Although the exact molecular actors are still being defined, it is clear proteases play an essential role in host cell invasion by the Apicomplexa (reviewed in (8)). The serine protease inhibitors DCI and AEBSF (9) and cysteine protease inhibitors (10) prevent T. gondii tachyzoites from penetrating host cells. Inhibition is not caused by a change in morphology or an effect on gliding motility of the parasite (9). Invasion by Plasmodium is prevented by a variety of serine and cysteine protease inhibitors that probably affect different steps in invasion (reviewed in (11,12)). Proteolytic processing of microneme secretory proteins, PfAMA1 and TgMIC2, and malaria surface protein MSP-1 have been shown to be essential for host cell invasion (13).
Proteolysis during microneme formation and secretion
Micronemes are the smallest of the secretory organelles. Involved in the early stages of invasion, they contain many adhesins that help in the tight attachment of the parasite to the host cell. Microneme secretion is a calcium-dependent process and is enhanced by manipulations that increase intracellular calcium (14,15).
Microneme proteins assemble as macromolecular adhesion complexes. Microneme proteins that are type I integral membrane proteins contain microneme targeting signals in their C-terminal domains (16,17). The membrane proteins form a complex with soluble microneme proteins (17). Proper assembly of these complexes is required for exit from the ER/Golgi and targeting to micronemes (17,18). The MIC2/M2AP complex appears to be a major adhesive complex mediating invasion (18,19) but others such as the MIC1/4/6 complex are not essential (17).
Most microneme complexes have one member that is proteolytically cleaved during transit through the secretory pathway. Proteolysis is required for trafficking of M2AP and MIC5 (cited in (20)). Proteolytic cleavage of MIC3 is required for its adhesive properties (21) and deletion of its prodomain results in MIC3 that is retained within the secretory pathway (22). Proteolysis within the secretory pathway appears to regulate maturation of microneme contents and formation of micronemes, much as it appears to regulate formation of secretory granules in other eukaryotes (4–6).
As invasion occurs, T. gondii microneme proteins are secreted onto the parasite's surface and move towards the posterior end before being shed from the surface by a number of unidentified proteases (8). The interaction of the treadmilling proteins with the actin–myosin complex provides the force for host cell invasion. Mutation of sites cleaved during invasion does not affect trafficking to micronemes (23). The adhesive complexes are shed from the parasite surface as invasion proceeds and proteolysis at this phase is hypothesized to be a mechanism to inactivate the adhesins (24). Studies with protease inhibitors suggest that different proteases are involved in each of these steps and implicate both cysteine and serine proteases.
Proteolysis during rhoptry formation
Rhoptries are long club-shaped organelles that secrete proteins through their elongated necks at the apical tip of the parasite. In Toxoplasma, secretion of the micronemes precedes rhoptry exocytosis (25), but biological triggers of rhoptry secretion have not been identified. In Plasmodium, the sequence of secretory events is less firmly established, but probably proceeds similarly. Secretion coincides with formation of the parasitophorous vacuole (PV), where the parasite resides inside of host cells. The PV is mainly composed of host cell lipids (26). Rhoptry proteins and lipids, however, modify the vacuole, enabling T. gondii to subsist in the host cell without fusing with other organelles or acidifying, and prevent the parasite from being degraded by host cell lysosomes (27). Although rhoptries are thought to be similar in all the Apicomplexa, there is little homology among the characterized rhoptry proteins of T. gondii and malaria.
The rhoptries are the only known acidified organelle in T. gondii (28). Rhoptries are composed of lipids and protein and have a high cholesterol lipid content compared with the T. gondii plasma membrane (29). Rhoptries have internal membranes and type I transmembrane proteins ROP2/3/4 and TgSUB2 localize to the lumen of rhoptries rather than their periphery (2,30). Orthologs of the trafficking machinery for multivesicular body formation are present in T. gondii and malaria species and appear to be functionally conserved (3).
T. gondii ROP1 was originally identified as a component of ‘penetration enhancing factor’, but no definite function has been attributed to it (31). ROP1 is not essential, but rhoptries of T. gondii tachyzoites lacking ROP1 have a denser appearance (32,33). ROP2, the founding member of the ROP2/3/4/8 family, has been shown to mediate recruitment of host cell mitochondria and ER (34). Despite the existence of multiple other family members, ROP2 appears to be essential (34).
ROP2 targeting to rhoptries involves the clathrin/AP1 machinery and is mediated by YXXΦ and LL motifs in its cytoplasmic tail (35). There appear to be redundant rhoptry targeting signals as the prodomain of ROP4 (a ROP2 family member), as well as multiple segments of ROP1, are sufficient to target heterologous proteins to rhoptries (22,36,37).
Most characterized T. gondii rhoptry proteins are proteolytically cleaved during transit in the secretory pathway. In T. gondii only the cleavage site of ROP1 has been definitively mapped (38). Surprisingly, trafficking of ROP1 does not seem to be dependent on proteolytic processing since unprocessed ROP1 is correctly trafficked to rhoptries and secreted into the parasitophorous vacuole during invasion (37,39). It is unknown whether proteolytic processing of other rhoptry proteins affects their trafficking.
Many Plasmodium rhoptry proteins are associated in macromolecular complexes in immunoprecipitation studies, including the RAP1/2/3 complex and the RhopH1/H2/H3 complex (reviewed in (40)). Trafficking of RAP2/3 to the rhoptries is dependent upon association with RAP1 C-terminus, but the RAP1/2/3 proteins are not essential (41). Several Plasmodium rhoptry proteins including RAP1, RhopH 1 and RAMA are proteolytically cleaved, but the significance of processing has not been explored. RAP1 is proteolytically cleaved during maturation. The cleavage site for RAP1 (after A190 (40)) does not have homology with the T. gondii ROP1 cleavage site.
Once the parasite safely resides in the PV, the third secretory organelles, dense granules, secrete. T. gondii dense granule secretion is constitutive, but is also likely to have a regulated component (25,42). Dense granules represent the default pathway for secretion (43). There are no known signals for release. Components of the dense granules modify the PV and also associate with a vesicular network within the PV (42). Proteolytic processing of dense granule contents of T. gondii has not been described.