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TRENDS in Microbiology Vol.10 No.4 April 2002

193

Intracellular vs extracellular
recognition of pathogens common
concepts in mammals and flies
Stephen E. Girardin, Philippe J. Sansonetti and Dana J. Philpott
There are common themes in innate immune defense systems across the
animal and plant kingdoms. Pathogen recognition is commonly based on the
identification of microbial molecular patterns by defined receptors and the
subsequent activation of signaling pathways that initiate a defense response
to fend off the invading microorganism. The existence of mammalian Toll-like
receptors (TLRs) and the recent identification of two mammalian nucleotidebinding site leucine-rich repeat (NBS-LRR) proteins (NOD1 and NOD2) as
intracellular sensors of bacterial products bring new insights into the
possibility of extracellular versus intracellular pathogen recognition and signal
transduction depending on the nature of the infection. The homology between
TLRs and the Toll system in Drosophila suggests that conserved defense
mechanisms are likely to be shared by diverse organisms.
Published online: 11 March 2002

Stephen E. Girardin
Philippe J. Sansonetti
Dana J. Philpott*
Pathognie Microbienne
Molculaire and
Immunit Inne et
Signalisation, Institut
Pasteur, 28 rue du Dr Roux,
Paris 75724 Cdex 15,
France.
*e-mail:
philpott@pasteur.fr

In vertebrates, the immune system achieves its goal

through the involvement of both innate and adaptive
immunity. The combination of these two systems
defends the organism against infection by pathogens.
Although both systems rely on the recognition of nonselfmolecular patterns by specialized receptors, the
adaptive and innate immune systems use distinct
mechanisms to achieve the final elimination of the
pathogens that carry these patterns. The adaptive
immune system depends on somatic gene
rearrangements for the generation of millions of
antigen receptors with random specificities that are
expressed on two cell populations of the hematopoietic
lineage, the B and T cells. By contrast, the innate
immune system uses a set of germline-encoded
receptors expressed by a broad range of cell populations,
such as macrophages, neutrophils and epithelial cells,
that are likely to come into contact with pathogens.
Innate immunity provides a first line of defense
against pathogens and can be activated rapidly
following infection. Recent advances have shown that
innate immunity also plays an instructive role in the
adaptive response to antigens by modulating the
threshold of activation of adaptive antigen-recognition
receptors and by inducing key co-stimulatory
molecules and cytokines [1,2]. Comparison of the
mechanisms as well as the molecular components
involved in innate and adaptive immunity has
revealed that innate immunity preceded adaptive
immunity in the evolution of the vertebrates [2,3]. For
this reason, Medzithov and Janeway have described
innate immunity as an ancient system of host
http://tim.trends.com

defense [2]. Accordingly, analysis of the innate defense

mechanisms of vertebrates, insects, worms and even
plants has revealed some striking elements of
functional and structural conservation among
lineages that diverged more than one billion years ago.
The activation of the innate immune system relies
on the recognition of pathogen-associated molecular
patterns (PAMPs) by specific pattern-recognition
receptors (PRRs). Owing to the inability to generate
an infinite range of PRRs, evolution has probably
favored the development of PRRs that recognize a
limited set of PAMPs corresponding to relatively
invariant structures of microorganisms, such
as lipopolysaccharide (LPS) or peptidoglycan.
A considerable advance in this field is the
determination of the SptzleTollCactus signaling
cascade that is triggered following exposure of
Drosophila to fungal infection [4]. Although the
transmembrane receptor Toll is not itself a PRR, it is a
key signaling molecule required for the production of
anti-fungal peptides following microbial infection of
flies. Several elements of the Toll pathway identified
in insects have now been characterized in mammals.
A large family of Toll-like receptors (TLRs), expressed
at the plasma membrane by various cell populations,
is responsible for the recognition of several PAMPs
[5,6]. Although evidence of direct PAMP recognition
by TLRs is still lacking, several reports support the
idea that TLRs are indeed true PRRs [79].
The position of TLRs on the cell surface indicates
that these receptors can recognize PAMPs presented
extracellularly. However, can they also distinguish
between intracellular and extracellular pathogens?
Recently, NOD1 and NOD2, two cytoplasmic proteins
that contain a nucleotide-binding site (NBS) and
a leucine-rich repeat (LRR) domain, have been
implicated in innate immune defense [1012]. Their
role in the recognition of LPS [12,13] and intracellular
pathogens [14] has recently been discovered. These
findings suggest that the innate immune system
of vertebrates might use a family of membrane
receptors involved in outside-in signaling (the TLRs)
and another set of cytoplasmic proteins that could be
responsible for inside-in signaling (the NBS-LRRs)
following PAMP recognition.
Plants can also mount a defensive response when
attacked by pathogens. This response is brought about

