International Journal of Medical Microbiology 308 (2018) 237–245

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International Journal of Medical Microbiology

journal homepage: www.elsevier.com/locate/ijmm

Regulation of innate immune functions by guanylate-binding proteins T

a,b,⁎
Gerrit J.K. Praefcke
a
Division of Haematology / Transfusion Medicine, Paul-Ehrlich-Institut, Langen, Germany
b
Institute for Genetics, University of Cologne, Cologne, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Guanylate-binding proteins (GBP) are a family of dynamin-related large GTPases which are expressed in re-
Guanylate-binding proteins sponse to interferons and other pro-inflammatory cytokines. GBPs mediate a broad spectrum of innate immune
Interferon-inducible GTPase functions against intracellular pathogens ranging from viruses to bacteria and protozoa. Several binding partners
Autophagy for individual GBPs have been identified and several different mechanisms of action have been proposed de-
Inflammasome
pending on the organisms, the cell type and the pathogen used. Many of these anti-pathogenic functions of GBPs
Immunity-related GTPase
involve the recruitment to and the subsequent destruction of pathogen containing vacuolar compartments, the
Intracellular pathogens
assembly of large oligomeric innate immune complexes such as the inflammasome, or the induction of autop-
hagy. Furthermore, GBPs often cooperate with immunity-related GTPases (IRGs), another family of dynamin-
related GTPases, to exert their anti-pathogenic function, but since most IRGs have been lost in the evolution of
higher primates, the anti-pathogenic function of human GBPs seems to be IRG-independent. GBPs and IRGs share
biochemical and structural properties with the other members of the dynamin superfamily such as low nu-
cleotide affinity and a high intrinsic GTPase activity which can be further enhanced by oligomerisation.
Furthermore, GBPs and IRGs can interact with lipid membranes. In the case of three human and murine GBP
isoforms this interaction is mediated by C-terminal isoprenylation. Based on cell biological studies, and in
analogy to the function of other dynamins in membrane scission events, it has been postulated that both GBPs
and IRGs might actively disrupt the outer membrane of pathogen-containing vacuole leading to the detection
and destruction of the pathogen by the cytosolic innate immune system of the host. Recent evidence, however,
indicates that GBPs might rather function by mediating membrane tethering events similar to the dynamin-
related atlastin and mitofusin proteins, which mediate fusion of the ER and mitochondria, respectively. The aim
of this review is to highlight the current knowledge on the function of GBPs in innate immunity and to combine it
with the recent progress in the biochemical characterisation of this protein family.

1. Introduction
Interferons are cytokines released upon viral or microbial infections
to induce the expression of several hundred antivirally or anti-
microbially active genes (MacMicking, 2012; Platanias, 2005; Boehm
et al., 1998). Of these, four families of GTPases are among the most
strongly induced genes: the guanylate-binding proteins (GBP), the im-
munity-related GTPases (IRG), the Mx-proteins and the very large in-
ducible GTPases (VLIG) (Li et al., 2009; MacMicking, 2012; Meunier
and Broz, 2016; Pilla-Moffett et al., 2016).
The guanylate-binding proteins were identified due to their strong
induction by type II interferons (IFN-γ) and their ability to interact with
guanine nucleotides (Cheng et al., 1983). The characterisation of the
GBP promotor was crucial for the elucidation of interferon-gamma
signalling (Darnell et al., 1994; Lew et al., 1991; Platanias, 2005) long
before the cellular functions of GBPs were identified. Cloning of the first
human and murine GBPs showed an unusual conservation of the ca-
nonical GTP-binding motifs. While humans express seven GBPs, the
murine genome codes for 11 genes (Kresse et al., 2008; Olszewski et al.,
2006). In both, humans and mice, three GBPs carry a C-terminal iso-
prenylation motif, the CaaX-box (Cheng et al., 1991). While the se-
quence homology between dynamin and the antiviral Mx GTPases had
been identified around that same time (Obar et al., 1990), the GBPs
were only included in the dynamin superfamily when the biochemical
and structural similarities became apparent and when other dynamin-
related protein families were identified (Ghosh et al., 2006; Praefcke
et al., 1999; Praefcke and McMahon, 2004; Prakash et al., 2000a;
Schwemmle and Staeheli, 1994)(Fig. 1). The common feature of dy-
namin GTPases include a high intrinsic GTPase activity, which is fur-
ther stimulated by oligomerisation, and the interaction with biological
membranes. Activation of the GTPases on membranes induces a change
in membrane conformation resulting in either fission or fusion (Daumke

⁎
Corresponding author at: Division of Haematology / Transfusion Medicine, Paul-Ehrlich-Institut, Langen, Germany.
E-mail address: gerrit.praefcke@pei.de.

https://doi.org/10.1016/j.ijmm.2017.10.013
Received 22 August 2017; Received in revised form 27 October 2017; Accepted 31 October 2017
1438-4221/ © 2017 The Author. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
G.J.K. Praefcke
domain.
and Praefcke, 2016). The closest homologues of the GBPs within the
dynamin superfamily are the atlastin proteins. Atlastins are conserved
from yeast to man and regulate dynamics of the endoplasmic reticulum
(ER) by tethering and fusion of ER tubules (Hu and Rapoport, 2016;
McNew et al., 2013).
