Gram-negative bacteria binding protein 3: Biological Overview | References
Gene name - Gram-negative bacteria binding protein 3
Cytological map position - 66E5-66E5
Function - pattern recognition receptor
Keywords - immune response to fungus, pattern recognition receptor, required for the detection of and reaction to fungal cell wall components
Symbol - GNBP3
FlyBase ID: FBgn0040321
Genetic map position - 3L: 8,948,121..8,949,651 [+]
Classification - O-Glycosyl hydrolase superfamily
Cellular location - secreted
The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and GNBP1 that cooperate to detect the presence of infections in the host. This study reports that GNBP3 is a pattern recognition receptor that is required for the detection of fungal cell wall components. Strikingly, there is a second, parallel pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host (Gottar, 2006).
Fungi represent a threat to insects in the wild, with more than 700 described entomopathogenic species. Insects must have evolved responses to handle these infections. This study used Drosophila to decipher the mechanisms that stimulate immune responses to fungal infections (Gottar, 2006).
The Drosophila host response includes both cellular and humoral arms. The analysis of the humoral immune response within the framework of a septic injury model has led to the current paradigm in which two distinct intracellular transduction pathways, immune deficiency (IMD) and Toll, regulate the transcription of hundreds of genes by controlling the nuclear uptake of the NF-κB transcription factors Relish and Dorsal-related immunity factor (DIF), respectively. The classical effector molecules of the systemic humoral response, the antimicrobial peptides, are synthesized in the fat body, a functional analog of the mammalian liver, and are released into the hemolymph, where they kill invading microorganisms. One of these peptides, Drosomycin, exhibits fungicidal activities at micromolar concentrations and is active mainly on filamentous fungi. Others, such as Cecropins, Attacins, Drosocin, and Diptericin, are active mostly on Gram-negative bacteria, whereas Defensin is effective against Gram-positive bacteria (Gottar, 2006).
The IMD pathway is required for the host response against Gram-negative bacteria. Mutants in this pathway fail to express antibacterial peptides and are highly sensitive to such infections yet resist fungal and Gram-positive bacterial infections as well as wild-type flies (Gottar, 2006).
Toll is the receptor of the second intracellular transduction pathway and is activated by the binding of a proteolytically cleaved form of the Spätzle (SPZ) cytokine. Toll pathway mutants are susceptible to infections by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae or by the opportunistic pathogen Aspergillus fumigatus. Toll is also required to resist some Gram-positive bacterial infections (Gottar, 2006).
The Drosophila immune response is adapted to the nature of the invading microorganism. The Toll pathway is induced by fungi and Gram-positive bacteria, whereas the IMD pathway is predominantly triggered upon Gram-negative bacterial challenges. These observations imply that several receptors mediate the discrimination between various types of microbial infections. Indeed, members of the peptidoglycan recognition protein (PGRP) family have been shown to be required for these distinct events (Ferrandon, 2004; Kaneko, 2005). PGRP-LC, a receptor of the IMD pathway, can be activated by meso-diaminopimelic acid peptidoglycan (PGN), a compound characteristic of the cell wall of Gram-negative bacteria and of Gram-positive bacilli. PGRP-LE, a secreted member of the PGRP family, is also involved in sensing Gram-negative bacteria (Kaneko, 2006). In contrast, the circulating PGRP-SA receptor activates the Toll pathway upon detection of lysine-type PGN, which is a major component of the cell wall of many Gram-positive bacterial strains. The Gram-negative binding protein 1 (GNBP1) associates with PGRP-SA, and this complex is both necessary and sufficient to activate the Toll pathway upon Gram-positive challenge (Gobert, 2003). The circulating PGRP-SA/GNBP1 complex activates a downstream proteolytic cascade that leads to the cleavage of the Spätzle cytokine, which then activates the Toll transmembrane receptor (Jang, 2006; Kambris, 2006). Thus, PGRP-SA and GNBP1 define a Gram-positive-specific branch of Toll receptor activation. PGRP-SD also belongs to this branch and is required for the detection of other Gram-positive bacterial strains (Bischoff, 2004; Gottar, 2006).
This study addresses the existence of a second branch devoted to the detection of fungal infections, which also activates Toll. Indeed, mutants for the persephone (psh) gene, which encodes a clip-prodomain-containing protease, are characterized by an increased sensitivity to natural infections with the entomopathogenic fungus B. bassiana, whereas they are resistant to bacterial infections (Ligoxygakis, 2002). The psh mutations had been originally isolated as suppressors of the necrotic (nec) phenotype. nec encodes a serine protease inhibitor of the serpin family, the absence of which leads to the constitutive, psh- dependent, activation of the Toll pathway (Levashina, 1999; Ligoxygakis, 2002). Thus, psh and nec define a fungal-specific branch of Toll receptor activation. By analogy to the Gram-positive branch, it is expected that an as-yet-unidentified immune receptor detects fungal infections and activates in turn the psh-dependent proteolytic cascade (Gottar, 2006).
GNBP1 belongs to the family of GNBP/β-glucan recognition proteins (βGRP) (Kim, 2000). Members of this family have been reported to bind to β-(1,3)-glucan, a major component of the fungal cell wall. In Drosophila, three members of this family, GNBP1 to GNBP3, have been described (Kim, 2000). Among these, GNBP3 shows the greatest degree of similarity to lepidopteran β-(1,3)-glucan recognition proteins and was therefore a good candidate for a fungal-specific sensor. This paper reports that GNBP3 is indeed required for Toll pathway activation in response to fungal infections. Strikingly, it was also found that psh is required in a distinct yet complementary detection pathway that can be activated by fungal virulence factors (Gottar, 2006).
The detection of infections is a crucial step in the timely initiation of an appropriate immune response. In Drosophila, the use of nonentomopathogenic bacteria such as M. luteus and E. coli has allowed the delineation of both intracellular signal transduction pathways as well as the identification of five innate receptors (PRRs), PGRP-LC and PGRP-LE for the IMD pathway and PGRP-SA/GNBP1/PGRP-SD for the Toll pathway. To elucidate the mechanisms involved in the detection of fungi, concentration was placed on a somewhat artificial infection system using an opportunistic human pathogenic yeast, C. albicans. Understanding of the system was refined by using the entomopathogenic fungi B. bassiana and M. anisopliae (Gottar, 2006).
