You are on page 1of 7


TRENDS in Microbiology Vol.10 No.4 April 2002


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*
Pathogénie Microbienne
Moléculaire and
Immunité Innée et
Signalisation, Institut
Pasteur, 28 rue du Dr Roux,
Paris 75724 Cédex 15,

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 ‘nonself’molecular 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

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 Spätzle–Toll–Cactus 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 [7–9].
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 [10–12]. 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

0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(02)02334-X

albeit with slower kinetics. Therefore. following recognition of a fungal PAMP in the hemolymph by an unknown PRR. it binds the secreted host protein Spätzle. In mammalian cells. The functional homolog of IRAK in Drosophila. In this review. Pelle [26]. the Toll receptor signals through the well-characterized Tube–Pelle–Cactus–Dorsal/Dif cassette to induce the synthesis of antifungal peptides [21]. platelet-derived growth factor BB (PDGF-BB) and human chorionic gonadotropin (hGC) [18]. the processing of Spätzle is negatively regulated by a protease inhibitor of the serpin family called Necrotic. how Tube interacts with the Toll receptor remains unknown. Following Spätzle binding. rather than recognizing a microorganismspecific product. it appears that. 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. Tube contains a death domain but. 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]. Recently. unlike MyD88.25]. the first intracellular event following LPS or IL-1 stimulation is the recruitment of the adaptor protein MyD88 to the receptor complex [23]. This is the first report of a TLR4-specific signaling protein independent of the IL-1 pathway. Mal. or specific gene products from a given pathogen. The antifungal response in seml mutant flies is intact. Accordingly. Toll and the IL-1 receptor (IL-1R) share a similar intracytoplasmic Toll–IL-1R (TIR) domain. IL-1R-associated kinase (IRAK). Toll signaling in Drosophila also requires an adaptor-type molecule to link receptor events to downstream kinases.17]. which leads to the activation of nuclear factor κB (NF-κB) in mammals [4]. Toll signaling can proceed via a mechanism involving the inactivation of Necrotic to give rise to an active Spätzle molecule. In fact. the gene semmelweiss (seml). These recognition proteins in plants share similarities with Drosophila Tolls. Dorsal/Dif and Cactus are functionally homologous to myeloid differentiation factor 88 (MyD88). respectively [21]. Genetic studies place DmMyD88 in the Toll pathway upstream of Tube and Pelle. which interact with species-specific receptors in the plant as defined by the so-called ‘gene-for-gene’ concept of plant resistance [15]. which can affect many different plant species. and mammalian TLRs and NBS-LRR proteins. 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. lacks a TIR domain.10 No. 1) [22]. Ten TLRs have been characterized so far in mammals. Using a genetic screen. the molecular mechanisms of the interactions between these effector proteins are still unclear. Spätzle requires proteolytic processing by extracellular serine proteases. was found to be involved in resistance to Gram-positive bacterial infection [20]. To be active. Thus. was identified in Drosophila [27. DmMyD88-deficient flies are highly susceptible to fungal and Grampositive bacterial infections but responses to Gramnegative bacterial infections are unaffected. a MyD88-like protein. DmMyD88. However. Recently. Recently. Mal recruits IRAK2 to the activated TLR4 complex [25]. The protein Tube appears to be an adaptor protein analogous to MyD88 as genetic studies place Tube between Toll and the downstream kinase. NF-κB and inhibitor of κB (IκB). flies deficient in necrotic constitutively express the antifungal gene drosomycin. ‘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]. suggesting the existence of a distinct PRR that is involved in the recognition of fungal PAMPs. and possibly explains why LPS-treated macrophages from MyD88-deficient mice still activate NF-κB. Pelle. Pelle. http://tim.trends. unless they are also deficient in either spätzle or toll [19]. a bone fide PRR was described in Drosophila that is involved in recognition of Grampositive bacteria. was cloned and shown to be involved in signaling downstream of TLR4 [24. Spätzle is a member of the cysteine-knot superfamily that includes growth factors and hormones such as nerve growth factor (NGF). transforming growth factor β (TGFβ). In addition. suggesting that a straightforward Toll–DmMyD88–Tube–Pelle pathway is involved in resistance to fungal or Grampositive bacterial infections [28].194 Review TRENDS in Microbiology Vol. MyD88 recruits the kinase IRAK to the TLR/IL-1R complex [29].28]. More recently. also known as TIRAP. 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. a MyD88 homolog. and they are likely to possess different specificities for PAMPs (Table 1). Tube. IL-1 signaling to NF-κB is not affected. reflecting the conservation of strategies used to combat pathogens in the animal and plant kingdoms. These proteins each form a unique 3-D cysteine-knot fold and dimerize to bind to their cognate receptors. which encodes a peptidoglycanrecognition protein. Although dominant-negative forms of Mal block NF-κB activation through LPS/TLR4. MyD88 interacts with the cytoplasmic domain of the TLR and IL-1R through homotypic TIR interactions. is recruited to the membrane following .4 April 2002 by either non-specific elicitors. toll was shown to control the immune response of the adult fly to fungal and Gram-positive bacterial infections [4. This signaling pathway presents striking homologies with the interleukin 1 (IL-1) pathway. Likewise. The Toll protein is not itself a PRR as.

