Emerging themes in IFN-γ-induced macrophage immunity by the p47 and p65 GTPase Families

doi:10.1016/j.imbio.2007.09.018

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Immunobiology. Author manuscript; available in PMC 2009 July 6.

Published in final edited form as:

Immunobiology. 2007; 212(9-10): 771–784.

Published online 2007 November 28. doi: 10.1016/j.imbio.2007.09.018.

PMCID: PMC2705969

NIHMSID: NIHMS37005

Emerging themes in IFN-γ-induced macrophage immunity by the p47 and p65 GTPase Families

Avinash R. Shenoy, Bae-Hoon Kim, Han-Pil Choi, Takeshi Matsuzawa, Sangeeta Tiwari, and John D. MacMicking

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06510, U.S.A

Correspondence: John D. MacMicking, Ph.D, Section of Microbial Pathogenesis, BCMM 349, Yale University School of Medicine, New Haven, C.T. 06510. U.S.A., Phone: (203) 737-1570, Fax: (203) 737-2630, E-mail: john.macmicking/at/yale.edu

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Abstract

Vertebrates have evolved complex immune specificity repertoires beyond the primordial components found in lower multi-cellular organisms to combat microbial infections. The type II interferon (IFN-γ) pathway represents one such system, bridging innate and acquired immunity and providing host protection in a cell-autonomous manner. Recent large-scale transcriptome analyses of IFN-γ-dependent gene expression in effector cells such as macrophages have highlighted the prominence of two families of GTPases - p47 IRGs and p65 GBPs – that are now beginning to emerge as major determinants of antimicrobial resistance. Here we discuss the recent clarification of known family members, their cellular biochemistry and host defense functions as a means to understanding the complex innate immune response engendered in higher vertebrates such as humans and mice.

Keywords: macrophage, IFN-γ, p47 IRG, GBP, GTPase, phagosome, autophagy

Introduction

Of the soluble stimuli sensed by mammalian macrophages to trigger antimicrobial activation, few match the effectiveness of interferon-gamma (IFN-γ) (Nathan et al., 1983). IFN-γ is over 100,000-fold more potent in aiding the oxidative burst of human mononuclear phagocytes, for example, than other macrophage-activating cytokines like tumor necrosis factor (TNF-α) or type I IFNs (IFN-α, IFN-β) (Nathan et al., 1984). This outcome is even more remarkable given the relative paucity of IFN-γ receptors on the cell surface - ~4,000–12,000 -well below the number found for most plasma membrane proteins (Pace et al., 1983; Finbloom et al., 1985).

In addition, IFN-γ priming heightens the macrophage response to microbial products such as lipopolysaccharide (LPS) that signal via Toll-like receptors (TLRs) (Schroder et al., 2006) while basal MyD88 activity in turn augments IFN-γ-induced gene expression and mRNA stability (Shi et al., 2003; Sun and Ding, 2006). Thus, IFN-γ can act singly or in synergy with host-derived cytokines or pathogen-derived products to restrict intracellular infections, a nexus that makes it one of the most critical of all vertebrate immune pathways enlisted in this fight (Schroder et al., 2006).

The ability of IFN-γ to endow macrophages with the capacity to kill ingested micro-organisms stems largely from the complex transcriptional programs it elicits within these cells (Ehrt et al., 2001). Here, over 1,000 genes may be engaged. Within this group are mRNA transcripts that encode host proteins with long-recognized antimicrobial activity, notably inducible nitric oxide synthase (iNOS/NOS2), phagocyte oxidase and natural resistance associated macrophage protein-1 (NRAMP1) (MacMicking et al., 1997; Nathan and Shiloh, 2000; Skamene et al., 1998).

In addition to these more established mechanisms, two new families of GTPases have emerged that provide cell-autonomous resistance against a variety of bacterial, eukaryotic and viral pathogens. On the basis of molecular mass and guanosine nucleotide-binding activity, these have been labeled the p47 immunity-related GTPases (p47 IRGs) and p65 guanylate-binding proteins (p65 GBPs), respectively (MacMicking, 2004; Taylor et al., 2004; MacMicking, 2005; Vestal, 2005; Martens and Howard, 2006). Members of both groups were initially isolated in piecemeal cloning efforts that spanned decades (see MacMicking, 2004; Vestal, 2005; Martens and Howard, 2006). More recently, however, the application of bioinformatics has begun to usher in a new era of understanding the rich genetic diversity and powerful biological functions of these novel host defense factors.

