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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: J Immunol. 2013 Jul 10;191(4):1765–1774. doi: 10.4049/jimmunol.1300641

Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells1

Helen M Springer *, Michael Schramm †, Gregory A Taylor ‡,§, Jonathan C Howard *,¶,2
PMCID: PMC3753587  NIHMSID: NIHMS494363  PMID: 23842753

Abstract

The interferon-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases (IRG) which function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1-deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes, on other organelles or as an inducer of autophagy. In this study we have re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1 using two different immunofluorescent staining techniques either in fibroblasts or in macrophages. We have demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria, however only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1−/− mice is reversed in Irgm1/Irgm3-double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the IRG protein family.

Introduction

There is increasing interest in cell-autonomous, mostly interferon-induced, effector mechanisms that confer basal non-adaptive resistance on mammals to a variety of different pathogens (1, 2).

The interferon-inducible protein Irgm1 (formerly called LRG-47) belongs to the family of immunity-related GTPases (IRG proteins) which function in cell-autonomous resistance against certain intracellular pathogens in mice (3, 4). Irgm1 is one of three mouse IRGM proteins, informally known as “GMS” IRG proteins (5) because they possess a distinctive non-canonical sequence, GxxxxGMS, in the P-loop of the GTP binding site. They can be distinguished from the second group in the IRG protein family, the “GKS” subfamily, that carry the canonical GxxxxGKS sequence (5, 6). In studies on resistance to the protozoal pathogen, T. gondii, principally the GKS proteins such as Irga6 or Irgb6 transfer from cytoplasmic compartments to the cytosolic face of the parasitophorous vacuole membrane (PVM) (7, 8) in a nucleotide-dependent (9) and cooperative manner (10). This process can lead to disruption of the PVM and death of the parasite (7, 11, 12). Of the GMS proteins, Irgm2 and Irgm3 are present at the T. gondii vacuole in low amounts and Irgm1 not at all (7, 10, 13). However the GMS proteins function as essential regulators of the GKS proteins by controlling their nucleotide-bound state in the cytosol and thereby preventing premature activation of the GKS IRG proteins before parasite entry: loss of GMS proteins causes premature activation of GKS proteins and unregulated oligomerisation in the cytosol instead of accumulation on the PVM (9).

Mice deficient in Irgm1 through gene targeting are highly susceptible to a number of protozoal pathogens, Toxoplasma gondii, Leishmania major or Trypanosoma cruzi (4, 14, 15) as well as intracellular bacteria such as Listeria monocytogenes, Chlamydia trachomatis, Mycobacterium avium, M. tuberculosis and Salmonella typhimurium (14, 16-19). In contrast, other IRG knock-out mice tested (14, 20, 21), including Irgm1−/−/Irgm3−/− and Irgm1−/−/IFN-γ−/− double knock-outs involving Irgm1 (22-24), are resistant to these bacteria, wherever tested, while retaining susceptibility to T. gondii and Chlamydia.

Models describing either cell-autonomous or systemic effects have been proposed to account for these Irgm1-associated phenotypes (25-28). The earliest, and still widely accepted, model proposes a cell-autonomous role for Irgm1 in phagocytosis of bacteria. Infection experiments using Mycobacterium tuberculosis attributed the resistance function of Irgm1 in wild-type mice to localization of the protein at the mycobacterial phagosome and to an Irgm1-facilitated accelerated acidification of the vacuole (16, 29, 30). This model gained some support from the finding that Irgm1 could be found at the phagocytic cup and phagolysosome of phagocytosed latex beads (31). Another related proposal arose from experiments suggesting that Irgm1 can stimulate the formation of autophagosomes and thereby participate in IFN-γ-dependent control of Mycobacteria (32, 33).

However, parallel studies noted that Irgm1-deficient mice infected with a number of different organisms such as M. avium, T. cruzi and S. typhimurium suffered a striking collapse of their lymphomyeloid systems (15, 17, 18) which could well be responsible for non-cell-autonomous, systemic susceptibility to many pathogens. Recently it has become clear that the effect of Irgm1 loss on the lymphomyeloid system is critically dependent on IFN-γ expression: trematodes, that excite exclusively a Th2 immunity without IFN-γ expression, do not cause collapse of the lymphomyeloid system in Irgm1-deficient mice and are resisted normally (22). Immune T lymphocytes from Irgm1-deficient mice undergo apoptosis-independent cell death when re-stimulated in vitro and this effect disappears if the responding cells are also deficient in IFN-γ expression (22, 34). Irgm1-deficient mice also show a marked constitutive haemopoietic stem cell defect which is abrogated in Irgm1−/−IFN-γR1−/−, and Irgm1−/−Stat1−/− double knock-out animals (35, 36).

These observations suggested an alternative explanation for the systemic failure of immunity in Irgm1-deficient mice, based on the role of Irgm1 as an essential regulator of the IRG protein-based resistance mechanism. As noted above, failure of regulation causes accumulation of the GKS subset of IRG proteins in cytoplasmic membrane-associated aggregates (9). These may have cytopathic effects in dividing lymphomyeloid cells (22, 27). In IFN-γ-induced Irgm1/Irgm3-double-deficient cells the concentrations of GKS IRG proteins are greatly reduced, plausibly reducing the hypothetical cytopathic effects and reversing the susceptible Irgm1 phenotype (23). An alternative interpretation is that Irgm1 normally opposes possible cytopathic effects of Irgm3 (23). It is highly likely that the marrow stem cell defect observed in Irgm1−/− animals has the same cause as the immunological defect, since this is also reversed in the Irgm1/Irgm3-double-deficient mouse (35, 36).

The view that Irgm1 can function directly as an effector in mycobacterial (and listerial) immunity has gained considerable support from successive observations of Irgm1 associated with mycobacterial and listerial phagosomes in IFN-γ-induced cells (16, 29, 30). In this study, we have re-examined this view in detail, as it is pivotal in assessing the various models concerning Irgm1 function. We document the existence and properties of two different isoforms of Irgm1 derived from differential splicing. With the help of new serological reagents which are able to detect short and long Irgm1 isoforms, we establish to the best of our ability that neither isoform of Irgm1 is in fact associated with either the listerial or the mycobacterial phagosome in mouse macrophages or primary fibroblasts.

