Molecular and cellular investigation of subtilisin -like serine proteases in Toxoplasma gondii

Abstract

Toxoplasma gondii is an intracellular parasite dependent on its ability to invade and replicate within host cells. We have characterized the function and specificity of TgSUB1 and TgSUB2, subtilisin-like serine proteases (subtilases) of Toxoplasma gondii.;TgSUB1 localizes to the micronemes, secretory organelles involved in host cell invasion. In addition to its signal peptide, prodomain, and catalytic domain, TgSUB1 contains a proline rich domain and GPI anchor. The prodomain appears to be essential for TgSUB1's microneme localization, and deletion of the prodomain leads to retention of TgSUB1 in the secretory pathway. Proper localization of TgSUB1 is also influenced by the promoter and proteolytic processing.;TgSUB1 knockout parasites invaded normally and had intracellular growth rates similar to control parasites. TgSUB1KO parasites were mildly attenuated, displaying a one-day delay in median time to death compared with control parasites. TgSUB1KO parasites show defects in extracellular microneme protein processing indicating that TgSUB1 is microneme processing protease 2 (or MPP2).;TgSUB2 is found in the rhoptries, organelles important for generation of the parasitophorous vacuole. Prior studies suggested that TgSUB2 is an essential protein critical for rhoptry organelle biogenesis. To define the substrate specificity of TgSUB2, we utilized three strategies. The ROP1 cleavage motif (SphiXE) has been identified in TgSUB2 and numerous other rhoptry proteins. Autocatalytic processing of TgSUB2 was affected by mutagenesis of these putative cleavage sites. Additionally, serpins based on the ROP1 cleavage site were tested for their ability to inhibit TgSUB2 processing. Serpins were found to affect T. gondii intravacuolar growth. Finally, we expressed TgSUB2 in the baculovirus expression system. The recombinant protein appears to be active, and attempts at purifying active enzyme are underway to test for cleavage of TgSUB2 and ROP1 based peptides.;In summary, we have characterized two serine proteases of T. gondii. TgSUB1 appears cleave other microneme proteins during host cell invasion while TgSUB2 appears to play a role in maturation of proteins found in rhoptries. Although further studies are needed, this work suggests that subtilases in T. gondii have important biological functions and could be important drug targets.