First Characterization of the Neospora caninum Dense Granule Protein GRA9

Figure 7

(a) shows that the full-length sequence of NcGRA9 contains an N-terminal signal peptide (red), an alpha helix with amphipathic characteristics (blue), a hydrophobic alpha helix (yellow), and a PEST motif at the C-terminus of the protein (light blue). An HA-tagged version of the full-length protein and two C-terminal truncations (201, Δ280) were stably expressed in N. caninum tachyzoites. In (b) the localization was analysed by confocal microscopy. Full-length NcGRA9-HA was secreted into the PV and is located near the PVM or in the vacuolar space near the tachyzoites (A, B). 280 NcGRA9-HA either retained inside the tachyzoites especially anterior of the nucleus (C, D, white arrows) or was secreted into the vacuolar space (E, F, white arrow). 201 NcGRA9-HA always retained inside the tachyzoites in the paranuclear space (G, H, white arrow). (c) shows the enumeration of 280 NcGRA9-HA localization in 100 PV in three independent immunofluorescence experiments. No significant differences were observed (n.s. = not significant). In (d) N. caninum tachyzoite growth upon additional expression of NcGRA9 variants in HFF was analysed. Therefore, quantitative real-time PCR was performed. No significant differences in parasite numbers were observed (n.s. = not significant). Green: anti-HA cy2; blue: DAPI; merge: anti-HA, DAPI, DIC (differential interference contrast).

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