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by either non-specific elicitors, which can affect many

different plant species, or specific gene products from
a given pathogen, which interact with species-specific
receptors in the plant as defined by the so-called
gene-for-gene concept of plant resistance [15]. These
recognition proteins in plants share similarities
with Drosophila Tolls, and mammalian TLRs and
NBS-LRR proteins, reflecting the conservation of
strategies used to combat pathogens in the animal and
plant kingdoms. In this review, we will focus on new
developments in the field of pathogen recognition and
signal transduction in the context of outside-in versus
inside-in systems of recognition.
Outside-in signaling
The crucial role of Toll and the TLRs

The product of the toll gene is a transmembrane

receptor with an LRR ectodomain that was originally
identified in the fruit fly for its crucial role in dorsalventral patterning during development [16]. More
recently, toll was shown to control the immune
response of the adult fly to fungal and Gram-positive
bacterial infections [4,17]. The Toll protein is not itself
a PRR as, rather than recognizing a microorganismspecific product, it binds the secreted host protein
Sptzle. Sptzle is a member of the cysteine-knot
superfamily that includes growth factors and
hormones such as nerve growth factor (NGF),
transforming growth factor (TGF), platelet-derived
growth factor BB (PDGF-BB) and human chorionic
gonadotropin (hGC) [18]. These proteins each form a
unique 3-D cysteine-knot fold and dimerize to bind to
their cognate receptors. To be active, Sptzle requires
proteolytic processing by extracellular serine
proteases. However, the processing of Sptzle is
negatively regulated by a protease inhibitor of the
serpin family called Necrotic. In fact, flies deficient
in necrotic constitutively express the antifungal
gene drosomycin, unless they are also deficient in
either sptzle or toll [19]. Therefore, it appears
that, following recognition of a fungal PAMP in the
hemolymph by an unknown PRR, Toll signaling can
proceed via a mechanism involving the inactivation of
Necrotic to give rise to an active Sptzle molecule.
Recently, a bone fide PRR was described in
Drosophila that is involved in recognition of Grampositive bacteria. Using a genetic screen, the gene
semmelweiss (seml), which encodes a peptidoglycanrecognition protein, was found to be involved in
resistance to Gram-positive bacterial infection [20].
The antifungal response in seml mutant flies is intact,
suggesting the existence of a distinct PRR that is
involved in the recognition of fungal PAMPs.
Following Sptzle binding, the Toll receptor
signals through the well-characterized
TubePelleCactusDorsal/Dif cassette to induce the
synthesis of antifungal peptides [21]. This signaling
pathway presents striking homologies with the
interleukin 1 (IL-1) pathway, which leads to the
activation of nuclear factor B (NF-B) in mammals [4].
http://tim.trends.com