Genes and genomes

Refined annotations suggest 23,000–26,000 genes in humans and 28,000–30,000 genes in mice (Venter et al., 2001; Waterston et al., 2002); ~5–10% are thought to subsume immune-related activities (Venter et al., 2001). A particularly striking feature of the latter category is the large number of multigene families or even superfamilies present (Venter et al., 2001). One common explanation for such preponderance is that strong evolutionary pressure leads to immune gene expansion in the face of microbial diversity (Sabeti et al., 2006). Both the p47 IRG and p65 GBP families may be good examples of this, especially in mice, while in humans alternative splicing appears to be the preferred mechanism for generating additional protein isoforms (MacMicking, 2004; Vestal, 2005; Martens and Howard, 2006; MacMicking et al., unpublished).

In silico analyses posit as many as 23 p47 Irg and 6 p65 Gbp loci in the euchromatic mouse genome (Bekpen et al., 2005). Humans, in contrast, appear to possess 2 IRG and 7 GBP genes (Bekpen et al., 2005) (Fig. 1

). Numerical disparity between the mouse and human IRG families can be explained in part by duplicative expansion of a common Irga ancestor on mouse chromosome 18 and the existence of multiply-spliced transcripts for human IRGs (eg. IRGM) (Bekpen et al., 2005; MacMicking et al., unpublished). In fact the sizeable mouse Irg family appears anomalous among vertebrates: rats, for example, possess only 7 Irg genes, with dogs, zebrafish and pufferfish harboring 9,11 and 2 members, respectively (Bekpen et al., 2005; MacMicking et al., unpublished). Reexamination of the current mouse genome also suggests that the total number of original Irg entries may be an over-estimate, while in the case of the Gbps, more members have arisen (MacMicking et al., unpublished).

Fig. 1

Phylogenetic analyses of p47 IRGs and p65 GBPs. G-domains of p47 IRGs, p65 GBPs and other representative GTPases were first aligned separately using ProbCons (Do et al., 2005) followed by two step profiles using TCoffee (Notredame et al., 2000) which (more ...)

For the p47 IRG family, pseudogenic ascriptions are now predicted for Irga1 (LOC546714) and Irga8 (LOC667597) while an additional Irg gene, Irg11, lies between Irgb7 and Iigp1/Irga6 genes on chromosome 18 (see Fig. 2

). Moreover, re-assemblage of mouse chromosome 11 indicates that the Irgb3/4 and Irgb5/6 regions are probably not duplicated as first thought (Bekpen et al., 2005) (Fig. 2

). Previous analysis also divided a number of GTPase bidomain proteins into smaller single domain Irg genes (Bekpen et al., 2005). Newer EST deposits, however, do not support this partition. Instead larger single domain proteins or even entire in-frame ORFs with tandem GTPase domains (e.g. AK145236) appears more likely. These larger Irg proteins could conceivably function as dimers or co-operatively regulate the activities of monomers within an oligomeric complex, biochemical behavior reminiscent to that already seen for Iigp1/Irga6 (Uthaiah et al., 2003). As such we have considered Irgb1/Irgb2, Irgb3/Irgb5 and Irgb8/Irgb9 groupings as bidomain or bigger single domain p47 Irg GTPases and thus refer to them here as Large-Irg proteins 1–3 (Lirg1-3) with a predicted M_r of ~95 kDa (Fig. 2

Fig. 2

Genomic organization of human and mouse p47 IRGs and p65 GBPs. (A) Schematic representation of the human genomic regions containing p47 IRG and p65 GBP genes on chromosomes 1, 5 and 19. Arrows represent 5′–3′ direction of mRNA (more ...)

For the GBP family, several additional mouse orthologs appear within pre-existing clusters on chromosome 5 (Fig. 2

). Hereafter these have been termed Gbps-7 through -12 (Figs. 1

, ,2).2

). On chromosome 3, an additional gene 3′ to Gbp2 appears intact and has been called Gbp13 (EG621605) (Figs. 1

, ,2).2

). Phylogenetic representation of the p65 GBP G-domains indicates that as a family they are more interrelated than are the p47 IRGs (Fig. 1

). Moreover, interspecies comparison between mouse and human GBPs suggests a relatively recent evolution of these highly homologous genes as compared to the p47 IRGs (Olszewski et al., 2006). Reasons for this divergence are unknown but could include specific pathogen-host range dependencies influencing one family or host species more than the other (Frank, 2002). Alternatively, a large number of disparate murine Irg genes may be needed if they also assume non-immune activities. To date such activities have not been found (see MacMicking, 2004; Taylor et al., 2004; Martens and Howard, 2006) but it remains an issue for investigation.