GNBP3 was found to be a PRR dedicated to the detection of fungi because (1) recombinant GNBP3 is able to bind in vitro to Candida and to polymeric chains of β-(1,3)-glucan; (2) it is required for the activation of the Toll pathway by polysaccharides of the fungal cell wall; (3) GNBP3 is required for resistance against yeast infections, including C. albicans, C. glabrata, and C. tropicalis, and against mold infections such as B. bassiana, M. anisopliae, and A. fumigatus; (4) GNBP3 triggers an adequate immune response; namely, it activates the antifungal Toll pathway in a spz-dependent manner. The possibility that another fungal receptor acts together with GNBP3 to activate the Toll antifungal host defense cannot be excluded (Gottar, 2006).
Of note is that fungi can induce the IMD pathway with short-term kinetics (Lemaitre, 2007). This induction was found to be dependent on PGRP-LC and not on GNBP3. One possibility is that a PGRP-LC coreceptor senses fungal microbial patterns. Alternatively, fungal cell wall constituents might bind directly to PGRP-LC. Interestingly, Lee (2003) has reported that a coleopteran PGRP is able, in addition to its liaison to PGN, to bind with high affinity to tetralaminariose, a tetramer of β-(1,3)-glucan (Gottar, 2006).
As is the case for members of the PGRP family, the GNBP/βGRP proteins have evolved to recognize distinct carbohydrate chains that form the cell wall of microorganisms. Given their distribution in the arthropod lineage, it is likely that these two families form an essential part of their immunity repertoire. Whereas PGRP homologs exist in mammals, βGRP members have not been reported in vertebrates. However, the phagocytic and signaling receptor Dectin-1 detects β-(1,3)-glucans (Brown, 2001) and may to some extent fulfill in mammals a primary function that is similar to that of GNBP3 in insects, i.e., the sensing of fungal infections (Gottar, 2006).
Because spz is required for Toll activation by GNBP3, it is proposed that the binding of GNBP3 to its microbial ligand leads to the activation of a proteolytic cascade that ultimately processes proSPZ into a functional Toll ligand. Because psh and GNBP3hades have distinct phenotypes as regards Toll pathway activation, and because the double mutant psh;GNBP3hades displays a stronger phenotype than either mutant alone when challenged with live fungi, PSH cannot belong exclusively to a proteolytic cascade activated by GNBP3. However, epistatic analysis reveals that the spz-dependent expression of Drosomycin induced by GNBP3 overexpression partly requires psh function. Taken together, these data indicate the existence of an alternative, psh-independent proteolytic cascade that mediates the GNBP3-dependent maturation of the Toll ligand Spätzle. This cascade is distinct from the one that activates Toll signaling during early embryogenesis (Gottar, 2006).
It is hypothesized that at least four distinct proteolytic cascades converge to process the Toll ligand Spätzle (SPZ). Dorsoventral (D/V) patterning occurs during early embryogenesis and involves the proteases Gastrulation Defective (GD), Snake (SNK), and Easter (EA): this proteolytic cascade is unlikely to be involved in the activation of Drosomycin expression by fungi. In addition to sensing virulence factors, PSH might function downstream of an unknown pattern recognition receptor (PRR). Indeed, epistatic analysis indicates that PSH partially functions downstream of GNBP3. PGN, peptidoglycan; SPE, Spätzle processing enzyme (Gottar, 2006).
An unexpected finding of this study is that the Toll pathway is normally induced in GNBP3hades mutants undergoing a B. bassiana infection. Yet, these mutants are more susceptible to this pathogen than wild-type flies. These observations suggest that GNBP3 fulfills other functions required in the host defense against fungal pathogens that are independent of its role in triggering the Toll pathway. Indeed, some biochemical evidence is available that GNBP3 is involved in other aspects of host defense (Gottar, 2006).
Many pathogens have adapted to their hosts and developed specific strategies to defeat their defenses. Fungi such as B. bassiana and M. anisopliae are able to infect insects following deposition of spores on the surface of the cuticle. To penetrate this physical barrier, they secrete several virulence factors such as chitinases and proteases. The PR1A protease is able to activate Drosomycin expression in the absence of infection when overexpressed in flies. This effect on Toll pathway activation is specific because it can be blocked in a psh background and depends on the proteolytic activity of PR1A. These data establish the proof of concept that a virulence factor can be detected by the innate immune system. Interestingly, the data indicate that PR1 can directly process PSH into its active form (Gottar, 2006).
PR1A is one of ten proteases in this subtilisin family and is expressed only during cuticle penetration (Bagga, 2004; Wang, 2005). A PR1A/PR1B-deficient strain is still able to induce Drosomycin expression in a GNBP3hades mutant background, presumably through other fungal PR1 proteases. Thus, further work will be required to understand the multiple pathogenic mechanisms taking place during a natural fungal infection (Gottar, 2006).
The data show that the detection of fungal infections relies on a two-pronged sensor system that constitutes a partially redundant recognition system. The psh;GNBP3 double mutant strain consistently yields a stronger phenotype than that of the respective single mutants. Since only GNBP3 is strictly required in the defense against opportunistic yeasts, it is likely that the recognition of fungal patterns represents an ancestral, basal mode of infection sensing. The psh-dependent system that monitors virulence factors may have evolved secondarily in response to the selective pressure exerted by entomopathogenic fungi. Indeed, if the psh-based and the GNBP3-based sensing systems were perfectly redundant, it would be expected that the deletion of one of these systems would not prevent the activation of the Toll pathway. This is indeed what was observed when infecting flies with live C. albicans or with M. anisopliae. In contrast, Drosomycin inducibility is abolished in psh mutants, but not in GNBP3 mutants, infected by B. bassiana. These data indicate that B. bassiana has evolved a strategy that allows it to escape or to block GNBP3 surveillance (Gottar, 2006).
Future studies will reveal whether or not similar systems of virulence factor detection exist also to sense infection by entomopathogenic bacteria (Gottar, 2006).
It is surmised that some pathogens have developed strategies to inactivate the GNBP basal sensor system of Drosophila and that this led to the selection of a novel host counterstrategy: the surveillance of virulence factor activity. This theme is a central tenet of the current understanding of plant innate immunity. In plants, basal sensor systems detect the presence of microbial elicitors and trigger an immune response. Some virulence factors of the plant pathogen inhibit the elicitor-induced signaling by manipulating host proteins that regulate the host basal response (Kim, 2005). In some plant cultivars, a surveillance system based on R proteins 'guards' the targets of virulence factors (coded by microbial avirulence [avr] genes) and triggers a strong immune response when under attack. One example is provided by Arabidopsis, in which the cleavage of the endogenous PBS1 kinase by the Pseudomonas syringae type III effector AvrPphB, a cysteine protease, leads to the activation of the hypersensitive response by the R protein RPS5 (Shao, 2003). A case possibly more relevant to fly immunity is provided by the tomato, in which the host protease Rcr3 is required for the recognition of the pathogen virulence factor Avr2 by the Cf-2 transmembrane receptor (Rooney, 2005; Gottar, 2006 and references therein).
