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ABSTRACT: Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with “non-self” PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on “self” organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of “self” IRGM proteins from these structures.
PLoS Pathogens 06/2013; · 9.13 Impact Factor
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ABSTRACT: The immunity-related GTPases (IRGs) are a family of proteins induced by interferon-γ that play a crucial role in innate resistance to intracellular pathogens. The M subfamily of IRG proteins (IRGM) plays a profound role in this context, in part because of the ability of its members to regulate the localization and expression of other IRG proteins. We present here evidence that IRGM proteins affect the localization of the guanylate-binding proteins (GBPs), a second family of interferon-induced GTP-binding proteins that also function in innate immunity. Absence of Irgm1 or Irgm3 led to accumulation of Gbp2 in intracellular compartments that were positive for both the macroautophagy (hereafter referred to as autophagy) marker LC3 and the autophagic adapter molecule p62/Sqstm1. Gbp2 was similarly relocalized in cells in which autophagy was impaired because of the absence of Atg5. Both in Atg5- and IRGM-deficient cells, the IRG protein Irga6 relocalized to the same compartments as Gbp2, raising the possibility of a common regulatory mechanism. However, other data indicated that Irga6, but not Gbp2, was ubiquitinated in IRGM-deficient cells. Similarly, coimmunoprecipitation studies indicated that although Irgm3 did interact directly with Irgb6, it did not interact with Gbp2. Collectively, these data suggest that IRGM proteins indirectly modulate the localization of GBPs through a distinct mechanism from that through which they regulate IRG protein localization. Further, these results suggest that a core function of IRGM proteins is to regulate autophagic flux, which influences the localization of GBPs and possibly other factors that instruct cell-autonomous immune resistance.
Journal of Biological Chemistry 07/2011; 286(35):30471-80. · 4.77 Impact Factor
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ABSTRACT: The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of bacterial sexually transmitted diseases in the United States. In women C. trachomatis can establish persistent genital infections that lead to pelvic inflammatory disease and sterility. In contrast to natural infections in humans, experimentally induced infections with C. trachomatis in mice are rapidly cleared. The cytokine interferon-γ (IFNγ) plays a critical role in the clearance of C. trachomatis infections in mice. Because IFNγ induces an antimicrobial defense system in mice but not in humans that is composed of a large family of Immunity Related GTPases (IRGs), we questioned whether mice deficient in IRG immunity would develop persistent infections with C. trachomatis as observed in human patients. We found that IRG-deficient Irgm1/m3((-/-)) mice transiently develop high bacterial burden post intrauterine infection, but subsequently clear the infection more efficiently than wildtype mice. We show that the delayed but highly effective clearance of intrauterine C. trachomatis infections in Irgm1/m3((-/-)) mice is dependent on an exacerbated CD4(+) T cell response. These findings indicate that the absence of the predominant murine innate effector mechanism restricting C. trachomatis growth inside epithelial cells results in a compensatory adaptive immune response, which is at least in part driven by CD4(+) T cells and prevents the establishment of a persistent infection in mice.
PLoS Pathogens 06/2011; 7(6):e1001346. · 9.13 Impact Factor
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Aliaksandr Khaminets,
Julia P Hunn,
Stephanie Könen-Waisman,
Yang O Zhao,
Daniela Preukschat, Jörn Coers,
Jon P Boyle,
Yi-Ching Ong,
John C Boothroyd,
Gabriela Reichmann,
Jonathan C Howard
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ABSTRACT: The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.
Cellular Microbiology 07/2010; 12(7):939-61. · 5.46 Impact Factor
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PLoS Pathogens 06/2009; 5(5):e1000333. · 9.13 Impact Factor
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Stanley C Henry,
Xiaoju G Daniell,
Ashley R Burroughs,
Maanasa Indaram,
David N Howell, Jörn Coers,
Michael N Starnbach,
Julia P Hunn,
Jonathan C Howard,
Carl G Feng,
Alan Sher,
Gregory A Taylor
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ABSTRACT: The immunity-related GTPases (IRG), also known as p47 GTPases, are a family of proteins that are tightly regulated by IFNs at the transcriptional level and serve as key mediators of IFN-regulated resistance to intracellular bacteria and protozoa. Among the IRG proteins, loss of Irgm1 has the most profound impact on IFN-gamma-induced host resistance at the physiological level. Surprisingly, the losses of host resistance seen in the absence of Irgm1 are sometimes more striking than those seen in the absence of IFN-gamma. In the current work, we address the underlying mechanism. We find that in several contexts, another protein in the IRG family, Irgm3, functions to counter the effects of Irgm1. By creating mice that lack Irgm1 and Irgm3, we show that several phenotypes important to host resistance that are caused by Irgm1 deficiency are reversed by coincident Irgm3 deficiency; these include resistance to Salmonella typhimurium in vivo, the ability to affect IFN-gamma-induced Salmonella killing in isolated macrophages, and the ability to regulate macrophage adhesion and motility in vitro. Other phenotypes that are caused by Irgm1 deficiency, including susceptibility to Toxoplasma gondii and the regulation of GKS IRG protein expression and localization, are not reversed but exacerbated when Irgm3 is also absent. These data suggest that members of the Irgm subfamily within the larger IRG family possess activities that can be opposing or cooperative depending on the context, and it is the balance of these activities that is pivotal in mediating IFN-gamma-regulated host resistance.
