p47 GTPases RegulateToxoplasma gondiiSurvival in Activated Macrophages

Abstract

The cytokine gamma interferon (IFN-␥) is critical for resistance to Toxoplasma gondii. IFN-␥ 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-␥-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-␥-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-␥-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.

Impaired Macrophage Function Underscores Susceptibility to Salmonella in Mice Lacking Irgm1 (LRG-47)

“…These data implicate Irgm3 in restricting Toxoplasma gondii (22,23) and C. trachomatis (16) survival; Irgm1 in restricting Mycobacterium tuberculosis (24), Trypanosoma cruzi (25), and Toxoplasma gondii (22); Irga6 in restricting T. gondii (21); and Irgd in restricting T. cruzi (26). Because IRG proteins often localize to pathogen-containing vacuoles as the pathogen enters the host cell and remain with the vacuole as it is processed (20,22), it has been suggested that the proteins regulate processing of pathogen-containing vacuoles, thereby governing growth and survival of the pathogen. At least three different mechanisms have been suggested through which IRG proteins may regulate survival of vacuolar pathogens, although these mechanisms are not necessarily exclusive.…”

“…Thus, the ability of Irgm1-deficient BMM to restrict the growth of S. typhimurium in response to IFN-␥ activation was compromised. Nevertheless, it should be noted that in these assays, Irgm1-deficienct BMM retain some ability to restrict the growth of S. typhimurium and, furthermore, that the impact on controlling the growth of S. typhimurium in BMM following activation with IFN-␥ was not as striking as the impact on controlling the growth of other pathogens such as T. gondii, where even at high doses of IFN-␥ (100 U/ml), the cytokine induced little pathogen growth restriction in absence of Irgm1 (22). This prompted further investigation into the functioning of Irgm1-deficient macrophages.…”

“…It has been shown previously that Irgm1 mediates the ability of IFN-␥ to restrict the growth of M. tuberculosis (24), T. cruzi (25), and T. gondii (22) in cultured macrophages. However, in studies involving M. avium, absence of Irgm1 did not have a significant effect on the ability of IFN-␥ to restrict bacterial growth (34), suggesting that the ability of Irgm1 to affect bacterial survival in macrophages may not be a general function of the protein in all contexts.…”

Metabolic Alterations Contribute to Enhanced Inflammatory Cytokine Production in Irgm1-deficient Macrophages

“…The reduced citrate availability would probably have the effect of decreasing fatty acid synthesis as well as promoting glycolysis by reducing the inhibitory effect of citrate on phosphofructokinase (85). It is plausible that Irgm1 could play a direct role in lipid homeostasis, because it is a dynamin-like GTPase that associates with specific intracellular membrane compartments, including the Golgi, where it may alter membrane trafficking (11,12,21,25,52,78,87). Additionally, IRGM proteins have been reported to play roles in lipid droplet maintenance (27,78,88).…”

Vacuolar and plasma membrane stripping and autophagic elimination of Toxoplasma gondii in primed effector macrophages

“…The p47 GTPase, IIGP, has been localized by immunofl uorescence microscopy to the PV in astrocytes (16), but whether IGTP is also found in the PV remains controversial (24). We therefore performed cryo-immunoelectron microscopy to visualize IGTP localization within primed and T. gondiiinfected WT macrophages.…”