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Proteolytic activity of cultured Pseudoperkinsus tapetis extracellular products
M Camino Ordás1, B Novoa, M Faisal
1Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello, 6, 36208 Vigo, Spain.
Abstract:
Several pathogenic protozoan release proteases are necessary for host invasion and initiation of infection. We have identified proteolytic activities in extracellular proteins secreted by the clam parasite Pseudoperkinsus tapetis (Mesomycetozoa) in vitro. The protein concentration of the P. tapetis extracellular products (ECP) increased only during the first week of culture. The appearance of new proteins of 10 and 157 kDa at the second week sample and of 12 kDa at the third week sample was shown by SDS-PAGE. The protease activity rapidly increased in the first 3 weeks of culture, and five clear bands of 23, 29, 60, 67 and 96 kDa with proteolytic activity were detected in the ECP on gelatin SDS-PAGE. Using inhibitors, the proteases were identified as members of the Ca(2+) dependent, serine protease family. Their optimum pH was higher than pH 9.4. The protease activity of the P. tapetis ECP was different than that described for Perkinsus marinus, an oyster pathogen very similar morphologically to the clam parasite and member of the genus in which P. tapetis had been initially included.
Insights
Researchers identified novel proteases secreted by the clam parasite Pseudoperkinsus tapetis. These proteases are calcium-dependent serine proteases with high optimal pH, crucial for parasite invasion.
Area of Science:
- Marine biology
- Parasitology
- Biochemistry
Background:
- Pathogenic protozoa utilize secreted proteases for host invasion.
- Understanding parasite proteases is key to controlling infections.
Purpose of the Study:
- To identify and characterize proteases secreted by the clam parasite Pseudoperkinsus tapetis.
- To compare these proteases with those of related oyster pathogens.
Main Methods:
- Culturing Pseudoperkinsus tapetis in vitro.
- Analyzing extracellular products (ECP) using SDS-PAGE and gelatin SDS-PAGE.
- Enzyme inhibition assays to determine protease family and optimal pH.
Main Results:
- Protease activity and protein concentration in ECP increased over 3 weeks.
- SDS-PAGE revealed new protein bands at 10, 12, and 157 kDa.
- Five active protease bands (23-96 kDa) were identified as Ca(2+)-dependent serine proteases with optimal pH > 9.4.
- Protease activity differed from Perkinsus marinus.
Conclusions:
- Pseudoperkinsus tapetis secretes unique Ca(2+)-dependent serine proteases.
- These proteases likely play a role in clam parasitism.
- The findings highlight distinct proteolytic mechanisms between related marine parasites.