Dorsal/Dif and Cactus are functionally homologous to MyD88. transforming growth factor-β-activated protein kinase 1. Like mammalian NOD proteins. (1) Activation of the IMD pathway in Drosophila by Gram-negative bacteria leads to the activation of the NF-κB family member Relish. nuclear factor κB. Pti. and Tube.trends. NF-κB. The IMD pathway shares some similarity with the mammalian TNF-α pathway. plant NBS-LRR proteins possess an amino-terminal LRR domain and a central NBS whereas the carboxyl terminus can be other protein–protein interaction domains such as TIR or LZ domains. NOD2. the caspase Dredd. 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]. and a regulatory subunit. leucine-rich repeat. death domain. leucine zipper. the IκBs. LRR. inhibitor of κB. respectively. Like IRAK. NBS. Toll-like receptors. LRR LRR DD ? DmIKK complex (Kenny/Ird5) LPS.. NF-κB and IκB. Spätzle binds to the Toll receptor. two members of the Drosophila IKK complex. which are a class of plant NBS-LRR proteins. Following oligomerization of either NOD1 or NOD2. CARD. In plants. The next steps in the pathway downstream of the TLRs and IL-1R in the mammalian system is the TRAF6-dependent activation of the IκB kinase (IKK) http://tim. IL-1R-associated kinase. IKK activation by TRAF6 relies on two independent activities in the complex. 1. TIR. This complex. The interaction between NOD1 and RICK recruits the IKK complex. (3) Intracellular recognition of pathogens in mammals and plants using proteins sharing conserved NBS-LRR domains. nucleotide-binding site. Flagellin. peptidoglycan. TRAF. This pathway involves IMD.. dTRAF2. N. immune deficiency.4 April 2002 Insects PAMPs: LPS. thus promoting the activation of NF-κB. avirulence proteins. CARD Cleavage IRAKs NBS Pelle DD dTAK1 MyD88 Pto. K. mammals and plants. dTAK1. NEMO (or IKKγ). In NOD1. PG. pathogen-associated molecular pattern. Relish is activated by the combination of a phosphorylation event (via Ird5 and Kenny) and a proteolytic cleavage (via Dredd). caspase recruitment domain. DD. This pathway has homologies with the IL-1 pathway in mammals. IκB. IKK.10 No. One function of TRAF6 is to . IKKα and IKKβ. PG. which consists of two kinases.. TNF-receptor-associated factor. Recent evidence has shown that Pelle then interacts with and phosphorylates a Drosophila TRAF protein. interleukin. which phosphorylates IκB. which activates NF-κB. In Drosophila. specific Avrs from intracellular pathogens are recognized by resistance (R) proteins. Abbreviations: Avr. LZ.RPS2. other PAMPs? TIR CARD DD TIR Tube Dredd Plants Mammals Spätzle (1) IMD pathway 195 X: TIR.. activation of the Toll pathway. IκB proteins are polyubiquitinated and targeted for degradation by the proteosome thus releasing NF-κB and allowing this transcription factor to translocate to the nucleus and mediate the expression of several genes. RICK is recruited to these molecules through homophilic CARD–CARD interactions. is responsible for the serine phosphorylation of the inhibitory molecules of NF-κB. Although genetic evidence is lacking. IRAK. kinase domain.. resulting in signal transduction through the wellcharacterized Tube–Pelle–Cactus–Dorsal/Dif cassette to induce the synthesis of antifungal peptides. (2) Extracellular recognition of pathogens in Drosophila and mammals relies on homologous Toll/TLR signaling to activate the NF-κB family of transcription factors. TAK1.. Once phosphorylated. IRAK. the carboxy-terminal LRR domain is likely to play a negative regulatory role. LRR LPS Toll (2) LPS receptor ? IMD TLRs (2) Relish TRAF6 dTRAF2 Phosphorylation Cactus IKK complex kinase? IκB Cactus Dif.Review TRENDS in Microbiology complex. IL. lipopolysaccharide. both the amino-terminal CARD domain and the NBS domains are necessary for NF-κB activation. TLRs. which is homologous to mammalian caspase-8. Activation of these proteins by plant pathogens results in the hypersensitive response in the susceptible plant. IκB kinase. Dorsal RICK LRR LRR RPP5. L6. Toll/IL-1 receptor domain. IMD. a step that precedes its release from the receptor complex. LPS.. which is the homolog of mammalian TRAF6 [31]. Strategies of innate immune defense in insects.. LZ K NF-κB Immune response genes TRENDS in Microbiology Fig. Pelle. Pelle is also autophosphorylated. and Ird5 and Kenny. PAMP. many of which are important for the inflammatory response. NBS-LRRs (3) NBS K Intracellular Avr K X NOD1.

