Targeting of the GTPase Irgm1 to the phagosomal membrane via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) promotes immunity to mycobacteria

Abstract

Vertebrate immunity to infection enlists a newly identified family of 47-kilodalton immunity-related GTPases (IRGs). One IRG in particular, Irgm1, is essential for macrophage host defense against phagosomal pathogens, including Mycobacterium tuberculosis (Mtb). Here we show that Irgm1 targets the mycobacterial phagosome through lipid-mediated interactions with phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and PtdIns(3,4,5)P(3). An isolated Irgm1 amphipathic helix conferred lipid binding in vitro and in vivo. Substitutions in this region blocked phagosome recruitment and failed to complement the antimicrobial defect in Irgm1(-/-) macrophages. Removal of PtdIns(3,4,5)P(3) or inhibition of class I phosphatidylinositol-3-OH kinase (PI(3)K) mimicked this effect in wild-type cells. Cooperation between Irgm1 and PI(3)K further facilitated the engagement of Irgm1 with its fusogenic effectors at the site of infection, thereby ensuring pathogen-directed responses during innate immunity.

Figure 1

Endogenous Irgm1 traffics from the cis-Golgi to MPGs in IFN-γ-activated macrophages. (a) IFN-γ-activated (24h, 100U/ml) RAW264.7 cells were stained with Alexa488-conjugated anti-Irgm1 plus Alexa594-conjugated anti-GM130 mAb (top) or Alexa594-conjugated anti-EEA-1 (bottom). Scale bar, 10μm. (b) BMMs (left) or RAW264.7 cells (right) were chased for 30 min with M. bovis BCG Phipps (MOI 2: 1), after which cells were stained with Alexa488-conjugated anti-Irgm1 and Alexa594-conjugated anti-Mtb complex to detect nascent MPGs. Arrowheads, MPGs; scale bar, 2μm; DIC, Differential Interference Contrast. Images selected from 3-4 independent experiments. (c) Left, RAW264.7 cells stained at 6 h p.i. with TO-PRO-3 to detect bacterial and host nuclear DNA and anti-Irgm1. Inset, Irgm1-“coated” MPG. Right, Percent of total MPGs that were Irgm1⁺ in IFN-γ-treated (filled circles) or untreated (open circles) macrophages at various times p.i. (>300 MPGs counted/time point). (d) IFN-γ-activated RAW264.7 cells expressing RFP-Irgm1 (pseudocolored green) were infected with EGFP-expressing M. bovis BCG Pasteur (pseudocolored red) and subjected to live imaging. Blue bar placed across the lower mycobacterium (depicted here in the first frame only) yielded fluorescence intensity plots for Irgm1 and M. bovis below. White arrows depict both lower and upper bacteria. Scale bar, 5μm. Representative of 8 movies collected. (e) EM of immuno-labeled cryosections showing 15nm BSA-gold fused M. bovis BCG phagolysosomes (red arrow) harboring endogenous Irgm1 (10nm Protein A-gold; black arrows) in IFN-γ-activated RAW264.7 cells at 6 h p.i. BSA-gold was pulse-chased into lysosomes overnight. Scale bar, 400nm.

