANKEL, J. (1999). Cell biology of Tetrahymena termophila. In Methods in Cell Biology. Vol. 62. Tetrahymena termophila (ed. Aai, D. J. & Forney, J. D. ), pp. 27–125. Academic Press, San Diego.
IGLESIAS, R., PARAMÁ, A., ÁLVAREZ, M. F., LEIRO, J., FERNÁNDEZ, J. & SANMARTÍN, M. L. (2001). Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW, Spain). Diseases of Aquatic Organisms 46, 47–55.CrossRefGoogle Scholar
IGLESIAS, R., PARAMÁ, A., ÁLVAREZ, M. F., LEIRO, J., AJA, C. & SANMARTÍN, M. L. (2003 a). In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora, Scuticociliatida). Veterinary Parasitology 111, 19–30.Google Scholar
IGLESIAS, R., PARAMÁ, A., ÁLVAREZ, M. F., LEIRO, J., UBEIRA, F. M. & SANMARTÍN, M. L. (2003 b). Philasterides dicentrarchi (Ciliophora: Scuticociliatida) expresses surface immobilization antigens that probably induce protective immune responses in turbot. Parasitology 126, 125–134.Google Scholar
JIMÉNEZ, J. C., UZCANGA, G., ZAMBRANO, A., DI PRISCO, M. C. & LYNCH, N. R. (2000). Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis. Journal of Parasitology 86, 859–862.CrossRefGoogle Scholar
KARANU, F. N., RURANGIRWA, F. R., McGUIRE, T. C. & JASMER, D. P. (1993). Haemonchus contortus: Identification of proteases with diverse characteristics in adult worm excretory–secretory products. Experimental Parasitology 77, 362–371.CrossRefGoogle Scholar
KEENE, W. E., HIDALGO, M. E., OROZCO, E. & McKERROW, J. H. (1990). Entamoeba histolytica: correlation of the cytopathic effect of virulent trophozoites with secretion of a cysteine proteinase. Experimental Parasitology 71, 199–206.CrossRefGoogle Scholar
McKERROW, J. H., ENGEL, J. C. & CAFFREY, C. R. (1999). Cysteine protease inhibitors as chemotherapy for parasitic infections. Bioorganic and Medicinal Chemistry 7, 639–644.CrossRefGoogle Scholar
McKERROW, J. H., SUN, E., ROSENTHAL, P. J. & BOUVIER, J. (1993). The proteases and pathogenicity of parasitic protozoa. Annual Review of Microbiology 47, 821–853.CrossRefGoogle Scholar
MUNDAY, B. L., O'DONOGHUE, P. J., WATTS, M., ROUGH, K. & HAWKESFORD, T. (1997). Fatal encephalitis due to the scuticociliate Uronema nigricans in sea-caged, southern bluefin tuna Thunnus maccoyii. Diseases of Aquatic Organisms 30, 17–25.CrossRefGoogle Scholar
MURRICANE, C. (1986). Purification of a proteinase and proteinase inhibitor from Tetrahymena pyriformis. International Journal of Biochemistry 18, 403–406.CrossRefGoogle Scholar
NORTH, M. J. (1997). Parasite proteinases. In Analytical Parasitology, Vol. 5 ( ed. Rogan, M. T. ), pp. 133–186. Springer-Verlag, Heidelberg.CrossRef
PARAMÁ, A., IGLESIAS, R., ÁLVAREZ, M. F., LEIRO, J., AJA, C., SANMARTÍN, M. L. (2003). P. dicentrarchi (Ciliophora, Scuticociliatida): experimental infection and possible routes of entry in farmed turbot (Scophthalmus maximus). Aquaculture 217, 73–80.Google Scholar
PERKINS, P. S., HALEY, D. & ROSENBLATT, R. (1997). Proteolytic enzymes in the blood-feeding parasitic copepod, Phrixicephalus cincinnatus. Journal of Parasitology 83, 6–12.CrossRefGoogle Scholar
QUE, X. & REED, S. L. (1997). The role of extracellular cysteine proteinases in pathogenesis of Entamoeba histolytica invasion. Parasitology Today 13, 190–194.CrossRefGoogle Scholar
RAWLINGS, N. D. & BARRETT, A. J. (1994). Families of cysteine peptidases. Methods in Enzymology 244, 461–538.CrossRefGoogle Scholar
REED, S. L., KEENE, W. E. & McKERROW, J. H. (1989). Thiol proteinase expression correlates with pathogenicity of Entamoeba histolytica. Journal of Clinical Microbiology 27, 2772–2777.Google Scholar
REED, S. L., BOUVIER, J., SIKES, A., ENGEL, J., BROWN, M., HIRATA, K., QUE, X., EAKIN, A., HAGBLOM, P., GILLIN, F. & McKERROW, J. (1993). Cloning of a virulence factor of Entamoeba histolytica: Pathogenic strains possess a unique cysteine proteinase gene. Journal of Clinical Investigation 91, 1532–1540.CrossRefGoogle Scholar
RHOADS, M. L. & FETTERER, R. H. (1997). Extracellular matrix: a tool for defining the extracorporeal function of parasite proteases. Parasitology Today 13, 119–122.CrossRefGoogle Scholar
ROSENTHAL, P. J. (1998). Proteases of malaria parasites: new targets for chemotherapy. Emerging Infectious Diseases 4, 49–57.CrossRefGoogle Scholar
ROSENTHAL, P. J. (1999). Proteases of protozoan parasites. Advances in Parasitology 43, 106–159.CrossRefGoogle Scholar
SELZER, P. M., SABINE, P., HSIEH, I., UGELE, B., CHAN, V. J., ENGEL, J. C., BOGYO, M., RUSSELL, D. G., SAKANARI, J. A. & McKERROW, J. H. (1999). Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target. Proceedings of the National Academy of Sciences, USA 96, 11015–11022.CrossRefGoogle Scholar
SKELLY, P. J. & SHOEMAKER, C. B. (2001). Schistosoma mansoni proteases Sm31 (cathepsin B) and Sm32 (legumain) are expressed in the cecum and protonephridia of cercariae. Journal of Parasitology 87, 1218–1221.CrossRefGoogle Scholar
SPEARE, D. J. (1998). Disorders associated with exposure to excess dissolved gases. In Fish Diseases and Disorders, Vol. 2. Non-Infectious Disorders (ed. Leatherland, J. F. & Woo, P. T. K. ), pp. 207–224. CABI Publishing, Oxon.
STRAUS, J. W., MIGAKI, G. & FINCH, M. T. (1992). An assessment of proteolytic enzymes in Tetrahymena thermophila. Journal of Protozoology 39, 655–662.CrossRefGoogle Scholar
SUZUKI, K. M., HAYASHI, N., HOSOYA, N., TAKAHASHI, T., KOSAKA, T. & HOSOYA, H. (1998). Secretion of tetrain, a Tetrahymena cysteine protease, as a mature enzyme and its identification as a member of the cathepsin L subfamily. European Journal of Biochemistry 254, 6–13.CrossRefGoogle Scholar
VÖLKEL, H., KURZ, U., LINDER, J., KLUMPP, S., GNAU, V., JUNG, G. & SCHULTZ, J. E. (1996). Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide. European Journal of Biochemistry 23, 198–206.Google Scholar
WILLIAMS, A. G. & COOMBS, G. H. (1995). Multiple protease activities in Giardia intestinalis trophozoites. International Journal for Parasitology 25, 771–778.CrossRefGoogle Scholar