Unveiling the role of conoid gliding protein in Toxoplasma gondii invasion and egress: insights from cryogenic correlative light and electron microscopy (cryo-CLEM)

Toxoplasma gondii exhibits numerous cellular adaptations facilitating the invasion of host cells and the subsequent egress from them after multiplication inside of parasitophorous vacuoles. The apical complex comprised of cytoskeletal machinery and secretory organelles is pivotal in these processes. While the overall morphology of the parasites and the apical complex have been extensively studied using various techniques [1-3], the mechanistic understanding and structural composition of these components remain elusive. In a previous study, we identified novel genes implicated in invasion and egress, notably cgp, which encodes the conoid gliding protein. This protein colocalizes with RNG2, an integral component of Toxoplasma"s conoid. Conditional knockout of the cgp gene resulted in significantly reduced egress rates compared to wild-type parasites. To elucidate the impact of cgp depletion on the structure of the conoid and to gain insight into the underlying molecular mechanisms, we developed cryogenic correlative light and electron microscopy (cryo-CLEM) workflows to target and visualize the molecular architecture of the conoid within intact, cryo-preserved parasites. Our in situ cryo-electron tomography analyses revealed a loss of PCR structure in cgp knockout parasites compared with the wild-type phenotype, while other components of the conoid remained unaffected. Our results show the structural role of cgp protein for proper PCR formation and demonstrate the potential of cryo-CLEM targeted in situ tomography for studying the architecture and life cycle of native apicomplexa at unprecedented detail.

1) Tosetti et al., Essential function of the alveolin network in the subpellicular microtubules and conoid assembly in Toxoplasma gondii, eLife, 2020

2) Koreny et al., Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species, PLoSBiol, 2021

3) Gui et al., Cryo-tomography reveals rigid-body motion and organization of apicomplexan invasion machinery, Nature Communications, 2023