Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse

Abstract

The intracellular parasite Toxoplasma gondii enjoys a wide host range and is adept at surviving in both naive and activated macrophages. Previous studies have emphasized the importance of the active serine-threonine protein kinase rhoptry protein 18 (ROP18), which targets immunity-related GTPases (IRGs), in mediating macrophage survival and acute virulence of T. gondii in mice. Here, we demonstrate that ROP18 exists in a complex with the pseudokinases rhoptry proteins 8 and 2 (ROP8/2) and dense granule protein 7 (GRA7). Individual deletion mutant gra7 or rop18 was partially attenuated for virulence in mice, whereas the combined gra7rop18 mutant was avirulent, suggesting these proteins act together in the same pathway. The virulence defect of the double mutant was mirrored by increased recruitment of IRGs and clearance of the parasite in IFN-γ-activated macrophages in vitro. GRA7 was shown to recognize a conserved feature of IRGs, binding directly to the active dimer of immunity-related GTPase a6 in a GTP-dependent manner. Binding of GRA7 to immunity-related GTPase a6 led to enhanced polymerization, rapid turnover, and eventual disassembly. Collectively, these studies suggest that ROP18 and GRA7 act in a complex to target IRGs by distinct mechanisms that are synergistic.

Keywords: cooperative polymerization; innate immunity; pathogenesis.

Fig. 1.

Interaction of ROP18 and GRA7 and generation of KO and complemented lines. (A) Co-IP of proteins with ROP18. Lysates from CTG or CTG + ROP18-Ty parasites were immunoprecipitated with mAb BB2 to Ty (Mo αTy). Western blotting (WB) of the pellet (Pell, 100% loaded) and supernatant (Sup, 25% loaded) fractions was probed for rabbit anti-ROP18 (Rb αROP18), rabbit anti-ROP2 (Rb αROP2), rabbit anti-GRA7 (Rb αGRA7), or mAb to GRA5 (Mo αGRA5). (B) Immunofluorescence localization of ROP18 and GRA7 in selectively permeabilized (digitonin) vs. fully permeabilized (Triton X-100) cells. Permeabilized cells were incubated with rabbit anti-GRA7 (red), mAb BB2 to Ty tag to detect ROP18-Ty (blue), and mAb DG52 to SAG1 (green). (Scale bars: 5 μm.) (C) Lysates from WT or mutant parasites were resolved by SDS/PAGE and probed with Rb αROP18 and rabbit anti-T. gondii actin (Rb αTgACT) as a loading control (Upper) or with Rb αROP2 and Rb αGRA7 (Lower). (D) Plaque assay of in vitro growth of the WT and mutant parasites.

Fig. 2.

Virulence of WT and mutant parasites in laboratory mice. (A) WT and deletion mutants were injected into CD-1 mice (100 parasites, s.c. inoculation), and survival was followed for 30 d (n = 10 mice for all strains except Δrop8/2 and Δgra7Δrop8/2, where n = 5 mice). ****P < 0.0001, Gehan–Breslow Wilcoxon test. (B) Dose-dependent mortality of Δgra7Δrop18 parasites. CD-1 mice (n = 5) were injected i.p with varying doses of parasites, and survival was followed for 30 d. (C) Δgra7Δrop18 parasites and their singly complemented strains (i.e., Δrop18/GRA7, Δgra7/ROP18-Ty) were injected into C57BL/6 mice (100 parasites, s.c. inoculation), and survival was monitored for 30 d (n = 10 mice for all strains). ****P < 0.0001, Gehan–Breslow Wilcoxon test. (D) Survival of WT C57BL/6 mice vs. IFN-γR^−/− mice challenged with Δgra7Δrop18 parasites. Mice (n = 10) were injected i.p. with 100 Δgra7Δrop18 parasites, and survival was followed for 30 d.

Fig. 3.

IRG recruitment and clearance in activated macrophages. (A) Immunofluorescence staining of Irga6 on parasite-containing vacuoles in activated RAW macrophages at 30 min postinfection stained with mAb 10D7 for Irga6 (green), rabbit polyclonal antibody to ROP5 (red), and DAPI (blue). (Scale bar: 5 μm.) (B) Quantification of Irga6 localized to the parasite vacuole in IFN-γ–activated RAW cells. Mean ± SD of the population (SDP) (n = 3 experiments). *P < 0.05 and **P < 0.001, Student t test. (C) Immunofluorescence staining of Irgb6 on parasite-containing vacuoles in IFN-γ–activated RAW macrophages at 30 min postinfection stained with rabbit polyclonal α-Irgb6 (green), mAb Tg17-113 to GRA5 (red), and DAPI (blue). (Scale bar: 5 μm.) (D) Quantification of Irgb6 localized to the parasite vacuole in activated RAW cells. Mean ± SDP (n = 3 experiments). *P < 0.05, Student t test. (E) Quantification of parasite survival in IFN-γ–activated RAW macrophages. Mean ± SD values for two experiments with three replicates each are given (n = 6). *P < 0.05 and **P < 0.001, Mann–Whitney test. (F) Survival of Irgm3^−/− vs. WT C57BL/6 mice injected s.c. with 100 parasites (n = 7 mice for Δrop18 and Δgra7Δrop18, n = 5 mice for Δgra7). ****P < 0.0001, Gehan–Breslow Wilcoxon test.

Fig. 4.

Interaction of GRA7 and Irga6 in vitro. (A) Oligomerization of 50 μM Irg6 ± 20 μM GRA7 in a 90° light scattering assay, in which 1 mM GTP was added at the location indicated by the arrow at 37 °C. AU, arbitrary units; PK, proteinase K-treated GRA7. (B) Oligomerization of 50 μM Irg6 ± GRA7 in a 90° light scattering assay in which 1 mM GTP was added at the location indicated by the arrow at 37 °C. (C) GTP-dependent binding of WT (GRA7) vs. double-point mutant (GRA7 MUT) to Irga6. Supernatants (S; 10%) and pellets (P; 50%) were resolved by SDS/PAGE and stained with SYPRO Ruby. His-tagged ALD (His-Ald) was included as a negative control. (D) Nucleotide-dependent binding of GRA7 to Irga6. Recombinant His-tagged GRA7 (His-GRA7) was incubated with WT or mutant Irga6 and various nucleotides for 10 min at 37 °C and then immunoprecipitated with Ni beads. The supernatants were resolved by SDS/PAGE and stained with SYPRO Ruby. (E) Phosphate release during oligomerization of Irga6 (50 μM); incubation ± 20 μM GRA7, 20 μM ALD (Ald), or PK-treated GRA7 (+PK); and addition of final 1 mM GTP (+GTP). [Pi], cumulative phosphate released (micromolar) (n = 3). **** P < 0.0001, two-way ANOVA test.