First Characterization of the Neospora caninum Dense Granule Protein GRA9

Figure 4

In (a) the localization of NcGRA9 was analysed by confocal microscopy to show that it belongs to the dense granule proteins of N. caninum. In extracellular tachyzoites NcGRA9 was detectable in a dotted pattern (white arrow, top panel) implying its localization in dense granules. In N. caninum infected host cells NcGRA9 was secreted into the PV. The protein localized either near the PVM (white arrow, middle panel) or in the vacuolar space near the tachyzoites (white arrow, lowest panel). (b) shows colocalization studies of NcGRA9 with NcGRA7 (PV/PVM marker), NcMIC1 (apical marker), and NcSAG1 (cell surface marker). NcGRA9 colocalizes partially with NcGRA7 and NcSAG1, but not with NcMIC1. In (c) cell fractionation by ultracentrifugation was performed with N. caninum infected HFF. It shows the targeting behaviour of NcGRA9 after secretion into the host cell. NcGRA9 was present in the low speed pellet (LSP) which includes host cell debris and intact parasites, but also in the low speed supernatant (LSS) which was further separated into a membranous fraction (high speed pellet, HSP) and a soluble fraction (high speed supernatant, HSS) by ultracentrifugation. Secreted NcGRA9 is membrane-bound (HSP) as well as soluble (HSS) in the vacuolar space. The control protein NcGRA7 behaves as TgGRA7 and is mainly membrane-bound after secretion. The surface antigen NcSAG1 is mainly detectable in the LSP fraction which contains the intact parasites, as expected. Green: anti-NcGRA9 cy2; blue: DAPI; merge: anti-NcGRA9, DAPI, DIC (differential interference contrast).

(a)

(b)

(c)