The first publication of an innate immune function of GBPs reported
an antiviral effect against vesicular stomatitis virus (VSV) and en-
cephaloymyocarditis virus (EMCV) (Anderson et al., 1999). The anti-
viral effect was not very prominent compared to the resistance medi-
ated by e.g. human MxA protein against influenza virus, which may be
due to the incomplete depletion of hGBP1 and −2 by the used antisense
RNA. A similar effect was found for murine GBP2 in a later study
(Carter et al., 2005). The first publications showing an anti-pathogenic
activity of GBPs against protozoa (Degrandi et al., 2013; Yamamoto
et al., 2012) and bacteria (Tietzel et al., 2009) appeared a decade late.
Since then is has become clear that GBPs are important for the defence
against a broad array of intracellular pathogens (Meunier and Broz,
2016; Pilla-Moffett et al., 2016).
The immunity-related GTPases (IRGs), were initially identified in
mice due to their strong induction by IFN-γ starting with IRGM1 under
the name LRG-47 (Gilly and Wall, 1992) followed by five other isoforms
(Boehm et al., 1998; Carlow et al., 1995; Lafuse et al., 1995; Sorace
et al., 1995; Taylor et al., 1996). Later it became apparent that the
genes coding for this protein family, which was referred to as p47
GTPases to distinguish them from the previously identified p65/67
GBPs, is evolving fast leading to more than 20 murine isoforms de-
pending on the mouse strain (Lilue et al., 2013). In contrast, IRGs are
almost completely lost in humans (Bekpen et al., 2005; Bekpen et al.,
2009). Biochemically, the best studied IRG protein is Irga6 (Ghosh
et al., 2004; Pawlowski et al., 2011; Schulte et al., 2016; Uthaiah et al.,
2003) which displays a GTP-dependent oligomerisation and a dimer-
isation induced stimulation of the GTPase activity. In cells Irga6 can be
N-terminally myristoylated (Martens et al., 2004; Papic et al., 2008).
Despite their later identification, the biological function of IRGs was
more intensively studies than the GBPs. The first report of an anti-pa-
thogenic activity of an IRG identified the apicomplexan parasite Tox-
oplasma gondii as a target pathogen (Taylor et al., 2000). The study of
murine IRG knock-out strains strongly accelerated the identification of
additional resistance phenotypes. Cell biological studies showed that a
subgroup of the IRGs, the so called GMS-proteins, serve as negative
regulators of the other IRG family members. (Hunn et al., 2008). In the
absence of pathogens these GMS-proteins reside on different en-
domembrane compartment and protect them from the attack by the
other IRGs, the so-called GKS proteins. Certain pathogens (e.g. T. gondii,
Chlamydia and E. cuniculi), enter the host cell by non-phagocytic me-
chanisms. During the active invasion by T. gondii, most transmembrane
proteins of the host are excluded from the membrane of the forming PV
(Mordue et al., 1999). The GKS proteins identify such PVs as non-self
structures by the absence of GMS-proteins and are recruited to them
resulting in the disruption of the PV and the destruction of the parasite
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International Journal of Medical Microbiology 308 (2018) 237–245
Fig. 1. Structural comparison of mammalian dy-
namin superfamily proteins. A: Structural compar-
ison of hGBP1 and two conformations of atlastin1.
The large GTPase (LG)-domains are shown in orange,
the middle domains in green and the C-terminal
α12/13-domain in hGBP1 in blue. Note that the
structures of atlastin only contain the N-terminal
part of the protein up to the first transmembrane
region. B: Structural comparison of dynamin1, MxA
and Irga6. The LG-domains are shown in orange, the
bundle signal element in red and the stalk in green.
The membrane binding PH-domain of dynamin1 and
the L4-region of MxA are shown in viridian green
and the N-terminal helical region of Irga6 in blue-
grey, respectively. Note that the structure of dy-
namin1 does not contain the C-terminal proline-rich
(Haldar et al., 2013; Khaminets et al., 2010; Zhao et al., 2009). The
current model for the mechanism of IRGs is based on analogy to other
dynamin-related proteins and on the careful observation of the se-
quential order of pathogen entry, IRG recruitment to the PV membrane
(PVM), shape change and finally permeabilisation of the PV membrane.
A direct disruption of the PVM by IRG proteins, however, has so far not
been formally proven (Zhao et al., 2009). Over time it became clear that
the deletion of individual regulatory GMS proteins can disrupt this
mutual regulatory network. In the case of Irgm1-/- mice, the mis-
localisation of GKS-proteins leads to a loss of lysosomal acidity and
autophagic processing resulting in IFN-γ-induced lymphopenia (Feng
et al., 2008; King et al., 2011; Maric-Biresev et al., 2016; Traver et al.,
2011). For some pathogens this effect has been interpreted as loss of
resistance (Feng et al., 2008), but against others, such as Chlamydia,
Irgm1 does mediate cell autonomous resistance (Coers et al., 2011). In
mice the recruitment of IRGs and GBPs to PVs are linked to proteins
from the autophagy pathway (Al-Zeer et al., 2009; Haldar et al., 2014;
Khaminets et al., 2010) (Fig. 2). After relocalisation to the PV of T.