In conjunction with the documented restoration of immunological competence in the Irgm1/Irgm3-double-deficient mouse (23), the results favor the view that the failure of Irgm1-deficient mice in mycobacterial and listerial immunity is indeed due to a complex regulatory failure and not to a direct cell-autonomous effect on the pathogen-containing compartment.

Material and Methods

Expression constructs

The following expression constructs were used: pGEX-4T2-Irgm1 and pGW1H-Irgm1 (31); the first 16 amino acids of pGEX-Irgm1-short (= Irgm1Δ1-16) were deleted using “QuikChange” site-directed mutagenesis kit protocol (Stratagene, La Jolla, CA) of pGEX-Irgm1 construct with the following primer 5′-ccccaggaattcccgggtcgaccaccatggcagagacccattatgctcccctgagc-3′. pGW1H-Irgm1-short was generated by subcloning Irgm1Δ1-16 fragment from pGEX-Irgm1-short into pGW1H (British Biotechnology, Oxford, U.K.) using SalI digestion.

Pfu-polymerase (Promega, Madison, WI) was used for PCR amplification, and primers were obtained from Operon Biotechnologies GmbH (Cologne, Germany). Restriction enzymes were purchased from New England Biolabs (Beverly, LA). All constructs were verified by sequencing.

Cell culture

The following cells and cell lines were used: RAW264.7 macrophages (ATCC), human embryonic kidney cells HEK293FT (kindly provided by Dr. Thomas Langer) C57BL/6 mouse embryonic fibroblasts (MEF) and Irgm1−/− T MEFs which are spontaneously transformed Irgm1−/− MEFs (13). All cells were cultured in DMEM, high glucose (Invitrogen Life Technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS, Biochrom AG, Berlin, Germany), 2 mM L-glutamine, 1 mM sodium pyruvate, 1x MEM non-essential amino acids, 100 U/ml penicillin and 100 mg/ml streptomycin (all PAA, Pasching, Austria). Primary bone marrow-derived macrophages (BMM) were prepared according to (18) with minor modifications and cultivated in RPMI (PAA) supplemented with 25% (v/v) FCS, 10% (v/v) L929 cell-conditioned medium, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (both PAA). Frozen BMMs were thawed for each experiment and seeded directly on glass slides.

Transient transfection of HEK293FT cells was conducted with 3 μg of DNA per 6 × 105 cells using FuGENE HD (Roche, Mannheim, Germany) according to the manufacturer's instructions. Cells were stimulated with 200 U/ml of mouse IFN-γ (PeproTech, Rocky Hill, NJ) for 24 h.

Immunological reagents

The following immunoreagents were used: supernatant of mouse hybridoma 1B2 cells against Irgm1 (13), rabbit anti-Irgm1 antiserum L115 raised against the peptides NESLKNSLGVRDDD and QTGSSRLPEVSRSTE (10), anti-LRG-47 A19 goat antiserum (#sc-11075, Lot no. I1004, E2107, L1610, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-LRG-47 P20 goat antiserum (#sc-11074, Lot no. I1004 Santa Cruz Biotechnology, Inc.), rabbit anti-Irgm1 antiserum rbMAE15 and chicken anti-Irgm1 chMAE15, both raised against the N-terminal peptide of the short isoform MAETHYAPLSSAFPC, rabbit anti-Irgm2 antiserum H53/3 (7, 10), mouse anti-IGTP monoclonal antibody Clone 7 (BD Transduction Science, now Santa Cruz Biotechnology, Inc.), rabbit anti-human IRGM polyclonal antibody (#ab69464 and 69465, Abcam plc, Cambridge, U.K.), rabbit anti-Calnexin antibody (Calbiochem Merck KGaA, Darmstadt, Germany), rat anti-LAMP1 1D4B monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa), mouse monoclonal anti-Gm130 (BD Bioscience, Heidelberg, Germany), mouse monoclonal Cytochrome C (BD Bioscience), mouse monoclonal anti-Complex II (#459200, Invitrogen Life Technology), mouse monoclonal anti-ActA antibody (kindly provided by Pascal Cossart and Edith Goudin), polyclonal rabbit anti-L. monocytogenes antibody (US Biologicals, Salem, MA), and polyclonal rabbit anti-M. tuberculosis (#B65601R, Meridian, Memphis, TN). Secondary antibodies were Alexa Fluor 488/555/647-labelled donkey anti-mouse, -rabbit, -chicken and -goat sera (all Molecular Probes, Invitrogen Life Technology), donkey anti-rabbit- (GE Healthcare, Freiburg, Germany), donkey anti-goat- (Santa Cruz Biotechnology, Inc.), and goat anti-mouse-HRP (horseradish peroxidase) (Pierce, Thermo Fisher Scientific, Bonn, Germany) antisera. 4′, 6-Diamidino-2-phenylindole (DAPI, Invitrogen Life Technology) was used for nuclear staining at a final concentration of 0.5 mg/ml.

Bacterial infection

Single colonies of L. monocytogenes, strain EGD, serotype 1/2a and the isogenic Δhly-deletion mutants of L. monocytogenes were expanded in brain-heart infusion (BHI) medium. For infection assay, L. monocytogenes were grown overnight in BHI medium, resuspended in fresh BHI medium and harvested during mid-log phase. M. bovis BCG were grown in Middlebrook 7H9 Broth supplemented with oleic albumin dextrose catalase (OADC) and 0.05% Tween80 and centrifuged at 25 g to remove clumps. After washing once with PBS, concentration of the bacteria was estimated by optical density measurement. M. bovis BCG were incubated with 1 mM TRITC isomer R -X succinimidyl ester in 0.1 M NaHCO3 in H2O, pH 9 for 1 h at room temperature (RT). Unbound dye was removed by repeated washing with PBS. The bacterial suspensions were diluted, L. monocytogenes was added to cells at a MOI of 5-10 (fibroblasts) or 0.5 (macrophages) and M. bovis BCG at an MOI of 5 (macrophages) in ice cold DMEM with 10% FCS. Adherence of bacteria was synchronized by centrifugation at 850 g, 4°C for 5 min. Subsequently, non-adherent L. monocytogenes were removed by triple washing with ice cold PBS. Infected cells were incubated in pre-warmed DMEM with 10% FCS. At specific times after infection, samples were fixed with 3% PFA in PBS for 20 min at RT followed by washing once with PBS.