Accordingly, Toll and the IL-1 receptor (IL-1R) share

a similar intracytoplasmic TollIL-1R (TIR) domain.
In addition, Tube, Pelle, Dorsal/Dif and Cactus are
functionally homologous to myeloid differentiation
factor 88 (MyD88), IL-1R-associated kinase (IRAK),
NF-B and inhibitor of B (IB), respectively [21]. The
identification of TLRs in mammals provides evidence
for general conservation between the Drosophila
Toll signaling pathway and the TLR/IL-1 pathway
in mammals (Fig. 1) [22]. Ten TLRs have been
characterized so far in mammals, and they are likely
to possess different specificities for PAMPs (Table 1).
The proximal events following activation of the
Toll and TLR/IL-1 pathways in Drosophila and
mammals involve similar mechanisms of protein
recruitment and subsequent activation. In
mammalian cells, the first intracellular event
following LPS or IL-1 stimulation is the recruitment
of the adaptor protein MyD88 to the receptor complex
[23]. MyD88 interacts with the cytoplasmic domain
of the TLR and IL-1R through homotypic TIR
interactions. Recently, a MyD88-like protein, Mal,
also known as TIRAP, was cloned and shown to be
involved in signaling downstream of TLR4 [24,25].
Although dominant-negative forms of Mal block
NF-B activation through LPS/TLR4, IL-1 signaling
to NF-B is not affected. This is the first report of a
TLR4-specific signaling protein independent of the
IL-1 pathway, and possibly explains why LPS-treated
macrophages from MyD88-deficient mice still
activate NF-B, albeit with slower kinetics.
Toll signaling in Drosophila also requires an
adaptor-type molecule to link receptor events to
downstream kinases. The protein Tube appears to be
an adaptor protein analogous to MyD88 as genetic
studies place Tube between Toll and the downstream
kinase, Pelle [26]. Tube contains a death domain but,
unlike MyD88, lacks a TIR domain. Thus, how Tube
interacts with the Toll receptor remains unknown.
Recently, a MyD88 homolog, DmMyD88, was
identified in Drosophila [27,28]. DmMyD88-deficient
flies are highly susceptible to fungal and Grampositive bacterial infections but responses to Gramnegative bacterial infections are unaffected. Genetic
studies place DmMyD88 in the Toll pathway
upstream of Tube and Pelle, suggesting that a
straightforward TollDmMyD88TubePelle
pathway is involved in resistance to fungal or Grampositive bacterial infections [28]. However, the
molecular mechanisms of the interactions between
these effector proteins are still unclear.
MyD88 recruits the kinase IRAK to the
TLR/IL-1R complex [29]. Likewise, Mal recruits
IRAK2 to the activated TLR4 complex [25]. IRAK is
autophosphorylated following activation and this
might facilitate its release from the receptor complex
and/or its association with and subsequent activation
of TRAF6 [30].
The functional homolog of IRAK in Drosophila,
Pelle, is recruited to the membrane following

Review

TRENDS in Microbiology Vol.10 No.4 April 2002

Insects

PAMPs:
LPS, PG, Flagellin...
LRR

LPS
Toll
(2)

LPS
receptor
?
IMD

TLRs
(2)

Relish

TRAF6
dTRAF2
Phosphorylation
Cactus
IKK complex
kinase?
IB
Cactus
Dif,
Dorsal

RICK

LRR
LRR

RPP5, L6,
N,RPS2...

NBS-LRRs
(3)

NBS

Intracellular
Avr

NOD1, NOD2...

CARD

Cleavage

IRAKs

NBS

Pelle

DD

dTAK1

MyD88

Pto, Pti...
LRR
LRR

DD

DmIKK
complex
(Kenny/Ird5)

LPS;
other PAMPs?