gondii and C. trachomatis, the GKS IRGs mediate recruitment of ubi-
quitin E3 ligases such as TRAF6 to the membrane. The ubiquitylation of
proteins on the PV, probably including the IRGs (Traver et al., 2011),
creates binding sites for the ubiquitin-binding protein p62/sequesto-
some (SQSTM1), which is itself a binding partner of mGBP2 (Haldar
et al., 2015) and mGBP1 (Kim et al., 2011). The recruitment of GBPs to
the T. gondii PV in murine cells requires GTP-binding but is independent
of GTP-hydrolysis and of isoprenylation (Kravets et al., 2012; Virreira
Winter et al., 2011). Accordingly, pathogen strains which express
virulence factors that disable the IRG system (Behnke et al., 2012;
Fentress et al., 2010; Fleckenstein et al., 2012; Steinfeldt et al., 2010)
also indirectly inactivate the GBPs and genetic deletions of GMS IRGs
resulting in mislocalisation of GKS IRGs also affect GBPs (Traver et al.,
2011). In human cells, IRGM is the only IRG isoform with an immune
function (Bekpen et al., 2009; Chauhan et al., 2016). The protein is
truncated compared to canonical IRG proteins and its expression if not
inducible by interferons. IRGM is involved in the regulation of autop-
hagy and polymorphisms in the gene are linked to Crohn’s disease and
the susceptibility to tuberculosis. So far, a connection to the function of
human GBPs has not been shown.
2. Structural and biochemical framework of GBPs
The domain architecture of GBPs consists of three parts (Fig. 1). The
N-terminal large GTPase (LG) domain harbours the GTP-binding site
which can be identified by three GTP-binding motifs: the phosphate
binding P-loop, the DxxG motif and the RD-motif, which deviates from
the N/TKxD motif in other GTP-binding proteins (Cheng et al., 1991;
Praefcke et al., 1999). Upon GTP-binding the GBPs dimerise, which
stimulates their enzymatic activity (Ghosh et al., 2006; Praefcke et al.,
2004; Prakash et al., 2000a; Prakash et al., 2000b; Wehner et al., 2012;
Wehner and Herrmann, 2010). The LG-domain is followed by a helical
G.J.K. Praefcke International Journal of Medical Microbiology 308 (2018) 237–245

Fig. 2. Model of the innate immunity function of GBPs. A: In human cells, cytoplasmic GBPs can either form large oligomers (1) or interact with membrane compartments such as the
plasma membrane, phagosomes (2) or the Golgi. GTP-hydrolysis maintains a dynamic exchange of monomers and may lead to membrane tethering (3). Certain viruses can be targeted by
GBPs (4). Chlamydia and Toxoplasma parasites residing in parasitophorous vacuoles (PV) are labelled with ubiquitin (5) and become surrounded by autophagosome-like structure positive
for LC3. The recruitment of GBPs to the PV does not occur in all cell types and seems not to be linked to the ubiquitylation of the PV. For intracellular gram-negative bacteria it is currently
unclear whether GBPs contribute to membrane destabilisation (6) or whether they are recruited later. In immune cells, GBPs activate inflammasomes in collaboration with LPS and other
bacterial patterns leading to pyroptosis (7). B: In murine cells, IRG proteins from the GKS group are recruited from their resting localisations, where they were in complex with GMS group
members (1). On the PV IRGs trigger ubiquitylation resulting in the binding of further E3 ligases (2). GBPs reside in large oligomeric structures (3) and on vesicles (4). From there they are
shuttled to the ubiquitinylated PV by the adaptor protein p62 (5). For the isoprenylated GBP this recruitment is counteracted by the interaction with RabGDIα. It has been suggested that
the formation of large complexes of IRGs and GBPs leads to the lysis of both the PV and the parasite membrane (6) resulting in death of the pathogen. The activation of the inflammasome
in murine immune cells by gram-negative bacteria seems to follow a similar mechanism as in human cells (7).

part which has been subdivided into the middle domain (MD) and the
C-terminal α12/13-domain. The middle domain consists of two three-
helix bundles which share one helix and extend away from the LG-
domain. The α12/13-domain runs along the middle domain and back to
the LG-domain, where these two domains make a few but mechan-
istically important interactions. During dimerisation of the LG-domains,
a conserved arginine residue in the phosphate-binding P-loop is posi-
tioned into the active site and stimulates the cleavage of the distal
phosphate group by stabilizing the transition state of the reaction. The
presence of a catalytic arginine in the P-loop and the RD-motif are also
found in atlastins (Daumke and Praefcke, 2016; Hu and Rapoport,
2016; McNew et al., 2013), but not in other dynamin-like proteins. A
peculiar feature of the GBPs, and hGBP1 in particular, is the ability to
cleave GTP to GDP and GMP (Abdullah et al., 2010; Ghosh et al., 2006;
Kravets et al., 2012; Kunzelmann et al., 2006; Neun et al., 1996;
Pandita et al., 2016; Schwemmle and Staeheli, 1994; Wehner and
Herrmann, 2010). The product ratio differs between 0% and > 85%
GMP for different isoforms and experimental conditions. So far, the
physiological function of this activity is not known. Based on gelfil-
tration studies we have postulated that hGBP1 dimers can assemble into
tetramers during GTP-hydrolysis or when bound to the transition state
analogue complex of GDP and aluminium fluoride (AlFx) (Prakash
et al., 2000a). Inhibition or reduction of dimer formation by mutations,
immobilisation or dilution to low concentrations leads to a strongly
reduced GTP turn-over and lower GMP production (Kunzelmann et al.,
2006; Praefcke et al., 2004; Syguda et al., 2012b). During GTP-hydro-
lysis the interaction between the LG- and the α12/13-domain is tran-
siently released in hGBP1 and the LG-domains of two hGBP1 molecules
enter into an interaction (Syguda et al., 2012a; Vöpel et al., 2009; Vöpel
et al., 2014). This conformational change leads to an increase of the
length of the hGBP1 complex from 120 Å to up to 240 Å (Vöpel et al.,
2014). Furthermore, a very recent study (Ince et al., 2017) has revealed
that these extended conformations of the dimers of both, hGBP1 and
hGBP5, with their increased hydrodynamic radius have falsely been
identified as tetramers in earlier studies (Pandita et al., 2016; Praefcke
et al., 2004; Prakash et al., 2000a; Shenoy et al., 2012; Vöpel et al.,
2010; Wehner and Herrmann, 2010). This underlines the importance of
shape-independent methods to determine the molecular weight of ex-
tended protein complexes in solution.