Figure 2

Irgm1 is a membrane-associated protein that binds PtsIns(3,4,5)P₃, PtdIns(3,4)P₂, and diphosphatidylglycerol (a) IFN-γ-activated macrophage sub-fractions were immunoblotted with Irgm1 or _p47 GTPase-specific antibodies. Irgm1 migrates slightly below its predicted M_r. One of three sub-fractionation experiments shown. (b) Protein gel overlay using 5μg/ml rGST-Irgm1, rGST-Irgm2, rGST-Irgm3 or rGST-Irgb6. 1, triglyceride; 2, diacylglycerol; 3, phosphatidic acid; 4, phosphatidylserine; 5, phosphatidylethanolamine; 6, phosphatidylcholine; 7, phosphatidylglycerol; 8, diphosphatidylglycerol; 9, phosphatidylinositol (PtdIns); 10, PtdIns(4)P; 11, PtdIns(3,4)P₂; 12, PtdIns(3,4,5)P₃; 13 cholesterol; 14, sphingomyelin; 15, sulfatide; 16, Solvent blank; 17, lysophoshatidic acid; 18, lysophosphocholine; 19, PI(3)P; 20, PtdIns(4)P; 21, PtdIns(5)P; 22, sphingosine-1-phosphate; 23, PtdIns(3,5)P₂; 24, PtdIns(4,5)P₂; 25, sphingosine; 26, phytosphingosine; 27, ceramide; 28, sphingomyelin; 29, sphingosylphosphatidylcholine; 30, myriocin; 31, monosialoganglioside G_M1; 32, disialoganglioside; 33, sphingosylgalactoside; 34, lysophosphatidylcholine. (c) Protein gel overlay using rGST alone, rGST-Irgm1 or rGST-Irgm1 pre-incubated with 15-fold molar excess of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃. rGST-Irgm1 bioactive lipids strip from (b) included for comparison. (d) Dose-responsive lipid binding to rGST fusion proteins. One of three gel overlay experiments shown. (e) rGST-Irgm1 was incubated with liposomes containing 5% mol/mol PtdIns(3,4)P₂ or PtdIns(3,4,5)P₃. Material was analyzed by immunoblot with anti-GST prior to (pre-pellet) and after (post-pellet) sedimentation by centrifugation. One of four sedimentation assays shown.

Figure 3

An Irgm1 C-terminal amphipathic helix confers membrane binding in vitro and in vivo. (a) Irgm1 structure based on dimeric Irgb6 (PDB code 1TQ2:2) depicting the membrane-accessible αK helix. Inset. Mutations generated within this region. (b) PtdIn binding profile for matched molar equivalents of rGST-Irgm1 (5μg/ml), rGSTμK (1.9μg/ml), rGSTIrgm1(F362E,R367E) (5μg/ml) and rGST-Irgm1(K350,R352,M354A,C356) (5μg/ml) in gel overlay. Derived from 2-3 independent overlay assays. (c) rGST-αK was incubated with liposomes containing no PtdIns (Control, C) or liposomes containing 5% w/w PtdIns(3,4)P₂ (P₂) or PtdIns(3,4,5)P₃ (P₃). Material was analyzed by immunoblot with anti-GST prior to (pre-pellet) or after (post-pellet) sedimentation by centrifugation. One of two sedimentation assays shown. (d) Lipid-binding profile of rGST-Irgm1(F362E,R367E) (labeled 362,367) (5μg/ml), rGSTIrgm1(K350,R352,M354A,C356) (labeled KMRC) (5μg/ml) and catalytically-active G-domain fragment (rGST-GD75-292; 3.8μg/ml). The membrane lipids strip from Fig. 2(b) included for comparison. (e) Intrinsic GTPase activity of lipid-binding and non-binding Irgm1 variants in $\tilde{\gamma }$[³²P]GTP hydrolysis assays. GST, heat-inactivated (HIA) GST-Irgm1 and catalytically-inactive rGST-Irgm1(S90N) were negative controls. Mean ± SD of triplicate samples. One of 4 independent assays shown. (f) COS1 or HeLa cells were nucleoporated with N-terminal EGFP-tagged Irgm1 variants. White arrows indicate Golgi membrane targeting. Scale bar, 10μm Images collected from ~150 transfected cells examined per plasmid in 2 separate experiments.