Fungal proteases secreted by entomopathogenic fungi have to cross the structurally invariant cuticular barrier of the insect host that thus conditions the type of proteolytic activity required to degrade the cuticular proteins. This phenomenon may have been exploited by Drosophila to detect entomopathogenic infections in a mechanism that is hence conceptually related to the guard hypothesis of plants, although in this case PSH would monitor indirectly a passive defense mechanism, the protection provided by the bodily armor. To date, the analysis of the immune response in Drosophila has been largely limited to the study of laboratory strains in a controlled environment. By analogy to plant-pathogen interactions that involve avr genes and their cognate plant R resistance genes, a major challenge for the coming years will be to determine if the insect-pathogen interactions in a natural environment involve several distinct virulence factors and their associated host detection systems (Gottar, 2006).
The discovery of a host sensor system dedicated to the detection of virulence factor activity begs the question of the relevance of such a system to mammalian innate immunity. It has been reported that virulence factors such as the cholesterol-dependent cytolysin or pertussis toxin are able to induce immune responses through TLR4. In these cases, the possibility remains open that TLR4 functions as a coreceptor needed for intracellular signal transduction and that the actual recognition is mediated by unknown receptors. A second class of interest is that of the protease-activated receptors. Indeed, PAR2 has been implicated in the induction of the HB2 defensin by bacterial proteases in epithelial cells. Similarly, Citrobacter rodentium induces the intestinal release of host proteases that activate the PAR2 receptor and subsequent colonic inflammation. Finally, virulence factors from Salmonella and Yersinia have been shown to inhibit NF-κB and MAPK signaling. Thus, it is legitimate to ask if receptors dedicated to the perception of virulence factor activity have been selected during the evolution of the mammalian innate immune system (Gottar, 2006).
The Drosophila Toll receptor does not interact directly with microbial determinants, but is instead activated by a cleaved form of the cytokine-like molecule Spätzle. During the immune response, Spätzle is processed by complex cascades of serine proteases, which are activated by secreted pattern-recognition receptors. This study demonstrates the essential role of ModSP, a modular serine protease, in the activation of the Toll pathway by gram-positive bacteria and fungi. ModSP integrates signals originating from the circulating recognition molecules GNBP3 and PGRP-SA and connects them to the Grass-SPE-Spätzle extracellular pathway upstream of the Toll receptor. It also reveals the conserved role of modular serine proteases in the activation of insect immune reactions (Buchon, 2009).
Sequential activation of extracellular serine protease (SP) cascades regulates important innate immune reactions, like blood clotting in arthropods and complement activation in vertebrates. In Drosophila, proteolytic cascades are also involved in the regulation of the Toll pathway, which mediates resistance to Gram-positive bacteria and fungi. Unlike mammalian Toll-like receptors, the Drosophila Toll receptor does not interact directly with microbial determinants and is instead activated by a cleaved form of the secreted cytokine-like molecule Spätzle (Spz). The immune-induced cleavage of Spz is triggered by proteolytic cascades, conceptually similar to vertebrate blood coagulation or complement activation cascades. These proteolytic cascades have a functional core consisting of several SP that undergo zymogen activation, upon cleavage by an upstream protease. Spätzle processing enzyme (SPE), an immune-regulated SP with a Clip-domain, has been identified as the terminal SP that maturates Spz (Jang, 2006; Kambris, 2006 and Bouchon, 2009 and references therein).
Genetic analysis supports the existence of several complex cascades of SPs that link microbial recognition to activation of SPE. Pattern-recognition receptors (PRRs) are thought to be present in the hemolymph where they sense microbial-derived molecules. Detection of Gram-positive bacteria is mediated through the recognition of peptidoglycan by the peptidoglycan-recognition protein-SA (PGRP-SA) with the help of Gram-negative binding protein1 (GNBP1). Activation of the Toll pathway by fungi is in part mediated by GNBP3 through the sensing of β-1,3-glucan (Gottar, 2006). RNAi and loss of function analyses have shown that activation of SPE by either PGRP-SA/GNBP1 or GNBP3 requires Grass, a Clip-domain SP (Kambris, 2006; El Chamy, 2008; Bouchon, 2009 and references therein).
Spores of entomopathogenic fungi such as Beauveria bassiana have the capacity to germinate on the fly cuticle and generate hyphae, which can penetrate the cuticle of insects and reach the hemolymph. It has been proposed that the presence of B. bassiana is detected, in a GNBP3-independent manner, through direct activation of the Toll pathway by a fungal protease PR1 (Gottar, 2006). PR1 would directly cleave the host SP, Persephone (Psh), which triggers Toll pathway activation (Gottar, 2006). Recently, this mode of activation was extended to the sensing of proteases produced by various bacteria (El Chamy, 2008). Surprisingly, tracheal melanization in mutant larvae lacking the serpin Spn77Ba also activates the Toll pathway in a Psh-dependent manner (Tang, 2008). This suggests that Psh-dependent Toll pathway activation is induced by a host factor derived from melanization. This also points to a possible cross-talk between the proteolytic cascades that regulate the Toll pathway and those regulating the melanization reaction (Bouchon, 2009).
Despite these recent studies, the extracellular events that lead to the activation of the Toll pathway remain poorly characterized. For instance, the apical protease linking recognition by PRRs to the cleavage of Spz is not known, leaving an important gap in knowledge of Toll pathway activation. The in vitro reconstruction of a similar cascade with purified proteases in the beetle Tenebrio molitor, suggests an important role for a modular SP (Tm-MSP). In this insect, binding of peptidoglycan to the PGRP-SA/GNBP1 complex induces activation of the Tm-MSP zymogen that in turn activates another SP (Tm-SAE) to cleave Tm-SPE, the Tenebrio homolog of SPE, resulting in both activation of Spz and melanization (Kan, 2008; Kim, 2008; Bouchon, 2009 and references therein).