All biochemical studies described so far have been performed with
GBPs lacking the lipid modification. However, when expressed in
mammalian cells, three of the seven human GBPs and three of the
eleven murine GBPs, the isoforms GBP1, −2 and −5, are post-trans-
lationally modified by attachment of a C15 (farnesyl) or a C20 (ger-
anylgeranyl) isoprene moiety (Nantais et al., 1996; Olszewski et al.,
2006; Stickney and Buss, 2000). HGBP1 is isoprenylated with a C15
farnesyl lipid moiety and, in addition, possesses a polybasic region di-
rectly preceding the CaaX-motif of the protein, which could enhance
membrane affinity (Britzen-Laurent et al., 2010). Using a bacterial co-
expression system, we were able to produce farnesylated recombinant
hGBP1 in E. coli. In contrast to most other isoprenylated proteins, the
lipid modified form of hGBP1 did not stably interact with membranes in
cosedimentation assays except when bound to the GDP*AlFx transition
state analogue complex (Fres et al., 2010). In contrast, when the
binding of farnesylated hGBP1 to giant unilamellar vesicles (GUVs) was
directly imaged, the protein was also recruited to the membranes in the
presence of GTP or non-hydrolysable GTP-analogues (Shydlovskyi
et al., 2017). The interaction with membranes in the presence of GTP
was transient and hGBP1 dissociated from the membrane when the
substrate was consumed. Accordingly, a mutant of the catalytic arginine
displayed a stable interaction with membranes. In contrast to most
other dynamin-related proteins, membrane binding did not accelerate
the rate of GTP-hydrolysis. In the absence of membranes, however,
GTP-bound farnesylated hGBP1 formed ring-like polymers which could

239
G.J.K. Praefcke
further assemble into longer forms. Another mutant with a strongly
reduced GMP production showed no evidence for such a polymerisa-
tion. Stimulation of the GTP-hydrolysis resulted in the disassembly of
the polymers, which is reminiscent of the behaviour of Irga6 in the
presence of GTP (Ghosh et al., 2004; Pawlowski et al., 2011; Uthaiah
et al., 2003). While we found no evidence for a disruption of the
membrane of the GUVs, we observed a nucleotide dependent tethering
activity of farnesylated hGBP1 (Shydlovskyi et al., 2017). Although we
did not observe evidence for membrane fusion, these findings imply a
mechanistic similarity between GBPs and the atlastins and other dy-
namin-related proteins involved in membrane effusion events and add a
new aspect to the search for the function of GBPs on cellular mem-
branes.
3. Cellular localisation of GBPs
In interferon-induced cells and in the absence of pathogens, human
GBP1 displays a granular or vesicular cytosolic distribution (Britzen-
Laurent et al., 2010). In cell fractionations it was mainly found in the
cytosolic fraction despite its lipid modification (Nantais et al., 1996).
This can be explained by the transient membrane localisation and
polymerisation which we have described (Shydlovskyi et al., 2017).
Addition of AlFx induces the redistribution of IFN-γ induced hGBP1 to
the Golgi complex in a farnesylation-dependent manner (Modiano
et al., 2005). Tripal and colleagues studied the localisation of fluores-
cently labelled hGBP1 in IFN-γ treated human endothelial cells (Tripal
et al., 2007) confirming these results. Furthermore, the localisation of
hGBP2 followed the same pattern with additional nuclear staining
which was also seen for hGBP4, while hGBP3 was exclusively cyto-
plasmic and hGBP5 constitutively Golgi-associated. Not surprisingly,
localisation of the human CaaX-GBPs is prenylation dependent (Britzen-
Laurent et al., 2010) and the three prenylated isoforms are able to re-
cruit the non-prenylated isoforms hGBP3 and hGBP4 to their respective
localisation by heterodimerisation. Furthermore, a double mutation of
the contact between the LG-domain and the α12/13-domain (R227E/
K228E) results in enhanced membrane binding of hGBP1 and its re-
cruitment to the plasma membrane in vivo, while a protein mutant
impaired in nucleotide-binding is exclusively cytosolic (Britzen-Laurent
et al., 2010; Vöpel et al., 2010). Localisation of hGBP1 to the plasma
membrane has also been seen in intestinal epithelial cells, where it
localised at tight junctions to regulate the barrier function (Schnoor
et al., 2009). We have recently analysed the nature of the punctate
cytoplasmic hGBP1 structures in HeLa cells and found a partially co-
localisation with marker proteins of the endolysosomal pathway such as
Rab5, EEA1, late Rab7 and LAMP1 (Shydlovskyi et al., 2017). Fur-
thermore, endogenous IFN-γ-induced as well as recombinant mCherry-
labelled hGBP1 was recruited to latex bead phagosomes where it co-
localised with F-actin. Photobleaching experiments revealed that the
localisation to latex bead phagosomes and to the other cytosolic vesicle-
like structures is very dynamic.