Immunocytochemistry

Immunocytochemistry was performed on paraformaldehyde-fixed cells as described earlier (7, 31, 37). The differential staining was performed as follows: firstly exclusively extracellular bacteria were stained by blocking with 3% BSA (Roth, Karlsruhe, Germany) in PBS (blocking buffer 1) for 1 h at RT followed by staining the unpermeabilized cells with the primary antibody against bacteria as well as Alexa Fluor 647-conjugated secondary antibody diluted in blocking buffer 1 for 30 min at RT. Between all steps cells were triple washed with PBS. Thereafter intra- and extracellular bacteria as well as the protein of interest (Irgm1) were stained by permeabilization and blocking in blocking buffer 2 (3% BSA and 0.1% saponin in PBS) for 1 h at RT, followed by staining with both primary antibodies diluted in blocking buffer 2 for 1 h at RT as well as Alexa Fluor 488/555-conjugated secondary antibodies diluted in blocking buffer 2 for 30 min at RT. Between all steps cells were triple washed with 0.1% saponin in PBS and then mounted on glass microscopic slides in ProLong Gold anti-fade reagent (Invitrogen Life Technology). The images were taken with an Axioplan II fluorescence microscope and AxioCam MRm camera and processed by Axiovision 4.7. Confocal Images were taken with the Zeiss Meta Confocal microscope and processed by Zen 2011 software (all Zeiss, Oberkochen, Germany).

SDS-PAGE and Western blot

MEFs and L929 cells were lysed in 0.5% NP-40, 140 mM NaCl, 20 mM Tris/HCl (pH 7.6), 2 mM MgCl2 supplemented with Complete Mini Protease Inhibitor Cocktail, EDTA-free (Roche) 1h on ice. Post-nuclear supernatants of MEFs and L929 cells as well as whole cell lysates of HEK293FT were boiled 5 min at 95°C in SDS-PAGE sample buffer (1% SDS, 50 mM Tris/HCl (pH 6.8), 5% Glycerol, 0.0025% bromophenol blue, 0.7% β-mercaptoethanol). Equal amounts were subjected to 8-13, 5% gradient SDS-PAGE and Western blot. Protein transfer was confirmed by staining the nitrocellulose membranes with Ponceau S solution [0.2% Ponceau S (Roth) and 3% acetic acid in dH2O]. Membranes were blocked in 5% non-fat dry milk in PBS and probed for IRG proteins with the indicated primary and HRP-coupled secondary antibodies.

Immunoprecipitation

Irgm1 was immunoprecipitated from lysates of L929 and C57BL/6 MEFs which were stimulated 24 h with IFN-γ. Cells were lysed as above and postnuclear supernatants of 107 cells were incubated with 30μl rbMAE15 antisera overnight followed by 2 h incubation with 150 μl of protein A–Sepharose (GE Healthcare) suspension both at 4°C. Beads were washed three times with ice-cold lysis buffer without detergent and boiled in SDS-PAGE sample buffer for 5 min at 95°C. Proteins were resolved on 8-13, 5% gradient SDS-PAGE and silver-stained as described earlier (38, 39). Three independent immunoprecipitation experiments generated 10 samples of SDS bands; in all samples Irgm1 could be identified with a sequence coverage ranging from 43-82%. Irgm1−/− MEFs served as negative control.

Nano-LC coupled ESI mass spectrometry

2-4 bands with an approximate size of 40 kDa were cut out and samples were subjected to tryptic in-gel digest and nano-LC ESI-MS/MS as described earlier with minor modifications (38).

Sequest as implemented in the Proteome Discoverer 1.3 software (Thermo Scientific) was used for protein identification by searching the Uniprot database of Mus musculus using carbamidomethylation at cysteine and oxidation at methionine and phosphorylation at serine, threonine and tyrosine residues as variable modifications. Since the Mascot algorithm (version 2.2, Matrix Science) allows to set N-terminal modifications this search engine was used with acetylation at the protein N-terminus in addition to the above mentioned modifications. Mass tolerance for intact peptide masses was 10 ppm for Orbitrap data and 0.8 Da for fragment ions detected in the linear trap. Search results were filtered to contain only high confident rank 1 peptides (false discovery rate ≤1%) with a mass accuracy of ≤5 ppm, and a peptide length of ≥6 amino acid residues. In case of Sequest results peptides had to match score versus charge state criteria (2.0 for charge state 2, 2.25 for charge state 3 and 2.5 for charge state 4), in case of Mascot results the peptide score had to be at least 20 and the expectation value ≤ 0.05.

Results

Long and short isoforms of Irgm1

The mouse Irgm1 gene is located on chromosome 11 and contains two introns and three exons. Two different mRNAs of Irgm1 are generated through alternative splicing (6). The long mRNA has the initiator codon for the first methionine at the 3’-end of the second exon and translation gives rise to a full-length Irgm1 protein of 409 amino acids. In the alternative shorter splice variant the second exon is skipped leading to usage of an initiator codon in the third exon. The first methionine of the short isoform corresponds to amino acid position 17 of the long isoform (Fig. 1A). Transcriptome sequencing of interferon-induced diaphragm-derived cells from C57BL/6 mice showed that transcripts of both splice variants are present at the same ratio (unpublished data, Jingtao Lilue). Likewise, expressed sequence tags (ESTs) for both splice variants can be found abundantly in the NCBI database in different mouse strains (www.ncbi.nlm.nih.gov.com). Clearly both splice variants are generated at the transcript level.