TIR

CARD

DD

TIR
Tube

Dredd

Plants

Mammals

Sptzle
(1) IMD
pathway

195

X: TIR, LZ

NF-B
Immune response
genes
TRENDS in Microbiology

Fig. 1. Strategies of innate immune defense in insects, mammals and plants. (1) Activation of the IMD pathway in Drosophila by Gram-negative
bacteria leads to the activation of the NF-B family member Relish. This pathway involves IMD, dTAK1, the caspase Dredd, which is homologous
to mammalian caspase-8, and Ird5 and Kenny, two members of the Drosophila IKK complex. Relish is activated by the combination of a
phosphorylation event (via Ird5 and Kenny) and a proteolytic cleavage (via Dredd). The IMD pathway shares some similarity with the mammalian
TNF- pathway. (2) Extracellular recognition of pathogens in Drosophila and mammals relies on homologous Toll/TLR signaling to activate the
NF-B family of transcription factors. In Drosophila, Sptzle binds to the Toll receptor, resulting in signal transduction through the wellcharacterized TubePelleCactusDorsal/Dif cassette to induce the synthesis of antifungal peptides. This pathway has homologies with the IL-1
pathway in mammals, which activates NF-B, and Tube, Pelle, Dorsal/Dif and Cactus are functionally homologous to MyD88, IRAK, NF-B and IB,
respectively. (3) Intracellular recognition of pathogens in mammals and plants using proteins sharing conserved NBS-LRR domains. In NOD1, both
the amino-terminal CARD domain and the NBS domains are necessary for NF-B activation; the carboxy-terminal LRR domain is likely to play a
negative regulatory role. Following oligomerization of either NOD1 or NOD2, RICK is recruited to these molecules through homophilic CARDCARD
interactions. The interaction between NOD1 and RICK recruits the IKK complex, which phosphorylates IB, thus promoting the activation of NF-B.
In plants, specific Avrs from intracellular pathogens are recognized by resistance (R) proteins, which are a class of plant NBS-LRR proteins. Like
mammalian NOD proteins, plant NBS-LRR proteins possess an amino-terminal LRR domain and a central NBS whereas the carboxyl terminus
can be other proteinprotein interaction domains such as TIR or LZ domains. Activation of these proteins by plant pathogens results in the
hypersensitive response in the susceptible plant. Abbreviations: Avr, avirulence proteins; CARD, caspase recruitment domain; DD, death
domain; IB, inhibitor of B; IKK, IB kinase; IL, interleukin; IMD, immune deficiency; IRAK, IL-1R-associated kinase; K, kinase domain; LPS,
lipopolysaccharide; LRR, leucine-rich repeat; LZ, leucine zipper; NBS, nucleotide-binding site; NF-B, nuclear factor B; PAMP, pathogen-associated
molecular pattern; PG, peptidoglycan; TAK1, transforming growth factor--activated protein kinase 1; TIR, Toll/IL-1 receptor domain; TLRs, Toll-like
receptors; TRAF, TNF-receptor-associated factor.

activation of the Toll pathway. Like IRAK, Pelle is also

autophosphorylated, a step that precedes its release
from the receptor complex. Recent evidence has shown
that Pelle then interacts with and phosphorylates a
Drosophila TRAF protein, dTRAF2, which is the
homolog of mammalian TRAF6 [31]. Although genetic
evidence is lacking, these findings suggest that
dTRAF2 is the functional target of Pelle and could be a
link in the pathway to the activation of Dorsal [31].
The next steps in the pathway downstream of the
TLRs and IL-1R in the mammalian system is the
TRAF6-dependent activation of the IB kinase (IKK)
http://tim.trends.com

complex. This complex, which consists of two kinases,

IKK and IKK, and a regulatory subunit, NEMO (or
IKK), is responsible for the serine phosphorylation
of the inhibitory molecules of NF-B, the IBs. Once
phosphorylated, IB proteins are polyubiquitinated
and targeted for degradation by the proteosome thus
releasing NF-B and allowing this transcription
factor to translocate to the nucleus and mediate the
expression of several genes, many of which are
important for the inflammatory response. IKK
activation by TRAF6 relies on two independent
activities in the complex. One function of TRAF6 is to

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Table 1. Various products recognized by the family

of mammalian Toll-like receptors
TLR

Product recognized

TLR1
TLR2

?
PG, BLP, MALP2, Zymosan
Leptospira interrogans LPS
dsRNA
LPS, LTA, viral protein, Taxol, HSP60
Flagellin
PG (with TLR2), MALP2 (with TLR2),
Zymosan (with TLR2)
Imidazoquinoline compounds
?
CpG DNA
?

TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
TLR10

Refs
[5156]
[57]
[51,5862]
[63]
[54,55]
[64]
[65]

Abbreviations: BLP, bacterial lipoproteins; HSP, heat-shock protein;

LPS, lipopolysaccharide, LTA, lipoteichoic acid; MALP2, mycoplasma
lipopeptide-2; PG, peptidoglycan; TLR, Toll-like receptor.

recruit ubiquitin-conjugating enzymes that mediate

the assembly of polyubiquitin chains onto the IKK
complex. Ubiquitination in this case does not involve
proteolysis of the targeted protein but appears to
regulate its kinase activity [32]. The other function of
TRAF6 is to recruit and activate the mitogen-activated
protein kinase kinase kinase (MAP3K) TAK1 and its
associated proteins, TAB1 and TAB2. Once activated,
TAK1 can directly phosphorylate and activate IKK as
well as MKK6, leading to the induction of both NF-B
and c-Jun amino-terminal kinase (JNK) [33].
In Drosophila, the steps between Pelle and/or
dTRAF2 activation and the phosphorylation and/or
degradation of Cactus, the fly IB homolog, are
still unknown. Similar to mammals, the ultimate
step in the activation of Dorsal and Dif is the
phosphorylation and degradation of Cactus, which
then releases these transcription factors. The kinase
upstream of Cactus remains to be identified.
The IMD pathway in Drosophila