Stretches and structures

At the primary sequence level, both the p47 IRGs and p65 GBPs are defined by a single stretch of 200–300 amino acids encompassing the guanosine nucleotide-binding or G-domain (Fig. 3A and B

). p47 IRGs share ~40–80% similarity within this domain while GBPs exhibit ~55–80% similarity across the corresponding 280 residue stretch. Interfamily relatedness, however, is only ~20–25% over the first 4 motifs (G1-G4) of the G-domain, a difference that may help account for their disparate nucleotide-binding specificities.

Fig. 3

Structures and sequence relationship within the p47 IRGs and p65 GBPs. (A) A computer rendering of full-length dimeric Iigp1/Irgb6 (pdb code 1tq2) crystallized in the presence of phosphoaminophosphonic acid-guanylate ester (GNP; shown in red stick representation). (more ...)

Prototypic G-domains exemplified by Ras and the heterotrimeric G-proteins act as molecular switches with distinct conformations in their GTP- and GDP-bound states (Vetter and Wittinghofer, 2001). Proteins are often active in their GTP-bound form and can be inactivated by specific GTPase activating proteins (GAPs) that accelerate the conversion of GTP to GDP. Attendant conformational changes in the G-domain are then transmitted to proximal domains or interacting effector proteins, bringing about GTPase-dependent regulation of output function(s). The p47 IRGs hydrolyze GTP to GDP while certain GBPs (hGBP1 and hGBP2) convert GDP further to GMP (Schwemmle and Staeheli, 1994; Neun et al., 1996; Taylor et al., 1996; Carlow et al., 1998; Han et al., 1998; Uthaiah et al., 1999; Tiwari and MacMicking, unpublished). Two-step hydrolysis is unusual for a G-domain and insights into the reaction mechanism have been obtained from crystal structures of transition-state analog-bound hGBP1 (Prakash et al., 2000a; Prakash et al., 2000b) and its isolated catalytic region (Ghosh et al., 2006) (Fig. 3A and B

). An ability to bind GMP is a unique feature of the GBPs compared with other GTPases, although the physiological relevance of this nucleotide preference is still unclear.

Several evolutionarily conserved sequence motifs - labeled G1 to G5 - are involved in binding the phosphate moieties (G1,G2,G3) or purine ring (G4,G5) of GTP, co-ordinating Mg²⁺ at the active site (G1,G2,G3) and regulating effector functions (G2 and G3) (Sprang, 1997; Vetter and Wittinghofer, 2001). Each in turn is considered below.

The G1-motif, also called the glycine-rich loop, P-loop or the Walker-A motif, corresponds to GxxGx^K/_MS and GXXRxKS in the p47 IRGs and p65 GBPs, respectively (Fig. 3C

). Mutation of the last G1 lysine or serine residue leads to profound loss of GTPase activity as noted for hGBP1 (Lys₅₁, Ser₅₂) (Praefcke et al., 2004). This is because Lys₅₁ binds the β/γ-phosphate of GTP whereas in its absence a polar bond with Thr₉₈ (G3) is formed. Other G1 residues also contribute to activity: hGBP1 Arg₄₈ is required for transactivation and cooperative GTPase activity of the homodimeric structure (Ghosh et al., 2006). This mechanism of activation is analogous to that brought about by arginine-fingers in GAPs (Vetter and Wittinghofer, 2001; Scheffzek and Ahmadian, 2005). The crystal structure of Iigp1/Irga6 also highlights the requirement for Lys₇₈ in binding the γ-phosphate and Mg²⁺ (Ghosh et al., 2004). Interestingly, 3 mouse Irg proteins (Lrg-47/Irgm1, Gtpi/Irgm2, Igtp/Irgm3) and 1 putative human ortholog (IRGM) possess methionine instead of lysine at this position although any detrimental effects of this substitution on enzyme activity have not been found (Taylor et al., 1996; Tiwari and MacMicking, unpublished).

A role for the G2 loop in folding back on the active site nucleotide to form a phosphate cap is seen for GBPs but not p47 IRGs (see Fig. 3B

; Prakash et al., 2000b). The invariant threonine normally involved in binding the γ-phosphate and Mg²⁺ as part of a switch-I region is missing in IRGs. Instead, residues within the G3 motif (Iigp1/Irga6 Asp₁₂₆ for example) may take on the role of co-ordinating Mg²⁺ (Ghosh et al., 2004). This residue is conserved in hGBP1 (Thr₇₅; switch-I) and its mutation leads to near-inactivation of the enzyme (Praefcke et al., 2004). Another critical catalytic residue adjacent to the G3 motif (Gln₆₀ in Ras) is absent in both p47s and GBPs, pointing to a divergent reaction mechanism in these GTPases (Praefcke et al., 2004).