Figure 1. Long and short Irgm1 isoforms and their detection by immunological reagents.

Figure 1

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(A) The Irgm1 gene gives rise to a long and short isoform due to alternative splicing of the second exon. In (B) identified N-terminal peptides specific for long and short Irgm1 isoform as well as the posttranslational modification such as N-terminal acetylation and phosphorylation are listed. The amino acid range of the found peptide is according to the sequence of the long Irgm1 isoform. IFN- γ-induced Irgm1 from C57BL/6 MEFs or L929 cells was immunoprecipitated followed by tryptic digestion and nano-LC ESI-MS/MS. Analysis with Sequest algorithm (using carbamidomethylation at cysteine and oxidation at methionine and phosphorylation at serine, threonine and tyrosine residues as variable modifications) as well with the Mascot algorithm (using N-terminal acetylation in addition to the above mentioned modifications) was used for protein identification by searching the Uniprot database of Mus musculus. Three independent immunprecipitations were performed. (C) Immunological reagents used for the detection of Irgm1 and their epitopes on the Irgm1 protein. The immunizing peptides used for the commercial goat polyclonal antibodies A19 and P20 (Santa Cruz Biotechnology, Inc.) map near the N-terminus (dashed lines). The immunizing peptides of anti-human IRGM rabbit polyclonal antibodies (ab69464/5, Abcam plc) map as shown on human IRGM (dotted line). (D) Mouse embryonic fibroblasts (MEFs) from wild type C57BL/6 mice and Irgm1-deficient mice were treated with 200 U/ml IFN-γ for 24 h and lysed in 1% Triton X-100. Additionally, HEK293FT cells were transiently transfected with pGW1H-Irgm1-long or pGW1H-Irgm1-short for 24 h and lysed with 2x SDS-sample buffer. Proteins were separated by 12% SDS-PAGE, Western blots were probed with different anti-Irgm1 antibodies (1B2, L115, rbMAE15 chMAE15, A19 and P20). Calnexin served as loading control. The asterisk marks an unspecific band. 1B2, A19 and P20 detect only the long Irgm1 isoform whereas L115, rbMAE15 and chMAE15 detect both isoforms of Irgm1.

To answer the question whether both isoforms are also translated, we immunoprecipitated endogenous Irgm1 from IFN-γ stimulated C57BL/6 mouse embryonic fibroblasts (MEFs) or L929 (C3N/An) cells and analyzed silver-stained bands from SDS-PAGE (see Supplemental Fig. 1A) by mass spectrometry (MS). In bands with an approximate size of 40kDa Irgm1 was the most abundant protein and could be recovered with sequence coverage ranging from 43-82%. The tryptic digest of Irgm1 generates N-terminal peptides that can discriminate long (38aa peptide) and short Irgm1 (24aa peptide) isoforms. Both isoform-specific N-terminal peptides were detected in our MS analysis showing the presence of both endogenous Irgm1 isoforms. The N-terminal peptide of the short Irgm1 isoform was found repeatedly and in three different states: unmodified, N-terminally acetylated, or N-terminally acetylated and phosphorylated (Fig. 1B, Supplemental Table I). In contrast, the N-terminal peptide of the long Irgm1 isoform was found only once, as an acetylated peptide, maybe due to the large size of this peptide. However the existence of abundant endogenous long isoform could be confirmed with specific antibodies (see below). The existence of two splice forms of Irgm1, encoding proteins that differ substantially at the N-terminus, has not been considered in earlier studies. Since a phosphorylation site of Irgm1 at Serine 202 has been proposed at the phagosome of IFN-γ stimulated RAW264.7 mouse macrophages (40), we wanted to know whether we could determine additional phosphorylation sites of Irgm1 Besides the N-terminally acetylated and phosphorylated peptides of the short isoform described above, we found another phosphorylated peptide that matched our search criteria. CID fragmentation of phospho-serine- and –threonine results in favorite neutral loss of phosphoric acid. The MS/MS spectrum is characterized by a peak corresponding to the loss of phosphoric acid from the parent mass and fewer low-intense fragments making it difficult to determine the exact site of phosphorylation. The phosphoRS algorithm implemented in the Proteome Discoverer software calculates probabilities (see Supplemental Table I for possible phosphorylation sites).

Immunological reagents to detect long and short Irgm1 isoforms

Several immunological reagents against Irgm1 protein have been produced by us and others over the years. Their epitopes on Irgm1 protein are shown in Fig. 1C. The mouse monoclonal antibody 1B2 was raised against the peptide CEAAPLLPNMAETHY (residues 8-22) near the N-terminus of Irgm1 (13). This peptide crosses the differential splice site at residue 17, and only the C-terminal 6 residues (-MAETHY) of the immunising peptide are present in the short Irgm1 isoform. 1B2 antibody is therefore expected to have a preference for the long Irgm1 isoform. The first rabbit polyclonal antiserum was raised against the immunising peptide YNTGSSRLPEVSRSTE (residues 36-50) which is shared by both isoforms (14). The second rabbit polyclonal antiserum L115 was raised against a combination of two peptides, again QTGSSRLPEVSRSTE (residues 36-50) and NESLKNSLGVRDDD (residues 284-298) of Irgm1 (10). These two rabbit polyclonal antisera both showed an unspecific band at about 50kDa perhaps due to cross-reactivity from the shared peptide N-terminal immunogen (see Fig. 1D and (14)). Two goat polyclonal antibodies from Santa Cruz Biotechnology, Inc., A19 and P20, were raised against peptides from N-terminal regions of Irgm1, and though the exact sequences of the immunising peptides are not available, the A19 antibody was described as having been raised and purified against a 15-25aa peptide derived from a region between amino acids 20 and 70 of Irgm1 (personal communication C. Gernemann, Santa Cruz Biotechnology, Inc.). Two further rabbit polyclonal antibodies from Abcam plc (ab69464 and ab69465) were produced by immunisation with a peptide from the human homologue IRGM and were described as cross-reactive on mouse IRGM proteins (e.g. used to detect Irgm1 in (41)). We have now also produced two new reagents against the N-terminal peptide of the short isoform, MAETHYAPLSSAFPC (residues 17-31), in rabbit (rbMAE15) and in chicken (chMAE15). These antisera should detect the short isoform but may also recognize the long isoform of Irgm1, depending on the degree of immunodominance of the free N-terminal methionine.

