Figure 1.
Gbp1 is recruited to vacuoles containing T. gondii in a ROP5 and ROP18-dependent manner.
(A) Immunofluorescence localization of Gbp1 on the PVM in IFN-γ-activated BMM (10 U/ml IFN-γ, 0.1 ng/ml LPS). Images shown are 2 hr post infection with GT-1 (type I), CTG (type III), CTG+ROP18 (type III expressing ROP18) and CTG+ROP18 D/A (type III expressing kinase-dead ROP18) parasites. Gbp1 was localized with rabbit polyclonal sera followed by goat anti-rabbit IgG conjugated to Alexa Fluor 488. The vacuole marker GRA5 was detected with mAb Tg 17–113 followed by goat anti-rabbit IgG conjugated Alexa Fluor 594. Scale bar = 5 µm. (B) Quantification of Gbp1 localization to the PVM surrounding the indicated parasite strains in IFN-γ-activated BMMs examined at 2 hr post infection. (C) Immunofluorescence localization of Gbp1 on the PVM in IFN-γ-activated BMM (100 U/ml IFN-γ, 0.1 ng/ml LPS) examined at 2 hr post infection. Comparison of type I parasites lacking ROP18 (RHΔku80Δrop18) or ROP5 (RHΔku80Δrop5) vs. the respective complemented strains (RHΔku80Δrop18/ROP18) and (RHΔku80Δrop5/ROP5). Gbp1 was localized with rabbit polyclonal sera followed by goat anti-rabbit IgG conjugated to Alexa Fluor 488. The vacuole marker GRA5 was detected with a mAb Tg 17–113 followed by goat anti-rabbit IgG conjugated Alexa Fluor 594. (D) Quantification of Gbp1 localization to the PVM in IFN-γ-activated BMMs macrophages examined at 2 hr post infection. Strains indicated in (A) compared to the type I parent (RHΔku80). Mean ± S.D.P., n = 3 experiments. (*P<0.05, Student's t test).
Figure 2.
Recruitment of Gbp1 to the parasitophorous vacuole membrane is associated with vesiculation.
Cryo-immuno-EM localization of Gbp1 in IFN-γ-activated RAW 264.7 macrophages (10 U/ml IFN-γ, 0.1 ng/ml LPS). (A) Distribution of Gbp1 in activated macrophages infected with wild type T. gondii (T) parasites (RHΔku80). Arrow indicates PVM. (B) Enlargement of A showing PVM (arrow). (C–F) Distribution of Gbp1 in IFN-γ-activated macrophages infected with ROP18-deficient parasites (RHΔku80Δrop18). Arrow indicates PVM. (D) Enlargement of C showing PVM (arrow). (E,F) Distribution of Gbp1 in membrane vesicles in the vicinity of the PVM. Cells were fixed at 2 hr post-infection and immunostained using rabbit anti-Gbp1 (Rab α Gbp1) polyclonal sera followed by goat anti-rabbit IgG conjugated to 18 nm gold. Scale bars = 500 nm.
Figure 3.
Ultrastructural features of the parasitophorous vacuole membrane in IFN-γ-activated cells.
(A–D) Ultrastructural features of the parasitophorous vacuole (PV) T. gondii (T) in IFN-γ-activated (100 U/ml IFN-γ, 0.1 ng/ml LPS) BMM from Gbp1−/− or wild type mice infected with ROP5-deficient (Δrop5) or ROP18-deficient (Δrop18) parasites. (E–F) Enlargement of the PV surrounding parasites in IFN-γ-activated BMM from wild type mice shows the highly scalloped appearance of the membrane (marked by black arrowheads). Scale bars = 500 nm in A–D, 100 nm in E,F.
Figure 4.
Gbp1 localization is altered in Atg5-deficient cells.
Immunofluorescence localization of Gbp1 and Irga6 in wild type (WT) (A) or Atg5-deficient (Atg5 KO) (D,E) MEFs activated with IFN-γ (10 U/ml IFN-γ, 0.1 ng/ml LPS). Gbp1 was localized with rabbit polyclonal sera followed by goat anti-rabbit IgG conjugated to Alexa fluor 488. Irga6 was localized with mAb 10D7 followed by goat anti-mouse IgG conjugated to Alexa fluor 594. Transmission electron microscopy of wild type (WT) (B) or Atg5-deficient (Atg5 KO) (F,G) IFN-γ-activated MEFs. Scale bar = 200 nm. Cryo-immuno-EM of IFN-γ-activated wild type (WT) (C), or Atg5-deficient (Atg5 KO) (H,I), MEFs. Gbp1 was detected with rabbit polyclonal sera (Rab α Gbp1) followed by goat-anti-rabbit IgG conjugated to 18 nm gold beads and protein disulfide isomerase using mAb antibody 1D3 (Mo α PDI) followed by goat anti-mouse IgG conjugated to 12 nm gold beads. Scale bar = 200 nm. Immunofluorescence of Gbp1 co-localized with p62 (J), ubiquitin (K), or LAMP1 (L) in IFN-γ-activated MEFs from Atg5-deficient mice (Atg5KO). Gbp1 was localized with rabbit polyclonal sera followed by goat anti-rabbit IgG conjugated to Alexa fluor 488. p62 was localized with guinea pig polyclonal sera followed by goat anti-guinea pig IgG conjugated to Alexa Fluor 594. Ubiquitin was localized with mAbFK2 followed by goat anti-mouse IgG conjugated to Alexa fluor 594. LAMP1 was localized with rat mAB 1D4B followed by goat anti-rat 1gG conjugated to Alexa fluor 594. Image in L is a deconvolved Z slice, all other fluorescence images are wide field. Scale bars = 5 µm.