Lastly, the G4 region in p47 IRGs and p65 GBPs can be represented as ^Y/_DFFVWTxxD with non-canonical substitutions outside the classical ^N/_KTxD motif giving rise to different substrate-binding capacities (Fig. 3C

). In Iigp1/Irga6, G4 Lys₁₈₄ and Asp₁₈₆ make specific contacts with the nucleotide (Ghosh et al., 2004). For hGBP1, additional conserved residues in the G4 motif termed the arginine-aspartate (‘RD’) pair also contribute to GTP binding (Fig. 3B

). A loose G5 motif (SA^K/_L) corresponding to Ser₂₃₁-Lys₂₃₃ is found in Iigp1/Irga6 (Ghosh et al., 2004) but is absent in the GBPs. The latter instead employ a unique guanine cap composed of two short helices to allow for tight substrate binding (Fig. 3

) (Ghosh et al., 2006).

These differences in the GTP-binding pocket of GBPs lead to a significant change in the active site nucleotide N-glycosidic bond angle (~ −75°) compared to that in Ras (~ −112°) (Ghosh et al., 2006). Moreover, the torsion angle at the C4′–C5′ bond in the ribose moiety undergoes a major change to the extent that the α-phosphate in the GMP/AlF₃-bound structure is in a position occupied by β-phosphate in the GDP/AlF₄⁻ bound structure (Ghosh et al., 2006). The implications of this conformational change are twofold: first, flexibility brought about in the nucleotide may allow GBPs to convert GTP to GMP, and second, GDP hydrolysis involves the same reaction mechanism as the hydrolysis of GTP (Ghosh et al., 2006).

Apart from the G-domain, motifs for post-translational lipid modifications are noted for several members of both the p47 IRG and p65 GBP families. Here a C-terminal CaaX motif targets human and mouse GBPs1, 2 and 5 for isoprenylation (Vestal, 2005) and an N-terminal myristoylation site (MGxxxS) is predicted in 11 mouse Irga subfamily proteins (Bekpen et al., 2005). At least in the case for Iigp1/Irga6, mutation of the second N-terminal glycine residue leads to diminished Golgi membrane binding (Martens et al., 2004).

Regulation and regulators

The p47 IRGs and p65 GBPs are among the most abundant genes activated upon exposure of cells to IFN-γ. For the p47 IRGs, Lrg-47/Irgm1 and Iigp1/Irga6 can also respond directly to IFN-α/β (Sorace et al., 1995; Zerrahn et al., 2002), while these two members plus Igtp/Irgm3 and Iigp1/Irga6 are solicited by indirect signaling with lipopolysaccharide (LPS) (Zerrahn et al., 2002; Lapaque et al., 2006). To date most p47 proteins are not induced by other inflammatory cytokines such as interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6 and TNF-α (see MacMicking, 2004; Martens and Howard, 2006). STAT1-dependent induction of p47 members after IFN stimulation is based on the identification of GAS sites (γIFN-activated sites; GAS, Fig. 4

) in their promoters and is supported by studies from STAT1^−/− (Collazo et al., 2002; Gavrilescu et al., 2004; Lieberman et al., 2004) and phosphorylation-deficient STAT^S727A mice (Varinou et al., 2003).

Fig. 4

IFN-inducible transcription factor-binding sites within p47 IRG and p65 GBP promoters. Results from earlier studies of known genes are summarized schematically (Bekpen et al., 2005; Olszewski et al., 2006). Analyses of newly identified members were carried (more ...)

Many p47 IRG promoters also contain the interferon stimulated response elements (ISRE) bound by the heterotrimeric STAT-1/STAT-2/IRF-9 (interferon-regulatory factor-9) transcription factor complex (called interferon stimulated gene factor (ISGF)-3 (Honda and Taniguchi, 2006; Fig. 4

). To date only the mouse Irg-47/Irgd promoter ISRE and YY1 motifs have been verified biochemically to recruit this transcription factor complex (Gilly et al., 1996). The ISRE is indistinguishable from the binding site for the IRF-1 transcription factor (Honda and Taniguchi, 2006), and in the absence of IRF-1 expression, several p47 IRGs are compromised (Boehm et al., 1998). ISGF3 binding may also require phosphorylation of STAT1 via an IkB kinase-ε (IKK-ε) signaling cascade as seen for Irg-47/Irgd, Iigp1/Irga6 and Gtp1/Irgm2 in Ikbke^−/− fibroblasts (tenOever et al., 2007). For the recently-cloned human IRGs no evidence exists regarding immunologic induction and the canonical transcription factor binding sites outlined above appear absent within 1–2kb upstream of the start site (Belkpen et al., 2005; MacMicking et al, unpublished). Thus the human and mouse p47 IRGs diverge considerably in terms of their induction.

