The IFN-inducible Golgi- and endoplasmic reticulum- associated 47-kDa GTPase IIGP is transiently expressed during listeriosis.

Members of the 47-kDa GTPase family are implicated in an IFN-gamma-induced, as yet unclear, mechanism that confers innate resistance against infection with intracellular pathogens. Overt immunological parameters are apparently uncompromised in mice deficient for individual members and the prototype of this family, IGTP, localizes to the endoplasmic reticulum. This suggests that these GTPases are involved in intracellular defense. We analyzed the expression of the 47-kDa GTPase cognate, IIGP, in splenic sections from mice infected with the intracellular pathogen Listeria monocytogenes by immunohistochemistry. An early transient IIGP induction was observed revealing the IFN-gamma responsiveness of cellular subcompartments within the spleen in early listeriosis. Marginal metallophilic macrophages and endothelial cells within the red and white pulp strongly expressed IIGP, while other splenocytes remained negative. In vitro analyses show that both type I and type II IFNs are prime stimuli for IIGP induction in various cells, including L. monocytogenes-infected or LPS-stimulated macrophages, endothelial cells, and activated T cells. Contrary to the subcellular localization of IGTP, IIGP was predominantly associated with the Golgi apparatus and also localizes to the endoplasmic reticulum. We conclude that IIGP exerts a distinct role in IFN-induced intracellular membrane trafficking or processing

Methionine deficiency does not increase polyamine turnover through depletion of hepatic S-adenosylmethionine in juvenile Atlantic salmon

During the last few decades, plant protein ingredients such as soya proteins have replaced fishmeal in the diets of aquacultured species. This may affect the requirement and metabolism of methionine as soya contains less methionine compared with fishmeal. To assess whether methionine limitation affects decarboxylated S-adenosylmethionine availability and polyamine status, in the present study, juvenile Atlantic salmon were fed a methionine-deficient plant protein-based diet or the same diet supplemented with dl-methionine for 8 weeks. The test diets were compared with a fishmeal-based control diet to assess their effects on the growth performance of fish. Methionine limitation reduced growth and protein accretion, but when fish were fed the dl-methionine-supplemented diet their growth and protein accretion equalled those of fish fed the fishmeal-based control diet. Methionine limitation reduced free methionine concentrations in the plasma and muscle, while those in the liver were not affected. S-adenosylmethionine (SAM) concentrations were higher in the liver of fish fed the methionine-deficient diet, while S-adenosylhomocysteine concentrations were not affected. Putrescine concentrations were higher and spermine concentrations were lower in the liver of fish fed the methionine-deficient diet, while the gene expression of SAM decarboxylase (SAMdc) and the rate-limiting enzyme of polyamine synthesis ornithine decarboxylase (ODC) was not affected. Polyamine turnover, as assessed by spermine/spermidine acetyltransferase (SSAT) abundance, activity and gene expression, was not affected by treatment. However, the gene expression of the cytokine TNF-α increased in fish fed the methionine-deficient diet, indicative of stressful conditions in the liver. Even though taurine concentrations in the liver were not affected by treatment, methionine and taurine concentrations in muscle decreased due to methionine deficiency. Concomitantly, liver phospholipid and cholesterol concentrations were reduced, while NEFA concentrations were elevated. In conclusion, methionine deficiency did not increase polyamine turnover through depletion of hepatic SAM, as assessed by SSAT activity and abundance.publishedVersio

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

Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii

Members of the immunity-related GTPase (IRG) family are interferon-inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide-dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide-binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide-bound state of the GKS proteins, preventing their GTP-dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen's elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction

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

Choline supplementation increased total body lipid gain, while surplus methionine improved growth and amino acid retention in adult Atlantic salmon (Salmo salar)

Methionine–choline-deficient (MCD) mammals are known to accumulate liver TAG probably due to phosphatidylcholine (PC) deficiency and thus assembly of VLDL and transport of lipids from liver to peripheral organs. To assess whether supplementation of choline could spare methionine and secure a healthy liver metabolism, by reducing the endogenous PC synthesis without interfering with lipid transport and distribution, Atlantic salmon with initial BW of 700 g were fed adequate (1.9 g Met/16 gN) or surplus methionine (2.5 g Met/16 gN) diets of which were supplemented with choline or not for a period of 19 weeks. Fish fed the low-methionine diets had reduced growth (p = .013) due to reduced protein gain (p = .007), while lipid gain slightly improved in fish fed the choline-supplemented diets (p = .047). Also, feed conversion improved when fed surplus methionine (p < .001), while choline supplementation had no impact on feed conversion. No interaction between choline and methionine on growth performance or retention existed. Phospholipid status in liver and muscle was not affected by treatments, and no liver TAG accumulation occurred at the methionine levels used. Gene expression of ApoB100 necessary for assembling VLDL or pemt necessary for endogenous PC synthesis was unaffected by treatments. Capacity of methylation (MAT, BHMT) within the liver was not affected by treatment nor was the gene expression of enzymes in liver transsulfuration (CBS or CDO). Methionine status within liver was unaffected by treatments, while free methionine reduced in those fish fed the low-methionine diets in muscle and plasma. Cystathionine and taurine were elevated when fed surplus methionine. Choline supplementation had no impact on sulphur amino acid metabolites in either tissue. Neither did choline supplementation improve TAG mobilization from liver to muscle. To conclude, choline does not improve endogenous phospholipid synthesis or transport of TAG from liver to muscle depot when added to diets containing 1.9 g Met/16 gN, while surplus methionine improved growth and protein retention, indicating that 1.9 g Met/16 gN is enough to support a healthy liver metabolism, but too low to support muscle protein deposition in adult salmon fed high plant protein diets for longer periods of time.submittedVersio