IFN-Inducible Gtpases in Host Cell Defense

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IFN-Inducible GTPases in Host Cell Defense

Bae-Hoon Kim,1 Avinash R. Shenoy,1 Pradeep Kumar,1 Clinton J. Bradﬁeld,1 and John D. MacMicking1,* 1Department of Microbial Pathogenesis, Boyer Centre for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2012.09.007

From plants to humans, the ability to control infection at the level of an individual cell—a process termed cell- autonomous immunity—equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell’s interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic, and inflammasome-related antimicrobial activities within the cytosol, as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of guanylate binding proteins (GBPs) with tuberculosis susceptibility and Crohn’s colitis.

Introduction IFNs serve as powerful signals for marshaling host defense Essentially all diploid cells are endowed with a capacity for self throughout the vertebrate kingdom. These cytokines induce defense. This type of host resistance, called cell-autonomous numerous gene families that encode effectors and associated immunity, is ubiquitous throughout both plant and animal king- regulators to direct each protein’s microbicidal or static activity doms (Beutler et al., 2006). It is operationally defined by a minimal (MacMicking, 2012). Prominent within this group is an IFN- set of germline-encoded antimicrobial defense factors that inducible GTPase superfamily that operates against several enables an infected host cell to resist a variety of prokaryotic microbial classes (MacMicking, 2004; Martens and Howard, and eukaryotic pathogens. In this respect, it functions not only 2006; Shenoy et al., 2007; Taylor et al., 2004). Over the past in cells of the immune system, but also in histogenetic lineages few years, we have begun to understand how some of these as diverse as keratinocytes, endothelia, cardiac myocytes, new IFN-inducible GTPases execute their functions as part of hepatocytes, enterocytes, astrocytes, tonsillar epithelia, fibro- a potent host defense program in several vertebrate species. blasts, and neurons. Most importantly, cell-autonomous immu- Likewise, disease-associated genetic variants have recently nity spans both the sensory apparatus used for microbial surfaced within the human population that may predispose to detection and the effector mechanisms directly responsible for tuberculosis (TB) and Crohn’s disease. This updated review microbial killing (MacMicking, 2012; Yan and Chen, 2012). highlights progress on these IFN-inducible GTPases and exam- Given its evolutionary significance, it is remarkable how often ines their cell-autonomous role at the interface of several host- this form of somatic self-defense is either overlooked or under- pathogen relationships. appreciated, but it remains obligate for host survival, especially in lower organisms, where it serves as the primary if not sole The IFN-Inducible GTPase Superfamily form of pathogen resistance (Medzhitov et al., 2012; Ronald Bioinformatic analysis coupled to chromosomal mapping and Beutler, 2010). Much of that resistance relies on a pre- recently led to assembly of the complete superfamily in two existing RNA and protein repertoire mobilized within minutes of mammalian species—humans and mice (Kim et al., 2011) infection (Yan and Chen, 2012). This basal repertoire is capable (Figure 1). This particular effort utilized pair-hidden Markov of conferring cell-autonomous protection against a variety of model algorithms plus cosmid and BAC fragment hybridization phytopathogens and a more limited set of metazoan pathogens to validate members of this new family. It built on the pioneering (Ronald and Beutler, 2010). work of Bekpen et al. (2005) and some excellent in silico annota- Once we move to higher species such as mammals, a second tions for two IFN-inducible GTPase subfamilies, notably the mode of cell-autonomous immunity begins to emerge as a likely immunity-related GTPases (IRGs) and guanylate binding pro- consequence of the broader microbial challenge faced by many teins (GBPs) (Degrandi et al., 2007; Kresse et al., 2008; chordates such as humans (Sabeti et al., 2006). This gene reper- Olszewski et al., 2006; Shenoy et al., 2007). Besides humans toire differs from the hard-wired circuitry of its predecessor by and mice, IFN-inducible GTPase annotations have now been the characteristic of inducibility. Here a new set of proteins are compiled for other primates, plus cows, dogs, rats, opossums, transcriptionally elicited by activating stimuli such as cytokines cartilaginous fish, frogs, lizards, birds, and cephalochordates and microbial products, a response that is often more sustained such as Brachiostomata (Bekpen et al., 2005; Li et al., 2009). and expansive than that of its earlier counterpart, enlisting Further studies have unearthed sporadic ancestral orthologs hundreds of antimicrobial genes (MacMicking, 2012; Yan and as far back as Arabidopsis (e.g., AEC09593) (Lin et al., 1999), Chen, 2012). suggesting resistance functions in these lower organisms that Chief among the activating stimuli that remodel the mamma- have been refashioned in vertebrates to respond to cytokine lian cell’s transcriptome is the interferon (IFN) cytokine family. stimulation (MacMicking, 2012).

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Li et al., 2009; Olszewski et al., 2006; Shenoy et al., 2007). An exception is the mouse IRG cluster on chromosome 11, where only a single shortened IRGM gene is present on human chromosome 5q33.1 after deletion of the remaining family members by insertion of an ERV9 U3 LTR retroelement (Bekpen et al., 2005, 2009). Here three mouse Irgm loci (Irgm1–Irgm3) are replaced by a related prosimian precursor, IRGM9, which through a series of truncation and resurrection events yield the IRGM locus in humans and anthropomorphs such as gorillas and chimpanzees (Bekpen et al., 2009)(Figure 1). Hence, the hominoid IRG genes have been heavily trimmed during evolution, although alterna- tive mRNA splicing may partially compensate for this loss by generating several 21 kDa IRGM isoforms (IRGMa, IRGMb, IRGMc/e, IRGMd in humans) with discrete functions (Brest et al., 2011; Singh et al., 2010). The human GVINP1 locus has also undergone some degree of pseudogenization compared with the corresponding murine Gvin genes (Klamp et al., 2003). In most other cases, however, gene expansion rather than contraction predominates, probably driven by duplicative events in response to the evolutionary pressure exerted by intracellular pathogens (Shenoy et al., 2007). This has led to high degrees of homology within subfamilies, especially across the catalytic G domain.

