Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

Abstract

Limited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondii accompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasma tachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.Members of the phylum Apicomplexa are obligate intracellular parasites that replicate in a variety of cell types. Some, including the human pathogens Plasmodium and Babesia, replicate primarily in the bloodstream, whereas others such as Cryptosporidium, a cause of chronic gastritis among the immune-compromised, and Eimeria, an agricultural parasite, replicate in the intestinal epithelium. Only parasites in the isosporoid coccidian clade, which includes Toxoplasma gondii, the causative agent of toxoplasmosis, replicate in deep tissues (2). Thus, it can be expected that each clade has its own complement of proteins facilitating survival in a specific habitat, along with conserved proteins that play fundamental roles in events common to all apicomplexans.Many invasion studies have been performed in T. gondii, as it is more amenable to in vitro manipulation than other members of the Apicomplexa. Upon contact with a host cell, T. gondii tachyzoites discharge the contents of apically localized microneme (MIC) organelles (13). MIC adhesive proteins contribute to binding host cell receptors, and blocking micronemal secretion dramatically reduces invasion (9). These proteins, which often cluster into multiunit complexes, are transiently deployed to the apical surface during apical attachment and invasion. Transmembrane (TM) MIC proteins, such as MIC2, MIC6, and MIC8, are thought to act as bridging molecules that connect host receptors with the parasite's motility apparatus, the glideosome (37). By translocating MIC-receptor complexes backwards toward the posterior end, the parasite "pulls" itself into the target cell, invaginating the host plasma membrane to form the nascent parasitophorous vacuole (PV). As they treadmill posteriorly,...

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

“…A distinct role in immune modulation has also been recently proposed for TgSUB1 processing of TgMIC4, which generates a galactosebinding product (TgMIC4 20 ) from the C terminus of TgMIC4 (42). Earlier work revealed a function for TgMIC5 in regulating TgSUB1 activity (16), but the molecular mechanism of regulation remained unclear. Our current findings strongly suggest that TgMIC5 suppresses TgSUB1 activity directly by mimicking the inhibitory mechanism of a subtilisin propeptide.…”

“…Several other unidentified proteins also seem to be degraded upon secretion from TgMIC5-deficient parasites. Additionally, whereas TgSUB1 processes TgMIC4 into TgMIC4 50 and TgMIC4 20 in WT parasites, it hyperprocesses TgMIC4 into a new species, TgMIC4 18 , in the absence of TgMIC5 (16). That TgMIC5 parasites do not appear to have an invasion or virulence phenotype might be the result of offsetting effects: higher efficiency trimming of the major adhesin TgMIC2 promotes parasite attachment and invasion whereas hyperprocessing or degradation of other substrates diminishes their functionality.…”

“…TgMIC5 Is Associated with the Parasite Surface via Its Interaction with TgSUB1-TgMIC5 resides on the parasite surface during cell invasion despite not possessing a membrane anchor (16,17), raising the question of how TgMIC5 is retained on the parasite surface. Our finding that TgMIC5 interacts with and inhibits TgSUB1 suggests a possible mechanism of surface retention via TgSUB1.…”

“…Using gene knock-out and proteome profiling Brydges et al (16) showed that the protease activity of TgSUB1 is negatively regulated by another microneme protein, TgMIC5. TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by the signal peptidase before being further processed in a post-Golgi compartment to the mature protein (17).…”

“…Immunoprecipitation experiments failed to identify partners of TgMIC5, thus limiting further mechanistic insight of its role in regulating TgSUB1 proteolysis (16).…”