This result prompted an investigation of the role of the Drosophila homolog of Tm-MSP, which is encoded by the CG31217 gene. A null mutation was generated in CG31217 by homologous recombination and its essential role in the activation of Toll by secreted PRRs was demonstrated (Bouchon, 2009).
modSP deficient flies fail to activate the Toll pathway in response to either Gram-positive bacteria or yeast. Epistatic analyses demonstrated that ModSP acts downstream of PGRP-SA, but upstream of the SP Grass. Importantly, ModSP was shown not to participate in the Psh-dependent branch of the Toll pathway as shown by the wild-type activation of this pathway in modSP1 flies upon injection of bacterial proteases. The modSP1 phenotype was very similar to that of a loss of function Grass mutant, suggesting that these SP act in a linear pathway connecting microbe recognition by PRR to the activation of Spz by SPE. The analysis of psh1; modSP1 double mutant flies indicates a synergistic action of the ModSP and Psh pathways in the response against filamentous fungi, which might be detected through both host PRRs and their virulence factors. Collectively, these results confirm the model of an activation of Toll by 2 extracellular pathways: A PRR-dependent pathway and a Psh-dependent pathway (El Chamy, 2008). This model was extended by showing that Grass and ModSP function in a common SP cascade. In addition, the apical position of ModSP suggests a direct branching of signals from secreted PRRs to the ModSP-Grass pathway. Biochemical analyses in T. molitor indicate that Tm-MSP interacts directly with the PRR complexes involved in the sensing of peptidoglycan (Kim, 2008) or glucan (Roh, 2009). Although it was not formally demonstrated in the present study, it seems likely that Drosophila ModSP acts as the most upstream SP directly activated by secreted PRRs (Bouchon, 2009).
ModSP has a number of structural features that make it unique among Drosophila SP and suggest its critical role in the initial events leading to the activation of the proteolytic cascade upstream of Toll. First, ModSP does not contain a Clip-domain in contrast to Grass and SPE. The function of the Clip-domain is still unknown but its presence in many SP that function in signaling cascades suggests a regulatory role (Piao, 2005). This indicates that ModSP activation differs from the chain reaction activating Grass or SPE. Second, ModSP contains additional domains such as the CCP and LDLa motifs in its N-terminal extremity. Consistent with the presence of the LDLa domain, a ModSP-GFP fusion protein was secreted and found bound to the surface of lipid vesicles that circulate in the hemolymph. It is speculated that the association of ModSP to vesicles is important to nucleate the activation of downstream SP (Bouchon, 2009).
In the beetle, T. molitor, Tm-MSP is activated upon formation of a tri-molecular complex composed of PGRP-SA, GNBP1, and lysine-type peptidoglycan (Kim, 2008). Although the possibility of a SP other than ModSP functioning as the initial protease in the Drosophila Toll pathway cannot be ruled out, epistatic analysis suggests an apical role of ModSP. Overexpression of a full-length version of ModSP is sufficient to reach a high level of Toll activation, in contrast to other SP that generally require the overexpression of a preactivated form to fully induce the cascade. These observations favor a model in which ModSP directly interacts with GNBP3 or GNBP1/PGRP-SA recognition complexes. Recruitment of ModSP by PRRs would increase its local concentration, a situation sufficient for its autoproteolysis. In agreement with this hypothesis, a recombinant form of ModSP produced in baculovirus appears to be unstable as a zymogen, presumably because of a high level of autoactivation. Unfortunately, this high level of autoproteolysis did not permit in vitro reconstitution experiments using ModSP, GNBP1, and PGRP-SA as performed with Tm-MSP. Nevertheless, a higher level of ModSP activation was observed when the protein was incubated with GNBP1 and PGRP-SA. The most parsimonious model is that ModSP would interact with GNBP1 or GNBP3, the common protein family members found in the 2 recognition complexes. The exact contribution of GNBP1 to sensing Gram-positive bacteria is currently debated. It was recently proposed that GNBP1 functions upstream of PGRP-SA by cleaving peptidoglycan, a step required for an optimal binding of PGRP-SA to peptidoglycan. In contrast, T. molitor GNBP1 is recruited subsequent to the binding of PGRP-SA to peptidoglycan and is required for the interaction with Tm-MSP. The implication of ModSP in sensing of fungi through GNBP3 and peptidoglycan through GNBP1/PGRP-SA suggests a similar mechanism of activation of this SP and Tm-MSP. This would favor a role of GNBP1 as a linker between PGRP-SA and ModSP. In accordance with this model, neither recombinant full-length Drosophila nor Tenebrio GNBP1 exhibited any enzymatic activity toward peptidoglycan in vitro (Bouchon, 2009).
The participation of ModSP and SPE in an extracellular pathway linking PRR recognition to Spz activation in both T. molitor (Coleoptera) and D. melanogaster (Diptera), which diverged ~250 million years ago, demonstrates the conservation of this extracellular signaling module in insects. In the lepidopteran Manduca sexta, the hemolymph protein 14 (Ms-HP14) contains a domain arrangement very similar to that of ModSP and regulates the melanization cascade in response to microbial infection. Collectively, biochemical studies performed in T. molitor and M. sexta, and genetic analysis in D. melanogaster, reveal striking similarities in the mechanisms underlying SP activation by PRRs. All involve the sequential activation of an apical modular SP (that displays a certain level of autoactivation) and clip-domain proteases. Interestingly, a similar organization is also observed in the proteolytic cascade that regulates Toll during dorso-ventral patterning of the embryo. Gastrulation Defective, the apical SP, contains von Willebrand domains and is also thought to be autoactivated, although the precise mechanism that triggers its activation has not been determined. These features are also reminiscent of the complement activation by the lectin pathway in mammals in which the recognition of carbohydrate by the mannose binding lectin (MBL) leads to the autoactivation of MBL-associated serine proteases (MASPs). MASPs are also modular proteases with CUB, CCP, and EGF domains in their N terminus. Thus, genetic and biochemical analyses now reveal a similar level of organization for various proteolytic cascades in insects and increase understanding as to how these cascades have evolved to fulfill diverse developmental or immune functions (Bouchon, 2009 and references therein).
A complete understanding of the precise sequence of events leading to Toll pathway activation is still in the future, because the task is complicated by the high number of SP encoded in the Drosophila genome (Ross, 2003). The analysis described in this paper strongly suggests the existence of an intermediate SP acting between ModSP and Grass. Future works should identify this SP and determine the interaction between Grass and SPE. To date, no serpin regulating SP involved in the PRR-dependent pathways has been identified despite the critical role of this family in the negative control of proteolytic cascades. Further experiments combining genetics, biochemistry, and cell biology are required to identify additional components of this cascade and to clarify in vivo how and where proteolytic cascades downstream of PGRP-SA or GNBP3 are activated in the hemolymph compartment (Bouchon, 2009).
The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-kappaB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Three serine proteases have been identified mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component beta-1,3-glucan of fungi, and their order of activation have not been characterized yet. This study has identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, it was shown that injection of beta-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal beta-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation (Roh, 2009).
