An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma (2022)
Figure 2

Fig 2. TgCRMPa and TgCRMPb are essential for rhoptry secretion and host cell invasion in Toxoplasma.

Immunoblot with anti-HA Abs of lysates from parental (Ctrl) and tagged lines (TgCRMPa-HA3 and TgCRMPb-HA3) together with inducible-knockdown lines (TgCRMPa-HA3_iKD and TgCRMPb-HA3_iKD) treated with ATc for 0, 24 or 48h. TgROP5 was used as loading control. Two close bands around 300kDa were detected for TgCRMPa. A ~820kDa protein, corresponding to the predicted size for TgCRMPb, was observed together with a ~130kDa band.

Immunofluorescence microscopy of intracellular parasites (untagged, TgCRMPa-HA3, TgCRMPb-HA3, and TgCRMPs-depleted (iKD) lines). Parasites were labeled with anti-HA and anti-AMA1 Abs to visualize CRMPs-HA3 and micronemes, respectively. The nuclei (DNA) are stained with Hoechst. DIC: differential interference contrast. TgCRMPs-HA3 shows a heterogeneous distribution within the parasite cytosol, occasionally showing a microneme-like apical gradient (yellow boxes), highlighted in (C). Shown are single focal planes

Images in yellow boxes shown in (B) with increased contrast and brightness.

Confocal immunofluorescence images of TgCRMPb-depleted (iKD) intracellular tachyzoites. Parasites were stained with anti-HA and with anti-AMA1 and anti-MIC2 Abs to visualize TgCRMPb and micronemes, respectively. The nuclei (DNA) are stained with Hoechst. Shown are single focal planes.

Quantification of plaques areas for control, TgCRMPa_iKD and TgCRMPb_iKD in absence of ATc, and upon 24 and 48h ATc treatment for TgCRMPb and TgCRMPa, respectively. Values are reported as mean ± SD (n=3 biological replicates, each with 3 technical replicates). The biological replicates are represented by different symbols.

Invasion of TgCRMPa- and TgCRMPb-depleted tachyzoites upon 48 and 24h treatment with ATc, respectively. Data are reported as in (E) (n=3 biological replicates, each with 3 technical replicates). The biological replicates are represented by different symbols.

Electron micrographs of TgCRMPa_iKD and TgCRMPb_iKD intravacuolar parasites treated with ATc for 48 and 24h, respectively. Micronemes (m) and rhoptries (Rh) appeared properly localized and shaped in both mutants.

Quantification of microneme secretion in TgCRMPa- and TgCRMPb-depleted tachyzoites was measured by detecting the processed form (arrowhead) of TgMIC2 (arrow) in the media. Control, TgCRMPa_iKD and TgCRMPb_iKD parasites, ATc-treated (+) and untreated (-), were stimulated with propranolol to release microneme contents. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.

Quantification of rhoptry secretion in TgCRMPa_iKD and TgCRMPb_iKD parasites upon 48 and 24h ATc-treatment, respectively, using the SeCreEt system (Koshy et al, 2010). Successful secretion of rhoptry proteins into the host causes a switch from red to green fluorescence in a reporter host cell line. CRMPs-depleted parasites were unable to efficiently deliver rhoptry content into the host cytosol. Data are represented as mean ± SD (n=3 biological replicates). The biological replicates are represented by different symbols. Data information: P values in E, F, I, were measured by two-tailed t-test.