In IFN-treated murine macrophages and fibroblasts, a similar
granular cytoplasmic distribution and partial localisation to vesicle-like
structures of heterogeneous sizes was found for the CaaX bearing iso-
form mGBP2 (Balasubramanian et al., 2011; Vestal et al., 2000), while
mGBP1 displayed a homogeneous cytoplasmic distribution. Also the
other murine GBPs displayed a discrete vesicle-like localisation which
has so far not been further analysed (Degrandi et al., 2007). Expression
of mutant versions of mGBP2 in IFN-γ-induced mGBP2-/- MEFs re-
vealed that GTP-binding and hydrolysis are important for the proper
localisation and also for the formation of multimers in the cellular
context (Kravets et al., 2012). These large complexes and also the ve-
sicle-like structures contain also heteromultimers of mGBP2 with other
murine GBPs and serve as cytoplasmic reservoir from where mGBPs are
recruited to parasitophorous vacuoles (Kravets et al., 2016).
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International Journal of Medical Microbiology 308 (2018) 237–245
4. Antiviral action of GBPs
As mentioned before, the antiviral activity of hGBP1 against VSV
and EMCV was the first evidence for a function of GBPs in pathogen
defence (Anderson et al., 1999). It should be noted that the reported
antiviral activity of GBPs is weak compared to that of Mx proteins for
their target viruses (Haller et al., 2015). So far, antiviral activity of
several GBPs against different viruses has been found and although
there is no general pattern, many of these viruses are positive sense
RNA (+RNA) viruses. This includes hepatitis C virus (HCV), a member
of the flaviviridae family which showed increased replication upon
knock-down of hGBP1 (Itsui et al., 2009). Overexpression of hGBP1
suppressed both replication and virus production of HCV, which is
mediated by the interaction of the LG-domain of hGBP1 with the finger
domain of non-structural protein 5B (NS5B) of HCV. Interestingly, the
interaction reduced the GTPase activity of hGBP1 and was blocked by a
mutation of the catalytic arginine residue in the P-loop and other re-
sidues in the LG-domain of hGBP1 important for the GTPase cycle (Itsui
et al., 2009; Pandita et al., 2016). These studies suggest that HCV-NS5B
interacts specifically with hGBP1 in its activated form. A very similar
scenario was also seen for classical swine fever virus (CSFV) (Li et al.,
2016). In the case of this flavivirus, the antiviral effect of porcine GBP1
(sGBP1) was also dependent on a functional LG-domain and binding of,
in this case, the NS5A protein to the LG-domain inhibited the GTPase
activity. Since also dengue virus, another human flavivirus, is inhibited
by hGBP1 (Pan et al., 2012) it seems likely that also other viruses from
that family might be targets of GBPs.
Several human GBPs have been found to interfere with influenza
virus (-ssRNA). Nordmann and colleagues reported that hGBP1, hGBP3
and especially a novel splice variant hGBP-3ΔC repressed the activity of
the viral polymerase complex (Nordmann et al., 2012). For this func-
tion, GTP-binding, but not hydrolysis was necessary. Binding of NS1
from influenza A virus (IAV) to hGBP1 resulted in a reduction in both
GTPase activity and the anti-IAV activity (Nordmann et al., 2012). For
mGBP5 an indirect effect on IAV was described by upregulating the
expression of IFN and other proinflammatory cytokines (Feng et al.,
2017). A direct interference with viral propagation was described for
hGBP5 and human immunodeficiency virus (HIV) by interfering with
processing and incorporation of the viral envelope glycoprotein (Krapp
et al., 2016). For this process, the isoprenylation-dependent Golgi lo-
calisation of hGBP5 was needed, but mutations impairing GTP-binding
and hydrolysis retained their activity against HIV. In pigs, poly-
morphisms in the GBP locus have been associated with the outcome of
porcine reproduction and respiratory syndrome virus (PRRSV,
+ssRNA) (Abella et al., 2016; Boddicker et al., 2012; Koltes et al.,
2015, 2015; Kommadath et al., 2017; Niu et al., 2016; Schroyen et al.,
2016). Animals homozygous for the unfavourable genotype pre-
dominantly produced a splice variant of hGBP5 coding for a C-terminal
truncation of 88 amino acids lacking the isoprenylation site (Koltes
et al., 2015; Schroyen et al., 2016). A similar splice variant was also
seen in human cutaneous T-cell lymphoma cells (Fellenberg et al.,
2004; Wehner and Herrmann, 2010). The only antiviral effect of a GBP
against a DNA virus has recently been shown for Kaposi’s sarcoma as-
sociated herpesvirus (KSHV) (Zou et al., 2017). In this study the authors
show that hGBP1 mediates the inhibition of the nuclear delivery of
KSHV virions by disrupting the formation of actin filaments. In this
context it is noteworthy to mention that upregulation of hGBP1 had
already been identified several years ago in vivo in endothelial cells
from Kaposi's sarcoma tissue (Guenzi et al., 2001). This study initiated
the investigation into the role of hGBP1 in the regulation of cell pro-
liferation in endothelial cells (Britzen-Laurent et al., 2016) which led to
the recent identification of hGBP1 as an actin binding protein (Ostler
et al., 2014).
