Disruption of Toxoplasma gondii Parasitophorous Vacuoles by the Mouse p47-Resistance GTPases

The p47 GTPases are essential for interferon-γ-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-γ-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-γ-mediated T. gondii growth restriction in mouse astrocytes

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen

IFN-γ-Mediated Growth Inhibition and Intracellular Killing of <i>T. gondii</i> Are Accompanied by Accumulation of p47 GTPases at the PV

<div><p>(A) Astrocytes were induced with the indicated concentrations of IFN-γ and infected with <i>T. gondii</i> 24 h later for 68 h. The growth of intracellular parasites was monitored by uracil incorporation assay. (Inset) Lysates of astrocytes induced with the indicated concentrations of IFN-γ for 24 h were probed for IIGP1 protein by Western blotting.</p><p>(B) Untreated or IFN-γ induced astrocytes were infected with <i>T. gondii.</i> After 2 h, extracellular parasites were washed away and cells were either fixed or incubated further for a total of 8 h or 24 h. Shown are the mean values of three independent counts representing a total number of 650–997 cells per time point.</p></div

The Vacuolar Accumulations of IIGP1 Do Not Reflect Host Cell ER Recruitment by the Parasite

<div><p>Astrocytes were induced with IFN-γ or left untreated and infected with <i>T. gondii</i> 24 h later for 2 h. Cells were fixed and stained for the indicated proteins. (A) Shows a cell that was stained for IIGP1 (red) and calnexin (green). The vacuolar calnexin signal is markedly less concentrated at the PV than IIGP1. (A') shows the same cell as in (A) but with an electronically enhanced IIGP1 signal to reveal its non-vacuolar ER localization. Note the dramatic difference in the ratio of the ER versus PV signal between IIGP1 and calnexin.</p><p>(B and C) Shows astrocytes stained for IIGP1- and the ER-localized PDI. No PDI accumulation at the PV was detected.</p><p>(D) Astrocytes were treated as above but stained for IIGP1 (red) and ERP60 (green). Nuclei were stained with DAPI.</p></div

Induction of Autophagosomes in Vicinity of Disrupted Vacuoles

<p>Astrocytes were transfected with pEGFP-C3-LC3 and induced with IFN-γ<i>.</i> Cells were infected with <i>T. gondi</i> 24 h later and fixed after 2 h (A) or 6 h (B, C, and D). In cells containing only smooth IIGP1 vacuoles GFP-LC3 remained diffusely distributed throughout the cytoplasm (A). In cells containing disrupted IIGP1 PVs GFP-LC3 localizes to vesicular and filamentous structures that are in close proximity to, but do not engulf the IIGP1-positive PVs (B and C). The arrowheads point to IIGP1-positive PVs. The images shown in (A) were processed by 2D deconvolution. (D) Shows maximum projections of 3D deconvoluted Z-series.</p