In order to detect the two isoforms of Irgm1 differentially we tested these antibodies in Western blot assays. As depicted in Fig. 1D, in Western blots of lysates of C57BL/6 MEFs all reagents detected an IFN-γ-inducible, diffuse band running at or just below 40kDa which was absent in MEFs from Irgm1-deficient mice. The diffused band in non-gradient SDS-polyacrylamide gel electrophoresis and its apparent molecular weight well below the expected 47kDa are characteristic of endogenous Irgm1 in our hands (see e.g. (10)). Gradient SDS-PAGE as shown in Fig. 1D did not markedly improve the resolution of distinct bands representing the two isoforms (e.g. see rbMAE15 antiserum). The reason for the diffuse running behavior and low apparent molecular weight of Irgm1 in SDS-PAGE is not known but might partially be explained with post-translational modifications as suggested by the MS analysis. The IFN-γ-insensitive, presumably irrelevant 50kDa band seen by L115 was discussed above.

In order to clarify the isoform specificity of the different immunological reagents, transient transfection of eukaryotic expression plasmids encoding the long and short Irgm1 isoforms under the control of the CMV promoter, both with native N- and C-termini, into HEK293FT human cells (there is no Irgm1 gene in the human genome) was carried out. Expression of both protein isoforms was possible (Fig. 1D) although the expression of the short form always appeared somewhat weaker. Transfection of the short Irgm1 isoform into Irgm1-deficient MEFs gave fewer detectably transfected cells compared to the long Irgm1 isoform and expression of the transfected short form overall was too low to be detected in Western blots (data not shown).

In the transfected HEK293FT cells, monoclonal antibody 1B2 detected indeed only the long form of Irgm1 but not the short isoform. The rabbit antiserum L115 detected long and short isoforms equally, as expected from the locations of the immunising peptides. However, A19 as well as P20 showed a very strong preference for the long Irgm1 isoform, suggesting that the immunogenic peptides for these sera must in fact have consisted largely of sequence N-terminal of the splice junction, despite the statement from the Santa Cruz Biotechnology that a peptide between residues 20 and 70 was used (see above). The two new antisera, rbMAE15 and chMAE15, raised against the N-terminal peptide of the short isoform, also detected both long and short Irgm1 isoforms, indicating that the predominant epitopes seen by both rabbit and chicken were not defined by the free N-terminus of the short form. The same results were obtained using bacterially expressed GST-Irgm1 long and short isoforms (data not shown). A disappointing conclusion from this result is that there is still no reagent that is specific for the short isoform of Irgm1. The commercial (Abcam plc) rabbit polyclonal antibodies ab69464 and ab69465 raised against human IRGM detected both isoforms of bacterially expressed GST-Irgm1 as well as, respectively, GST-Irgm2 or GST-Irgm3 (Supplemental Fig. 1B). These reagents therefore cannot be used to identify Irgm1 unambiguously in mouse cells.

The short Irgm1 isoform does not colocalize with lysosomal compartments but both isoforms partially colocalize with mitochondria

Both Golgi and endolysosomal localization of Irgm1 have been shown to depend on the amphipathic alpha helix K in the C-terminal domain of the protein (30, 31, 37), but in these published experiments the short isoform was not resolved. Since none of our antibody reagents detected only the short form of Irgm1 we transfected Irgm1 expression constructs encoding either the long isoform (Irgm1-long) or the short isoform (Irgm1-short) into Irgm1-deficient MEFs. As expected, because of the presence of the targeting K alpha helix in the short as well as the long Irgm1 isoform, the short expressed protein showed a typical adnuclear Golgi signal colocalizing with GM130 (Fig. 2A), and a widely distributed non-Golgi component comparable to that seen with the long isoform. The localization of Irgm1 outside the Golgi was shown previously to correspond at least in large part with a LAMP1 positive organelle, identifying a lysosomal or late endosomal compartment (Fig. 2B, (37). Unexpectedly, however, the non-Golgi signal of the transfected short Irgm1 isoform solidly failed to colocalize with LAMP1 (Fig. 2C) or with early or recycling endosomes identified with fluorescent transferrin (data not shown). Thus despite the presence of the K alpha helix in both long and short isoforms, the short differential sequence at the N-terminus of the long isoform is required for significant targeting of Irgm1 to the endolysosomal system.

Figure 2. Short Irgm1 isoform localizes to Golgi, but not to lysosomes.

Figure 2

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Irgm1-deficient MEFs were transfected with pGW1H-Irgm1-long or pGW1H-Irgm1-short for 24 h and fixed. Cells were stained for Irgm1 and Golgi marker protein GM130 (A) or late endosomal-lysosomal marker protein LAMP1 (B, C) using rbMAE15 and anti-GM130 or 1D4B immunoreagents. Nuclei were labeled with DAPI. Three independent experiments were performed.

Endogenous, interferon-induced mouse Irgm1 has been reported to be associated with mitochondria in RAW264.7 (30) and ML-14a hepatoma cells (41), using A19 and rabbit anti-human IRGM antisera respectively to detect Irgm1, and cardiolipin or O-N-nonyl acridine orange and Tom40 respectively as mitochondrial markers. A19 is a relatively weak reagent (Supplemental Fig. 2), and the rabbit anti-human IRGM serum used by Chang and colleagues (41) does not discriminate between Irgm1 and Irgm2 (see Supplemental Fig. 1B). We therefore examined the mitochondrial localization of mouse Irgm1 in MEFs with the new rabbit polyclonal antiserum, rbMAE15. In confocal images we could demonstrate that IFN-γ-induced Irgm1 as well as transfected long and short isoforms clearly colocalize in part with the mitochondrial markers Complex II and Cytochrome C (Fig. 3). However, even with this powerful serum the staining intensity on mitochondria is weak.

