| Toxoplasma gondii is an obligate intracellular parasite of medical importance to both humans and animals. Host cell invasion is a critical step in the life cycle and relies on the secretion of proteins found in the secretory micronemes. The microneme protein MIC2, which is found in a complex with MIC2-associated protein (M2AP), plays central roles in both adhesion and invasion. Genetic disruption of M2AP (m2apKO) caused MIC2 retention in the Golgi apparatus, which resulted in decreased MIC2 secretion from the micronemes, reduced invasion efficiency, and reduced virulence in vivo. The aberrant function of MIC2 in m2apKO parasites highlighted the importance of formation of the MIC2/M2AP complex. Thus, we undertook studies using deletion mutants to define the major domains required for this interaction. We found that the thrombspondin (TSP)-containing M-domain of MIC2 is required and the large globular beta-domain of M2AP is sufficient for complex formation in both mammalian cells and in T. gondii.; Full-length MIC2 is a demonstrated adhesin, although the contributions of the individual adhesive domains had not been previously assessed. Using recombinant protein we demonstrated that the integrin-like A-domain binds to heparin sulfate as a high molecular weight form, corroborating evidence that the parasite uses glycosaminoglycans for adherence.; A defining feature of M2AP is the presence of a propeptide that is removed beyond the medial-trans Golgi interface in a region approximating the compartment in which MIC2 is retained in m2apKO parasites. Therefore, we hypothesized that the propeptide and its processing influences complex trafficking. We generated parasites expressing M2APDeltapro, a mutant that lacks the propeptide, and parasites expressing M2AP P4-P4'(A), a mutant that is refractory to propeptide processing. We found that the absence of the propeptide results in secretory retention and, in the absence of proteolytic processing, complex assembly is impaired. Both mutant proteins cause impaired invasion and attenuated in vivo infections, emphasizing the importance of the propeptide and its processing. In addition to our analysis of the propeptide of M2AP, we characterized another microneme proprotein TgMIC11 that undergoes sequential proteolytic events to remove its internal propeptide. Because attachment and invasion are essential steps in the life cycle of T. gondii, we believe that proteins involved in these processes are promising drug targets. |