Journal of leukocyte biology 02/2009; 85(5):877-85. · 4.99 Impact Factor
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ABSTRACT: Chlamydiae are obligate intracellular bacterial pathogens that exhibit a broad range of host tropism. Differences in host tropism between Chlamydia species have been linked to host variations in IFN-gamma-mediated immune responses. In mouse cells, IFN-gamma can effectively restrict growth of the human pathogen Chlamydia trachomatis but fails to control growth of the closely related mouse pathogen Chlamydia muridarum. The ability of mouse cells to resist C. trachomatis replication is largely dependent on the induction of a family of IFN-gamma-inducible GTPases called immunity-related GTPases or IRGs. In this study we demonstrate that C. muridarum can specifically evade IRG-mediated host resistance. It has previously been suggested that C. muridarum inactivates the IRG protein Irga6 (Iigp1) to dampen the murine immune response. However, we show that Irga6 is dispensable for the control of C. trachomatis replication. Instead, an effective IFN-gamma response to C. trachomatis requires the IRG proteins Irgm1 (Lrg47), Irgm3 (Igtp), and Irgb10. Ectopic expression of Irgb10 in the absence of IFN-gamma is sufficient to reduce intracellular growth of C. trachomatis but fails to restrict growth of C. muridarum, indicating that C. muridarum can specifically evade Irgb10-driven host responses. Importantly, we find that Irgb10 protein intimately associates with inclusions harboring C. trachomatis but is absent from inclusions formed by C. muridarum. These data suggest that C. muridarum has evolved a mechanism to escape the murine IFN-gamma response by restricting access of Irgb10 and possibly other IRG proteins to the inclusion.
The Journal of Immunology 06/2008; 180(9):6237-45. · 5.79 Impact Factor
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ABSTRACT: Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.
Cellular Microbiology 11/2007; 9(10):2344-57. · 5.46 Impact Factor
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ABSTRACT: Infections caused by the bacteria Chlamydia trachomatis contribute to diverse pathologies in a variety of human populations. We previously used a systemic model of C. trachomatis infection in mice to map three quantitative trait loci that influence in vivo susceptibility differences between the C57BL/6J and C3H/HeJ inbred strains of mouse. One of these quantitative trait loci, Ctrq-3, influences an IFN-gamma-dependent susceptibility difference in primary embryonic fibroblasts isolated from these strains. Here we use fine structure mapping in congenic fibroblasts carrying DNA from the susceptible parent to localize the effect of Ctrq-3 to a 1.2-megabase interval of genomic DNA that contains Irgb10 and Igtp, two members of the IFN-gamma-inducible p47 family of GTPases. This class of proteins has been widely implicated in resistance to intracellular pathogens in mice. We analyzed expression of Irgb10 and Igtp in parental and congenic embryonic fibroblasts treated with IFN-gamma and found that relatively resistant fibroblasts express more Irgb10 than relatively susceptible fibroblasts. However, we also found that abolishing the expression of either Irgb10 or Igtp increases susceptibility of embryonic fibroblasts to C. trachomatis. Thus, we conclude that, although a difference in Irgb10 expression is likely responsible for the effect of Ctrq-3 on susceptibility to C. trachomatis, both genes play a role in intracellular resistance to C. trachomatis.
Proceedings of the National Academy of Sciences 10/2006; 103(38):14092-7. · 9.68 Impact Factor
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ABSTRACT: Thrombopoietin and its cognate receptor c-Mpl are the primary regulators of megakaryopoiesis and platelet production. They also play an important role in the maintenance of hematopoietic stem cells. Here, we have analyzed the function of a truncated Mpl receptor isoform (Mpl-tr), which results from alternative splicing. The mpl-tr variant is the only alternate mpl isoform conserved between mouse and humans, suggesting a relevant function in regulating Mpl signaling. Despite the presence of a signal peptide and the lack of a transmembrane domain, Mpl-tr is retained intracellularly. Our results provide evidence that Mpl-tr exerts a dominant-negative effect on thrombopoietin-dependent cell proliferation and survival. We demonstrate that this inhibitory effect is due to down-regulation of the full-length Mpl protein. The C terminus of Mpl-tr, consisting of 30 amino acids of unique sequence, is essential for the suppression of thrombopoietin-dependent proliferation and Mpl protein down-regulation. Cathepsin inhibitor-1 (CATI-1), an inhibitor of cathepsin-like cysteine proteases, counteracts the effect of Mpl-tr on Mpl protein expression, suggesting that Mpl-tr targets Mpl for lysosomal degradation. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function through a proteolytic process directed by a truncated isoform of the same receptor.
Journal of Biological Chemistry 09/2004; 279(35):36397-404. · 4.77 Impact Factor
