Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi(Ciliophora: Scuticociliatida)
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 g...
Published in: | Parasitology |
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Main Authors: | , , , , , |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
Cambridge University Press (CUP)
2004
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Subjects: | |
Online Access: | http://dx.doi.org/10.1017/s0031182004004883 https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0031182004004883 |
Summary: | 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 . |
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