Whereas LPS signaling is mediated by TLR4 in

mammals, the signaling pathway induced following
infection by Gram-negative bacteria in Drosophila is
independent of any known member of the Toll family
and requires an as-yet-unidentified LPS receptor.
Although several receptors closely related to Toll
are found in the Drosophila genome [34], it remains
unclear whether the LPS receptor belongs to this
family [21]. Flies that are mutant for the imd
(immune deficiency) gene display a compromised
response to Gram-negative bacteria but a normal
immunity to fungi, thus demonstrating that the Toll
and IMD pathways account for two independent
defense mechanisms triggered by distinct
microorganisms. The recent cloning of imd revealed
that this protein contains a death domain and is
homologous to the mammalian receptor-interacting
protein (RIP) [35]. The nature of the link between
IMD and the as-yet-unidentified LPS receptor
remains to be characterized.
http://tim.trends.com

Besides imd, genetic evidence has shown that this

pathway involves dTAK, the Drosophila homolog of
TGF-activated kinase 1 (TAK1), the caspase Dredd,
two members of the Drosophila IKK complex, Ird5 and
Kenny, and Relish, another homolog of NF-B found in
the fruit fly [21,36]. The protein encoded by dTAK1
functions downstream of IMD and regulates the
activation of Relish through the Ird5/Kenny IKK
complex [37]. In addition to this so far linear
IMDdTAK1DmIKKRelish pathway, it has recently
been proposed that the caspase Dredd, which is
homologous to mammalian caspase-8, is required to
resist Gram-negative infection through the proteolytic
activation of Relish [38,39]. Therefore, it appears that
in the IMD pathway, the NF-B homolog Relish is
activated by the combination of a phosphorylation
event (via Ird5/Kenny) and a proteolytic cleavage (via
Dredd). Together, the initial results obtained for the
Drosophila IMD pathway suggest that it shares some
similarity with the mammalian tumor necrosis factor
(TNF)- pathway. Indeed, this pathway involves a
death domain-containing molecule related to RIP
(IMD), a Drosophila homolog of TAK1 (dTAK1) that is
known to be activated by TNF- in mammalian cells,
and a caspase related to the mammalian caspase-8
(Dredd). In addition, the identification of a Drosophila
homolog of Fas-associated death domain (dFADD)
that interacts with Dredd [40] suggests that this death
domain-containing molecule might play a role in fruit
fly resistance to Gram-negative bacterial infection.
A conserved framework of outside-in signaling
following infection by pathogens is found in mammals
and Drosophila through the activation of the Toll/TLR
signaling cascade. The transcription factor NF-B
plays a pivotal role in regulating the synthesis of
antimicrobial peptides or proinflammatory factors in
insects and mammals, respectively. It is intriguing
that the fruit fly has evolved two parallel systems
of intracellular cell signaling, the Toll and IMD
pathways, that are triggered selectively depending
upon the type of pathogen presented extracellularly,
whereas in mammals the TLR/IL-1 signaling
pathway is so far thought to be activated downstream
of each TLR.
Inside-in signaling: a role for NBS-LRR molecules

Plant resistance to pathogen aggression is fulfilled

by a large family of genes called resistance genes
or R-genes. The majority of these genes encode
cytoplasmic resistance proteins although some
encode transmembrane receptors. R proteins fall into
five classes, based on the motifs they contain [41,42].
The largest family described so far groups proteins
sharing NBS and LRR domains. NBS-LRR resistance
proteins can be found in various plants such as
tomato (Prf), Arabidopsis (RPS2, RPM1, RPP5) and
Tobacco (N). Strikingly, some of these NBS-LRR
resistance proteins encode amino-terminal domains
with homology to the TIR domain found in Toll and
the IL-1R, suggesting conservation of a common

Review

Fig. 2. Schematic
representation of the
eight NBS-LRR proteins
described in humans so
far. Abbreviations: BIR,
baculovirus inhibitory
repeat; CARD, caspase
recruitment domain; LRR,
leucine-rich repeat; NBS,
nucleotide-binding site.

