Preparing for an invasion: charting the pathway of adhesion proteins to Toxoplasma micronemes

Toxoplasma gondii is an apicomplexan parasite capable of infecting a broad host range including humans. The tachyzoite lytic cycle begins with active invasion of host cells involving the release of adhesive proteins from apical secretory organelles called micronemes. A protein complex consisting of the transmembrane adhesin MIC2 and a tightly associated partner, M2AP, is abundantly released from the micronemes. Similar to many proteins in a regulated secretory pathway, T. gondii proteins destined for micronemes and rhoptries (another secretory organelle associated with invasion) undergo proteolytic maturation. M2AP contains a propeptide that is removed in a post-Golgi compartment. By expressing an M2AP propeptide deletion mutant in the M2AP knockout background, we show that the propeptide is required for the MIC2-M2AP complex to exit from the early endosome. Although a cleavage-resistant M2AP mutant was able to efficiently reach the micronemes, it was unable to rapidly mobilize from the micronemes to the parasite surface. Strikingly, both mutants were unable to support normal parasite invasion and were partially attenuated in virulence to a degree that is indistinguishable from M2AP knockout parasites. Conditional expression of MIC2 showed that it is also required for correct M2AP sorting to the micronemes. These parasites were severely impaired in invasion efficiency. They switched almost exclusively to a non-productive circular gliding motility and were incapable of establishing an infection in mice when inoculated at a normally lethal dose. These findings underscore the importance of correct trafficking of invasion-related proteins. Our results also serve as a basis for future studies aimed at defining the branch points of protein sorting in T. gondii and at a deeper understanding of the precise roles of M2AP propeptide and MIC2 targeting motifs in MIC protein trafficking.