Unlike the p47 IRGs that are primarily immediate-early genes, expression of the p65 GBPs require de novo protein synthesis and secondary transcription factors such as IRF-1 that recognize ISREs in their promoters (Fig. 4

). As compared to IFN-γ, type I interferons are stronger inducers of the ISGF-3 complex, which also bind ISREs; thus human p65 GBPs can be induced by IFNα/β (Colonno and Pang, 1982) although the response to this cytokine is much weaker in mouse macrophages (Boehm et al., 1998; Kim and MacMicking, unpublished). Induction of murine GBPs by IFN-γ can be augmented by TNF-α, IL-1 or LPS (Nguyen et al., 2002) and in the case of hGBP1, TNF-α and IL-1β were shown to act through the NF-κB binding site in its promoter (Naschberger et al., 2004).

Early studies using luciferase reporters in fibroblasts and differentiated embryonic stem cells from IRF-1^−/− mice demonstrated the requirement of IRF-1 for Gbp2 induction (Lew et al., 1991; Briken et al., 1995). More recent molecular efforts have dissected the contributions of STAT1 and IRF-1 in the transcriptional induction of GBPs. Here STAT1 dimers are responsible for binding and recruiting accessory transcription co-activators such as the histone acetyl transferase, CREB-binding protein (Varinou et al., 2003), histone deacetylase-1, a presumed helicase from the mini chromosome maintenance (MCM) complex (Snyder et al., 2005), and BRG1, an ATPase of the key SWI/SNF complex (Pattenden et al., 2002; Ni et al., 2005). These studies allude to the possibility that STAT1 binds the Gbp2 promoter in a BRG1-dependent manner and by recruiting HAT prepares the chromatin for subsequent IRF-1-driven transcription.

LPS-dependent induction of p47 IRG proteins is primarily via TLR4 and is MyD88-independent, enlisting the Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-β (TRIF), IKK-ε and TANK-binding kinases (TBK1), and lastly, IRF3 (Doyle et al., 2002; Shi et al., 2003; Hemmi et al., 2004; Weighardt et al., 2004). Such induction is also more responsive to classical LPS agonists that carry a bisphosphorylate pyranose moiety than non-classical lipid A species belonging to pathogens such as L. pneumophila RC1 or B. melitensis (Lapaque et al., 2006). Other bacterial cell wall products – lipoarabinomannans from Mycobacterium spp. or lipotiechoic acid from Gram-positive bacteria – also display stimulatory p47 IRG activity, most likely as an indirect result of type I IFN secretion (MacMicking et al., 2003; McCaffrey et al., 2004).

Residence and residents

IFN-γ-inducible GTPases reside within lipid microdomains or cytoskeletal scaffolds that may help partition signaling complexes involved in intracellular traffic. Here they could also recruit binding partners to fulfill their antimicrobial functions. Targeting to membrane-bound compartments enlists isoprenylation for hGBP1, mGbp1 and mGbp2, and myristoylation for Iigp1/Irga6 (Nantais et al., 1996; MacMicking, 2004; Martens et al., 2004; Modiano et al., 2005; Vestal, 2005). A reliance on post-translational modification presumably stems from the fact that neither p47 IRGs nor p65 GBPs contain predicted transmembrane regions, especially given that membrane-association is emerging as the rule for most members of both GTPase families.

Organellar membranes to which p47 IRGs have been found to localize include Igtp/Irgm3 to the endoplasmic reticulum (ER) (Taylor et al., 1997), Iigp1/Irga6 to the ER and Golgi (Zerrahn et al., 2002; Martens et al., 2004), and Lrg-47/Irm1 to the ER, cis-Golgi, phagocytic cups and plasma membrane (MacMicking et al., 2003; Martens et al., 2004). For the GBPs, hGBP1 resides in the ER and Golgi (Modiano et al., 2005) and mGBP2 on unidentified intracellular vesicles (Vestal et al., 2000). Additionally, several mouse GBPs localize to distinct subpopulations of intracellular vesicles not unlike autophagosomes (Kim and MacMicking, unpublished).