IFN-Inducible GTPases: Design and Distribution Crystal structures for several IFN-inducible GTPases reveal a bidomain architecture consisting of a globular N-terminal G domain followed by a C-terminal helical domain (CTHD) (Gao et al., 2010; 2011; Ghosh et al., 2004; Prakash et al., 2000) (Figure 1). This configuration allied with biochemical similarities leads to their being grouped together with members of the dynamin-like family of proteins, which in mammals includes dynamins, mitofusin, atlastins, optic atrophy-1 (OPA1), and other dynamin-like proteins (DRPs) (Ferguson and De Camilli, 2012; Figure 1. The IFN-inducible GTPase Superfamily in Humans and Praefcke and McMahon, 2004). This family governs a variety of Mice cellular functions, from budding and fusion of transport vesicles (A) Phylogenetic tree of the IFN-inducible GTPase superfamily encompassing the four subfamilies: 21–47 kDa IRGs, 65–73 kDa GBPs, 72–82 kDa MX to organelle division and cytokinesis. They normally exhibit high –1 proteins, and the 200–285 kDa GVINs. Bracketed names depict former intrinsic rates of GTPase activity (kcat 20–250 min ), low mM assignations prior to the currently adopted MGI nomenclature, except for Lirg substrate affinity, and nucleotide-dependent self-assembly to bidomain proteins. Two IRGM and Gbp5 isoforms are included, along with a Gbp1 (Gbp2ts1) transpliced isoform (Kim et al., 2011). Selected H-Ras and generate >0.5–1 MDa complexes in cells. Hence, they are dynamin GTPases are shown for G domain comparison (EGA package MEGA ‘‘large’’ GTPases that operate either as mechanoenzymes or v4.0.). The scale bar indicates substitutions/site. as assembly platforms to orchestrate disparate functions. In (B) Structural models of prototypic IRG, GBP, and MX proteins based on crystallographic deposits showing the overall bidomain organization com- contrast, the smaller 20–30 kDa H-Ras and heterotrimeric G posed of an N-terminal G domain (GD) and C-terminal helical domain (CTHD). proteins operate as molecular ‘‘switches,’’ helping to relay up- In the case of murine Irga6, a short intervening N-terminal helical domain stream signals to downstream effectors with each cycle of (NTHD) precedes the CTHD. Protein data bank (PBD) codes for the individual GTP hydrolysis (Ferguson and De Camilli, 2012; Praefcke and proteins are given below. McMahon, 2004). While many IFN-inducible GTPases adhere to the self- Currently, the IFN-inducible GTPase superfamily comprises at catalyzing and GTPase-dependent assembly principles of the least 47 members in humans and mice (Kim et al., 2011) dynamin family, some do not. For example, the smaller IRG (Figure 1). Each GTPase can be grouped into one of four subfam- proteins exhibit low intrinsic rates of GTP hydrolysis and are ilies on the basis of paralogy and molecular mass. These include often GDP bound, suggesting that they could enlist guanine the 21–47 kDa IRGs, 65–73 kDa GBPs, 72–82 kDa myxoma (MX) nucleotide exchange factors (GEFs) to displace GDP and resistance proteins, and 200–285 kDa very large inducible possibly GTPase-activating proteins (GAPs) to accelerate hydro- GTPases (VLIGs/GVINs) (MacMicking, 2004; Martens and lysis, like the H-Ras GTPases (Hunn et al., 2008; Papic et al., Howard, 2006). 2008; Tiwari et al., 2009; Uthaiah et al., 2003). Alternatively, For the IRG and GBP subfamilies, the majority of genes reside some IRGs may undergo G domain dimerization like other non- in chromosomal clusters that are syntenic across species Ras GTPases to elicit GAP activity (Gasper et al., 2009; Uthaiah (Bekpen et al., 2005; Degrandi et al., 2007; Kresse et al., 2008; et al., 2003). Specific IRG and GVIN genes also encode tandem

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duplicated GTPase domains (Klamp et al., 2003; Shenoy et al., bacterial phagosomes, chlamydial inclusion bodies, and proto- 2007), which in the case of GVINs lack some of the canonical zoan parasitophorous vacuoles. Detection of microbial, nonmi- nucleotide-contact sites normally used for engaging its GTP crobial, or even ‘‘altered/missing self’’ structures on these substrate (Figure 1). Lastly, certain GBPs (e.g., GBP5) undergo compartments would enable IRGs to mobilize their effectors to tetramerization in the absence of GTP hydrolysis, while other this site. Thus, the IRGs may bridge upstream sensory events GBPs remain monomeric despite robust catalytic activity with downstream microbicidal activities in a way that helps (Shenoy et al., 2012; Wehner and Herrmann, 2010). Quite a decode the language of vacuolar pathogen recognition. number of GBPs also generate GMP along with GDP, the only Support for this idea came from examining Irgm1 (formerly known mammalian GTPases to do so (Colicelli, 2004; Praefcke lipopolysaccharide-responsive gene of 47 kDa [LRG-47]) and McMahon, 2004). Hence, the IFN-inducible GTPases would in macrophages infected with Mycobacterium tuberculosis appear to defy simple enzymologic classification. They share (MacMicking et al., 2003). M. tuberculosis phagosomes were selected biochemical features of the dynamin protein family in found to recruit Irgm1 that facilitated IFN-g-induced fusion of addition to displaying a uniqueness all their own. this compartment with microbicidal lysosomes as shown by Within the C-terminal region, a series of amphipathic a helices a series of direct functional assays; such activity was impaired are used for protein-lipid and protein-protein interactions; this in Irgm1–/– cells (MacMicking et al., 2003). Thereafter, several has been shown for both the IRGs and GBPs (Kim et al., 2011; groups analyzed the traffic of other IRGs and IFN-inducible Martens et al., 2004; Tiwari et al., 2009). Specific GBPs also GTPases to different pathogen compartments. harbor C-terminal CaaX motifs (GBP1, GBP2, GBP5) that can Human IRGM along with its murine Irgm1 ortholog are be either completely or partially isoprenylated to provide anchor- now known to be targeted to Listeria monocytogenes, Mycobac- age to endomembranes on different organelles distributed within terium bovis, Mycobacterium tuberculosis, Salmonella typhiu- the host cell (Britzen-Laurent et al., 2010; Modiano et al., 2005; murium, and most likely Crohn’s disease-associated adherent Tripal et al., 2007). Mutation of these residues often results in invasive Escherichia coli (AIEC, which can replicate inside phag- dispersal throughout the cytoplasm and can interfere with anti- olysosomes) in monocytes, macrophages, and epithelial cells microbial function, depending on the pathogen and the type of (Figure 2)(Brest et al., 2011; Gutierrez et al., 2004; Jua´ rez niche in which it resides (Kim et al., 2011; Tietzel et al., 2009; et al., 2012; Lapaquette et al., 2010; McCarroll et al., 2008; Virreira Winter et al., 2011). N-terminal glycines in some IRGs Shenoy et al., 2007; Singh et al., 2006; Tiwari et al., 2009). This are also myristoylated to aid membranous attachment, although translocation increases when otherwise cytosolic bacteria whether this modification is obligate for cell-autonomous remain trapped inside vacuoles (e.g., Listeria mutants lacking immunity is as yet unknown (Martens et al., 2004; Papic et al., the pore-forming toxin, listeriolysin O) (Shenoy et al., 2007). 2008). Likewise, mouse Irgm3, Irga6, and Irgb10 target inclusion bodies All IFN-inducible GTPases with the exception of MX resistance harboring Chlamydia trachomatis and Chlamydia psittaci but not proteins are transcribed in response to type I (primarily IFN-a/b), Chlamydia muridarum, suggesting that the murine pathogen has type II (IFN-g), and type III IFNs (IFN-ls). MX proteins are elicited coevolved strategies to evade IRG pursuit (Al-Zeer et al., 2009; by type I and III IFNs but not IFN-g (Bekpen et al., 2005; Klamp Coers et al., 2008; Nelson et al., 2005). et al., 2003; Degrandi et al., 2007; Kim et al., 2011; Kresse Numerous IRGs also converge on vacuoles containing the et al., 2008). Indeed, these IFN-induced GTPases represent apicomplexan parasite, Toxoplasma gondii, in IFN-g-activated some of the most highly expressed IFN-induced target genes; mouse macrophages, fibroblasts, or astrocytes (Hunn et al., for example, the GBPs account for as much as 20% of all 2008; Ling et al., 2006; Khaminets et al., 2010; Martens et al., proteins induced by IFN-g (Martens and Howard, 2006). More- 2005). The Toxoplasma model has helped reveal sophisticated over, in keeping with their role in cell-autonomous immunity, molecular strategies used by both host and pathogen to gain the IFN-inducible GTPases are expressed in most mammalian ascendency over one another. IRG targeting is only seen, for cell lineages examined to date, a reflection in part of the wide- example, with less-virulent type II and III forms of T. gondii spread distribution of different IFN receptors (MacMicking, because virulent type I strains have acquired mechanisms to 2004, 2012; Taylor et al., 2004). Certain GBPs also respond interfere with IRG recruitment (Fentress et al., 2010; Fleckenstein to TNF-a and IL-1b in human endothelial cells and mouse et al., 2012; Niedelman et al., 2012; Steinfeldt et al., 2010; Zhao macrophages, further diversifying the inducible host response et al., 2009a, 2009b). Such interference relies on the combined (Degrandi et al., 2007; Tripal et al., 2007). This enables cell- action of two secreted rhoptry proteins, ROP5 and ROP18. autonomous defense to intracellular bacteria, protozoa, and The functional kinase ROP18 engages mouse Irga6 and Irgb6 viruses in diverse cell types and at selected stages of the path- via the pseudokinase ROP5 to maintain these IRGs in the non- ogen life cycle. Many of their antimicrobial effects are facilitated active GDP-bound conformation (Fentress et al., 2010; Flecken- by direct targeting to the pathogen compartment, a character- stein et al., 2012; Niedelman et al., 2012; Steinfeldt et al., 2010). istic first noted for members of the IRG subfamily. ROP5 binding in particular exposes active site threonines on the GTPase switch I loop that are then targeted by ROP18 for phos- IRGs Target the Pathogen Vacuole phorylation and inactivation, which prevent Irga6 and Irgb6 ‘‘Sensing’’ the Pathogen Compartment targeting to the parasitophorous vacuole (Fentress et al., 2010; An early model proposed that the 21–47 kDa IRGs respond Fleckenstein et al., 2012; Niedelman et al., 2012; Steinfeldt primarily to signals emanating from compartmentalized patho- et al., 2010). Thus, pathogen strategies directed against IRG gens (MacMicking, 2004; Taylor et al., 2004). Such com- recruitment underscores its importance for cell-autonomous partments were thought to be structurally diverse, spanning control in different vertebrate hosts.