Figure 3. Endogenous Irgm1 and both transfected Irgm1 isoforms partially colocalize with mitochondria.

Figure 3

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(A) MEFs from wild type C57BL/6 mice were treated with 200 U/ml IFN-γ for 24 h. (B) Irgm1-deficient MEFs were transiently transfected with pGW1H-Irgm1-long or pGW1H-Irgm1-short for 24 h. The cells were fixed and stained for Irgm1 and mitochondrial marker using rbMAE15 and anti-Cytochrome C or anti-Complex II immunoreagents. Nuclei were labeled with DAPI. Images were taken with a confocal microscope. Scale bars represent 10μm. Two (ComplexII) or three (CytC) independent experiments were performed.

In summary, the long isoform of Irgm1 can be detected with all immunological reagents (A19, 1B2, L115, rbMAE15 and chMAE15) and is associated strongly with Golgi, and weakly with mitochondrial and lysosomal membranes. In contrast, the short isoform of Irgm1 is not detected by 1B2 and A19, localizes strongly to the Golgi and weakly to mitochondrial membranes but does not localize to the endolysosomal compartment.

Irgm1 does not localize to listerial phagosomes

An association of Irgm1 with bacterial phagosomes was first proposed in a cell-fractionation protocol in macrophages infected with M. tuberculosis BCG (16). Subsequently, striking colocalization with listerial and mycobacterial phagosomes was reported in an immunofluorescence protocol for Irgm1 in RAW264.7 macrophages shortly after infection (29). A more recent paper again reported immunofluorescent colocalization of Irgm1 with mycobacterial phagosomes (30). In these experiments, A19 was used as the Irgm1 detection reagent. We wanted to confirm these results with better characterized immunological reagents, using a differential staining method to discriminate between intracellular and extracellular bacteria (see Materials and Methods), a control that was not apparent in the earlier publications. In Fig. 4, MEFs were treated with IFN-γ, infected with L. monocytogenes, and analyzed for Irgm1 localization by immunofluorescence with the antibodies 1B2, rbMAE15, and chMAE15. Strong Golgi-like staining and weaker cytoplasmic staining with all three anti-Irgm1 reagents was consistent with positive identification of Irgm1 (Fig. 4 A, see also Fig. 2, 3). Transects across cells containing intracellular bacteria were quantified for Irgm1 fluorescence intensity associated with bacteria. By these criteria, colocalization of Irgm1 was never detected at the phagosome of intracellular wild-type Listeria. The possibility that this result was due to early escape from the phagosome was excluded by applying the same techniques to IFN-γ-induced cells infected with the listeriolysin O-deficient L. monocytogenes (Δhly) which cannot escape from the phagosome (42). After 30 min, 60 min and 120 min of infection in MEF cells, 0% of the intracellular L. monocytogenes Δhly were found to be Irgm1-positive (Fig. 4 B). The experiment was repeated in RAW264.7 macrophages, again no intracellular bacterium could be colocalized with Irgm1 (Fig. 4B).

Figure 4. Endogenous Irgm1 is not detected at intracellular Listeria monocytogenes.

Figure 4

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(A) MEFs were treated with 200 U/ml IFN-γ for 24 h, and infected with Listeria monocytogenes wt for 15 minutes. The cells were washed, fixed and stained initially without permeabilization against Listeria with Alexa Fluor 647-conjugated secondary antibody. Thereafter, cells were permeabilized (0.1% saponin) and stained for Irgm1 using 1B2, rbMAE15 or chMAE15 with Alexa Fluor488-conjugated secondary antibody and for Listeria with Alexa Fluor555-conjugated secondary antibody. Nuclei were labeled with DAPI. Transects were drawn through extracellular bacteria (in far-red detection channel, shown in magenta) and intracellular bacteria (red). The profiles show the pixel intensity of the 4 different detection channels within this transect. No local increase in the Irgm1 (green) signal is detected associated with intracellular Listeria. (B) Quantification of (A), of the infection with RAW264.7 macrophages and of later time-points post infection. For the 30 min, 60 min and 120 min p.i, cells were infected with the Listeria monocytogenes mutant Δhly. 300 - 500 cell nuclei were evaluated per sample, n indicates number of replicated samples.

The DNA chromosome of ingested Listeria in phagolysosomes can be detected with DAPI in LAMP1-positive compartments. RAW264.7 macrophages infected with L. monocytogenes (Δhly) had ingested Listeria into LAMP1-positive compartments 2h after infection (Fig. 5 and Supplemental Fig. 3). These bacterium-containing LAMP1-positive compartments were never found to carry Irgm1 protein. Surprisingly in view of the repeated observation of Irgm1 on latex bead phagosomes (13, 31), Irgm1 was also never seen on phagosomes containing heat-killed L. monocytogenes (Supplemental Fig. 3A)

Figure 5. Endogenous Irgm1 does not localize to the phagolysosome of Listeria monocytogenes.

Figure 5

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RAW264.7 cells were treated with 200 U/ml IFN-γ for 24 h, and infected with the Listeria monocytogenes mutant Δhly for 2 h. The cells were washed, fixed and stained for Irgm1 and LAMP1 using rbMAE15 and 1D4B immunoreagents. Nuclei were labeled with DAPI. Irgm1 was not detected at the LAMP1-positive bacterial phagolysosome. Images were taken with a confocal microscope. The experiments were performed twice.

These results clearly differ from the results of the studies mentioned above using the A19 antibody (29, 30). It would in principle be possible to confirm immunolocalizations attributed to endogenous Irgm1 by using tagged constructs of the protein in a transfection protocol. Thus Irgm1 C-terminally tagged with EGFP has also been reported to associate with mycobacterial phagosomes (30, 43). However the significance of these observations is not clear in view of the striking mislocalization of both N- and C-terminally tagged Irgm1 (31, 37). In the present study transiently transfected EGFP-Irgm1 and Irgm1-EGFP and EGFP-Irgm1αK showed the same localizations in RAW264.7 cells as described earlier (30, 31, 37), but did not relocalize to intracellular L. monocytogenes in our experiments (data not shown).