Figure 4

Irgm1 recruitment to PtdIns-enriched membranes for phagolysosomal fusion requires its αK helix. (a) Triple-labeled live imaging of Irgm1 translocation to PtdIns(3,4,5)P_3-pseudopods internalizing BCG in IFN-γ-activated RAW264.7 cells. CFP-Irgm1 (pseudocolored green); YFP-GRP1-PH (pseudocolored red); Cy5-BCG. 3D surface intensity plots (white boxes) below each frame. (b) Percent of MPGs targeted by EGFP-αK, EGFP-αK(F362E,R367E) (labeled 362,367), EGFP-Irgm1(F362E,R367E) (labeled 362,367), EGFP-αI,αJ or EGFP-GD_75-292 (labeled G-domain) within IFN-γ-activated macrophages (60 min pulse, 3h p.i.). 250-440 MPGs were counted per Irgm1 variant from 3 transfection experiments. *, P < 0.012 (Student's t-test). (c) MPG targeting of Irgm1 variants in IFN-γ-activated RAW264.7 macrophages. White and blue arrows depict targeted and non-targeted PGs, respectively. Scale bar, 10μm. (d) Complementation of MPG-lysosome fusion (DQ-Red BSA dequenching) in IFN-γ-activated primary Irgm1^-/^- BMMs (24 h p.i.) by EGFP-Irgm1 but not EGFP or EGFP-Irgm1(362,367). More than 600 MPGs were counted for each transfection in 2 experiments. *, P < 0.029 (single factor analysis of variance, ANOVA).

Figure 5

Pik3ca-Pik3r1-r2 heterodimers furnish PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ on MPGs for Irgm1 recruitment. (a) PtdIns(3,4,5)P₃ was depleted from MPGs in IFN-γ-activated RAW264.7 cells using rapamycin (Rap)-induced dimerization of an FBP-tagged Inp54p phosphatase, which is targeted to PGs (white arrows) via its interactions with a FKBP-tagged Lyn motif. CFPInp54p-FRB (pseudocolored green). Cy5-BCG (pseudocolored red). Time after Rap treatment shown above. Top, representative movie. Scale bar, 5μm. Bottom left, DIC overlay of In54p-expressing IFN-γ-activated macrophages treated or not with wortmanin (Wort) to remove residual PtdIns(3,4,5)P₃. YFP-Irgm1 (green), Cy5-BCG (pseudocolored red; 30 min chase). CFP channel was turned off to reveal loss of Irgm1 translocation. Bottom right, Percent of MPGs containing Irgm1 in cells treated as indicated. 150-220 MPGs were counted per treatment in 3 separate experiments. *, P < 0.0002 (ANOVA). (b) Efficiency of siRNA silencing in IFN-γ-activated RAW264.7 macrophages. Indicated PI(3)Kr isoforms and SHIP1 were detected by immunoblot. γ-actin, loading control. (c) Effect of Class I PI(3)K isoform-specific inhibition via chemical compounds (left) or siRNA (right) on Irgm1 recruitment to MPGs. 280-320 MPGs were counted in 4 separate experiments. *, P < 0.0025 (ANOVA). (d). Lower magnification of siRNA- or chemically-treated IFN-γ-activated macrophages. Cells fixed in 3% PFA were stained with Alexa488-conjugated anti-Irgm1 and Alexa488-conjugated anti-Mtb complex. White arrows, Irgm1-containing MPGs; Blue arrows, MPGs lacking Irgm1. Scale bar, 10μm. (e) Triple-color plus DIC sCFM overlay showing endogenous PI(3)K subunits or SHIP1 with Irgm1 on phagocytic cups engulfing Cy5-M. bovis BCG. Scale bar, 2μm.