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Figure 2. IRGs Target Intracellular Pathogens (A) Irgm1 targets the mycobacterial vacuole. Endogenous Irgm1 (green) translocates to the nascent phagocytic cup (arrows) engulfing Myco- bacterium bovis BCG (red) in IFN-g-activated mouse bone marrow-derived primary (left) or RAW264.7 (right) macrophages shown by confocal microscopy. The scale bar represents 2 mm. DIC, differential interference contrast (Tiwari et al., 2009). (B) Irgm1 recruitment to the phagocytic cup occurs within minutes of mycobacterial uptake. Live meta confocal imaging of IFN-g-activated macrophages expressing fluorescent Irgm1 (green), the pleckstrin homology (PH) domain of general receptor for phosphoinositides-1 (GRP1, which detects PtdIns3,4,5P3; red), and M. bovis BCG (in blue with arrow). Boxed region, Irgm1- and PtdIns3,4,5P3-enriched pseudopod engulfing bacteria. (C) Left: Robust lipid binding of Irgm1 directly to PtdIns3,4,5P3 and PtdIns3,4P2 in a gel overlay assay that is lost for the Irgm1 aK helical mutant in which amphipathicity of this region is destroyed (Irgm1F362E,R367E)(Tiwari et al., 2009). Right: Computer-generated Irgm1 model based on crystallized dimeric Irga6 (PBD code ITQ6) showing the membrane-accessible aK helix and mutated amphipathic residues within it.

phages (Tiwari et al., 2009)(Figure 2). Here lipid binding also served a second function, helping to derepress an intra- molecular inhibition of the aK helix to facilitate GTP hydrolysis and transition state binding to its effector proteins once Irgm1 had reached the mycobacterial phagosome (Tiwari et al., 2009). Thus, certain host lipid species not only act as a recognition motif but may also trigger an allosteric switch in Irgm1 to start assembling the prefusogenic machinery on the bacterial vacuole. Evolution of a lipid-sensing domain in the IRGs ﬁts with other mammalian GTPases; inspection of 125 Ras, Rab, Arf, and Rho GTPases, for example, found that nearly one-third contained C-terminal amphipathic helices that

bound PtdIns3,4,5P3 and PtdIns4,5P2 for redirection to the plasma membrane Compartmental Signatures Recognized by IRGs (Heo et al., 2006). Because vacuolar pathogens like Salmonella What are the compartmental signatures sensed by IRGs? typhimurium secrete phosphatases (SopB) that dephosphor- To date, little is known about the molecular determinants of ylate these PtdIns in establishing their replicative niche vacuolar recognition, although work with Mycobacterium bovis (Hernandez et al., 2004), Irgm1 may be forced to recognize

BCG found that host phosphatidylinositide lipids (PtdIns3,4P2 other lipid species or use different interaction domains besides and PtdIns3,4,5P3) generated by class I phosphatidylinositide the aK helix to target such bacteria as part of a coevolutionary kinases (PI3Ks) can act as a spatial cue for soliciting Irgm1 to arms race (Kuijl and Neefjes, 2009). Consistent with this the phagocytic cup within minutes of bacterial uptake (Figure 2). idea, Irgm1 also binds diphosphatidylglycerol (DPG, or car-

Irgm1 directly binds PtdIns3,4,5P3 and PtdIns3,4P2 via diolipin) which is a common inner membrane constituent of a C-terminal amphipathic helix (aK); mutation of this region or many gram-negative and gram-positive bacteria that is found inhibition of class I PI3Ks impaired its targeting to plasma on their phagosomes (Matsumoto et al., 2006; Tiwari et al., membrane-derived phagosomes in IFN-g-activated macro- 2009).