TRENDS in Microbiology Vol.10 No.4 April 2002

197

953
NOD1

CARD

NBS

LRR(10)
1024

CARD12

CARD

NOD2

CARD

NBS

LRR(13)
1040

CARD

NBS

LRR(10)
1473

NALP1

Pyrin

NBS

LRR(12)

CARD
1062

NALP2

Pyrin

NBS

LRR(12)

1403
NAIP

BIR

BIR

BIR

NBS

LRR(6)

920
Cryopyrin

Pyrin

NBS

LRR(6)
TRENDS in Microbiology

means of pathogen detection between animal and

plant kingdoms (Fig. 1).
A new family of NBS-LRR molecules has recently
been characterized in mammals that shares
structural similarity with plant NBS-LRRcontaining proteins (Fig. 2). The homology between
these molecules and plant disease resistance
proteins, together with their ability to induce the
NF-B pathway when overexpressed (see later),
suggests that this class of molecules could represent
intracellular detectors of PAMPs [10,11,43].
Recently, NOD2 and Cryopyrin, two mammalian
proteins containing an NBS-LRR motif, have been
implicated in inflammation-related diseases
(Table 2) [12,4446]. Based on these observations,
we propose that these two domains together,
NBS-LRR, constitute the crucial structural domains

involved in mediating pathogen recognition

and/or inflammation.
Although little is known about the signaling
pathways induced downstream of plant NBS-LRRs,
recent advances have initiated studies of the role of
mammalian NBS-LRRs in intracellular signaling
(Fig. 1). The transient overexpression of NOD1 or
NOD2 is sufficient to induce the activation of NF-B
(Table 2) [10,11,43]. Functional domain analysis of
NOD1 shows that both the amino-terminal caspase
recruitment domain (CARD) and the NBS domains
are necessary for NF-B induction by NOD1 [11]. By
contrast, the carboxy-terminal LRR domain is likely
to play a negative regulatory role in the pathway, as
deletion of this domain results in enhanced activation
of NF-B [11]. Similar results have been described for
NOD2, except that in this case, both amino-terminal
a

Table 2. The mammalian family of NBS-LRR proteins

Protein

Other names

Role in apoptosis

Activation of
NF-
B

Putative role in inflammation/innate immunity

NOD1

CARD4

CARD12
NOD2

CLANA/IPAF
CARD15

+
+

NALP1
NALP2
NAIP
Cryopyrin

CARD7/DEFCAP/NAC
NBS1

+
?
?
?

?
?
?
?

LPS signaling, involved in the response to

Shigella flexneri
?
Mutated in Crohns disease, involved in Blau
syndrome, LPS signaling
?
?
?
Mutated in cold autoinflammatory syndrome
and in MuckleWells syndrome

Abbreviations: CARD, caspase recruitment domain; DEFCAP, death effector filament-forming ced-4-like apoptosis protein; IPAF, ICE-protease
activating factor; LPS, lipopolysaccharide; LRR, leucine-rich repeat; NAC, NB domain and CARD; NBS, nucleotide-binding site; NF-B,
nuclear factor B.

http://tim.trends.com

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Questions for future research

What are the ligands recognized by TLR1, TLR8 and TLR10?

What is the LPS receptor in the Drosophila IMD pathway?
Is there functional cooperativity among various TLRs for ligand recognition?
Is there an interaction between the TLR and NBS-LRR pathways following PAMP
recognition?
Do the various NBS-LRR proteins recognize distinct PAMPs? To what extent does
this family represent the intracellular counterpart of TLRs?
Is there a role for TLRs and NBS-LRRs in apoptotic responses following PAMP
recognition?
What are the co-factors involved in PAMP recognition and signal transduction by
NBS-LRR proteins?
What is the role of NBS-LRR proteins in pathogen-associated inflammation?
What is the molecular mechanism that implicates NOD2 mutations in the
pathogenesis of Crohns disease and Blau syndrome?

Acknowledgements
We would like to thank
Maria Mavris and Isabel
Fernandez for critical
reading of the manuscript
and Bruno Lemaitre and
Franois Leulier for
helpful discussion.

