p47 GTPases regulate Toxoplasma gondii survival in activated macrophages

Abstract

The cytokine gamma interferon (IFN-gamma) is critical for resistance to Toxoplasma gondii. IFN-gamma strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-gamma-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-gamma-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-gamma-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.

FIG. 1.

IFN-γ-stimulated expression of IGTP, LRG-47, and IRG-47 in BMM. WT BMM were cultured in the presence or absence of IFN-γ or were infected with T. gondii at an MOI of 2:1. After 24 h, total cellular RNA was prepared and used for Northern blotting with the indicated probes.

FIG. 2.

Loss of IFN-γ-mediated T. gondii growth suppression in IGTP KO macrophages. (A) Wild-type or IGTP KO BMM were cultured in the presence or absence of IFN-γ for 24 h. The cultures were subsequently infected with T. gondii at an MOI of 2:1 for 2 h or were left uninfected, and then cultures were pulsed with [³H]uracil. After 48 h, incorporated radioactivity was determined and expressed as cpm ± standard deviation. These results are representative of four independent experiments. (B) Wild-type or IGTP KO BMM that had been cultured in the presence or absence of IFN-γ for 24 h were infected for 2 h with T. gondii at an MOI of 2:1. When the host cells reached almost complete lysis, the resulting tachyzoites were enumerated microscopically. These results are representative of two independent experiments.

FIG. 3.

Differential effects of IGTP, LRG-47, and IRG-47 deficiency on IFN-γ-mediated T. gondii growth suppression in macrophages. WT BMM or cells that lacked the indicated GTPases were cultured in the presence or absence of IFN-γ for 24 h. The cultures were subsequently infected with T. gondii at an MOI of 2:1 for 2 h and then were pulsed with [³H]uracil. After 48 h, incorporated radioactivity was determined. (A) Shown is a representative experiment with the results expressed as cpm ± standard deviation. (B) Also shown is the average of four independent experiments in which the results indicate the effect of IFN-γ pretreatment on T. gondii growth, relative to growth under control conditions (no IFN-γ pretreatment). The asterisks (*) indicate statistically significant differences (P < 0.05).

FIG. 4.

Distribution of IGTP and LRG-47 among sucrose gradient fractions. Postnuclear supernatants were prepared from J774 macrophages that had been stimulated with IFN-γ. The supernatants were then applied to sucrose step gradients and separated into four fractions, with fraction 1 being the most buoyant and fraction 4 the densest. Equal amounts of protein from each fraction were used for Western blot analysis with the indicated probes. Densitometry was applied to the autoradiographs to quantify the protein detected in each fraction. These results are representative of four experiments.

FIG. 5.

LRG-47 and IGTP immunofluorescence in macrophages. BMM that had been activated with IFN-γ for 24 h were stained with the indicated primary antibodies and Alexa Fluor 488 (green)- or Alexa Fluor 594 (red)-conjugated secondary antibodies, as described in the text. The staining was then analyzed by confocal microscopy (magnification, ×900). The LRG-47 (red) and IGTP (green) localizations are largely exclusive. LRG-47 (red) colocalizes with the Golgi marker, GM130 (green). IGTP (red) and TRAPα (green) display colocalization within the endoplasmic reticulum. In the merged images (right column), colocalization of green and red staining appears yellow.

FIG. 6.

Distribution of LRG-47 and IGTP in macrophages containing latex bead vacuoles. J774 macrophages were stimulated with IFN-γ for 24 h and were then allowed to phagocytose latex beads for 20 min; they were then incubated for the indicated times in the absence of beads. Latex bead vacuoles were then isolated, as described in Materials and Methods, and equal amounts or vacuole-associated protein were used for Western blotting with LRG-47, IGTP, and LAMP1 antibodies. Shown are a representative Western blot (A) and the relative signals for each protein as assessed by densitometry (B), with the levels detected at each time point expressed as a percentage of the total protein detected (average levels from four separate experiments).

FIG. 7.

LRG-47 and IGTP immunofluorescence in macrophages containing latex bead vacuoles. BMM that had been activated with IFN-γ for 24 h were allowed to phagocytose latex beads for 20 min and then were incubated in the absence of beads for 30 min. The cells were then fixed and stained with the indicated antibodies. Staining was assessed by deconvolution microscopy (magnification, ×600).

FIG. 8.

LRG-47 and IGTP immunofluorescence in T. gondii-infected macrophages. BMM that had been activated with IFN-γ for 24 h were infected with live or heat-killed T. gondii, as indicated in the figure, for 1 h. Parasites appear green, and the GTPases and LAMP-1 appear red. Colocalization of heat-killed parasites with LAMP-1, indicating late endosomal/lysosomal localization, appears yellow.