An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma

Abstract

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.

A conserved complex of microneme proteins mediates rhoptry discharge inToxoplasma

“…Only two groups of surface-exposed proteins, MIC8 and the CRMPs, have been specifically associated with rhoptry discharge in T. gondii (Kessler et al , 2008; Singer et al , 2022; Sparvoli et al , 2022). We previously showed that CLAMP depletion does not affect secretion of the microneme protein MIC2 (Sidik et al , 2016b).…”

“…MIC8, a prototypical single-pass transmembrane microneme protein, was the first factor shown to be specifically required for rhoptry discharge in T. gondii (Kessler et al , 2008). More recently, two large multipass transmembrane proteins (CRMPa and CRMPb) were similarly associated with rhoptry discharge and shown to form a complex with two single-pass microneme proteins bearing thrombospondin domains (Singer et al , 2022; Sparvoli et al , 2022). Although the CRMP complex proteins do not localize to micronemes unambiguously (Singer et al , 2022; Barylyuk et al , 2020), they accumulated at the apical end of extracellular parasites (Sparvoli et al , 2022).…”

“…More recently, two large multipass transmembrane proteins (CRMPa and CRMPb) were similarly associated with rhoptry discharge and shown to form a complex with two single-pass microneme proteins bearing thrombospondin domains (Singer et al , 2022; Sparvoli et al , 2022). Although the CRMP complex proteins do not localize to micronemes unambiguously (Singer et al , 2022; Barylyuk et al , 2020), they accumulated at the apical end of extracellular parasites (Sparvoli et al , 2022). Despite extensive analysis through co-immunoprecipitation, there is no evidence that MIC8 and the CRMP complex interact with one another or with the CLAMP complex, suggesting the three independently contribute to rhoptry discharge.…”

“…Adhesion complexes are translocated rearward along the parasite surface to produce forward motility (Frénal et al , 2017). Some microneme proteins have dedicated roles during invasion; in T. gondii , the microneme protein MIC8 and the microneme-associated CRMP complex are required for rhoptry effector secretion, but not parasite motility (Sparvoli et al , 2022; Kessler et al , 2008; Singer et al , 2022). After secretion, some rhoptry effectors rewire host cell signaling to prevent innate immunity, while other effectors, notably the components of the rhoptry neck (RON) complex, prepare the site of rhoptry secretion for parasite invasion.…”

“…Apicomplexans, therefore, appear to have adapted this conserved secretion machinery to their parasitic lifestyles. The CRMP proteins—required for rhoptry discharge but not the assembly of the apical vesicle or RSA—are broadly conserved among Alveolata much like the Nd proteins (Sparvoli et al , 2022) and bind additional microneme proteins in T. gondii (Singer et al , 2022). MIC8, a microneme protein required for rhoptry discharge in T. gondii , is limited to coccidians (Kessler et al , 2008) suggesting apicomplexans have clade-specific determinants of rhoptry discharge that potentially tune the process to their respective niches.…”