The Rhoptry Proteins ROP18 and ROP5 Mediate Toxoplasma gondii Evasion of the Murine, But Not the Human, Interferon-Gamma Response
Figure 5
ROP5 interacts with Irga6, but not ROP18, and inhibits Irg6 oligomerization.
(A) ROP5 and HA were immunoprecipitated from IFNγ-stimulated or unstimulated MEFs infected with CEP or CEP + ROP18II-HA and inputs and immunoprecipitates were Western blotted for ROP5 and HA. (B) Kinase activity of ROP18-HA immunoprecipitated from IFNγ-stimulated or unstimulated MEFs infected with S22, S22 + ROP18II-HA or S22 + LC37 + ROP18II-HA parasite strains. Half of the immunoprecipitated protein was Western blotted with anti-HA and the relative amount of ROP18-HA from each strain was quantified using the ImageQuant (GE Healthcare Life Sciences) software. The remaining immunoprecipitated proteins were incubated with 32P-γ-ATP and a model peptide substrate (Lim, D., submitted) and spotted in quadruplicate onto phospho-cellulose paper where the 32P-γ-ATP incorporation was quantified by phosphorimage analysis. The kinase activity is expressed as fold change over the S22 strain and normalized to the relative amounts of ROP18-HA that was immunoprecipitated. This experiment was performed twice and the graph represents the mean from those experiments. (C) Unique peptides (yellow) and percent sequence coverage of Irga6 recovered from mass spectrometry of proteins co-immunoprecipitated with ROP5-CIII-HA. Briefly, HA-immunoprecipitated proteins from IFNγ-stimulated or unstimulated MEFs infected with Pru, Pru + ROP5-AIII-HA, Pru + ROP5-CIII-HA or RH + GRA15II-HA and lysed in the presence or absence of 0.5 mM GTPγS were separated by SDS-PAGE and analyzed by MS/MS. Irga6 peptides were recovered only in ROP5-CIII-HA samples lysed in the presence of GTPγS. (D) Oligomerization of 20 µM Irga6 with10 mM GTP at 37°C in the presence of MBP-tagged ROP5-C or MBP shown as predicted mean hydrodynamic radius of the particle population determined by dynamic light scattering.