Figure 5.
Cells lacking Atg5 are unable to recruit Irgb6 or Gbp1 to the parasitophorous vacuole or clear susceptible parasites.
(A) Quantification of Irgb6 localization to the parasitophorous vacuoles surrounding intracellular parasites in IFN-γ-activated BMM (50 U/ml IFN-γ, 0.1 ng/ml LPS) from Atg5flox/flox or Atg5flox/flox+LysMCre mice. Means ± S.D., n = 6 samples from 2 combined experiments. (**P<0.005, Student's t test). (B) Quantification of Gbp1 recruitment to the parasitophorous vacuoles surrounding intracellular parasites IFN-γ-activated BMM (100 U/ml IFN-γ, 0.1 ng/ml LPS) from Atg5flox/flox or Atg5flox/flox+LysMcre mice. Representative experiment from three independent experiments with similar outcomes. Mean ± S.D., n = 3 replicates per group. (*P<0.05, Student's t test). In vitro clearance of parasites in IFN-γ-activated BMM (50 U/ml IFN-γ, 0.1 ng/ml LPS) from Atg5flox/flox (C) or Atg5flox/flox+LysMcre (D) mice. Means ± S.D., n = 6 samples from 2 combined experiments. (*** P<0.001, Student's t test).
Figure 6.
Gbp1 is necessary for control of T. gondii.
(A) In vitro clearance of ROP18-deficient (RHΔku80Δrop18), ROP5-deficient (RHΔku80Δrop5) and wild type (RHΔku80) T. gondii in IFN-γ-activated BMM (50 U/ml IFN-γ, 10 ng/ml LPS) from Gbp1−/− or C57BL/6 mice. Parasites were visualized using mAb to SAG1 (DG52) directly conjugated to Alexa Fluor 594. Percent of cells infected at 20 hr compared to 30 min time point. Mean ± S.D.P., n = 3–4 experiments (*P<0.005, Student's t test). (B) Analysis of replication in IFN-γ-activated BMM (as in A) infected with wild type (RHΔku80) parasites and analyzed at 20 hr post-infection. Mean number of parasites per vacuole ± S.D. n = 2 (*P<0.05, **P<0.005, ***P<0.0005, **** P<0.000005, Student's t test). (C) Quantification of Irgb6 and Gbp1 localization to parasitophorous vacuoles of intracellular parasites in IFN-γ-activated BMM (100 U/ml IFN-γ, 0.1 ng/ml LPS) infected with ROP5-deficient (RHΔku80Δrop5) parasites. Mean ± S.D. n = 6 samples from 2 combined experiments. (D) Quantification of Irgb6 and Irga6 localization to parasitophorous vacuoles surrounding ROP18-deficient (RHΔku80Δrop18) parasites in C57BL/6 vs. Gbp1−/− BMM activated with 100 U/ml IFN-γ and 0.1 ng/ml LPS. Cells were infected for 30 min vs. 2 hr, fixed, permeabilized and stained using rabbit anti-Irgb6 and mAb against GRA5, or mAb 10D7 against Irga6 and rabbit anti-GRA7, and detected using corresponding Alexa Flour secondary antibodies. Representative of two experiments with similar outcomes. Mean ± S.D.P, n = 4. (*P<0.05, Student's t test). (E) Quantification of Gbp1, Irgb6 and Irga6 localization to parasitophorous vacuoles of ROP5-deficient parasites (RHΔku80Δrop5) in C57BL/6 vs. Irgm3−/− BMM activated with 100 U/ml IFN-γ and 0.1 ng/ml LPS. Representative experiment from 2–3 independent experiments with similar outcomes. Mean ± S.D., n = 3 replicates per group. (*P<0.05, **P<0.005, Student's t test).
Figure 7.
Gbp1-deficient animals are more susceptible to T. gondii infection.
(A) Survival of Gbp1−/− or C57BL/6 mice s.c. infected with 100 ROP18-deficient (RHΔku80Δrop18) or wild type (RHΔku80) parasites. Two independent experiments combined, n = 8 mice per parasite strain for C57BL/6 and n = 10 mice per parasite strain for Gbp1−/−. (***P<0.0005, Gehan-Breslow-Wilcoxon Test). (B) Survival of Gbp1−/− or Combination of two experiments where n = 8–9 animals per group. (** P<0.005, Gehan-Breslow-Wilcoxon Test). (C–F) H&E paraffin sections from the thalamus region of the brains from representative surviving mice (from B) at 60 days post infection. Scale bars = 100 nm.