In addition to this so far linear IMD–dTAK1–DmIKK–Relish pathway. leading to the induction of both NF-κB and c-Jun amino-terminal kinase (JNK) [33]. Strikingly. heat-shock protein. and a caspase related to the mammalian caspase-8 (Dredd). TAB1 and TAB2. The largest family described so far groups proteins sharing NBS and LRR domains. the ultimate step in the activation of Dorsal and Dif is the phosphorylation and degradation of Cactus. it appears that in the IMD pathway. mycoplasma lipopeptide-2.trends. peptidoglycan.10 No. 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. ‘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. Zymosan (with TLR2) Imidazoquinoline compounds ? CpG DNA ? TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 Refs [51–56] [57] [51. BLP. MALP2 (with TLR2). it has recently been proposed that the caspase Dredd.196 Review TRENDS in Microbiology Vol. MALP2. recruit ubiquitin-conjugating enzymes that mediate the assembly of polyubiquitin chains onto the IKK complex. RPP5) and Tobacco (N). a Drosophila homolog of TAK1 (dTAK1) that is known to be activated by TNF-α in mammalian cells. 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. The majority of these genes encode cytoplasmic resistance proteins although some encode transmembrane receptors. It is intriguing that the fruit fly has evolved two parallel systems of intracellular cell signaling. Although several receptors closely related to Toll are found in the Drosophila genome [34]. RPM1. that are triggered selectively depending upon the type of pathogen presented extracellularly. Arabidopsis (RPS2. the Toll and IMD pathways. bacterial lipoproteins. LPS. Indeed.4 April 2002 Table 1. which then releases these transcription factors. viral protein. lipopolysaccharide. The recent cloning of imd revealed that this protein contains a death domain and is homologous to the mammalian receptor-interacting protein (RIP) [35]. it remains unclear whether the LPS receptor belongs to this family [21]. are still unknown. the NF-κB homolog Relish is activated by the combination of a phosphorylation event (via Ird5/Kenny) and a proteolytic cleavage (via Dredd). another homolog of NF-κB found in the fruit fly [21. Toll-like receptor. and Relish. PG. The transcription factor NF-κB plays a pivotal role in regulating the synthesis of antimicrobial peptides or proinflammatory factors in insects and mammals. Ird5 and Kenny. Ubiquitination in this case does not involve proteolysis of the targeted protein but appears to regulate its kinase activity [32]. Flies that are mutant for the imd (immune deficiency) gene display a compromised response to Gram-negative bacteria but a normal immunity to fungi. HSP.39]. Zymosan Leptospira interrogans LPS dsRNA LPS. the caspase Dredd. genetic evidence has shown that this pathway involves dTAK.55] [64] [65] a Abbreviations: BLP. LTA. The other function of TRAF6 is to recruit and activate the mitogen-activated protein kinase kinase kinase (MAP3K) TAK1 and its associated proteins. LTA. the steps between Pelle and/or dTRAF2 activation and the phosphorylation and/or degradation of Cactus. this pathway involves a death domain-containing molecule related to RIP (IMD). TLR. two members of the Drosophila IKK complex. is required to resist Gram-negative infection through the proteolytic activation of Relish [38. whereas in mammals the TLR/IL-1 signaling pathway is so far thought to be activated downstream of each TLR. Taxol. the initial results