Irgm1 does not localize to the phagosome of Mycobacterium bovis BCG

The effector model attributing Irgm1 action to accelerated maturation of bacterial phagosomes is based partly on observations suggesting an association of Irgm1 with M. bovis BCG phagosomes in mouse macrophages. In the first report of this association, Irgm1 was detected in a Western blot of BCG phagosomes isolated 20 minutes after infection of mouse bone marrow-derived macrophages in vitro (16). Subsequent publications have illustrated colocalization of anti-Irgm1 antibodies or fluorescent Irgm1 fusion constructs with BCG phagosomes (29, 30, 33, 43, 44). In view of our inability to confirm the reported colocalization of Irgm1 with the listerial phagosome we decided to examine the mycobacterial phagosome with similar methods. We infected RAW264.7 cells with M. bovis BCG, differentiated serologically between intracellular and extracellular organisms and stained for Irgm1 using 1B2. Chicken MAE15 could not be used because it cross-reacted with the surface of extracellular Mycobacteria. In contrast to expectation from the published findings, Irgm1 detected by the monoclonal 1B2 antibody was absent at the mycobacterial phagosome at any analyzed time points (15 min till 4h p. i., Fig. 6). Rabbit MAE15 antiserum could not be used in combination with the rabbit anti-Mycobacterium antibody used to define intracellular organisms, but as a second method we infected with TRITC-labeled M. bovis BCG and visualized intracellular organisms with rbMAE15 via costaining with LAMP1. No Irgm1 was detectable at bacterial phagolysosomes from 1h to 4h p. i. in RAW264.7 cells (Fig. 7A, B and Supplemental Fig. S3D/E) or primary bone-marrow derived macrophages (BMM) (Fig. 7 C, D).

Figure 6. Endogenous Irgm1 is not detected at intracellular Mycobacteria bovis.

Figure 6

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RAW264.7 cells were treated with 200 U/ml IFN-γ (B, C) for 24 h or left untreated (A), and infected with Mycobacterium bovis BCG for 1 h (A, B) or 4 h (C). The cells were washed, fixed and stained, initially without permeabilization against Mycobacteria with Alexa Fluor647-conjugated secondary antibody. Thereafter, cells were permeabilized (0.1% saponin) and stained for Irgm1 using 1B2 with Alexa Fluor488-conjugated secondary antibody, and against Mycobacteria with Alexa Fluor555-conjugated secondary antibody. Nuclei were labeled with DAPI. Transects were drawn through extracellular bacteria (in far-red, shown in magenta) and intracellular bacteria (red). The profiles show the pixel intensity of the different channels within this transect. No Irgm1 (green) signal is associated with intracellular Mycobacteria. At least three independent experiments were performed.

Figure 7. Endogenous Irgm1 does not localize to the phagolysosome of Mycobacterium bovis.

Figure 7

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RAW264.7 cells (A, B) or bone marrow-derived macrophages from wild type C57BL/6 mice (C) or Irgm1-deficient mice (D) were treated with 200 U/ml IFN-γ for 24 h, and infected with TRITC-labeled Mycobacterium bovis BCG for 1 h (A, C, D) or 4 h (B). The cells were washed, fixed and stained with Irgm1 and LAMP1 using rbMAE15 and 1D4B immunoreagents. Nuclei were labeled with DAPI. Transects were drawn through bacteria (in red) and LAMP1 (in far-red, shown in magenta) to demonstrate their residence in the phagolysosome, profiles show the pixel intensity of the different channels within this transect. No Irgm1 signal (green) is associated with the mycobacterial phagosomes. The experiment was performed twice.

Discussion

Functional analysis of the interferon-γ-inducible immunity-related GTPase, Irgm1, has followed two tracks since the observation that Irgm1-deficient mice are susceptible to Mycobacteria (16, 17). One track holds that Irgm1 is an effector of resistance at the bacterial phagosome, either by accelerating phagosome-lysosome fusion (16, 29, 30) by the stimulation of autophagy (32, 33), or possibly by some other effect mediated through other organellar systems (28). The second track stresses the role of Irgm1 as a regulator of other GKS IRG GTPases (27) or another GMS IRG protein, Irgm3 (23, 24, 36). Failure of regulation of the IRG system as a result of loss of Irgm1 leads, for unclear reasons, to an IFN-γ-dependent collapse of the lymphomyeloid system, causing general susceptibility to pathogens (15, 17, 18). That Irgm1 cannot be a general effector of resistance is already suggested by the recovery of immune competence to all organisms tested except T. gondii when a second IRG protein, Irgm3, is also absent (23, 24). In this latter model, therefore, IFN-γ-inducible resistance occurs without the necessity for Irgm1 to be located at the bacterial phagosome or, indeed, present at all. This is the context in which we here explore in some detail whether Irgm1 is in fact located on bacterial phagosomes, as reported earlier (29, 30, 43, 44). Our experiments do not directly address other potential modes of action of Irgm1 that could impact bacterial survival, such as control of Golgi, lysosomal traffic or mitochondrial function. Our study was directed at this specific aspect of Irgm1 phenomenology because of the numerous complexities associated with colocalization experiments conducted by immunofluorescence microscopy. To these problems one may add the localization artefacts associated with transfected, tagged constructs used in some experimental models, that have been documented previously for Irgm1 (31, 37). The localization of Irgm1 in uninfected, IFN-γ-induced cells is not simple. Initial experiments on this topic established the strong Golgi localization due to an amphipathic targeting helix (13, 31) since confirmed (30, 37). However it was noted even in the first report that there is a significant cytoplasmic signal outside the Golgi, and this was subsequently attributed at least in part to colocalization with LAMP1-positive organelles (37). A further colocalization of endogenous Irgm1 with mitochondria has also been observed (30, 41). In the present report we can confirm all of these localizations in fibroblasts using new reagents. It may however be of interest that only the long Irgm1 isoform was detected at endolysosomal organelles. A possible functional difference for the two Irgm1 isoforms associated with their different intracellular localization behavior remains to be investigated. Thus, while earlier accounts of individual endogenous Irgm1 localizations in uninfected, IFN-γ-induced cells were certainly incomplete, there is no significant discrepancy in the results as they now stand.

