Untangling the functional hierarchal architecture of the Toxoplasma gondii apical annuli proteins

The Toxoplasma gondii cytoskeleton contains a network of intermediate filaments, supporting a quilt of alveolar membrane compartments, together known as the inner membrane complex (IMC). Embedded within the IMC are the apical annuli: 5-6 donut shaped pores that facilitate secretion of dense granules. Annuli component LMBD3 spans from IMC to plasma membrane, as do several annuli localized SNARE proteins (Stx1, Stx20, Stx21). Here we describe a novel apical annuli protein, AAP7, which we functionally dissected by conditional knock-down (AAP7-cKD). AAP7 localizes to the annuli of mature parasites only, appearing when new daughters emerge. AAP7 is present in the second largest donut with an average diameter of 300 nm. However, in other parasites AAP7 is present in smaller diameter donuts, which hints at two distinct annuli pore states. Depletion of AAP7 causes a severe fitness defect but ~10% of parasites survive and develop a compensatory phenotype. To follow up on this resistance mechanism, we performed scRNA-seq to reveal any gene candidates that could be associated with overcoming the loss of AAP7. To further probe apical annuli function, we created a lethal double annuli deletion (DAD) strain, combining AAP7-cKD with a direct knock-out of AAP4 (AAP4-KO), the largest and most conserved annuli protein. DAD parasites have a slow death phenotype, and we are currently deciphering how the DAD parasites die. Dense granule secretion is reduced in the absence of AAP7, AAP4, and in the DAD parasites. In the absence of AAP4, annuli component Centrin2 is not recruited to the apical annuli. AAP7 is mis-localized in the AAP4-KO background, demonstrating AAP4 recruitment of AAP7. AAP2 is unaffected by the loss of AAP4, indicating independent recruitment. In the absence of AAP7, Centrin2 and AAP4 localize normally. Overall, our results demonstrate that Centrin2 and AAP7 recruitment to the annuli require AAP4, which is not essential to parasite viability. Both AAP4 and AAP7 are needed for the essential function of the apical annuli. Hence, we have provided insights into the functional hierarchy of apical annuli architecture and established the tool needed to probe the fitness-conferring mechanism of the T. gondii apical annuli.