obtained for the Drosophila IMD pathway suggest that it shares some similarity with the mammalian tumor necrosis factor (TNF)-α pathway. the Drosophila homolog of TGFβ-activated kinase 1 (TAK1). In addition.58–62] [63] [54. some of these NBS-LRR resistance proteins encode amino-terminal domains with homology to the TIR domain found in Toll and the IL-1R. thus demonstrating that the Toll and IMD pathways account for two independent defense mechanisms triggered by distinct microorganisms. which is homologous to mammalian caspase-8. R proteins fall into five classes. Therefore. Together. Various products recognized by the family of mammalian Toll-like receptors TLR Product recognized TLR1 TLR2 ? PG. respectively. MALP2. The protein encoded by dTAK1 functions downstream of IMD and regulates the activation of Relish through the Ird5/Kenny IKK complex [37]. the fly IκB homolog.42]. The nature of the link between IMD and the as-yet-unidentified LPS receptor remains to be characterized. TAK1 can directly phosphorylate and activate IKKβ as well as MKK6. http://tim. In Drosophila. HSP60 Flagellin PG (with TLR2). 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 Besides imd.36]. The kinase upstream of Cactus remains to be identified. suggesting conservation of a common . Similar to mammals. based on the motifs they contain [41. NBS-LRR resistance proteins can be found in various plants such as tomato (Prf). lipoteichoic acid. Once activated. The IMD pathway in Drosophila Whereas LPS signaling is mediated by TLR4 in mammals.

LPS. 1). Based on these observations. NB domain and CARD. ICE-protease activating factor. LPS signaling ? ? ? Mutated in cold autoinflammatory syndrome and in Muckle–Wells syndrome a Abbreviations: CARD. Similar results have been described for NOD2. we propose that these two domains together. nucleotide-binding site.11. two mammalian proteins containing an NBS-LRR motif. caspase recruitment domain. suggests that this class of molecules could represent intracellular detectors of PAMPs [10. leucine-rich repeat. NBS-LRR. death effector filament-forming ced-4-like apoptosis protein. except that in this case. nucleotide-binding site. the carboxy-terminal LRR domain is likely to play a negative regulatory role in the pathway. leucine-rich repeat. NBS. have been implicated in inflammation-related diseases (Table 2) [12. NF-␬B. lipopolysaccharide. 2. 2). Schematic representation of the eight NBS-LRR proteins described in humans so far. http://tim. constitute the crucial structural domains involved in mediating pathogen recognition and/or inflammation.11. both amino-terminal a Table 2. IPAF. caspase recruitment domain. The homology between these molecules and plant disease resistance proteins. involved in Blau syndrome. as deletion of this domain results in enhanced activation of NF-κB [11].10 No. 1). TRENDS in Microbiology Vol. LRR.Review Fig.44–46]. baculovirus inhibitory repeat. NOD2 and Cryopyrin. Although little is known about the signaling pathways induced downstream of plant NBS-LRRs. nuclear factor ␬B. recent advances have initiated studies of the role of mammalian NBS-LRRs in intracellular signaling (Fig.43]. 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. NBS. Functional domain analysis of NOD1 shows that both the amino-terminal caspase recruitment domain (CARD) and the NBS domains are necessary for NF-κB induction by NOD1 [11]. A new family of NBS-LRR molecules has recently been characterized in mammals that shares structural similarity with plant NBS-LRRcontaining proteins ( . together with their ability to induce the NF-κB pathway when overexpressed (see later). LRR. CARD.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.trends. involved in the response to Shigella flexneri ? Mutated in Crohn’s disease. The transient overexpression of NOD1 or NOD2 is sufficient to induce the activation of NF-κB (Table 2) [10. Abbreviations: BIR.43]. DEFCAP. By contrast. NAC. Recently.

