SummaryHost cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with fulllength TgSUB1 restores processing, complementation of Dsub1 parasites with TgSUB1 lacking the GPI anchor (Dsub1::DGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Dsub1 and Dsub1::DGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.

Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.

The Apicomplexan parasites Toxoplasma gondii and Plasmodium species are obligate intracellular parasites that rely upon unique secretory organelles for invasion and other specialized functions. Data is emerging that proteases are critical for the biogenesis of micronemes and rhoptries, regulated secretory organelles reminiscent of dense core granules and secretory lysosomes of higher eukaryotes. Proteases targeted to the Plasmodium food vacuole, a unique organelle dedicated to hemoglobin degradation, are also critical to parasite survival. Thus study of the targeting and function of the proteases of the Apicomplexa provides a fascinating model system to understand regulated secretion and secretory organelle biogenesis.

Subtilisin‐like proteases have been proposed to play an important role for parasite survival in Toxoplasma gondii (Tg) and Plasmodium falciparum. The T. gondii subtilase TgSUB1 is located in the microneme, an apical secretory organelle whose contents mediate adhesion to the host during invasion. TgSUB1 is predicted to contain a glycosyl‐phosphatidylinositol (GPI) anchor. This is unusual as Toxoplasma GPI‐anchored proteins are targeted to the parasite’s surface. In this study, we report that the subtilase TgSUB1 is indeed a GPI‐anchored protein but contains dominant microneme targeting signals. Accurate targeting of TgSUB1 to the micronemes is dependent upon several factors including promoter strength and timing, accurate processing and folding. We analyzed the targeting domains of TgSUB1 using TgSUB1 deletion constructs and chimeras made between TgSUB1 and reporter proteins. The TgSUB1 prodomain is responsible for trafficking to the micronemes and is sufficient for targeting a reporter protein to the micronemes. Trafficking is dependent upon correct folding or other context‐dependent conformation as the prodomain expressed alone is unable to reach the micromenes. Therefore, TgSUB1 is a novel example of a GPI‐anchored protein in T. gondii that bypasses the GPI‐dependent surface trafficking pathway to traffic to micronemes, specialized regulated secretory organelles.