Protease activity and host cell binding of the 42-kDa rhoptry protein from Toxoplasma gondii after secretion
1Department of Parasitology and Catholic Institute of Parasitic Diseases, Catholic University of Korea, Seoul, Korea.
Abstract:
Three proteases were identified in the excretory/secretory proteins (ESP) from Toxoplasma gondii by the gelatin acrylamide gel electrophoresis (GAGE), of which the molecular masses were 80, 70, and 42 kDa. One of the proteases with 42 kDa was reactive to a monoclonal antibody (mAb), Tg786 clone, which was localized in the rhoptry of T. gondii by immunohistochemistry. The protease was maximally active at the pH range between 7.5 and 8.5, and was sensitive to inhibition by TPCK and EGTA. The gelatinolytic activity of the protease was dependent on the concentration of calcium ion. The protease was active only in the millimolar ranges of calcium but not in micromolar ranges, implicating that the secretion is critical event for the activation of the protease. The secreted protease was shown to bind to the host cells upon Western blot and immunofluorescence analysis. It is suggested that the protease may target to the plasma membrane of the host cells, which provides appropriate environment for the entry of the parasite into host cells. The mAb (Tg786) of T. gondii also reacted with a protein of the same size and equivalent locality of rhoptry in Neospora caninum, a similar Apicomplexan protozoa, suggesting that secreted protease mediates a common function in the mechanism of entry into host cells.
Insights
Researchers identified three proteases in Toxoplasma gondii excretory/secretory proteins (ESP). A 42 kDa protease, localized to the rhoptry, showed calcium-dependent activity and bound host cells, suggesting a role in parasite invasion.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Toxoplasma gondii is an Apicomplexan parasite that invades host cells.
- The invasion mechanism involves parasite secreted proteins, including proteases.
- Understanding these proteases is crucial for developing anti-parasitic strategies.
Purpose of the Study:
- To identify and characterize proteases in T. gondii excretory/secretory proteins (ESP).
- To investigate the role of a specific 42 kDa rhoptry protease in host cell interaction and invasion.
Main Methods:
- Gelatin acrylamide gel electrophoresis (GAGE) for protease identification.
- Monoclonal antibody (mAb) reactivity and immunohistochemistry for localization.
- Enzyme activity assays (pH, inhibitors, calcium dependence).
- Western blot and immunofluorescence for host cell binding analysis.
Main Results:
- Three proteases (80, 70, 42 kDa) identified in T. gondii ESP.
- A 42 kDa protease, reactive to mAb Tg786, localized to the rhoptry.
- This protease exhibited optimal activity at pH 7.5-8.5, was inhibited by TPCK and EGTA, and required millimolar calcium concentrations.
- The secreted protease bound to host cells, suggesting interaction with the plasma membrane.
- Homologous protein detected in Neospora caninum rhoptry.
Conclusions:
- A calcium-dependent 42 kDa rhoptry protease is secreted by T. gondii.
- This protease likely plays a role in host cell binding and invasion.
- The findings suggest a conserved mechanism of host cell entry among Apicomplexan parasites.