Inohara and co-workers have shown that NOD1 confers responsiveness to bacterial LPS [13]. thus promoting the activation of NF-κB [48]. we have presented evidence that NOD1 is required to mediate NF-κB activation following infection of epithelial cells by an invasive strain of the Gram-negative pathogen Shigella flexneri [14]. which are committed to respond to infection with the production of antifungal peptides. most of which remain to be characterized (Fig. CARD domains together with the NBS domain are necessary to mediate NF-κB activation [43]. the link between these genetic data and the functional role of NOD2 in monocytes remain elusive and awaits further investigation. In addition. The interaction between NOD1 and RICK then induces the recruitment of the IKK complex. Nevertheless. recognize distinct proteins from pathogens specific to that particular plant species.trends. 2). 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. some evidence exists for the direct recognition of PAMPs [49. However. In addition to these observations. which phosphorylates IκB. infection by this invasive pathogen is sufficient to induce NOD1 oligomerization and the transient recruitment of RICK/IKK to the NOD1 oligomers. the physical separation between the hemolymph.10 No. where the recognition of the fungus occurs. Recently. thus leading to the dysregulated onset of inflammation [12. However. 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. the detection of an interaction between NOD1 and LPS suggests that NOD1 could play a role in LPS sensing [13]. and the cells of the fat body. The NBS-LRR family of mammalian proteins could comprise >30 members based on the human genome sequence.45]. An important question for the future is to understand better the different strategies used to recognize pathogens by the different innate immune systems.43. the general organization of flies. most of the R proteins. Accordingly. RICK (also known as RIP2 or CARDIAK) is recruited to these molecules through homophilic CARD–CARD interactions [43. plants or mammals could have dictated the distinct evolution of these recognition systems depending on the context of interaction between host and pathogen.50]. . The determination of this novel NOD1–RICK–IKK signaling pathway upstream of NF-κB was based on overexpression studies and no physiological stimulus was associated with this pathway. 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 Crohn’s 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 François Leulier for helpful identification of NOD2 as the first susceptibility gene for Crohn’s disease can be interpreted as a compromised response of the individuals carrying these mutations to the bacterial environment. For instance. if other mammalian NBS-LRR proteins apart from NOD1 and NOD2. flexneri-induced activation of NF-κB. the possibility has emerged that this family might represent the intracellular counterpart of extracellular pathogen detection mediated by TLRs. In plants. rather than recognizing a general molecular pattern associated with a microorganism. The activation of NF-κB by NOD1 or NOD2 requires the NBS-dependent oligomerization of the molecule through a mechanism similar to that described for Apaf-1. whereas in insects there is no evidence so far implicating any Toll protein in direct PAMP recognition. Firstly.47].Review 198 TRENDS in Microbiology Vol.48]. thus providing the first example of a physiological activation of the NOD1–RICK–IKK pathway [14]. 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. For example. the recent http://tim. could have contributed to the selection of a unique defense strategy through amplification using a proteolytic cascade of diffusable factors. With the recent characterization of intracellular PAMP detection by mammalian NBS-LRR proteins (NOD1 and NOD2). in mammals. the TLRs might specifically recognize a limited set of PAMPs. however. act as intracellular sensors for PAMPs. several reports have suggested that the NOD1 and NOD2 signaling pathway is likely to be activated following PAMP recognition. a pro-apoptotic mammalian protein [11. the mechanism of such responsiveness remains unclear as the LPS was delivered extracellularly. Overexpression of a mutated form of NOD1 deleted for its CARD domain acts as a dominant-negative for S. An exciting challenge for the coming years will be to determine if these genes are indeed general regulators of inflammation and further. However.4 April 2002 Questions for future research • • • • • • • • • What are the ligands recognized by TLR1. in Drosophila. The mechanism by which the LRR domain of NOD1 or NOD2 inhibits downstream signaling to NF-κB is still unclear. Following oligomerization of either NOD1 or NOD2.