Members of the p47 IRG family can translocate to specific pathogen compartments from their resting locations as first noted for Lrg-47/Irgm1 which travels via a brefeldin A-sensitive pathway to the M. tuberculosis phagosome (MacMicking et al., 2003). Here it is thought to facilitate the cytokine-stimulated phagosome maturation cascade which may include recruitment of the autophagic machinery (MacMicking et al., 2003; Gutierrez et al., 2004). Later studies have also observed Lrg-47/Irgm1 on nascent Listeria monocytogenes phagosomes within 10–15 min after uptake (Matsuzawa and MacMicking, unpublished; see Fig. 5

) and the presence of Igtp/Irgm3, Gtpi/Irgm2, Iigp1/irga6 and Tgtp/Irgb6 on Toxoplasma gondii parasitophorous vacuoles (Martens et al., 2005; Martens and Howard, 2006).

Fig. 5

Recruitment of endogenous Lrg-47/Irgm1 to the bacterial phagosome. Upper panel depicts single-channel and merged (overlay) confocal images of Lrg-47/Irgm1 on the mycobacterial phagosome (M. bovis BCG) in IFN-γ-activated RAW264.7 murine macrophages. (more ...)

Steady-state organellar residence and phagosome recruitment relies on a bidomain structure for p47 IRGs. An isolated G-domain of Lrg-47/Irgm1 and Iigp1/Irga6 can localize to the plasma membrane, unlike the full-length protein that is retained in the ER/Golgi (Martens et al., 2004). The region responsible for Lrg-47/Irgm1 retention lies in a C-terminal amphipathic helical domain that is shared with Gtpi/Irgm2 and Igtp/Irgm3 and can confer correct localization on GFP chimeras (Martens et al., 2004). Amino acid substitutions which destroy amphipathicity of this helix prevent the LRG-47/Irgm1 αK fragment or holoenzyme from targeting the cis-Golgi (Martens et al., 2004). These same mutations interfere with binding of Lrg-47/Irgm1 to specific phosphoinositols generated on internal membranes, providing an additional reason for failure to localize properly (Tiwari and MacMicking, unpublished). In contrast, the Iigp1/Irga6 αK does not seem to contain membrane-targeting sequences; instead, an N-terminal myristoylation site when mutated leads to complete partitioning into the aqueous phase (Martens et al., 2004).

Golgi/ER localization of Lrg-47/irgm1, Iigp1/Irga6 and Igtp/Irgm3 appears independent of GTP hydrolysis as seen from proteins with null mutations in the G1 motif (Taylor et al., 1997; Martens et al., 2004). Lrg-47/Irgm1 relocation to the plasma membrane during phagocytosis and to mycobacterial phagosomes, however, may require GTP binding, as does targeting of Iigp1/Irga6 to T. gondii vacuoles (Martens et al., 2004; Martens and Howard, 2006; MacMicking, unpublished). Remarkably, none of the aforementioned trafficking events appear to enlist IFN-γ-induced accessory factors.

Compared with the p47 GTPases, p65 GBPs differ in that hGBP1 requires GTP binding, other IFN-γ-stimulated proteins and isoprenyl modification for Golgi residence (Modiano et al., 2005). Moreover, oligomerization status of the native, GTPase- and farnesylation-mutant proteins affects localization of hGBP1 in these cells. For mGbp2, the need for enzyme activity in organelle targeting is less clear. Here a presumed inactive (S52N) mutant localizes to punctuate vesicles in a manner similar to that of the parent protein (Gorbacheva et al., 2002). Additionally, our own studies in murine macrophages suggest that several other GBPs localize to distinct intracellular vesicles and are likely to be membrane bound despite lacking canonical isoprenylation or palmitoylation/myristoylation motifs in their primary sequence (Fig. 5

; Kim and MacMicking, unpublished).

Identifying binding partners or interactions with resident proteins will undoubtedly provide some insight into the sub-cellular behavior of these GTPases. Yeast two-hybrid efforts have retrieved the Golgi protein hook3 as an interacting partner for Iigp1/Irga6 and the fatty-acid binding protein ADRP for Irg-47/Irgd (Kaiser et al., 2004; Yamaguchi et al., 2006). Hook3 interaction with Iigp1/Irga6 required the latter to be in a GTP-bound conformation as demonstrated by the G1 motif S83N mutant. Hook3 itself is a novel microtubule binding protein with four coiled-coiled domains, the fourth of which associates with Iigp1/Irga6, and treatment of cells with the microtubule-disrupting agent nocodozole abolished in vivo binding (Kaiser et al., 2004). The functional consequences of this interaction have not been explored further; however, it is known that the Salmonella type III secretion system effector, SpiC, targets hook3 to prevent phagolysosome fusion (Shotland et al., 2003). Thus Iigp1/Irga6 may be part the host cellular machinery needed to control trafficking or remodeling of this organelle.