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Human IRGMd likewise binds DPG (Singh et al., 2010). Here in human alveolar macrophages (Jua´ rez et al., 2012). Lastly, the regions used to engage this phospholipid are different, murine Irga6 binds the microtubule-binding protein Hook3 however, as it lacks the Irgm1-related CTHD (Bekpen et al., (Kaiser et al., 2004) that is otherwise disabled by the Salmonella 2005, 2009). IRGMd may instead enlist N-terminal helices or type III secretion effector, SpiC, to prevent phagolysosome even acquire a lipid-sensing domain in trans via its partners, fusion (Shotland et al., 2003); whether this binding restores since IFN-inducible GTPases interact with each other to regulate bacterial killing, however, has yet to be tested. Several IRGs trafﬁc to vacuolar compartments (Hunn et al., 2008; Khaminets thus appear to help deliver phagocytosed bacteria via a number et al., 2010; Traver et al., 2011; Virreira Winter et al., 2011). of interaction partners and pathways to lysosomes. Whether other host or pathogen lipid species besides For Chlamydia serovars, mouse Irgm3 acts as a noncanonical

PtdIns3,4,5P3, PtdIns4,5P2, and DPG serve as intracellular guanine nucleotide dissociation inhibitor (GDI) that releases PAMPs to recruit IRGs remains to be tested. So, too, does the membranolytic GKS-containing IRGs like Irga6 and Irgb10 to possibility that specific lipids impose membrane curvature directly disrupt the Chlamydial inclusion body (Al-Zeer et al., profiles that act as a second level of IRG recognition for sensing 2009; Coers et al., 2008). Additionally, Irgm3 and Irga6 intercept and discriminating between different pathogen compartments. sphingomyelin-containing exocytic vesicles (Nelson et al., 2005) Consequences of IRG Recruitment or bind to adipophilin/ADRP to regulate the stability of lipid Once IRGs reach the pathogen vacuole, what are the conse- bodies (Bougne` res et al., 2009), which store neutral lipids and quences for cell-autonomous immunity? Studies of AIEC, Chla- normally provide nutrition for the chlamydial inclusion (Saka mydia, Mycobacteria, Salmonella, and Toxoplasma have begun and Valdivia, 2012). Loss of these IRG activities renders to shed light on this issue (Al-Zeer et al., 2009; Brest et al., Irgm3–/–, Irga6–/–, and Irgb10–/– fibroblasts susceptible to infec- 2011; Hunn et al., 2008; Khaminets et al., 2010; Lapaquette tion with C. trachomatis or C. psittaci (Bernstein-Hanley et al., et al., 2010; Nelson et al., 2005; Singh et al., 2006; Tiwari et al., 2006; Coers et al., 2008; Miyairi et al., 2007). 2009; Zhao et al., 2008). IRGs serve distinct roles depending The regulatory functions of IRGM proteins extend to Toxo- on which biochemical class—GKS or GMS—they belong to. plamsa gondii tachyzoites where a hierarchical model for inter- This functional classification developed by Hunn and coworkers actions among IRGs themselves was first uncovered (Hunn (2008) is based on the presence of canonical (lysine, K) or non- et al., 2008). Here Irgm1–Irgm3 allow release and sequential canonical (methionine, M) G1 motifs (GX4G[K/M]S/T) within the accumulation of GKS IRGs—specifically, Irgb6, Irgb10, Irga6, conserved N-terminal catalytic G domain. Lysine substitution and Irgd in that order—on the parasitophorous vacuole (Hunn by methionine does not appear to adversely affect enzyme et al., 2008; Khaminets et al., 2010). These functions are partly activity (Taylor et al., 1996; Tiwari et al., 2009) but nonetheless shared with the autophagy protein, ATG5, and members of the leads to divergent biological activities. IRGs of the GMS GBP family that facilitates IFN-g or IFN-g/LPS-induced Irga6/ subclass (IRGMa–IRGMd in humans, Irgm1–Irgm3 in mice) Irgb6 trafficking to the T. gondii vacuole in a noncanonical appear to be intrinsic regulators, helping control the antimicro- fashion (Khaminets et al., 2010; Yamamoto et al., 2012; Zhao bial activities of their respective effectors, which include SNARE et al., 2008). IRG targeting becomes detectable over a 10– adaptor and autophagy proteins as well as GKS-containing IRGs 20 min period before progressive accumulation up to 90 min, and possibly even GBPs (Al-Zeer et al., 2009; Hunn et al., 2008; when the GTPase-driven oligomerization of different GKS IRGs Khaminets et al., 2010; Lapaquette et al., 2010; Nelson et al., leads to membrane vesiculation and disruption akin to the tubu- 2005; Singh et al., 2006; Tiwari et al., 2009; Traver et al., 2011; lating activities of other dynamin-like proteins (Ling et al., 2006; Zhao et al., 2008). Martens et al., 2005; Zhao et al., 2009a, 2009b). This membrane During infection with AIEC, Mycobacteria or Salmonella disruption is reported to have different cell-autonomous fates serovars, human IRGM or its murine Irgm1 ortholog recruit auto- depending on host cell type. In activated fibroblasts it leads to phagic (e.g., MAP1/LC3) and SNARE adaptor (e.g., Snapin) necroptosis (Zhao et al., 2009b), whereas in cytokine-stimulated proteins to assemble fusogenic and possibly biogenesis of macrophages the damaged parasite organelle appears to lysosome-related organelle complexes (BLOCs) in a pathogen- undergo autophagic elimination (Ling et al., 2006). specific fashion for lysosomal targeting, since IFN-g-induced GMS-containing IRGs thus coordinate multiple classes of autophagosome formation appears ostensibly normal in unin- effectors—SNARE and BLOC adaptors, autophagy and lipid fected Irgm1–/– macrophages (Brest et al., 2011; Jua´ rez et al., droplet proteins, GKS-containing IRGs, and even GBPs—to 2012; Gutierrez et al., 2004; Lapaquette et al., 2010; MacMicking promote cell-autonomous immunity to intracellular pathogens et al., 2003; Matsuzawa et al., 2012; Singh et al., 2006, 2010; occupying different vacuolar habitats. Such widespread involve- Tiwari et al., 2009; Traver et al., 2011). The human IRGM locus ment likely contributes to the pronounced susceptibility pheno- generates at least five messenger RNA (mRNA) transcripts en- types seen in several IRGM-deficient human and mouse hosts coding four IRGM isoforms: IRGMa–IRGMd (Bekpen et al., (see MacMicking, 2012). 2005; 2009). IRGMd is posited to translocate to mitochondria where it causes fission to elicit autophagy against mycobacterial GBPs Target Both Vacuolar and Cytosolic Pathogens infection (Singh et al., 2010), a process that could underlie auto- Patrolling the Host Cell’s Interior phagic responses to Salmonella typhimurium and AIEC, as well Interactions within and outside the GBP family enable these (Brest et al., 2011; Lapaquette et al., 2010; McCarroll et al., 65–73 kDa GTPases not only to patrol membrane-enclosed 2008). Other human IRGM isoforms appear to target the pathogens like Chlamydia trachomatis, Mycobacterium bovis, M. tuberculosis phagosome directly when elicited through the and Toxoplasma gondii but also alert the innate immune system innate immune receptor NOD2 by its ligand muramyl dipeptide to cytosolically escaped microbes. GBPs normally partition

