Structural insights into an atypical secretory pathway kinase crucial for Toxoplasma gondii invasion

Abstract

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.

Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways

“…PKA-C1 is thought to antagonize Ca 2+ signaling in T. gondii ( Jia et al, 2017 ; Uboldi et al, 2018 ). RON13 is a secretory-pathway kinase that phosphorylates substrates in apicomplexan invasion-associated organelles called rhoptries ( Lentini et al, 2021 ). The function of the remaining two proteins is unknown, although TGGT1_286710 contains a zinc finger domain and TGGT1_309290, annotated as a hypothetical protein, contains an HD/PDEase domain—both contribute to parasite fitness in cell culture ( Sidik et al, 2016a ).…”

“…TGGT1_286710 and TGGT1_309290 localized to the parasite nucleus, with TGGT1_286710 exhibiting additional staining in the residual body. Finally, RON13 appeared to stain the rhoptries ( Lentini et al, 2021 ). To validate the Ca 2+ -dependent stability of individual candidates, we prepared parasite extracts as described earlier, but relied on an immunoblot readout instead of MS.…”

Ceramide biosynthesis is critical for establishment of the intracellular niche of Toxoplasma gondii

“…Using antibodies specific to phosphorylated STAT6 (STAT6-P), its detection in infected host nuclei confirmed the results obtained with RON4 ( Figures 3 L and S5 D). Finally, the rhoptry protein toxofilin, fused to β-lactamase (BLA) and a hemagglutinin (HA) epitope tag, was expressed in Tgspt1-ko and in the RH parental strain to assess rhoptry discharge via fluorescence resonance energy transfer (FRET)-based BLA assay, as described previously ( Lentini et al., 2021 ; Lodoen et al., 2010 ). The transgenic parasites were characterized by WB and IFA to confirm expression of toxofilin BLA-HA ( Figures S5 E and S5F).…”

De novo mapping of the apicomplexan Ca2+-responsive proteome

“…PKA-C1 is thought to antagonize Ca 2+ signaling in T. gondii (Jia et al ., 2017; Uboldi et al ., 2018b) . RON13 is a secretory-pathway kinase that phosphorylates substrates in apicomplexan invasion-associated organelles called rhoptries (Lentini et al, 2021). The function of the remaining two proteins is unknown, although TGGT1_286710 contains a zinc finger domain and TGGT1_309290, annotated as a hypothetical protein, contains an HD/PDEase domain—both were indispensable for parasite survival (Sidik et al, 2016a).…”