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.

Apicomplexa are obligatory intracellular parasites that sense and actively invade host cells. Invasion is a conserved process that relies on the timely and spatially controlled exocytosis of unique specialized secretory organelles termed micronemes and rhoptries. Microneme exocytosis starts first and likely controls the intricate mechanism of rhoptry secretion. To assemble the invasion machinery, micronemal proteins—associated with the surface of the parasite—interact and form complexes with rhoptry proteins, which in turn are targeted into the host cell. This review covers the molecular advances regarding microneme and rhoptry exocytosis and focuses on how the proteins discharged from these two compartments work in synergy to drive a successful invasion event. Particular emphasis is given to the structure and molecular components of the rhoptry secretion apparatus, and to the current conceptual framework of rhoptry exocytosis that may constitute an unconventional eukaryotic secretory machinery closely related to the one described in ciliates. Expected final online publication date for the Annual Review of Microbiology, Volume 76 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Apicomplexan parasites secrete contents of the rhoptries, club-shaped organelles in the apical region, into host cells to permit their invasion and establishment of infection. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium . It is unknown if a similar molecular machinery exists in the malaria parasite Plasmodium . In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P . falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct apical vesicle docking the tips of the two rhoptries to the RSA. We also discover two additional rhoptry organizations that lack the apical vesicle. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to different rhoptry organizations. Our results highlight previously unknown steps in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.