Ionophore-stimulation promotes re-organization of the invasion machinery of Toxoplasma gondii
Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa, is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of Mixed Scale Dense networks for image-processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry, that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization. Significance StatementParasites in the phylum Apicomplexa are responsible for some of the most important parasitic diseases of humans, such as malaria and toxoplasmosis. Invasion by these obligatory, intracellular parasites depends on protein injection into the host cell. Using cryogenic electron tomography, we reveal evolutionarily conserved features shared by the invasive forms of Plasmodium falciparum and Toxoplasma gondii. By comparing resting Toxoplasma tachyzoites with those primed to invade we also gain new insight into the very first steps in invasion. For this work, we take an interdisciplinary approach, adopting a mixed-scale dense neural network that enables efficient and objective processing of the data. Combined, the results provide new information on how these important parasites accomplish the essential step of invasion.