In situ ultrastructures of two evolutionarily distant apicomplexan rhoptry secretion systems

Abstract

Parasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite’s apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.

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

“…Closer inspection of the IMT-aligned AVs revealed a density partially surrounding the vesicle in the majority (75%) of the tomograms, with a similar density also observed surrounding the AVs positioned anterior to the IMTs (S7A-D Fig). Using the tomographic data to examine this structure more carefully revealed that in at least 31 out of 48 tomograms the density partially coats the AVs, and in 10 instances it resembled the rosette that was previously characterized as part of the RSA found between the rhoptry-interacting AV and the parasite’s plasma membrane [19] (S8 A-D Fig). The difference in our data from those previously reported is that we observe these rosette-like structures associated with more than one AV in a single AC and even around AVs that are IMT-associated, not just those that are anterior-most or interacting with a rhoptry tip.…”

“…The role of the AVs in rhoptry secretion has long been speculated [14, 30] and they were previously described in other apicomplexans, e.g. Cryptosporidium, Eimeria, Sarcocystis , and Besnoitia [15–17, 19, 31]. Although no direct connection was detected between the IMTs or their associated AVs and the conoid fibrils, we did observe that the AVs are not centered or positioned randomly in the intraconoid space; instead, most were positioned to the side, as viewed from the top of the specimen.…”

“…This method can visualize membranes and protein-based cellular structures in a near-native, frozen, hydrated state but it can be severely limited by the thickness and the inherently low contrast of biological samples. Recently, cryo-ET has proven successful in revealing the 3D organization of the subcellular organelles in the AC of Toxoplasma [8, 15, 19]; however, due to the transient nature of host cell invasion and the limited throughput of cryo-ET, it has not yet been possible to use this method to capture the invasion process itself. As an alternative to observing parasites in direct interaction with host cells, one can observe the biological machinery of extracellular parasites under internal calcium flux via stimulation with calcium ionophore that, among other things, primes the parasites for invasion by initiating protrusion of the conoid and microneme secretion.…”

Discovery of New Microneme Proteins in Cryptosporidium parvum and Implication of the Roles of a Rhomboid Membrane Protein (CpROM1) in Host–Parasite Interaction

“…and Toxoplasma gondii, sporozoites and merozoites are the invasive stages of C. parvum and possess a number of specialized subcellular structures, including rhoptries, micronemes, and dense granules (3)(4)(5)(6). Cryptosporidium parasites possess a secretory machinery composed of a single rhoptry, numerous micronemes, and a number of dense granules that are discharged during invasion (3,4,(7)(8)(9). The morphologies of the three secretory organelles in Cryptosporidium sporozoites and merozoites have been studied using transmission electron microscopy (TEM) (3,7,(9)(10)(11)(12).…”

“…Cryptosporidium parasites possess a secretory machinery composed of a single rhoptry, numerous micronemes, and a number of dense granules that are discharged during invasion (3,4,(7)(8)(9). The morphologies of the three secretory organelles in Cryptosporidium sporozoites and merozoites have been studied using transmission electron microscopy (TEM) (3,7,(9)(10)(11)(12). The rhoptry is a single flaskshaped organelle whose tip is attached to the apical end of the parasite, with its end extending to slightly less than a third of the zoite.…”