Only two studies of antiviral activity of murine GBPs have so far
been published. Carter and colleagues found that mGBP2 inhibited the
replication of both VSV (-ssRNA) and EMCV (+ssRNA) (Carter et al.,
G.J.K. Praefcke
2005) but with different requirements. While a functional GTP binding
motif was required for inhibition of EMCV, it was dispensable for in-
hibition of VSV. Recently, the targeting of the viral replication centres
of murine norovirus (MNV, +ssRNA) by the microtubule-associated-
protein-1-light-chain-3 (LC3) followed by the recruitment of both IRGs
and GBPs has been described (Biering et al., 2017). The fact that both
LC3 and the IFN-inducible GTPases were necessary to inhibit MNV re-
plication in mice and in human cells is highly reminiscent of the de-
fence against T. gondii and C. trachomatis.
5. Antibacterial function
In recent years, the antimicrobial function of the murine GBPs has
been established by several groups. Specificity towards different pa-
thogens and the underlying molecular mechanisms have remained
controversial. The direct comparison of the different observations is
impeded by the use of different pathogens, host cell types and the use of
murine or human model systems.
In proteomic studies several murine GBPs were enriched on latex
bead phagosomes (Jutras et al., 2008; Trost et al., 2009). Tietzel and
colleagues provided the first evidence for an antibacterial activity of
human GBPs (Tietzel et al., 2009). They showed that ectopically ex-
pressed hGBP1 and hGBP2 are recruited to the inclusion membrane of
C. trachomatis in HeLa cells and inhibit growth of the bacteria, while
down regulation of hGBP1 and −2 alleviated the inhibitory effect of
interferon-γ. Recruitment of GBP1 to Chlamydia was also observed in
murine dendritic cells (Fiegl et al., 2013) and a human macrophage cell
line (Al-Zeer et al., 2013). The latter study also showed that the anti-
chlamydial action of hGBP1 and −2 involved changes in the autophagy
pathway leading to the rerouting of the typically non-fusogenic bac-
terial inclusions to lysosomal degradation. Recently, it has been shown
that also other murine GBPs are recruited to the PV of Chlamydia or T.
gondii to variable degrees (Lindenberg et al., 2017).
Kim et al. have reported that murine GBPs are critical for control of
Listeria monocytogenes and Mycobacteria bovis infections (Kim et al.,
2011). They proposed that mGBP1 and mGBP7 enhance the formation
of reactive oxygen by recruitment of NADPH oxidase to phagosomes
and induce autophagy via the interaction with p62/SQSTM1 and Atg4b.
The same group also reported that human and murine GBP5 promote
the assembly and activation of the NLRP3 inflammasome to control
Listeria infections (Shenoy et al., 2012). However, Yamamoto et al.
found no effect on the proliferation of Listeria in mice lacking a region
of chromosome 3 containing mGBP1, mGBP2, mGBP3, mGBP5 and
mGBP7 and also no effect on the recruitment of Atg8/LC3 and the
formation of reactive oxygen species (Yamamoto et al., 2012). Using
mice lacking mGBP2 Degrandi and colleagues also found no effect
against Listeria (Degrandi et al., 2013).
A series of studies has investigated the cooperation of GBPs and
IRGs in murine cells at the vacuoles of both T. gondii and Chlamydia. For
the IRGs it had already previously been shown that the loading on PVs
requires an intact autophagy system (Al-Zeer et al., 2009; Khaminets
et al., 2010; Zhao et al., 2008). Haldar and colleagues showed that the
localisation of murine GBPs depends on an intact IRG system (Haldar
et al., 2013). Subsequently, the same group showed that the docking of
IRGs and mGBPs to the PV depends on the expression of the autophagy
proteins ATG3 and ATG5, the E2 enzyme and a subunit of the E3
complex for the LC3 lipidation, respectively (Haldar et al., 2015). The
link between these events is the ubiquitylation of the PV triggered by
the recruitment of E3-ubiquitin ligases to the PV by IRG proteins
(Haldar et al., 2015). The following decoration of the PV with ubiquitin
then creates binding sites for p62/SQSTM1 which enhances the ubi-
quitylation of the PV and recruits GBPs. As a result, the IFN-mediated
and cell-autonomous resistance to C. trachomatis requires both families
of IFN-induced GTPases, the GBPs and the IRGs, and a functional au-
tophagy pathway. In human cells, which lack most IRG proteins, the
mechanism seems to be different. Ubiquitylation of PVs was observed
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International Journal of Medical Microbiology 308 (2018) 237–245
for C. muridarum infecting human endothelial cells followed by the
recruitment of p62, LC3 and hGBP1 and finally elimination of the in-
clusions. The human-specific pathogen C. trachomatis, however, was
resistant to ubiquitylation and elimination (Haldar et al., 2016). Fur-
thermore, the mechanisms by which cells respond to bacterial infec-
tions is also cell-type specific. In human macrophages, GBPs are not
recruited to C. muridarum inclusions (Finethy et al., 2015). Instead,
GBPs activate NLRP3- and AIM2-inflammasomes leading to the release
of inflammatory cytokines and pyroptosis of the macrophage. Similar
observations have also been made for GBP-dependent induction of
pyroptosis by infection of murine macrophages with Legionella pneu-
moniae (Pilla et al., 2014). This study also showed that the induction of
pyroptosis by the mGBPs on chromosome 3 depends on the presence of
LPS in the cytoplasm. Another study, however, indicated that these
GBPs are required upstream of LPS sensing (Meunier et al., 2014). In-
stead the authors found reduced levels of Salmonella in cells that are
positive for galectin-8, a marker for lysed vacuoles (Thurston et al.,
2012). Since galectin-8 is also a binding partner for the autophagy
adaptor protein NDP52, lysed vacuoles could be detected by the au-
