Functional analysis of the apical polar ring its role in secretion and motility of Toxoplasma parasites

© 2017 Dr Nicholas Jeremy KatrisHuman parasites Toxoplasma and Plasmodium species belong to the phylum Apicomplexa and are some of the most successful groups of human parasites on the planet. Part of this success can be attributed to the cytoskeletal components that afford them structural stability and flexibility required to efficiently attach to and invade host cells. As members of the superfamily Alveolata, they possess a pellicle comprised of a set of flattened vacuoles pressed up against the plasma membrane, with proteinaceous support network and actin actin-based motility system. In addition to this, Toxoplasma also possesses an apical complex which is a tubulin based structure comprised of a set of apical polar rings and a conoid, which is a tight-knit tubulin based structure that is evolutionarily derived from ancestral flagella components. The apical complex is biologically significant because it is the entry point the parasite uses to enter a host cell in order to parasitize it, and this process is conserved in Plasmodium species. However, unlike other organelles, the proteins of the apical complex have no known conserved targeting signals so identification of proteins that target here has been slow to progress. A Toxoplasma protein homologous to a predicted cytoskeletal Tetrahymena thermophila protein was identified and localized to the apical complex, which we call RNG2. RNG2 was functionally characterized by inducible knock down and found that RNG2 played a role in the cGMP signalling pathway upstream of calcium dependent activation of CDPKs, which severely impacted microneme secretion, conoid extrusion and even other downstream processes, particularly internal calcium release. In addition to this, I used various calcium and cyclic di-nucleotide signalling agonists and inhibitors to investigate novel regulation patterns of micronemes and dense granules. RNG2 and other cGMP and calcium signalling proteins, PKG, CDPK1 and CDPK3 all show altered secretion of dense granules showing for the first time a regulatory mechanism of dense granules based on calcium