Toxoplasma gondii is a single-celled obligate intracellular parasite which is estimated to infect 30% of the global human population. While T. gondii infections are typically asymptomatic in healthy individuals, they can cause life-threatening complications in immunocompromised individuals and congenitally infected neonates. As current treatments are toxic and do not clear the lifelong chronic infection, a deeper understanding of parasite biology is needed to identify novel targets for therapeutic intervention. As a member of the phylum Apicomplexa, T. gondii contains several unique organelles which play critical roles in facilitating the parasite’s lytic cycle, which causes the acute phase of disease. One of these unique apicomplexan organelles is the inner membrane complex (IMC), which lies directly underneath the parasite’s plasma membrane and is composed of a series of flattened vesicles supported by an underlying cytoskeletal network. The IMC plays essential roles throughout the T. gondii lytic cycle by serving as the platform for the molecular machinery that controls parasite motility, stabilizing the apical complex which facilitates host cell invasion, and acting as a scaffold for developing daughter cells during parasite replication.
T. gondii replicates using a unique form of internal budding called endodyogeny, in which two daughter buds are formed within the cytoplasm of a single maternal cell. Endodyogeny can be divided into four steps: bud initiation, elongation, constriction, and maturation. During this process, IMC components are added to the developing daughter cell scaffold in a tightly regulated, sequential manner. While many of the components of the IMC are known to be important for parasite fitness, most are maintained in mature parasites and play critical roles in other phases of the lytic cycle besides replication. Several IMC proteins have been identified which are found only in the IMC of daughter buds, but most are dispensable or have only moderate impacts on parasite fitness. The identification of IMC32, a daughter-specific IMC protein which recruits during bud initiation and is essential for endodyogeny, led us to hypothesize that other unidentified daughter-specific IMC proteins coordinate with IMC32 to lay the foundation of the daughter cell scaffold.
Here, we report the discovery of an essential daughter bud assembly complex which lays the foundation for the daughter IMC in T. gondii. Using proximity labelling and protein-protein interaction screens, we identify two novel proteins, IMC43 and BCC0, as binding partners of IMC32. We analyze the function of each of these proteins using conditional knockdown systems which reveal that both IMC43 and BCC0 are essential for endodyogeny. We additionally use deletion analyses and functional complementation to identify which regions of IMC43 and BCC0 are essential for localization and function. By employing pairwise yeast two-hybrid assays, we determine which regions of IMC32, IMC43, and BCC0 are involved in complex formation. Finally, we assess how loss of each complex component affects each of the others. These data allow us to develop a hierarchical model for complex assembly in which BCC0 depends on IMC32 for its localization during bud initiation, and IMC32 in turn depends on IMC43 for its localization later during bud elongation. Together, this work expands our understanding of how nascent daughter buds are assembled during endodyogeny and yields ample opportunities for future studies into the function and regulation of these essential proteins in both T. gondii and other parasites.