tophagic machinery. The early role of GBPs in the activation of in-
flammatory signals prompted Meunier and colleagues to propose that
GBPs might promote the lysis of vacuoles thereby triggering the de-
tection of pathogen associated molecular patterns (PAMPs) (Meunier
et al., 2014). A following study suggested that GBPs might not only be
involved in the attack on the PV but also on the pathogen itself
(Meunier and Broz, 2015) since the number of lysed cells of Francisella
novicida in murine macrophages lacking the GBPs on chromosome 3
was higher than in macrophages from wild-type mice. In this context,
mGBP2 and mGBP5 also triggered pyroptosis but via the activation of
the AIM2-inflammasome. Recent evidence points towards a cooperative
function of GBPs and IRGs, however in a different order than previously
described for infections with C. trachomatis since Irgb10 was recruited
to F. novicida cells in a GBP-dependent manner (Man et al., 2016). The
subsequent lysis of the bacteria releases LPS and DNA which then ac-
tivate caspase-11, AIM2 and NLRP3. A very recent study indicates that
the effect of GBP against F. novicida is not only mediated by the AIM2
inflammasome. In interferon primed macrophages expressing high le-
vels of several GBPs the authors found that GBPs control also several
other apoptotic pathways and control killing of intracellular bacteria in
an inflammasome-independent manner (Wallet et al., 2017). The link
between galectins, GBPs and activation of inflammasomes was further
devised by several recent studies. In cells infected with Yersinia or Le-
gionella, the presence of bacterial secretion systems recruited galectin-3
to the PVs followed by delivery of GBP1 and GBP2 (Feeley et al., 2017).
Recently, it has been reported that GBPs can also mediate caspase-11-
dependent pyroptosis in response to LPS which is delivered to cells by
outer membrane vesicles of gram-negative bacteria (Finethy et al.,
2017). In zebrafish, GBP4 contains a C-terminal CARD domain. By di-
merisation with the CARD-domain of the inflammasome adaptor ASC it
mediates the clearance of Salmonella thyphimurium infections (Tyrkalska
et al., 2016). In case of cytosolic Shigella flexneri, the recruitment of
several human GBPs seems to be independent of galectin-8 (Wandel
et al., 2017). Instead, hGBP1 is recruited first and depending on its
catalytic activity. Other GBPs follow in an hierarchical order to form a
dense coat which reduces the actin-based motility and spreading of the
bacteria. This effect is counteracted by a bacterial E3 ubiquitin ligase
which targets hGBP1 and other GBPs for degradation.
6. Defence against protozoa
The analysis of the function of GBPs in innate immunity against
protozoan pathogens has been following the study of the IRG proteins
and has almost exclusively been focused on the apicomplexan parasites
T. gondii and Neospora caninum. Initially, only colocalisation of GBPs
with the PV of T. gondii was shown (Degrandi et al., 2007; Kravets et al.,
2012; Virreira Winter et al., 2011), which, in the case of the CaaX-GBPs,
G.J.K. Praefcke
is depended on the isoprenylation and a functional GTPase-domain. The
proof for an activity of GBPs against Toxoplasma infections was made
possible by the generation of mouse strains lacking either individual
GBPs (Degrandi et al., 2013; Selleck et al., 2013) or the GBP locus on
chromosome 3 containing mGBP1, mGBP2, mGBP3, mGBP5 and
mGBP7 (Yamamoto et al., 2012). The availability of these model sys-
tems has been a milestone in the study of GBP function. Since then, the
hierarchy of events at the PV of T. gondii in murine cells was elaborated
in parallel to the investigation of the activity against Chlamydia de-
scribed above. The possible sequence of events begins when the PV of
actively invading T. gondii cells is recognised by the GKS IRG proteins
due to the missing GMS IRGs (Hunn et al., 2008; Khaminets et al., 2010)
and also by proteins of the autophagy pathway leading to the deposi-
tion of LC3 on the PV (Choi et al., 2014). Several ubiquitin ligases such
as TRAF6 (Haldar et al., 2015) and TRIM21 (Foltz et al., 2017) are
recruited to the PV, which they decorate with ubiquitin, probably on
the IRGs (Traver et al., 2011). This initial ubiquitylation creates binding
site for the ubiquitin-binding adaptor protein p62 which escorts mGBP2
to the PV (Pilla-Moffett et al., 2016). Before their relocalisation to the
PV, GBPs reside in high molecular weight complexes and on vesicle-like
structures (Kravets et al., 2016). On the PV they from even larger
complexes and after disruption of the PV they relocalise further to the
parasite membrane. Upon disruption of its membrane the parasite dies,
followed by necrotic cell death of the host cell (Zhao et al., 2009). Also
as in the case of Chlamydia, intracellular T. gondii parasites trigger the
activation of the inflammasome in macrophages (Ewald et al., 2014;
Gorfu et al., 2014). It should be noted, however, that there are data
supporting alternative scenarios. Lee and colleagues, for example, come
to the conclusion that ubiquitylation of the PV and recruitment of p62
occur after the disruption of the PV by IFN-γ-inducible GTPases and are
needed to activate the acquired immune response (Lee et al., 2015). The
recruitment of GBP to the PV of T. gondii can also be negatively regu-
lated. Besides the indirect effect of mistargeting or inactivation of IRGs
by virulence factors (Alaganan et al., 2014; Behnke et al., 2012;
Etheridge et al., 2014; Fentress et al., 2010; Fleckenstein et al., 2012;
Hermanns et al., 2016; Howard et al., 2011; Niedelman et al., 2012;
Steinfeldt et al., 2010), T. gondii have also evolved effectors which di-
rectly target GBPs (Kim et al., 2016). Furthermore, also the host cells
control the membrane recruitment of mGBP2 by the interaction with
the isoprenyl-binding protein RabGDIα (Ohshima et al., 2015).