This study reports three novel findings regarding the β-1,3-glucan recognition signaling pathway in T. molitor larvae. This study shows the following: (1) Tenebrio MSP functions apically downstream of GNBP3; (2) the β-glucan recognition signal is transferred via the sequential activation of the three Tenebrio serine proteases, namely modular serine protease (MSP), Spätzle-processing enzyme-activating enzyme (SAE), and Spätzle-processing enzyme (SPE), and that this results in the processing of pro-Spätzle to its mature form; and (3) the activation of the GNBP3/MSP/SAE/SPE/Spätzle module cascade regulates the expression of two AMPs, Tenecin 1 and Tenecin 2. Collectively, these results demonstrate that the Tenebrio GNBP3/MSP/SAE/SPE/Spätzle cascade is an essential unit involved in the recognition of fungi and in the activation of Toll-mediated antimicrobial defense (Roh, 2009).
The current work supports a model in which Lys-type PG and β-glucan activate a common set of three serine protease zymogens sequentially. This three-step proteolytic cascade-dependent processing of the extracellular pro-Spätzle produces active Spätzle, which then binds to the Toll receptor, resulting in the induction of AMP expression in the Tenebrio larval hemocytes. Each of the three serine proteases has unique biochemical properties to regulate activation of the Tenebrio Toll pathway. The initial enzyme pro-MSP is an 82-kDa serine protease zymogen with an N-terminal chymotrypsin-like cleavage site and a C-terminal chymotrypsin-like catalytic serine protease domain. Pro-SAE is a 41-kDa serine protease with an N-terminal chymotrypsin cleavage site and a C-terminal trypsin-like catalytic serine protease domain, and pro-SPE is a 44-kDa protein with an N-terminal trypsin-like cleavage site and a C-terminal trypsin-like catalytic serine protease domain. These three different serine protease combinations enhance the specificity of the proteolytic serine protease cascade and prevent nonspecific cleavage of these serine protease zymogens (Roh, 2009).
Recent genetic studies using either in vivo RNAi strategy or loss-of-function mutation have demonstrated that several Drosophila serine proteases, such as Persephone, Grass, and Spirit, function upstream of the Drosophila SPE during activation of Toll. Furthermore, Drosophila Grass functions as a signaling component required for both the detection of fungi by GNBP3 and the sensing of Gram-positive bacteria by PGRP-SA. Also, it has been suggested that another Drosophila serine protease, Persephone, previously shown to be specific for fungal detection, is required for the sensing of proteases that are released by fungi and bacteria. This indicates that Drosophila Persephone defines a parallel proteolytic cascade activated by virulence factors in the hemolymph. However, Drosophila Grass and Persephone genes do not show any homology with Tenebrio MSP, which functions as an apical serine protease common to both Tenebrio GNBP3 and PGRP-SA/GNBP1 complex. These results suggest the existence of significant differences in the proteolytic cascades working upstream of Toll in Tenebrio and Drosophila. In addition to this diversity of serine proteases, the gene expression sites of Tenebrio AMPs are also different from those of Drosophila AMPs. The Tenebrio Tenecin 1 gene was strongly induced in hemocytes of the Tenebrio larvae, while Drosophila AMPs are mainly produced by the fat body. Taken together, the differences in the organization of the serine protease cascade, in the type of AMPs and in the site of AMP synthesis between Drosophila and Tenebrio could represent adaptation mechanisms to fight specific environmental pathogens or may be linked to differences in their physiology. Analyzing differences and similarities in the mechanisms used by insects to fight microbial infection should shed light on the evolution of the immune system in this important phylum (Roh, 2009).
Recently, biochemical evidence has been provided that the activated Tenebrio SPE converted both the 79-kDa Tenebrio pro-phenoloxidase and clip-domain serine protease homologue 1 zymogen to their matured forms to generate an active melanization complex. This complex, consisting of a 76-kDa Tenebrio-activated phenoloxidase and a 43-kDa serine protease homologue 1, efficiently produced melanin on the surface of bacteria, and this activity had a strong bactericidal effect. Because Tenebrio SPE also participates in the activation of the Toll pathway by GNBP3 upon β-1,3-glucan sensing and by PGRP-SA upon Lys-type PG recognition, the results indicate that both the melanization and the Toll pathway immune modules are sharing a common serine protease cascade for the regulation of these two major innate immune responses (Roh, 2009).
In summary, these biochemical studies shed light on the molecular mechanism regulating the Toll pathway by fungi. This work supports a model in which bacterial Lys-type PG and β-1,3-glucan recognition activate a common proteolytic cascade involving three different serine protease zymogens that are sequentially processed. This three-step proteolytic cascade leads to the maturation of Spätzle and the activation of the Toll intracellular signaling that control the synthesis of AMPs. A greater understanding of these two cascades could also facilitate the development of novel kits to detect bacteria and fungi in clinical and food products rapidly and sensitively (Roh, 2009).
The Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-kappaB-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. This study showed that the recombinant N-terminal domain of the mealworm beetle Tenebrio molitor GNBP3 binds to beta-1,3-glucan, but does not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocks GNBP3-mediated serine protease cascade activation in vitro and also inhibits beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation (Lee, 2009).
Gram-negative binding protein 3 (GNBP3), a pattern recognition receptor that circulates in the hemolymph of Drosophila, is responsible for sensing fungal infection and triggering Toll pathway activation. This study reports that GNBP3 N-terminal domain binds to fungi upon identifying long chains of beta-1,3-glucans in the fungal cell wall as a major ligand. Interestingly, this domain fails to interact strongly with short oligosaccharides. The crystal structure of GNBP3-Nter reveals an immunoglobulin-like fold in which the glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long chain structured polysaccharides. This represents a novel mechanism for beta-glucan recognition (Mishima, 2009).
The discrimination between host and microbe-associated molecules is crucial to the function of PRRs. Short oligosaccharide chains may not constitute an ideal target for PRRs as they might also be displayed by host cells and, thus, may not represent a bona fide microbial signature. Therefore, it is likely that the host selected PRRs able to sense long glucan chains idiosyncratic to most fungal cell walls. This study reports that the glucan binding domain of GNBP3 binds preferentially to long β-1,3-glucan chains (Mishima, 2009).
The N-terminal domain of GNBP3 binds to the cell wall of C. albicans and most likely to β-glucans as indicated by the preferential binding to growing cell buds and bud scars, a pattern evocative of that of Dectin-1. Indeed, the recombinant protein binds to the cell wall alkali-insoluble polysaccharide fraction of S. cerevisiae and A. fumigatus. The latter induces a GNBP3-dependent activation of the Toll pathway when injected into Drosophila. These data indicate that the relevant biochemical moiety of these fungal cell wall alkali-insoluble (AI) extracts are β-1,3-glucan chains. The longer the glucan chain, the more efficient is the competition. Efficient binding to GNBP3-Nter is observed with polymeric chains that incorporate more than 16 glucan units. In keeping with this result, it has previously been shown that injection in Drosophila of the alkali-insoluble fraction of the A. fumigatus cell wall, which consists of long polysaccharides including β-1,3-glucans, induces a strong activation of the Toll pathway. Short laminarioligosaccharides with a degree of polymerization (DP) ranging from 2 to 7 failed to induce Toll pathway activation when injected into flies. β-1,6-Branching in the linear chain of β-1,3-glucans does not appear to be required for recognition by GNBP3-Nter since strong binding was not observed with schizophyllan, a highly β-1,6-branched β-1,3-glucan from Schizophyllum commune. Interestingly, the glucan binding properties of the mammalian fungal receptor Dectin-1 have been reported to be fairly similar, with a minimum degree of polymerization of 11 required for β-glucan binding (Mishima, 2009).