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between the cytosol and endomembranes but upon infection are tions among family members (Britzen-Laurent et al., 2010; redirected to the pathogen compartment and to Listeria mono- Virreira Winter et al., 2011) or nonspecific intergenic effects being cytogenes, Shigella flexneri, and a subpopulation of Salmonella associated with the 173 kb deletion in Gbpchr3 mice (Yamamoto typhimurium that have escaped their vacuole to replicate in the et al., 2012). Clean chromosomal deletions of each Gbp loci will cytosol (Degrandi et al., 2007; Kim et al., 2011; Kravets et al., thus probably be needed to correctly ascribe functions to 2012; MacMicking, 2012; Tietzel et al., 2009). individual members. GBP translocation requires nucleotide binding and catalytic Protein-protein interaction screens found Gbp1 and Gbp7 activity to help self-assemble these larger GTPases along with assembled oxidative and autophagic complexes for bacterial their effector proteins (Kim et al., 2011; Kravets et al., 2012; elimination (Kim et al., 2011)(Figure 3). Gbp7 bound NADPH MacMicking, 2012; Tietzel et al., 2009; Virreira Winter et al., oxidase, a membrane complex that generates superoxide – 2011). Isoprenylation of human and mouse GBP1 and GBP2 is (O2 ), and Atg4b, a cysteine protease required for autophagy, also required to recruit these two GTPases, for example, to while Gbp1 engaged the ubiquitin-binding protein p62/ M. bovis BCG, L. monocytogenes, and S. typhimurium but Sequestosome-1. Gbp7 could help form the NADPH oxidase not to T. gondii parasitophorous vacuoles (Kim et al., 2011; by acting as a bridging protein between the membrane-associ- MacMicking, 2012; Virreira Winter et al., 2011). This selectivity ated heterodimer, p22 phox-gp91phox, and cytosolic p67phox to is further evident for the kinetoplastid parasite Trypanosoma assemble the holoenzyme on bacterial phagosomes and endo- cruzi, which fails to recruit mouse Gbp1 altogether in IFN-g- somal vesicles targeting the pathogen vacuole (Kim et al., activated macrophages (Virreira Winter et al., 2011). Such differ- 2011). Gbp7 also recruits Atg4b to help drive the extension of ential targeting probably reflects the membrane composition of autophagic membranes around bacteria within damaged distinct compartments inhabited by specific pathogens as well compartments, an activity that is likely to require Gbp7 self- as the presence or absence of specific host components on assembly. Notably, Atg4b was found to be recruited to the bacterial envelope once they enter the cytosol (MacMicking, T. gondii vacuoles, although this was not affected in Gbpchr3 2012). Again, targeting of GBPs to the site of pathogen peritoneal macrophages, similar to that seen for the oxidative replication often appears requisite for subsequent antimicrobial burst (Kim et al., 2011; Yamamoto et al., 2012). Such differences activity. may arise from pathogen-specific responses for Gbp7 against GBPs Assemble Pathogen-Specific Defense Complexes Mycobacterium versus Toxoplasma, elicited versus resting cells, What do the GBPs assemble on replicative vacuoles or deliver to or the loss-of-function strategy employed (Dupont and Hunter, cytosolic pathogens? Recent work indicates that different 2012). Gbp1 bound p62/Sequestosome-1 to help deliver ubiqui- GBPs galvanize specific defense complexes that confer cell- tinylated cargo to autolysosomes and possibly respond to autonomous resistance in both locations. Identification of these bacteria ‘‘marked’’ by ubiquitin in the cytosol, where it solicits new GBP-mediated functions represent a major breakthrough other GBPs as effectors (Kim et al., 2011; MacMicking, 2012). for the field of host defense, since the molecular mechanisms This interdependence between GBP family members is simi- employed by the atypical 65–73 kDa GTPases had remained larly noted against T. gondii, where Gbp1 can recruit Gbp2 and elusive for over two decades. Gbp5 as downstream partners (Virreira Winter et al., 2011). A A family-wide screen combining three independent loss-of- pan-Gbp1-5 antibody also retrieved endogenous Irgb6, sug- function approaches—siRNA silencing, dominant-negative gesting that certain GKS IRGs are recruited as potential inhibition and chromosomal deletion—recently uncovered path- membranolytic effectors (Yamamoto et al., 2012), although their ogen-specific involvement for four members against M. bovis absence from the human genome indicates that human GBPs BCG and L. monocytogenes (Kim et al., 2011). Here Gbp1, probably operate via different mechanisms in IFN-g-activated Gbp6, Gbp7, and Gbp10 promoted cell-autonomous defense cells (Bekpen et al., 2005; Niedelman et al., 2012). Notably, in IFN-g-activated macrophages while the generation of Gbp1- virulent type I T. gondii strains strongly interfere with GBP deficient mice revealed loss of bacterial control for these two recruitment, a process that enlists the parasite rhoptry proteins pathogens in vivo (Kim et al., 2011). ROP16, ROP18, and the parasite-dense granule protein, Subsequent studies have also reported Toxoplasma suscepti- GRA15 (Degrandi et al., 2007; Virreira Winter et al., 2011). bility in mice with single deletions at the Gbp2 locus and en bloc Whether ROP16 and ROP18 kinases phosphorylate the analo- deletion the entire Gbp cluster (Gbp2ps, Gbp2, Gbp1, Gbp3, gous switch I region in GBPs to turn off this class of host defense Gbp7, Gbp5) on chromosome 3H1 (Gbpchr3)(Kravets et al., GTPases akin to the IRGs awaits experimental validation. 2012; Yamamoto et al., 2012)(Figure 3). Despite lacking Gbp2, Besides bacteria and protozoa, GBPs also promote cell- experiments in Gbpchr3 mice suggested Gbp1, Gbp5, and autonomous defense against viruses. This antimicrobial profile Gbp7 are more important based on reconstitution of individual differs from the IRGs that appear to lack intrinsic antiviral activity Gbps in Gbpchr3 cells (Yamamoto et al., 2012). Likewise, suscep- but resembles the related IFN-inducible MX GTPases that are tibility to L. monocytogenes in Gbp1–/– and Gbp5–/– mice was potent viral restriction factors, especially against orthomyxovi- absent from Gbpchr3 animals that also lack both Gbps; here ruses such as influenza A (Haller and Kochs, 2011). Human differences likely reflect the adminstrative route used, namely, GBP1, GBP3, and a C-terminally truncated GBP3 splice isoform oral versus peritoneal inoculation (Kim et al., 2011; Shenoy designated GBP3DC were recently found to inhibit influenza A in et al., 2012; Yamamoto et al., 2012). Nonetheless, the fact that IFN-g-activated lung epithelia as well (Nordmann et al., 2012). Gbp1–/–, Gbp2–/–, and Gbp5–/– single knockouts differ from Antiviral activity required the G domain and was dependent on Gbpchr3 mice against some of the same pathogens (T. gondii, GTP binding but not hydrolysis, suggesting that oligomerization L. monocytogenes) may indicate disrupted regulatory interac- was essential for repression of the influenza PB1, PB2, and PA