C-terminal binding of Irg-47/Irgd to ADRP is suggestive of a role for p47 IRGs in intracellular lipolysis although little overlap within cells was noted for these two proteins (Yamaguchi et al., 2006). Involvement of the Irg-47/Irgd C-terminus may also argue against a common mechanism shared by other p47 IRGs since this region is the least conserved across family members. Further experiments are therefore needed to establish the relevance of this partner for p47 IRG-mediated antimicrobial activity. At the time of writing, no binding partners for members of the p65 GBP family have been isolated.

Microbes and macrophages

A single vertebrate species can serve as host to an estimated 1,400 phylogenetically distinct pathogens (Mascie-Taylor and Karim, 2003). Pathogen discrimination via TLRs and nucleotide-oligomerization domain-leucine rich repeat (NOD-LRR) proteins help reduce this complexity, yet recognition alone does not guarantee microbial clearance. This is because many of the effector proteins needed for killing are invoked by a different set of signals. Such is the case in M. tuberculosis-infected macrophages (Shi et al., 2003). Here, TLR stimulation itself has no direct effect on bacterial viability although it can enhance IFN-γ-induced activation of genes that subsequently curtail growth (Shi et al., 2003). Thus PRRs are often required to work in tandem with IFN-γ-responsive proteins to limit infection at the level of the individual cell (Schroder et al., 2006).

Both the p47 IRGs and p65 GBPs fall into this latter category; their over-expression confers cell autonomous protection while their removal renders IFN-γ-activated macrophages more permissive for microbial replication. Initial evidence for a role of p47 IRGs in macrophage-mediated killing came from studies of the classic facultative intracellular pathogen, Mycobacterium tuberculosis (MacMicking et al., 2003). Here, genetic removal of Lrg-47/Irgm1 led to cell-autonomous defects at the level of the activated macrophage which have been corroborated using siRNAs against the murine gene and possibly a related human orthologue, IRGM, although the latter needs independent confirmation (Singh et al., 2006).

Other pathogens targeted by the Lrg-47/Irgm1 pathway within macrophages include the protozoan parasites Toxoplasma gondii (Butcher et al., 2005) and Trypanosoma cruzi (Santiago et al., 2005). Additional members of the p47 IRG family operate to curtail T. gondii growth including Igtp/Irgm3 and Iigp1/Irga6 (Halonen et al., 2001; Bernstein-Hanley et al., 2006; Ling et al., 2006) while both p47 IRGs help restrict Chlamydia trachomatis replication in epithelia and embryonic fibroblasts, respectively (Nelson et al., 2005; Bernstein-Hanley et al., 2006). For the latter bacterium, a third p47 IRG gene - Irgb10 – was recently mapped to the susceptibility locus Ctrq-3 and can complement resistance in trans to C3H/HeJ susceptible host cells (Bernstein-Hanley et al., 2006). Thus members of the p47 IRG family may act in concert to provide effective host defense.

Defects in cell-autonomous p47 IRG activity are also manifested at the level of the whole organism. Lrg-47/Irgm1^−/− mice, for example, fail to control M. tuberculosis, M. avium, L. monocytogenes, S. typhimurium, T. gondii, T. cruzi, and L. major infections; Igtp/Irgm3^−/− mice are vulnerable to T. gondii and C. trachomatis while Irg-47/Irgd and possibly Iigp1/Irga6 are needed for resistance to T. gondii as well (reviewed in MacMicking, 2004; Taylor et al., 2004; Martens and Howard, 2006). Interestingly, none of these deficiencies led to compromised anti-viral activity, although MCMV and Ebola have been the only viruses examined to date (Taylor et al., 2000; Collazo et al., 2001). Earlier over-expression studies had implicated a role for Tgtp/Irgb6 against VSV (Carlow et al., 1998) and Igtp/Irgm3 against coxsackievirus B3 (Zhang et al., 2003) but it was unclear whether these effects were due to direct antiviral activity or enhanced non-cytopathic cell survival.