In human cells, it was believed that tryptophan-depletion by the
IFN-inducible indoleamine 2,3-dioxygenase (IDO) (Pfefferkorn, 1984;
Pfefferkorn et al., 1986) and the generation of nitric oxide by inducible
nitric oxide synthase (iNOS) (Fox et al., 2004; Scharton-Kersten et al.,
1997) would mediate the effect against T. gondii infections (Yarovinsky,
2014). Within the recent years it has become apparent that the de-
pendence of the innate immune response against T. gondii in human cell
shows a great variability between different cell-types and pathogen
strains. In IFN-stimulated fibroblasts T. gondii is regulated by cell death
independently of hGBP1 and hGBP2 (Niedelman et al., 2013), whereas
hGBP1 restricted replication of T. gondii in epithelial cells but did not
localise to the PV (Johnston et al., 2016). Nevertheless, vacuoles of
certain T. gondii strains in HUVECs were targeted by Lys63-linked
ubiquitin chains and recruited both p62 and NDP52, but not the au-
tophagy proteins Atg16L1, GABARAP and LC3B. The ubiquitylated
vacuoles were then destroyed in LAMP1-positive endo-lysosomal com-
partments. In contrast to that, HeLa cells, which are a human epithelial
cancer cell line, restricted T. gondii by non-canonical autophagy
(Clough and Frickel, 2017). These results from endothelial cells are in
contrast to results obtained with the near haploid HAP1 cells, which are
derived from a leukaemia cell line. In these cells, GBPs did localise to
the PV of T. gondii but deletion of all seven human GBPs had no effect
on the IFN-γ mediated suppression of T. gondii replication (Ohshima
et al., 2014). Again a different setting and outcome was reported for of
human mesenchymal stromal cells. Depending on the T. gondii strain
between 10% and 20% of the vacuoles were recruiting hGBP1 and
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International Journal of Medical Microbiology 308 (2018) 237–245
knock-down of hGBP1 reduced the IFN-γ dependent resistance (Qin
et al., 2017).
7. Outlook
Research on GBP and IRG function has developed from the begin-
ning by mutual stimulation where similar questions were asked and
addressed with similar experimental approaches. Now it has become
clear that the two IFN-inducible GTPase families are much more closely
interconnected than their primary sequence has indicated. In both fa-
milies, subsets of several isoforms act at distinct cellular compartments
and are recruited to their points of action in a controlled and hier-
archical manner. Still the question remains if they all share the same
fundamental mechanism to control intracellular pathogens. The bio-
chemical and structural analysis of the GBPs and IRGs has provided a
deep understanding of the conformational changes during GTP-binding
and hydrolysis. However, we should remember that we do actually only
have detailed information about some of the family members and re-
cent results show that posttranslational modifications or the presence of
another isoforms may have dramatic effects on the biochemical activ-
ities. Furthermore, we should be cautious with mechanistic models
which are based on circumstantial evidence and not direct proof. On the
positive side is the availability of novel tools and models in which we
can test our hypotheses. The CRISPR/Cas technology could be used to
introduce site-specific mutations into the genes of individual GBPs to
address the physiological significance of GMP-production, the inter-
and intramolecular interactions and the heterooligomerisation without
interfering with their endogenous IFN-stimulated expression. It will
also be interesting to find out whether the defence of viruses involves
similar pathways as the defence against bacteria and protozoa and
whether the functions of GBPs in cell proliferation, motility and tumour
suppression (Britzen-Laurent et al., 2016) are independent from the role
in innate immunity. The recent report that the processing and secretion
of hGBP1 depends on inflammatory caspase activity is a hint that these
“moonlighting” functions may actually be functionally linked
(Naschberger et al., 2017).
Funding
This work was funded by the German Research Foundation, Priority
Programme 1580, Intracellular Compartments as Places of Pathogen-
Host Interactions, grant Pr 1090/3-1.
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