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Figure 3. Members of the GBP Family Assemble Defense Complexes (A) Chromosomal configuration of human and mouse GBP families showing inverted and direct synteny of mouse chromosome 3H1 and 5E5 with human chromosome 1q22.1 (Kim et al., 2011). BAC clones spanning GBP clusters that were used for assembly are listed in blue font. Red circles designate centromeric orientation. (B) Gbp7 (green) also targets M. bovis BCG (red) within 30 min as seen via live imaging in IFN-g-activated mouse macrophages. Relative fluorescent units (RFU) for boxed insets are shown below. (C) Left: Retrieval of endogenous NADPH oxidase membrane-spanning gp91phox and p22phox subunits by the Gbp7 C-terminal helical domain (CTHD) specifically from IFN-g-activated macrophage lysates in silver-stained gels. Individual mass spectrometric peptides are shown alongside in red, with overlapping peptides in green. Right: Gbp7 (red) and p22phox (blue) on vesicles targeting M. bovis (green) in IFN-g-activated macrophages (inset squares shown below). The scale bar represents 10 mm(Kim et al., 2011). polymerase complex that leads to reduced viral RNA and protein GBP5-Mediated Inflammasome Activation synthesis (Nordmann et al., 2012). Thus, a recurring biochemical Another recent discovery is the involvement of GBP5 in assem- feature of this superfamily—self-assembly—appears important bling the NLRP3 inflammasome, a complex that senses micro- for the antimicrobial activity of many GBPs against at least three bial molecules and stimulates caspase-1 activation and proin- pathogen classes: bacteria, protozoa, and viruses. flammatory cytokine production. Interestingly, GBP5 mediated

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Figure 4. GBP5 as an Inflammasome Activator During Cell-Autonomous Host Defense (A) Schematic model of IFN-g-induced GBP5 that helps assemble the NLRP3 inflammasome in response to whole bacteria, their TSS-secreted products, and cell wall fragments (lipopolysaccharide [LPS], muramyl dipeptide [MDP], and g-D-glutamyl-meso-diaminopimelic acid [ieDAP]) within the cytosol. These activating signals are accompanied by potassium efflux (K+) and mobilize tetrameric GBP5 bound via its G domain (GD) to the pyrin domain of NLRP3 (NLRP3PYD) to begin assembling the inflammasome complex that includes the adaptor protein, Asc, which bridges NLRP3 and the zymogen, procaspase-1. This GBP5-dependent assembly ultimately leads to caspase-1 autoproteolysis and activation to cleave its procytokine substrates, interleukin-1b and interleukin-18 (Shenoy et al., 2012; Caffrey and Fitzgerald, 2012). (B) Formation of a GBP5 scaffold around the NLRP3-ASC inflammasome in response to LPS and the K+ ionophore, nigericin, in human HeLa cells reconstituted with ASC (blue), GBP5 (red), and NLRP3PYD (green) (Shenoy et al., 2012). The scale bar represents 10 mm. Boxed insets are shown below.

gested that interactions between the GBPs and inflammasome-related NLRs could exist in higher species such as mammals, except that here the relevant domains would be separated onto seemingly unrelated proteins through evolutionary divergence. Probing of the interdomian profiles of lower organisms thus enabled functional relationships to be established in higher species that would otherwise remain imperceptible because they operate over large protein distances. Subsequent analysis found human GBP5 and its murine Gbp5 ortholog were critical for NLRP3 but not AIM2 or flagellin-dependent NLRC4 inflammasome activation in IFN-g-activated macrophages and in Gbp5-deficient mice (Figure 4). The stimulus-dependent role of GBP5 encompassed live Salmo- nella and Listeria infection, as well as NLRP3 assembly specifically in response to infectious agents their cell wall components (LPS, MDP), but not crystalline like bacteria and their cell-wall components but not to sterile agents that also activate the NLRP3 inflammasome via agonists or adjuvants such as alum (Shenoy et al., 2012) a different pathway involving lysosomal rupture (Shenoy (Figure 4). GBP5 is the first noncanonical protein to exhibit this et al., 2012; Caffrey and Fitzgerald, 2012). How this selectivity activity, leading to a reappraisal of the way in which inflamma- is imposed remains unknown, but it leads to NLRP3 assembly somes are regulated in immunologically activated cells to by GBP5 tetramerization as a bound partner to activate combat infection (Caffrey and Fitzgerald, 2012). procaspase-1 (Shenoy et al., 2012)(Figure 4). Thus, GBP5 Evidence of this relationship came from large-scale in silico represents a unique ligand-specific rheostat for inflammasome screens designed to mine interdomain similarities across activation that links earlier observations on its role during 574 GBP-related sequences from 91 taxa: it retrieved domains Salmonella typhimurium-induced pyroptosis to help restrict found in NOD-like receptor (NLR) proteins fused to ancestral infection in a cell-autonomous manner (Rupper and Cardelli, GBPs from lower organisms (Shenoy et al., 2012). This sug- 2008).