On the basis of the above susceptibility studies it is clear that p47 IRGs can respond to pathogens inhabiting a range of membrane-bound compartments -phagosomes, parasitophorous vacuoles and inclusion bodies. An emerging consensus is therefore one in which the activities of these small GTPases impact the biogenesis or remodeling of the pathogen-containing niche. Studies from several groups have documented the presence of endogenous p47 IRGs on such organelles: Lrg-47/Irgm1 to M. tuberculosis and L. monocytogenes phagosomes (MacMicking et al., 2003; Matsuzawa and MacMicking, unpublished; see Fig. 5

), Gtpi/Irgm2, Igtp/Irgm1, Iigp1/Irga6 and Tgtp/Irgb6 to T. gondii parasitophorous vacuoles (Butcher et al., 2005; Martens et al., 2005; Ling et al., 2006) and Iigp1/Irga6 to C. trachomatis inclusion bodies (Nelson et al., 2005). Where tested, p47 IRG recruitment correlated with host protection.

Once relocated to the site of microbial replication, p47 IRGs may help facilitate fusion with lysosomes by soliciting components of the autophagic machinery (MacMicking et al., 2003; Gutierrez et al., 2004; Ling et al., 2006; Singh et al., 2006), disrupt the vacuolar membrane (Martens et al., 2005; Ling et al., 2006) or target lipid membrane intermediates (Nelson et al., 2005). Each could reflect contingent steps in a series of events, for example, membrane disruption invoking autophagic engulfment prior to eventual fusion with lysosomes. That the p47 IRGs participate in this cascade specifically during infection is reinforced by recent experiments on mannose receptor-mediated latex bead internalization; here, general phagocytosis did not require Lrg-47/Irgm1, Igtp/Irgm3 or Irg-47/Irgd (Yates et al., 2007).

Precise modes of p47 IRG action could also be dictated by the specific pathogen encountered or the presence of additional p47 IRGs in the vicinity. Antimicrobial activity requires GTP hydrolysis, at least in the case of Iigp1/Irga6 and Lrg-47/Irgm1, suggesting they are likely to engage other proteins or even members of the same family to bring about these changes (Martens et al., 2005; Martens and Howard, 2006; Choi and MacMicking, unpublished). Because the few p47 IRG-interacting partners isolated so far span several trafficking pathways, it is conceivable that a given p47 GTPase fulfills more than one task, from postER/Golgi cargo transport to assembly of pre-fusion pore complexes on the nascent phagosomal membrane.

An absence of binding partners for the p65 GBPs and a penchant for self-assembly implies a different mechanism may be employed for this family. The antimicrobial profile of p65 GBPs is also less well characterized, to date having been restricted to viruses. In ectopic over-expression studies, hGBP1 inhibited VSV, hepatitis C and encephalomyocarditis viruses by up to 50% (Anderson et al., 1999; Carter et al., 2005). hGBP1 siRNAs have also yielded an ~60% contribution to the control of hepatitis C virus (Itsui et al., 2006). Importantly, neither cell viability nor signal transduction cascades targeting ISRE, GAS, AP-1 or NF-κB transcription factor binding sites were affected by siRNA treatment.

mGbp2 also exerted a similar effect against encephalomyocarditis virus that appeared dissociated from its GTPase activity (Carter et al., 2005). This latter finding is surprising in light of biochemical evidence that demonstrates self-stimulated GTPase activity arising from nucleotide-dependent dimeric and tetrameric assembly (Kunzelmann et al., 2005). Perhaps the p65 GBPs do not act as mechanoenzymes in a manner analogous to the dynamin superfamily to inhibit microbial growth. Alternative explanations for their antiviral activity, however, remain to be proffered.

Summary

Vertebrates are endowed with at least two classes of IFN-γ-induced GTPases -p47 IRGs and p65 GBPs - that combat intracellular infections as part of the cell-autonomous response during innate host defense. Pathogen specificity is exemplified by p47 IRGs that are effective against vacuolarized bacteria and protozoa, whereas the p65 GBPs can also act against cytosolic rhabdoviruses and flaviviruses. Control of microbial replication at the level of the infected cell may involve GTPase-assisted fusogenic complex formation on the phagosomal membrane or viral assembly site, or regulation of the vesicular cargo that traffics to and from that compartment. A major challenge for the future, therefore, will be a complete molecular description of the trafficking events governed by these GTPases, a task made more complex by the recent discoveries of yet more family members within the activated macrophage.

Acknowledgments

Grant support for work described herein has been provided by NIH NIAID (R01 AI068041-01A1), Edward R. Mallinckrodt Foundation (R06152), Searle Foundation Scholars Program (05-F-114), Cancer Research Institute Investigator Award Program, W.W. Winchester Foundation (to J.D.M.), Yale University School of Medicine Cox-Browne Fellowship (A.R.S.) and Japanese Society for the Promotion of Science (to T.M.).

Footnotes

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