Figure 6

Spatial Irgm1 and PI(3)K convergence confers direct cross-regulatory functions. (a,b) HEK cells transfected with indicated Flag-tagged or Myc-tagged constructs were subject to immunoprecipitation and immunoblot. Arrows indicate bound partner. HC, IgG₁ heavy chain, LC, IgG₁ light chain. One of 3 experiments shown. (c,d) GST pulldown (Pd) of Flag-Pik3ca or EGFP-Pik3r1 expressed in HEK cells by GST-Irgm1 or a positive control, GST-Rab5a (black arrows). GST protein captured on Sepharose 4B beads is shown. First lane, beads alone. One of 2 experiments shown (e) Binding of wild-type and mutant Irgm1 to Pik3ca in HEK cell lysates was determined as in panel a. E-I (362,367), EGFP-Irgm1(362,367). (f) Lipid kinase activityof Flag-Pik3ca-Flag-Pi in the presence or absence of Irgm1. Positive controls, HA-tagged p21^Ras GTPase or HA-tagged p21^RasQ61L. Top, immunoblot of rate-limiting catalytic 110kDa Pik3ca subunit captured on Protein G-agarose. Bottom, mean ± SD of triplicate samples. One of 3 assays shown. *, P < 0.027 (ANOVA). G-1(S90N), GST-Irgm1(S90N). (g) Single turnover γ-[³²P]GTP hydrolysis by rGST-Irgm1 bound to rPik3r1 or rPik3r1(R274A). rGST-Rab5a (positive control). Mean ± SD of triplicate samples. Data are from two independent assays. *, P < 0.048 (Students t-test). (h) Continuous $\tilde{\gamma }$[³²P]GTP hydrolysis by rGST-Irgm1 or rGST-Irgm1(F362E,R367E) bound to 100nm PE:PC liposomes containing PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂. Mean ± SD of triplicate samples from two independent assays. *, P < 0.014 (ANOVA). PC:PE control liposomes not containing PtdIns. (i) Fold-change in GTPase activity of Irgm1 proteins bound to PtdIns(3,4,5)P₃ (P₃)-containing or PtdIns(3,4)P₂ (P₂)-containing lipid bilayers, versus Irgm1 proteins bound to control (C) PtdIns-deficient bilayers.

Figure 7

PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ production promotes Irgm1 effector binding at MPGs.(a) The t-SNARE adaptor snapin as an interacting partner of Irgm1. Yeast AH109 co-transformation (72 h; horizontal dilutions) in 2-hybrid analysis on SD-AHLT plates. 1, pGBKT7-cI + pACT2-cI (positive control); 2, pGBKT7-Irgm1 + pACT2-Snapin; 3, pGBKT7-Irgm1 + pACT2-Tmed10; 4, pGBKT7-Irgm1 + pACT2-cI; 5, pGBKT7-Irgm1 + pACT2; 6, pGBKT7-cI + pACT2-Snapin; 7, pGBKT7-cI + pACT2-Tmed10. Conditions 4-7 served as individual negative controls. U, undiluted. (b) Co-immunoprecipitation of Myc-Snapin by Flag-Irgm1 in HEK cells. Arrow depicts Snapin. HC, heavy chain; LC, light chain. Flag-Snap23 (positive control). One of 4 independent experiments shown. (c) Conformation-dependent association of GST-Irgm1 with His₆-Snapin in the free, GTP (1mM), GTP-γ-S (1mM) or GDP (0.1μM) + AIF_x (100μM AlCl₃/15mM NaF)-bound state. Compounds were added 60 min at 4°C prior to incubation of GST-Irgm1 with His₆-Snapin. GST, negative control. GST proteins bound to Snapin (1:6 stoichiometry, Supplementary Fig. 9 online) were immunoblotted with anti-His_6-Snapin One of 3 pulldown experiments shown. (d) Protein gel overlay of untagged Snapin (5μg/ml) detected by anti-Snapin. Lipid numbers match those in Fig. 2b. (e,f) Impaired binding of Snapin to Irgm1 in HEK cells treated with class I PI(3)K inhibitors (I; 15e + TXG-221 + 252424) or expressing EGFP-Irgm1(362,367). (g) Primary IFN-γ-activated wild-type and Irgm1-/^- macrophages were treated or not treated with class I PI(3)K inhibitors, and were infected with Cy5-labeled M. bovis. Three hours after infection cells were stained with anti-Snapin. Arrows, PGs. Scale bar, 10μm.