et al. Y. and Lemaitre. T. 1099–1103 64 Hemmi. 189. Natl. U. 29. 835–841 25 Fitzgerald. (2001) Mal (MyD88adapter-like) is required for Toll-like receptor-4 signal transduction. et al. 83–92 27 Horng. 16. R.M. N. B. H. Nature 411. N. J. 276. (2001) The lipopolysaccharides of the phytopathogen Xanthomonas campestris pv. Chem. Med. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. 398–401 61 Kawasaki. 13. Nat. (1998) MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. et al. et al. 276. (2000) Activation of the Drosophila NF-κB factor Relish by rapid endoproteolytic cleavage. et al. 933–940 55 Ozinsky. (2001) The evolution and genetics of innate immunity. S. Acad. et al. Genet.L. A. A. Biol. A. Immunol. D. K. and Beutler. O. (2001) TIRAP: an adapter molecule in the Toll signaling pathway. Cell 86. et al. 3. (2000) Mouse toll-like receptor 4MD-2 complex mediates lipopolysaccharidemimetic signal transduction by Taxol. J. et al. Med. J. 260–264 22 Kimbrell. et al. Genes Dev. S. an Apaf-1-like activator of caspase-9 and nuclear factor-κB. et al.V. 304–311 35 Georgel. 443–451 52 Brightbill. A. K. (2000) Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. C. J. (2001) Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. (2001) Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. and Boller. Nature 411. 11. Science 285. Science 276. (2000) The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Science 285. Natl.A. (1999) Toll. TRENDS in Microbiology Vol. Chem. Nat. Sci. EMBO Rep.A. et al. F. U. 253–258 24 Horng. Opin. Nature 412. E. L. S. Immunol. S. Nature 413. Cell 103. (2000) Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitinconjugating enzyme complex and a unique polyubiquitin chain. Mol. Planta 213. 1. J. et al. (2000) dFADD. (2001) Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. and Medzhitov. Curr. et al. Trends Genet. 91–97 29 Wesche.Review References 1 Fearon. et al. Proc. 1917–1919 20 Michel.dependent signaling pathway. (1999) Nod1. 973–983 5 Medzhitov. 347–352 40 Hu. 2321–2342 37 Vidal. and Yeager. Sci. Curr. (2001) TAK1 is a ubiquitindependent kinase of MKK and IKK. A. et al. F. J. (2002) Small anti-viral compounds activate immune cells via the TLR7 MyD88. 13. Rev. S. 97. 2. et al. 19–20 45 Hugot. Invest. 777–786 4 Lemaitre. a novel death domain-containing adapter protein for the Drosophila caspase DREDD. 2251–2254 62 Ohashi. Biol. EMBO Rep. 14560–14567 12 Ogura. S. (2001) Drosophila immunity: two paths to NF-κB. (2001) Drosophila MyD88 is an adapter in the Toll signaling pathway. 736–739 54 Takeuchi. J. B. Chem. (2001) Nod2. et al. 605–609 7 Poltorak. Immunol.P.D. (2002) Drosophila MyD88 is required for the response to fungal and Gram. O. 837–847 30 Qian. (1999) Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-κB. 2085–2088 59 Qureshi. Mol. R. Trends Immunol. Chem. and Maniatis. (2000) An induced proximity model for NF-κ B activation in the Nod1/RICK and RIP signaling pathways. K. H. 214–222 50 Gomez-Gomez. (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. 442–449 18 Mizuguchi. Bioessays 19. Biol. 97. 4812–4818 44 Miceli-Richard. EMBO Rep. N.A. Immunol. Nature 410. et al. (2001) Discrimination of bacterial lipoproteins by Toll-like receptor 6. Nat. Immunol. (1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. (2001) IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1-induced activation of NFκB. 726–733 42 Cohn. (2000) Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide. 55–62 43 Ogura. 15. 41661–41667 31 Shen. R. (1999) Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). (2000) Physical contact between lipopolysaccharide and Toll-like receptor 4 revealed by genetic receptor 3. (1997) Signaling in plant–microbe interactions. (2001) Toll receptors in innate immunity. 50–53 2 Medzhitov. (2000) The Toll receptor family and microbial recognition.T. 15. (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. E. Science 272. J. Biol. et al.trends. Cell 2.A. Jr. a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-κB. A. Chem. Biol. 599–603 199 46 Hoffman. (2000) The Drosophila caspase Dredd is required to resist Gram-negative bacterial infection. Trends Cell Biol. 276. 