CARD domains together with the NBS domain are

necessary to mediate NF-B activation [43]. The
activation of NF-B by NOD1 or NOD2 requires the
NBS-dependent oligomerization of the molecule
through a mechanism similar to that described
for Apaf-1, a pro-apoptotic mammalian protein
[11,43,47]. The mechanism by which the LRR domain
of NOD1 or NOD2 inhibits downstream signaling
to NF-B is still unclear. It is possible that the
interaction of the LRR domain with an as-yetunidentified ligand is necessary to unfold the
molecule and to stabilize it in an active conformation.
Following oligomerization of either NOD1 or NOD2,
RICK (also known as RIP2 or CARDIAK) is recruited
to these molecules through homophilic CARDCARD
interactions [43,48]. The interaction between NOD1
and RICK then induces the recruitment of the IKK
complex, which phosphorylates IB, thus promoting
the activation of NF-B [48]. The determination of
this novel NOD1RICKIKK signaling pathway
upstream of NF-B was based on overexpression
studies and no physiological stimulus was associated
with this pathway. Recently, several reports have
suggested that the NOD1 and NOD2 signaling
pathway is likely to be activated following PAMP
recognition. Firstly, Inohara and co-workers
have shown that NOD1 confers responsiveness to
bacterial LPS [13]. However, the mechanism of such
responsiveness remains unclear as the LPS was
delivered extracellularly. Nevertheless, the detection
of an interaction between NOD1 and LPS suggests
that NOD1 could play a role in LPS sensing [13].
Accordingly, we have presented evidence that NOD1
is required to mediate NF-B activation following
infection of epithelial cells by an invasive strain of
the Gram-negative pathogen Shigella flexneri [14].
Overexpression of a mutated form of NOD1 deleted
for its CARD domain acts as a dominant-negative for
S. flexneri-induced activation of NF-B. In addition,
infection by this invasive pathogen is sufficient to
induce NOD1 oligomerization and the transient
recruitment of RICK/IKK to the NOD1 oligomers,
thus providing the first example of a physiological
activation of the NOD1RICKIKK pathway [14].
In addition to these observations, the recent
http://tim.trends.com

identification of NOD2 as the first susceptibility

gene for Crohns disease can be interpreted as a
compromised response of the individuals carrying
these mutations to the bacterial environment, thus
leading to the dysregulated onset of inflammation
[12,45]. However, the link between these genetic data
and the functional role of NOD2 in monocytes remain
elusive and awaits further investigation.
The NBS-LRR family of mammalian proteins
could comprise >30 members based on the human
genome sequence, most of which remain to be
characterized (Fig. 2). An exciting challenge for the
coming years will be to determine if these genes are
indeed general regulators of inflammation and
further, if other mammalian NBS-LRR proteins
apart from NOD1 and NOD2, act as intracellular
sensors for PAMPs.
Conclusions and perspectives

Conserved mechanisms of pathogen detection and

cell signaling exist throughout the animal and plant
kingdoms that rely on the expression of sets of extraand intracellular receptors. However, the general
organization of flies, plants or mammals could have
dictated the distinct evolution of these recognition
systems depending on the context of interaction
between host and pathogen. For example, in
Drosophila, the physical separation between the
hemolymph, where the recognition of the fungus occurs,
and the cells of the fat body, which are committed to
respond to infection with the production of antifungal
peptides, could have contributed to the selection of a
unique defense strategy through amplification using a
proteolytic cascade of diffusable factors.
An important question for the future is to
understand better the different strategies used to
recognize pathogens by the different innate immune
systems. For instance, in mammals, the TLRs might
specifically recognize a limited set of PAMPs, whereas
in insects there is no evidence so far implicating any
Toll protein in direct PAMP recognition. In plants, some
evidence exists for the direct recognition of PAMPs
[49,50], however, most of the R proteins, rather than
recognizing a general molecular pattern associated
with a microorganism, recognize distinct proteins from
pathogens specific to that particular plant species.
With the recent characterization of intracellular
PAMP detection by mammalian NBS-LRR proteins
(NOD1 and NOD2), the possibility has emerged
that this family might represent the intracellular
counterpart of extracellular pathogen detection
mediated by TLRs. Are TLRs and NBS-LRR proteins
selectively expressed in different cell types depending
on the environment and the pathogens that are
encountered? What is the relative role of TLRs
versus NBS-LRR proteins in the mammalian innate
immune response? Further characterization of the
mammalian NBS-LRR family is necessary to define
clearly the role of these proteins in inflammation
and/or infectious processes.

Review

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