The present data, however, do not support claims that Irgm1, whether endogenous or transfected, colocalizes with bacterial phagosomes. The first report on this topic (16) did not strictly show colocalization, but rather copurification via a procedure intended to purify phagosomes from cells infected by Mycobacteria. However the now known association of endogenous Irgm1 with Golgi membranes, LAMP1-positive compartments and mitochondria renders the conclusion from these experiments questionable in the absence of formal exclusion of all these compartments from the putative phagosomal fraction. The most direct support for a colocalization of Irgm1 with both listerial and mycobacterial phagosomes has been an immunolocalisation study from Shenoy and colleagues (29). These authors showed effectively 100% coincidence of intense immunofluorescent signals from organisms and Irgm1 in IFN-γ-induced RAW264.7 macrophages infected in vitro with either Mycobacterium bovis BCG or Listeria monocytogenes. The discrepancy with our own results here on the same bacteria is not reconcilable except via technical differences. Our results are based on the analysis exclusively of intracellular organisms, defined by a two-stage staining protocol. Only those organisms that failed to stain without permeabilization of the cells but stained after permeabilization were used for analysis. Shenoy and colleagues did not distinguish between extracellular and intracellular organisms and in our hands extracellular organisms are in the majority in such preparations. In the case of Listeria we were able to employ a new, high-titred and highly specific rabbit antiserum, rbMAE15, directed against an N-terminal peptide of Irgm1 as well as the mouse monoclonal antibody, 1B2, to show in repeated transect scans that there is no local concentration of Irgm1 at the listerial phagosome, either shortly after infection in the case of wild-type L. monocytogenes, or up to 120 minutes after infection with the listeriolysin O-deficient strain, Δhly, which is unable to escape from the phagosome. We replicated this result in RAW264.7 cells and mouse embryonic fibroblasts. We also identified mycobacterial and listerial phagosomes carrying LAMP1 followed by analysis of local Irgm1 distribution using rbMAE15. In no case did we detect any accumulation of Irgm1 associated with these LAMP1-positive vacuoles. Moreover, and perhaps surprisingly, accumulation of Irgm1 could also not be detected at phagocytosed heat-killed Listeria in contrast to the repeatedly observed Irgm1 localisation at phagocytosed latex beads (13, 31, 37). Our conclusion from these experiments is that Irgm1 does not accumulate at either listerial or mycobacterial phagosomes.

Irgm1 has also been implicated in the facilitation of autophagy of Mycobacteria in IFN-γ-induced macrophages and macrophage cell lines (32, 33). Although these experiments could have yielded data on colocalization of Irgm1 with mycobacterial phagosomes and autophagosomes, no such observations were in fact made. Furthermore, the protocols used involved overexpression of a GFP fusion construct (not explicit, but probably Irgm1-GFP C-terminal fusion) which in our hands clearly mislocalises (37). A general role for Irgm1 as a pro-autophagic agent in mice has been uncertain for some time, since the interferon-dependent lymphomyeloid collapse associated with Irgm1-deficiency is accompanied by increased rather than decreased autophagy in T cells (22, 35) and haemopoietic stem cells (36). Very recently a role for Irgm1 in IFN-γ-dependent autophagy has been put in further doubt by the observation of normal autophagy in macrophages from Irgm1-deficient mice (45).

In summary, it appears that two roles attributed to Irgm1 in IFN-γ-induced cells in mice have not stood up to closer investigation. Neither a direct effector role of Irgm1 on the phagosomal membrane in accelerated acidification, nor a role in autophagy appear to be relevant to resistance to Mycobacteria or, probably, many other organisms. The only property of Irgm1 that appears to be robust is its role as a negative regulator of a specific subset of other IRG protein family members, namely the IRG proteins with the canonical G1 motif, the “GKS” subset (9), that probably act as effectors in the destruction of the Toxoplasma gondii parasitophorous vacuole membrane (7, 11, 12). It is not yet known why loss of Irgm1 causes damage to proliferating lymphomyeloid cells following IFN-γ induction. The hypothesis that it is an indirect consequence of the off-target activation of IRG effector proteins (9) has not yet been put to the test. Whatever the explanation for these phenomena may be, we would argue that the data presented here suggest that the loss of resistance to Mycobacteria, Listeria and many other organisms caused by Irgm1-deficiency is due to lymphomyeloid collapse and not to loss of a specific effector function of Irgm1 on the microbial phagosome.

Supplementary Material

1

Acknowledgement

We thank Paul Hasselgren of Innovagen for his advice and assistance during the preparation of the chicken and rabbit MAE15 reagents. We thank Pascal Cossart and Edith Goudin for providing the ActA antibody and Thomas Langer for providing HEK293FT cells as well as the mitochondrial markers. We thank Rita Lange for cloning Irgm1 constructs and Astrid Schauss for her technical assistance with the confocal microscopes. We thank Tobias Lamkemeyer and Denise Ungrue for performing MS analysis. We thank Miriam Dutra and Tobias Steinfeldt for critical comments on the manuscript.

Abbreviations

BCG

Bacillus Calmette-Guérin

LAMP1

lysosome associated membrane protein

GM130

cis-Golgi matrix protein

WT

wildtype

Footnotes

1

Financial support for this project was provided by the Sonderforschungsbereich SFB670 of the Deutsche Forschungsgemeinschaft (to H.M.S., M.S. and J.C.H.) as well as NIH grant AI57831 and a VA Merit Review grant (to G.A.T.).

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