Protease activity and host cell binding of the 42-kDa rhoptry protein fromToxoplasma gondii after secretion

Three proteases were identified in the excretory/secretory proteins (ESP) f rom Toxoplasma gondii by the gelatin acrylamide gel electrophoresis (GAGE), of which the molecular masses were 80, 70, and 42 kDa. One of the protease s with 42 kDa was reactive to a monoclonal antibody (mAb), Tg786 clone, whi ch was localized in the rhoptry of T. gondii by immunohistochemistry. The p rotease was maximally active at the pH range between 7.5 and 8.5, and was s ensitive to inhibition by TPCK and EGTA. The gelatinolytic activity of the protease was dependent on the concentration of calcium ion. The protease wa s active only in the millimolar ranges of calcium but not in micromolar ran ges, implicating that the secretion is critical event for the activation of the protease. The secreted protease was shown to bind to the host cells up on Western blot and immunofluorescence analysis. It is suggested that the p rotease 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 t he mechanism of entry into host cells. (C) 2001 Academic Press.