Figure 7
GRA15 expression in N. caninum upregulates the NF-κB pathway members and increases IL-12p40 production. (A) Diagram of the expression cassette encoding GRA15 fused to BirA*, plus a C-terminal 3xHA epitope tag, driven by the GRA7 promoter, (B) IFA of GRA15-BirA*-expressing parasites, grown for 48 h with or without biotin. GRA15-BirA* localizes to the parasite dense granules and biotinylates proteins in a biotin-dependent manner. Endogenously biotinylated proteins in the apicoplast are detectable, even in the absence of biotin. Bottom panel, Green, mouse anti-HA antibody; red, streptavidin-Alexa 594. Microscope magnification: 63x, (C) Western blot comparing the profile of biotinylated proteins from lysates cells infected with parental and GRA15-BirA* parasites, (D) Diagram and heat map of the mass spectrometry results of the biotinylated proteins analyzed by the Pathway Studio software shows the upregulation of the NF-κB pathway, (E) NcLiv_GRA15 induces the upregulation of IL- 12p40 in macrophages. Values are representative of two independent experiments indicating mean ± SEM of cytokine levels in relation the standard curve (*P < 0.05; ANOVA and Bonferroni multiple comparison post-test). Full-length blots are available in Supplementary dataset file.