The GNBP3-Nter crystal structure was solved, providing structural insight into the β-1,3-glucan recognition protein (βGRP) family. The overall structure displays an immunoglobulin-like fold similar to that of the fibronectin III superfamily. Although no solvent-exposed aromatic patch is present on GNBP3-Nter, Tyr-75, Trp-77, and Tyr-79 are good candidates to constitute such a binding platform after a structural rearrangement of the loop located between strands C and C'. In keeping with this hypothesis, it was found that the binding to curdlan was strongly impaired, and the binding to laminarin was completely abolished with the W77A mutant, thus underscoring the importance of this initially buried residue for glucan binding (Mishima, 2009).
The essential role of the C-C' loop in terms of binding and discrimination between short and long chains of β-glucan was confirmed by mutagenesis. To free access to the binding site, the C-C' loop should fold back toward the C-terminal domain of GNBP3. Tyr-79 may act as a primary determinant that anchors β-glucan polymers. Then the negatively charged patch formed by the four glutamic acids on the top of the loop may be expulsed by the vicinity of a large sugar surface, unmasking the rest of the binding site (Tyr-75 and Trp-77). After the lid opening, the side chain of Tyr-75 is free to re-orientate toward Trp-77. Like this, the relative spacing between the three residues would not stretch beyond a distance required to accommodate a disaccharide and, thus, would be very comparable with that of starch binding domains. The internal hydrophobic surface of the lid could hardly be fully-exposed to solvent in the open conformation and may probably interact with the ligand. This putative interaction between the lid and the ligand may explain the results obtained for the short-loop mutant. Namely, this mutant does not appear to bind efficiently to long-chain oligosaccharides, possibly because the two conserved hydrophobic residues in the lid, which are missing in the mutant, no longer stabilize the interaction (Mishima, 2009).
Both the sequence of the C-C' loop and that of the putative binding site are conserved in βGRP family members that have been reported to bind to β-glucans. Noticeably, these sequences are not conserved in Drosophila melanogaster GNBP1 (and GNBP1 of other Drosophila species), a member of the family required in the host defense against Gram-positive bacteria. It is inferred that the GNBP1 N-terminal domain will not bind significantly to β-1,3-glucans, even though some studies have reported some binding of full-length GNBP1 to curdlan. Thus, the sequences of the C-C' loop and of the glucan binding site may be useful predictors of the function of uncharacterized GNBP/βGRP family members. Using this criterion, it is predicted that the function of the funding member of the GNBP family, B. mori p50, is not involved in defense against fungi, at least by a GNBP3/βGRP-like mechanism. In any case, GNBP full-length proteins, with their glucanase-like domains, are likely to have emergent properties not displayed by the Nter domain alone. These may include the activation of downstream proteolytic cascades (for Toll pathway and prophenol oxidase activation) and, obviously, agglutination, which requires two sugar binding domains in the protein (Mishima, 2009).
An intriguing feature of the GNBP3-Nter domain is its capacity to discriminate between short and long chains of β-glucans. Many PRRs activate an immune response only when bound to long chains of carbohydrates through the use of spatially arranged multiple subunits or multimers. Yet, in striking contrast to GNBP3, the individual domains involved in carbohydrate recognition can bind to monomeric or short carbohydrate polymers. For instance, PGRP-SA, which binds to PGN muropeptide monomers as single molecules, requires the formation of PGRP-SA clusters on longer chains to trigger downstream proteolytic cascades. A similar case is presented by Factor G of the Japanese horseshoe crab where a laminariheptaose is required to activate the coagulation cascade even though it binds well to laminaribiose with an affinity that is only three times lower. Interestingly, the recognition domain that binds to β-glucans is actually made up of two carbohydrate binding subunits arranged in a tandem repeat. Only the tandem repeat, and not each individual subunit, is able to bind to the disaccharide. Another example of the importance of the spatial arrangements of multiple carbohydrate recognition domain (CRD) is provided by the mannose-binding lectin whereby each CRD head binds to a single sugar residue (mannose or fucose). Activation only occurs when the multiple heads arranged in a bouquet-like structure of trimers bind to an array of sugar residues present on the microbial but not the host cell surface. This study proposes that the lid of the GNBP3-Nter domain that masks the carbohydrate binding site is displaced only by long chains of β-glucans. In this respect, the structures of laminarins and curdlan as triple helices in an aqueous environment may be an essential feature that triggers the opening of the carbohydrate binding site and the recognition of the fibrillar fungal structure (Mishima, 2009).
The Drosophila Toll-signaling pathway controls the systemic antifungal host response. Gram-negative binding protein 3 (GNBP3), a member of the beta-glucan recognition protein family senses fungal infections and activates this pathway. A second detection system perceives the activity of proteolytic fungal virulence factors and redundantly activates Toll. GNBP3(hades) mutant flies succumb more rapidly to Candida albicans and to entomopathogenic fungal infections than WT flies, despite normal triggering of the Toll pathway via the virulence detection system. These observations suggest that GNBP3 triggers antifungal defenses that are not dependent on activation of the Toll pathway. This study shows that GNBP3 agglutinates fungal cells. Furthermore, it can activate melanization in a Toll-independent manner. Melanization is likely to be an essential defense against some fungal infections given that the entomopathogenic fungus Beauveria bassiana inhibits the activity of the main melanization enzymes, the phenol oxidases. Finally, GNBP3 was shown to assemble 'attack complexes', which comprise phenoloxidase and the necrotic serpin. It is proposed that Drosophila GNBP3 targets fungi immediately at the inception of the infection by bringing effector molecules in direct contact with the invading microorganisms (Matskevich, 2010).
