Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion

Abstract

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there is little data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites. We discovered that conoid fibre number varied, but there was a fixed spacing between fibres, leading to conoids of different sizes. Associated apical structures varied in size to accommodate a larger or smaller conoid diameter. However, the number of subpellicular microtubules on the apical polar ring surrounding the conoid did not vary, suggesting a control of apical complex size. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins (ROPs) may be trafficked between the parasite and the host cytoplasm

Rhoptry secretion system structure and priming in Plasmodium falciparum revealed using in situ cryo-electron tomography

“…1 and Extended Data Fig. 6 ) compared to that of other apicomplexan parasites 8 , 9 , 23 , 26 . Recent comparative analyses between the apicomplexan rhoptry secretion system and the extrusome system in the related, free-living Ciliata phylum 27 suggests that the AV is an adaptation in Apicomplexa for parasitism 7 , 8 .…”

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

“…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.…”

“…In Apicomplexa, an AV is sandwiched between the rosette and the rhoptries and it was recently suggested that these AVs facilitate the docking of a rhoptry to the parasite membrane through a newly described entity named the rhoptry secretory apparatus (RSA) [19]. The role of the AVs in rhoptry secretion has long been speculated [14, 34], and they were previously described in Toxoplasma, Cryptosporidium, Eimeria, Sarcocystis, and Besnoitia [15–17, 19, 35]; here, we extend their detection to Plasmodium . We observed that upon treatment of Toxoplasma tachyzoites with a calcium-ionophore, a commonly used stimulator of egress and invasion, a distended AV was seen fused with a rhoptry tip, and this connection appears to be stable enough to be captured in most such tomograms.…”

An apical membrane complex controls rhoptry exocytosis and invasion in Toxoplasma

“…Delivery requires docking and fusion of the organelle with the parasite plasma membrane (PPM); this process of exocytosis is coupled with the translocation of rhoptry content across the host plasma membrane (HPM). The latter likely involves the formation of a pore at the junction between the PPM and HPM (Burrell et al, 2021; Dubremetz, 1998; Hanssen et al, 2013; Nichols et al, 1983; Suss-Toby et al, 1996), but its nature and composition are unknown. Excitingly, recent studies revealed new insights into structure and molecular players essential for the exocytic step (Aquilini et al, 2021; Mageswaran et al, 2021; Suarez et al, 2019).…”