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Figure 5. Disease-Associated Polymorphisms in the Human IRGM Locus (A) Early observations of pulmonary TB susceptibility in experimental animals harboring a chromosomal deletion of the Irgm1 locus after aerogenic infection with M. tuberculosis (MacMicking et al., 2003) are now emerging for the IRGM locus in the human population (see B). Wild-type and Ifngr1 (ligand-binding chain of the IFN-g receptor)-deficient mice served as positive and negative controls, respectively, in these initial experiments. The scale bar represents 1 cm. (B) Configuration of the IRGM locus on human chromosome 5q33.1 that yields five splice mRNA isoforms (IRGMa–IRGMe) predicted to encode four different IRGM proteins (IRGMa, IRGMb, IRGMc/e, and IRGMd) (Bekpen et al., 2005; 2009). Upstream ERV9 retroelement insertion plus Alu repeats shown together with the 20.1 kb deletion polymorphism in linkage disequilibrium with a coding SNP rs10065172 that results in reduced IRGM expression and susceptibility to both TB and CD. Haplotypes at this nucleotide (313T/c) are selectively targeted by miRNA-196 to destabilize IRGM transcripts in intestinal epithelia and certain nonintestinal cell types (McCarroll et al., 2008; Brest et al., 2011). Other disease-associated polymorphisms and their respective SNPs across the IRGM locus are depicted.

Atomic Insights into Flu-Resisting MX Proteins bilize them (Haller and Kochs, 2011). An appealing feature of IFN-inducible MX GTPases differ from the IRGs and GBPs in that this structural model is that it accommodates the known ability they direct their antimicrobial activities almost exclusively to of MX proteins to target different viruses independently of viral viruses. Originally identified as an inherited trait conferring resis- nucleotide or sequence composition. tance to influenza A as shown by susceptible mouse strains that harbor deletions or nonsense mutations in the murine Mx1 Human Genetics of the IFN-Inducible GTPases: (Myxoma resistance gene 1) locus, the MX proteins are now Tuberculosis, Crohn’s, and MicroRNAs established broad-spectrum antiviral proteins effective against Recent human genetic studies implicate IRGM and several flu and influenza-like togaviruses, as well as bunyaviruses, GBPs in the protective or inflammatory immune response to rhabdoviruses, and Thogoto, Coxsackie, and Hepatitis B viruses clinical tuberculosis (TB), findings consistent with the experi- (Haller and Kochs, 2011; MacMicking, 2012). Indeed, their mentally determined in vivo roles for Irgm1 and Gbp1 against importance is perhaps best exemplified by the ability of a human M. tuberculosis and its close relative, M. bovis BCG (Feng MX1 transgene to completely rescue mice lacking IFN type I et al., 2004; Kim et al., 2011; MacMicking et al., 2003)(Figure 5). (IFNa/b) receptors from influenza infection, indicating that MX1 Examination of IRGM variants in over 2,000 pulmonary TB is sufficient to act autonomously without other type I IFN- patients and their unaffected controls found increased risk induced restriction factors (see Haller and Kochs, 2011). frequencies associated with the IRGM 2621TT genotype against More recently, crystallographic analysis has begun to unveil M. tuberculosis but not M. africanum clades in a Ghanese pop- the molecular mechanisms used by human MX1 at the atomic ulation (Intemann et al., 2009). TB susceptibility in African level (Gao et al., 2010, 2011). Nucleotide-free structures of this Americans also segregates with single-nucleotide polymor- GTPase found three topographical domains: a globular phisms (SNPs) in IRGM (rs13361189 T/C and rs10065172 C/T) N-terminal G domain, an extended three helical bundle termed that are in linkage disequilibrium with a 20 kb upstream deletion the central bundle signaling element (BSE), and a stalk spanning (King et al., 2011), while a À1208 A/G allelic promoter polymor- the middle and C-terminal GTPase effector domains (GEDs) phism within this region match smear and/or culture-positive responsible for tetrameric self-assembly. Cooperative functions TB in Beijing cohorts (Che et al., 2010)(Figure 5). Hence, alter- between the BSE and GED promote oligomerization akin to the ations in the IRGM locus may yield candidate susceptibility dynamin family and are essential for antiviral function (Gao profiles in diverse human populations against different myco- et al., 2010, 2011). Hinges each side of the BSE enable MX1 bacterial species. mechanoenzyme movement that on the basis of minireplicon For the GBPs, recent genome-wide transcriptional profiling systems are likely to assemble oligomeric rings around viral found that patients undergoing active TB mobilized strong mod- nucleocapsid proteins bound by the MX1 stalk region to immo- ular signatures within neutrophils dominated by IFN-induced

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Figure 6. IFN-Inducible GTPases Confer Pathogen-Specific Host Defense Naturally occurring and genetically engineered loss-of-function mutations in humans and mice reveal patterns of pathogen-directed immunity among the IFN-inducible GTPase subgroups. IRG proteins appear to be heavily biased toward vacuolar bacteria and protozoa, while MX proteins almost exclusively inhibit viruses. In contrast, the GBPs are required for cell-autonomous resistance against all three pathogen classes. Abbreviations are as follows: HBV57, hepatitis B virus 57; HCV, hepatitis C virus; Mx1D390–631, congenital mouse Mx1 locus deletion encoding amino acids 390–631. genes such as GBP1, GBP2, and GBP5 that are also prominent in silencing points to a complex regulatory circuit for IRGM in CD the lungs of TB-infected animals (Berry et al., 2010; Marquis et al., pathogenesis. 2011). Extrapolating from studies of Gbp1–/– and Gbp5–/– mice, Similarly complex roles emerge for the GBPs as well. such responses are likely to be protective overall; however, For example, GBP1 localizes to gap junctions in polarized human persistent inflammasome activation may lead to pulmonary intestinal epithelial cells to inhibit enteropathogenic E. coli- damage (Kim et al., 2011; Shenoy et al., 2012). Whether distinct induced apoptosis (Mirpuri et al., 2010; Schnoor et al., 2009). A GBP mutations influence human TB susceptibility like IRGM in more recent report, however, suggests that this GTPase can different ethnic groups or geographical locations has yet to be also block epithelial cell proliferation by interfering with b-catenin investigated. signaling (Capaldo et al., 2012). Definitive chromosomal dele- Comprehensive genome-wide association analysis has also tions of the human GBP1 locus may help resolve some of these recently shown IRGM acts as a major predisposing locus for issues as they relate to host defense operating within the gastro- Crohn’s disease (CD) (Craddock et al., 2010; Wellcome Trust intestinal mucosa. Case Control Consortium, 2007). Suprisingly, SNPs immediately flanking IRGM on chromosome 5q33.1 have the strongest repli- Concluding Remarks cating frequency and are associated with the upstream 20 kb GTPases serve protean roles in every mammalian organ system, deletion (Parkes et al., 2007), a finding confirmed in multiple from synaptic vesicle trafficking to neutrophil chemotaxis to ion CD studies (Khor et al., 2011)(Figure 5). This structural deletion channel transport for glomerular filtration in the kidney (Colicelli, is in linkage disequilibrium with a coding SNP that affects 2004). These and other physiological activities are subsumed by IRGM expression. Nucleotide 313T coding haplotypes are >180 GTPases in humans encompassing the RAS, RAC, RAB, ‘‘risk’’ alleles that largely abolish IRGM expression in colonic RHO, ARF, dynamin, and atlastin families (Colicelli, 2004). epithelia, which coincides with autophagy defects against Emergence of an IFN-inducible GTPase superfamily with dedi- S. typhimurium or colitogenic AIEC, whereas the 313C ‘‘protec- cated cell-autonomous defense functions adds another dimen- tive’’ haplotype generally exhibits higher IRGM levels and intact sion to the sphere of biological reactions driven by guanine autophagy responses (McCarroll et al., 2008). IRGM thus nucleotide binding and hydrolysis. That the IFN-inducible appears to be necessary for cell-autonomous defense against GTPases seem to have evolved highly pathogen-specific and enteropathogens, although its expression is tightly controlled. subclass-dependent activities to defend the cell’s interior is Recently, a specific microRNA, miR-196, was found to limit a remarkable adaptation on the part of larger vertebrate hosts IRGM expression in intestinal epithelia by binding the ‘‘protec- (Figure 6). Presumably, such adaptations come with a fitness tive’’ 313C allele since unchecked IRGM expression can lead cost, however, since most immune GTPases are placed under to dysregulated autophagic immunity against AIEC and induce strict transcriptional, epigenetic, and posttranslational control apoptosis (Brest et al., 2011)(Figure 5). Hence, epigenetic (MacMicking, 2012).

