Coupling cryo-electron tomography with mixed-scale dense neural networks reveals re-organization of the invasion machinery of Toxoplasma gondii upon ionophore-stimulation

Abstract

Host cell invasion by intracellular, eukaryotic parasites, like the many important species within the phylum Apicomplexa, is a remarkable and active process involving the coordinated action of many apical organelles and other structures. To date, capturing how these various structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to generate images of the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through addition of a calcium ionophore. Using AI-based image-processing we were able to annotate 48 tomograms to identify and extract densities of the relevant subcellular organelles and accurately analyze features in 3D. We describe an interaction between an anteriorly located apical vesicle and a rhoptry tip that occurs only in the ionophore-stimulated parasites and that is associated with dramatic changes in the vesicle's shape in what appears to be a stalled fusion event. We also present information to support the presumption that this vesicle originates from the well-described vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. Lastly, we show that a previously described rosette is found associated with more than just the anterior-most apical vesicle, indicating that multiple such vesicles are primed to enable rhoptry secretion.

A central protein complex essential for Invasion inToxoplasma gondii

“…More recently the rhoptry secretion system has been characterized with electron tomography in multiple species of apicomplexa 19 , 20 , 21 , but integration of this structural information and single protein function is still in its infancy.…”

How Apicomplexa Parasites Secrete and Build Their Invasion Machinery

“…In Toxoplasma, micronemes accumulate beneath the conoid-an apical truncated-cone-shaped structure (99)-or at the lateral periphery. A few micronemes are also visible engaged inside the conoid (42,57,88,125) and one microneme vesicle has been recently shown by cryo-electron tomography (cryo-ET) (57) to be docked at the exocytic site, close to but distinct from the rhoptry exocytic site described below (see the section titled Structural Elements of the Rhoptry Secretion Apparatus Pinpointed by Cryo-ET). Microneme exocytosis is likely driven by a fusion process during which the vesicles completely merge with the PPM.…”

“…In Cryptosporidium, the single rhoptry can be imaged either in a prefusion state with the AV in the absence of any exocytosis trigger or not fused at all (88). In Toxoplasma, the AV shape changes upon calcium ionophore treatment, which might suggest a prefusion state (125). In Plasmodium merozoites, two additional states have been observed by cryo-ET (90), both with the AV absent.…”

“…In T. gondii, three to six vesicles accumulate within the conoid in close association with a pair of intraconoidal microtubules (ICMTs) (57,88,101,125) (Figure 2a). Referred to as microtubuleassociated vesicles (MVs), they are similar in shape and size to the AV and are coated with a discontinuous layer of protein density, similar to the one observed on the AV ( 88).…”

“…Recent technical breakthroughs have shed light on the structure of the RSA (88) but also more broadly on the organization of the apex of the parasites (21,24,57,125,139,156). Some molecular players of the rhoptry exocytosis process have been identified, but further mapping of these factors onto the in situ structure of the RSA will be necessary to provide an insightful understanding of this unique eukaryotic secretion machinery.…”