Subtilisin-like proteases TgSUB1 and TgSUB2 in Toxoplasma gondii: Involvement in specialized parasite secretory organelles used for host cell invasion
Miller, Steven Andrew
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Toxoplasma gondii is an obligate intracellular protozoan parasite causing clinically significant disease in immunocompromised hosts. Host cell invasion by T. gondii involves sequential secretion from organellar micronemes, rhoptries, and dense granules. Several rhoptry and microneme proteins are proteolytically processed prior to or during invasion. Serine protease inhibitors block invasion of host cells by T. gondii and the related malaria parasite Plasmodium falciparum, and may represent a new class of chemotherapeutic agents for these infections.;We have identified two subtilisin-type serine proteases in T. gondii. TgSUB1 is cleaved early within the ER, packaged to micronemes, and secreted during attachment to host cells. Further proteolytic processing of TgSUB1 occurs coincident with secretion and is sensitive to serine protease inhibitors. TgSUB1 is a candidate microneme protein maturase.;The second T. gondii subtilisin protease, TgSUB2, is processed intracellularly and found in rhoptries which secrete during formation of the parasitophorous vacuole. Attempts to knock out the TgSUB2 gene were unsuccessful indicating that this may be an essential gene. Antisense reduction of TgSUB2 protein levels resulted in an increased replication time although these parasites were able to invade host cells normally. Ultrastructure of parasites transformed with antisense TgSUB2 revealed a block in early stages of cell division with abnormal morphology. These parasites contain few rhoptries and the observed structural changes are consistent with a lag in the cell cycle due to delayed rhoptry formation. These data indicate a role for TgSUB2 in rhoptry protein maturation and rhoptry biogenesis.