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A number of outstanding questions remain regarding the Bougne` res, L., Helft, J., Tiwari, S., Vargas, P., Chang, B.H., Chan, L., Campisi, biology of these new proteins, chief among them being the L., Lauvau, G., Hugues, S., Kumar, P., et al. (2009). A role for lipid bodies in the cross-presentation of phagocytosed antigens by MHC class I in dendritic cells. precise manner in which patterns of intracellular discrimination Immunity 31, 232–244. and defense are deployed. For example, what are the surface Brest, P., Lapaquette, P., Souidi, M., Lebrigand, K., Cesaro, A., Vouret-Cra- structures recognized by IRGs, GBPs, MX proteins, and possibly viari, V., Mari, B., Barbry, P., Mosnier, J.F., He´ buterne, X., et al. (2011). A GVINs on membrane compartments harboring different synonymous variant in IRGM alters a binding site for miR-196 and causes bacteria, protozoa and viruses? Similarly, how do IFN-inducible deregulation of IRGM-dependent xenophagy in Crohn’s disease. Nat. Genet. 43, 242–245. GBPs detect cytosolically escaped bacteria, and does detection uniformly lead to inflammasome activation or autophagic engulf- Britzen-Laurent,N., Bauer, M., Berton, V., Fischer, N., Syguda, A., Reipschla¨ ger, S., Naschberger, E., Herrmann, C., and Stu¨ rzl, M. (2010). Intracellular trafficking ment? What are the host effector pathways solicited by different of guanylate-binding proteins is regulated by heterodimerization in a hierarchical GTPase family members to restrict microbial replication, and, of manner. PLoS ONE 5, e14246. equal importance, what are the pathogen-encoded tactics used Caffrey, D.R., and Fitzgerald, K.A. (2012). Immunology. Select inflammasome to evade them? Recent evidence in Toxoplasma and Chlamydia assembly. Science 336, 420–421. and in HCV, HIV-1, and Measles virus point to multiple mechanisms for avoiding or manipulating the antimicrobial action of Capaldo, C.T., Beeman, N., Hilgarth, R.S., Nava, P., Louis, N.A., Naschberger, E., Stu¨ rzl, M., Parkos, C.A., and Nusrat, A. (2012). IFN-g and TNF-a-induced at least some IRGs and GBPs in their natural hosts (Fentress GBP-1 inhibits epithelial cell proliferation through suppression of b-catenin/ et al., 2010; Fleckenstein et al., 2012; Gre´ goire et al., 2011; Itsui TCF signaling. Mucosal Immunol. et al., 2009; Niedelman et al., 2012; Steinfeldt et al., 2010; Virreira Che, N., Li, S., Gao, T., Zhang, Z., Han, Y., Zhang, X., Sun, Y., Liu, Y., Sun, Z., Winter et al., 2011). Future efforts should unearth others. Zhang, J., et al. (2010). Identification of a novel IRGM promoter single nucleo- Lastly, the acquisition of such microbial strategies implies that tide polymorphism associated with tuberculosis. Clin. Chim. Acta 411, 1645– 1649. a strong selective pressure is placed by the IFN-inducible GTPases on intracellular pathogens irrespective of their replica- Coers, J., Bernstein-Hanley, I., Grotsky, D., Parvanova, I., Howard, J.C., Tay- tive lifestyle. Harnessing that power for clinical benefit would lor, G.A., Dietrich, W.F., and Starnbach, M.N. (2008). Chlamydia muridarum evades growth restriction by the IFN-g-inducible host resistance factor represent a ‘‘first’’ in the field of cell-autonomous immunity and Irgb10. J. Immunol. 180, 6237–6245. help answer the call for fresh approaches to anti-infective thera- Colicelli, J. (2004). Human RAS superfamily proteins and related GTPases. Sci. pies (Nathan, 2012). STKE 2004, RE13.

Craddock, N., Hurles, M.E., Cardin, N., Pearson, R.D., Plagnol, V., Robson, S., ACKNOWLEDGMENTS Vukcevic, D., Barnes, C., Conrad, D.F., Giannoulatou, E., et al.; Wellcome Trust Case Control Consortium. (2010). Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls. We apologize to colleagues whose work has not cited due to space limitations Nature 464, 713–720. and acknowledge support for some of the discoveries described in this review from the following sources: Yale Brown-Coxe and Anna Fuller postdoctoral Degrandi, D., Konermann, C., Beuter-Gunia, C., Kresse, A., Wu¨ rthner, J., fellowships (A.R.S.), National Institutes of Health grant AI068041-06, Bur- Kurig, S., Beer, S., and Pfeffer, K. (2007). Extensive characterization of IFN- roughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease induced GTPases mGBP1 to mGBP10 involved in host defense. J. Immunol. Award (1007845), Searle Foundation Scholars Program (05-F-114), Cancer 179, 7729–7740. Research Institute Investigator Award Program (CRI06-10), Crohn’s and Colitis Foundation of America Senior Investigator Award (R09928), and W.W. Dupont, C.D., and Hunter, C.A. (2012). Guanylate-binding proteins: niche Winchester Foundation (J.D.M.). recruiters for antimicrobial effectors. Immunity 37, 191–193.

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