This report investigated the defense mechanisms that rely on the fungal sensor GNBP3 independently of GNBP3's role in Toll-pathway activation. Several mechanisms have been found that may account, at least partially, for the increased sensitivity of GNBP3 mutants to fungal infections. Glucan recognition protein (GRP) from the lepidopteran Plodia interpunctella contains two glucan-binding domains that are required for efficient agglutination of microorganisms. This study found that GNBP3 is both necessary and sufficient for extensive agglutination of fungi, a property that may be linked to its potential role as a bifunctional sugar cross-linking agent. It has been proposed that agglutination of microorganisms may help in triggering or amplifying a cellular response. However, no evidence was found in Drosophila that phagocytosis plays a major role in the control of C. albicans infections, whether in a WT or sensitized background, although the yeasts were ingested in vivo (Matskevich, 2010).
It is proposed that agglutination is a mechanism that limits the initial phase of the infection process until the systemic response provides a more effective killing mechanism. It may do so by limiting the dissemination of the invading fungus in the hemocoel. The Toll-dependent systemic response is strictly required since most Toll-pathway mutants succumb earlier to fungal infections, although GNBP3 is present in these mutants (Matskevich, 2010).
Prophenoloxidases (POs) are widely distributed in invertebrates. The PO-mediated reactions are also believed to release ROS that may participate in the killing of microorganisms. Biochemical studies on several insect and crustacean models have led to a model of PO activation, in which the proteolytic cleavage of PO is mediated by dedicated proPO-activating enzymes (PAE) and their cofactors. These proteases would themselves be activated by pattern recognition receptors (PRR), including PGRP and GRP, upon the detection of the presence of microorganisms in the hemocoel. An interesting twist was provided by the genetic analysis of PO activation in Drosophila: the activation of PAE lead to the depletion of serpin SPN27A, which is a suicide inhibitor of at least one PAE in the PO activation cascade. As a result of complex degradation, the endogenous pool of SPN27A becomes depleted and this SPN is no longer detected in the hemolymph. The degradation of spn27A depends on new protein synthesis and requires a functional Toll pathway. Toll-pathway activation would lead to the production of either a PAE or a cofactor, which would first deplete SPN27A and then activate PO. These studies suggested a strict requirement for Toll-pathway activation for full maturation of PO to its active form, yet they did not address whether the activation of the Toll intracellular signaling cascade is sufficient to trigger PO processing. An RNAi screen was performed for suppressors of the SPN27A melanization phenotypes and two serine proteases, MP1 (CG1102) and MP2 (CG3066), were identified. MP1 acts downstream of MP2 to activate PO activity and thus is a likely proPO-activating enzyme. Activated forms of MP1 and MP2 lacking their Pro-domains activate the PO cascade independently of the Toll pathway, suggesting that they directly act in the hemolymph to ultimately process ProPO. This study uncoupled the activation of PO from that of the Toll pathway by analyzing GNBP3hades mutants. The significant cleavage of PO that was observed in this mutant is due to Toll signaling and not to an unknown PRR that would function redundantly with GNBP3 to activate the PAE cascade since PO cleavage is abolished in the psh; GNBP3hades double-mutant combination. Nevertheless, GNBP3 is required for full PO activation since PO activity is decreased by 50% in the cognate mutant. This observation reflects a requirement for GNBP3 in directly initiating the PAE cascade in a Toll-independent manner, as in other insects. Indeed, PO can be activated by GNBP3 in a Toll-pathway-independent manner by overexpressing GNBP3 in a spz background. Toll signaling is indeed blocked in this genetic combination, which prevents the immune challenge-independent expression of Drosomycin induced by the overexpression of GNBP3. Overexpressing GNBP3 resulted in only a 10-15% activation of PO in the absence of a septic injury (Matskevich, 2010).
This observation suggests that a wound is required for full PO activation, either through the contact with oxygen, coagulation, or the sensing of cuticle damage (Matskevich, 2010).
The PRR involved in Toll-pathway activation after Gram-positive bacteria infection, namely GNBP1 and PGRP-SA, are also required for full PO activation. Indeed, mutations in GNBP1 (osiris) or PGRP-SA (semmelweiss (PGRP-SA mutant)) reduced significantly PO activity after M. luteus infection. Strikingly, the PO activity is not totally blocked in the double mutant GNBP1/PGRP-SA. As was observed with GNBP3, PO can be activated by the GNBP1 and PGRP-SA complex in a Toll-pathway-independent manner by overexpressing the complex in a spz background (Matskevich, 2010).
It is concluded from these experiments that in a WT setting PO activation occurs both through Toll-pathway activation and through GNBP3-dependent direct PO activation, likely via the activation of the PAE proteolytic cascade. It is proposed that the PRR-dependent activation of the PO cascade plays its role immediately at the site of pathogen entry while a sustained activation of the PO system would rely on the systemic activation of the Toll pathway to overcome SPN27A or SPN28D inhibition (Matskevich, 2010).
An unexpected finding of this study is the partial association of GNBP3 with proPO and PO, in nonchallenged and challenged flies, respectively. This link is not due to nonspecific PO adhesion to GNBP3 since the binding of GNBP3 to PO is no longer observed after a B. bassiana natural infection. This may represent a means to concentrate proPO rapidly in the vicinity of the invading fungus. In this manner, reactive compounds like ROS may be generated only in close proximity to the microorganisms while at the same time limiting the exposure of the host to potentially toxic compounds. This strategy may be widespread in insects. Indeed, it has been reported in Manduca sexta that on the one hand the auxiliary cofactor serine protease homolog copurifies with the microbial lectin receptor immulectin-2, whereas on the other hand it forms a ternary complex with proPO and its activating protease. In the mosquito Armigeres subalbatus and in the coleopteran T. molitor, PO is deposited on microorganisms such as E. coli or M. luteus. Interestingly, T. molitor serine protease homolog links PO to the surface of bacteria, a function taken in Drosophila by GNBP3 as regards fungi. The observation that the entomopathogenic fungus B. bassiana is able to disrupt the association between PO and GNBP3, possibly through the expression of dedicated virulence factors, suggests that this complex effectively thwarts common fungal infections. There is a controversy as to whether B. bassiana triggers the PO cascade and plays a role in host defense against this pathogen. This study also failed to detect any PO activation. It is possible that the B. bassiana strain used in a previous study does not express the virulence factor that would dissociate the PO-GNBP3 complex, and thus explaining the partial sensitivity of PAE mutants to their strain. In another study, a cuticular melanization reaction was observed when larvae were naturally infected with B. bassiana but no melanization of trachea exposed to this fungus was observed either in vivo or ex vivo, whereas an ex vivo challenge with bacteria induced a strong melanization of trachea. These latter observations are compatible with an inhibition of PO activation by B. bassiana (Matskevich, 2010).