274. 21129–21135 9 Lien. J. H. 8596–8601 32 Deng. C. Cell 42. et al. 164. 12–15 3 Hughes. S. (2001) Innate immunity in plants.10 No. et al. et al. 8. 732–738 58 Poltorak. Biol. 615–625 60 Kurt-Jones. B. U. J. 826–833 16 Anderson. 78–83 26 Drier. Transfer from CD14 to TLR4 and MD-2. P. C. J. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in the TLR4 gene. 13766–13771 56 Werts. 1900–1912 38 Leulier. 353–358 39 Stoven. 12955–12958 11 Inohara. 351–361 33 Wang. (2001) Human Nod1 confers responsiveness to bacterial lipopolysaccharides. Genet. et al. 736–742 15 Dangl. Dev.S. Jr (1998) An ancient system of host defense. Acad. J. et al. Acad. (2001) Physical and functional interactions between Drosophila TRAF2 and Pelle kinase contribute to Dorsal activation. U. et al. and Yang. Nat. (1997) Molecular evolution of the vertebrate immune system. Natl. 346–352 57 Alexopoulou. B. K. Sci. (2001) Mutations in the Drosophila dTAK1 gene reveal a conserved function for MAPKKKs in the control of rel/NF-κB-dependent innate immune responses. 98. (2001) CARD4/Nod1 mediates NF-κB and JNK activation by invasive Shigella flexneri. J. 29. 275. 949–957 48 Inohara. et al. et al.positive bacterial infections. Sci. Chem. K. Y.A. Chem. et al. (2001) Recognition of double-stranded RNA and activation of NF-κB by Toll. Mol.L. Nat. R. and Janeway. (2000) Genes that fight infection: what the Drosophila genome says about animal immunity. Nature 411. Exp. 732–736 53 Aliprantis. 98. et al. 30761–30764 41 Baker. A. L. (2001) Plant pathogens and integrated defence responses to infection. Proc. Immunity 7.M. J. J. et al. et al. et al.L. T.. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. et al. M. Cell 1. Sci. 452–456 6 Wright. and Locksley. R. 256–267 http://tim. Genet. et al. (1999) Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Nat. Trends Microbiol. Cancer Biol. R. 275. N. Clin. 791–798 17 Khush. R.A. Chem. 196–200 65 Hemmi. 2. 740–745 . A. Nature 414.D. Natl. 1. 239–242 19 Levashina. L. et al. and Steward. 274. and Hoffmann. 756–759 21 Khush. (2000) Cutting edge: heat shock protein 60 is a putative endogenous ligand of the Toll-like receptor-4 complex. Science 282. S. a new piece in the puzzle of innate immunity. et al. 189. Immunol. Biol. 301–305 47 Srinivasula. et al. et al. Nat. 603–606 13 Inohara. (2001) NF-κB signaling pathways in mammalian and insect innate immunity. R. H.A. et al. J.. Nature 408. and Janeway. Cell 1. Immunol.E. 2. Semin. 497–504 10 Bertin. (2001) CARD15 mutations in Blau syndrome. C. (1998) Getting knotted: a model for the structure and activation of Spatzle. (1999) Constitutive activation of Toll-mediated antifungal defense in serpindeficient Drosophila. Genes Dev.D. (2001) Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. E. S. (1997) The dorsoventral signal transduction pathway and the Rel-like transcription factors in Drosophila. Exp. et al. Trends Biochem. 23. 10. et al. et al. D. 22. C. E. S. Int. Immunol. Chem. and Jones. J. 1. et al. A. 2.4 April 2002 23 Medzhitov. B. Nature 413. 3. (1999) Cell activation and apoptosis by bacterial lipoproteins through Tolllike receptor 2. (1996) The instructive role of innate immunity in the acquired immune response. (1999) Host defense mechanisms triggered by microbial lipoproteins through Toll-like receptors. 105. J. Acad. Science 285.S. (2001) Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Proc. Proc. et al. 276. 346–351 34 Imler. X. Nat. S. et al. et al. (2000) A Toll-like receptor recognizes bacterial DNA. Cell 5. et al. et al. J. et al. T. J. (1997) MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. 1003–1011 51 Takeuchi. et al. Immunol.M. T. 558–561 63 Hayashi. (2001) The innate immune response to bacterial flagellin is mediated by Tolllike receptor 5. campestris induce an oxidative burst reaction in cell cultures of Nicotiana tabacum. 27823–27831 49 Meyer. T. Opin. 8. A. H. S. 503–514 36 Silverman. Y. 12654–12658 28 Tauszig-Delamasure. Immunity 11. Biol. (1985) Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product. 275. Biol. 2163–2167 8 da Silva Correia. et al. 2551–2554 14 Girardin.