GMS proteins shield endocellular membranes from accumulation of effector Immunity-Related GTPases

GMS proteins shield endocellular membranes from accumulation of effector Immunity-Related GTPases Jelena Maric-Biresev Immunity-related GTPases (IRGs) play an important role in host immune response to a variety of intracellular pathogens by accumulation at the membranes of the pathogens and their disruption. However, it was never understood how exactly IRG proteins distinguish the membranes of the pathogen from the host membranes. Previously, it has been reported that regulatory IRG proteins, named GMS proteins, keep the effector IRG proteins, named GKS proteins, in the inactive GDP-bound state. In this study, it has been shown that GMS proteins also play an important role in protection of the endocellular compartments from GKS protein off-target activation. In the absence of the GMS protein Irgm1, which is localized at the lysosomes, GKS proteins Irga6, Irgb6, Irgb10 and Irgd accumulate and activate at these organelles. In the absence of Irgm3, a GMS protein which is localized at the endoplasmic reticulum (ER), GKS protein Irga6 accumulates at the ER. However, in the cells that lack Irgm1 and Irgm3, GKS proteins accumulate only to lipid droplets, but not to lysosomes or ER, indicating that GKS structures do not accumulate at every GMS-free membrane. In the second part of the study, the consequences Irgm1 accumulation to the lysosomes are investigated. Irgm1 KO mice undergo leukopenia and succumb after a variety of infections and inflammatory states. The results of this study suggest that the failure of Irgm1 KO mice is rather an indirect consequence of the off-target action of the GKS proteins, than the direct consequence of the Irgm1 response to a variety of pathogens. The GKS proteins, that accumulate at the lysosomes in Irgm1 KO cells, affect the acidity of these organelles and therefore lysosomal ability to process autophagosomes. In IFNγ-induced Irgm1 KO mouse embryonic fibroblasts (MEFs) the mature autophagosome marker LC3-II level is enhanced, the number of autophagosomes is increased and the co-localization between autophagosomes and lysosomes is higher than in the non-induced cells. Therefore, in this study, it is proposed that the autophagic flux of IFNγ-induced Irgm1 KO MEFs is impaired. The lymphocytes of the Irgm1 KO mice, that are stimulated to proliferate as a response to infection, could be the cells that are most affected by autophagic flux arrest and lysosomal acidification impairment. Thus, this effect could be the cause of described defects in lymphocyte proliferation and lymphocyte necrosis, which cause the death of the Irgm1 KO mous

Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection

The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.Deutscher Akademischer Austausch Dienst (DAAD); International Graduate School in Development Health and Disease (IGS-DHD); Deutsche For-schungsgemeinschaft (SFBs 635, 670, 680); Max-Planck-Gesellschaft (Max Planck Fellowship)

Additional file 1: Figure S1. of Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection

Activated Irga6 co-localizes with the lysosomes. There is no cross-reactivity between 10D7 and 1D4B antibodies. Irgm1 −/− mouse embryonic fibroblasts were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with mouse antibody 10D7, which stains the Irga6 in GTP-bound state, and rat antibody 1D4B, which stains LAMP1 lysosomal marker. The antibodies were visualized with secondary antibodies Alexa 488 donkey-anti-mouse and Alexa 555 goat-anti-rat. To test the cross-reactivity, one of the primary or secondary reagents was omitted in each sample. The images of stained cells were taken. Representative microscopic images of GTP-bound Irga6 and LAMP1 are shown, with omitted antibody being annotated. (TIF 14983 kb