The association of GNBP3 with the Necrotic serpin (NEC SPN) is also surprising, since NEC is thought to be involved in the control of the PSH branch of fungal detection, which appears to be mostly independent from the GNBP3 branch. However, the function of nec may not be limited to the regulation of the PSH proteolytic cascade. One possibility is that this SPN, which is induced during the immune response, may also inhibit fungal proteases. The observation that GNBP3 associates with PO and NEC, as well as similar observations for GNBP1, lead to a proposal that one function of GNBP is to assemble a functional attack complex that will target invading spores, thus maximizing the efficiency and the speed of the response while limiting deleterious effects to the host. The rapid containment of microorganisms before they have the time to multiply may be critical in the host defense against pathogens (Matskevich, 2010).
Genetic analysis suggests that GNBP3 has Toll-independent functions required to fight fungal infections. This study has analyzed several mechanisms which may represent such functions, namely agglutination, PO activation, and recruitment of an effector complex. At present, it is not possible to tell which one is the most important in a physiological setting. A careful structure/function analysis of the full length GNBP3 will be required to resolve this issue. Future studies in Drosophila and other insects will determine whether GNBP/GRP have evolved from primarily effector molecules into microbial sensors recruited later in the evolution of brachycera flies for the activation of the Toll pathway (Matskevich, 2010).
Search PubMed for articles about Drosophila GNBP3
Bagga, S., et al. (2004). Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene 324: 159-169. PubMed ID: 14693381
Bischoff, V., et al. (2004). Function of the Drosophila pattern-recognition receptor PGRP-SD in the detection of Gram-positive bacteria. Nat. Immunol. 5: 1175-1180. PubMed ID: 15448690
Brown, G. D. and Gordon, S. (2001). Immune recognition. A new receptor for beta-glucans. Nature 413: 36-37. PubMed ID: 11544516
Buchon, N., Poidevin, M., Kwon, H. M., Guillou, A., Sottas, V., Lee, B. L., Lemaitre, B. (2009). A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway. Proc. Natl. Acad. Sci. 106(30): 12442-12447. PubMed ID: 19590012
El Chamy, L., Leclerc, V., Caldelari, I. and Reichhart, J. M. (2008). Sensing of danger signals and pathogen-associated molecular patterns defines binary signaling pathways 'upstream' of Toll. Nat. Immunol. 9: 1165-1170. PubMed ID: 18724373
Ferrandon, D., Imler, J. L. and Hoffmann, J. A. (2004). Sensing infection in Drosophila: Toll and beyond. Semin. Immunol. 16: 43-53. PubMed ID: 14751763
Gobert, V., et al. (2003). Dual activation of the Drosophila Toll pathway by two pattern recognition receptors. Science 302: 2126-2130. PubMed ID: 14684822
Gottar, M., et al. (2006). Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 127: 1425-1437. PubMed ID: 17190605
Jang, I. H., et al. (2006). A Spatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity. Dev. Cell 10: 45-55. PubMed ID: 16399077
Kambris, Z., et al. (2006). Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation. Curr. Biol. 16: 808-813. PubMed ID: 16631589
Kan, H., et al. (2008). Molecular control of phenoloxidase-induced melanin synthesis in an insect. J. Biol. Chem. 283: 25316-25323. PubMed ID: 18628205
Kaneko, T. and Silverman, N. (2005). Bacterial recognition and signalling by the Drosophila IMD pathway. Cell. Microbiol. 7: 461-469. PubMed ID: 15760446
Kaneko, T., et al. (2006). PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila immune response to monomeric DAP-type peptidoglycan. Nat. Immunol. 7: 715-723. PubMed ID: 16767093
Kim, C. H., et al. (2008). A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect. J. Biol. Chem. 283: 7599-7607. PubMed ID: 18195005
Kim, M. G., et al. (2005). Two Pseudomonas syringae type III effectors inhibit RIN4-regulated basal defense in Arabidopsis. Cell 121: 749-759. PubMed ID: 15935761
Kim, Y. S., et al. (2000). Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells. J. Biol. Chem. 275: 32721-32727. PubMed ID: 10827089
Lee, M. H., et al. (2003). Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect. J. Biol. Chem. 279: 3218-3227. PubMed ID: 14583608
Lee, H., et al. (2009). N-terminal GNBP homology domain of Gram-negative binding protein 3 functions as a beta-1,3-glucan binding motif in Tenebrio molitor. BMB Rep. 42(8): 506-10. PubMed ID: 19712587
Lemaitre, B., Reichhart, J. M. and Hoffmann, J. A. (2007). Drosophila host defense: differential display of antimicrobial peptide genes after infection by various classes of microorganisms. Proc. Natl. Acad. Sci. 94: 14614-14619. PubMed ID: 9405661
Levashina, E. A., et al. (1999). Constitutive activation of Toll-mediated antifungal defense in serpin- deficient Drosophila. Science 285: 1917-1919. PubMed ID: 10489372
Ligoxygakis, P., et al. (2002). Activation of Drosophila Toll during fungal infection by a blood serine protease. Science 297: 114-116. PubMed ID: 12098703
Matskevich, A. A., Quintin, J. and Ferrandon, D. (2010). The Drosophila PRR GNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll-pathway activation function. Eur. J. Immunol. 40(5): 1244-1254. PubMed ID: 20201042
Mishima, Y., et al. (2009). The N-terminal domain of Drosophila Gram-negative binding protein 3 (GNBP3) defines a novel family of fungal pattern recognition receptors. J. Biol. Chem. 284(42): 28687-97. PubMed ID: 19692333
Piao, S., et al. (2005). Crystal structure of a clip-domain serine protease and functional roles of the clip domains. EMBO J. 24: 4404-4414. PubMed ID: 16362048
Roh, K. B., et al. (2009). Proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component. J. Biol. Chem. 284(29): 19474-81. PubMed ID: 19473968
Rooney, H. C., et al. (2005). Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2-dependent disease resistance. Science 308: 1783-1786. PubMed ID: 15845874
Ross, J., Jiang, H., Kanost, M. R. and Wang, Y. (2003). Serine proteases and their homologs in the Drosophila melanogaster genome: An initial analysis of sequence conservation and phylogenetic relationships. Gene 304: 117-131. PubMed ID: 12568721
Shao, F., et al. (2003). Cleavage of Arabidopsis PBS1 by a bacterial type III effector. Science 301(5637): 1230-3. PubMed ID: 12947197
Tang, H., Kambris, Z., Lemaitre, B. and Hashimoto, C. (2008). A serpin that regulates immune melanization in the respiratory system of Drosophila. Dev Cell 15: 617-626. PubMed ID: 18854145
Wang, C., Hu, G. and St Leger, R. (2005). Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation. Fungal Genet. Biol. 42: 704-718. PubMed ID: 15914043
date revised: 30 April 2010
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