Toll and IMD Signaling Pathways after Bacterial or Fungal Infection
Activation and regulation of Drosophila Toll and IMD signaling pathways Classical deletion-mapping studies on the cecropin A1 and diptericin genes began in 1990, to answer the question of how insect AMP proteins are strongly induced or upregulated by microbial infection. The unique DNA sequences (GGGGATTYYT) identified in the cecropin A1 and diptericin promoter regions are specifically recognized by the Relish family of transcription factors, which belong to the NF-kB protein family. This discovery led to the detection of this DNA motif in all other AMP genes (Engstrom et al., 1993). Based on these Drosophila genome sequences, three different Relish family genes, Dorsal, Dif (Dorsal-related immunity factor), and Relish, were targeted for mutagenesis screening (Hetru and Hoffmann, 2009). Dif was shown to be essential for the induction of several Drosophila genes when flies were challenged with Gram-positive bacteria and fungi. Namely, Dif mutant flies strongly reduced the expression of drosomycin and defensin genes after microbial infection. The Relish gene was also induced in infected flies. The Relish protein contains two domains, an N-terminal Rel domain and a C-terminal iKB-like ankyrin repeat domain. Upon infection, Relish was rapidly processed by the caspase Dredd molecule into two peptides: the Relish homology domain, which is translocated to the nucleus, and the ankyrin repeat domain, which remains cytosolic. This processing is essential for the expression of Drosophila AMPs. Dorsal-mutant larvae and flies exhibited a normal response to bacterial infection challenges. These basic studies provided important clues regarding the key roles the Relish family transcription factors play in the regulation of insect AMP genes via the Toll and IMD pathways.
The Toll pathway, named for the Toll transmembrane-associated receptor, was first genetically characterized for its role in the establishment of dorso-ventral polarity during Drosophila embryo development (Belvin and Anderson, 1996). The breakthrough genetic evidence of this pathway in insect immunology was reported between 2001 and 2003 by the group of Jules Hoffmann, which screened flies with mutant Drosophila PGRP-SA encoded by the gene semmelweis, and |-1,3-glucan recognition protein (|GRP)/Gram-negative binding protein-1 (GNBP1) encoded by the gene osiris (Michel et al., 2001; Gobert et al., 2003). Unexpectedly, loss-of-function mutations in either the PRGP-SA or the GNBP1 gene showed very similar phenotypes of compromised survival to Gram-positive bacterial infection, indicating that these two hemolymph proteins cooperate to sense Gram-positive bacteria and are essential for the activation of proteolytic enzymes(s) that cleave Spatzle, a Toll-receptor ligand. Conversely, when these two genes were overexpressed together, flies induced the activation of the Toll pathway even in the absence of a bacterial challenge (Gob-ert et al., 2003). Hoffman’s group also demonstrated that activation of Toll by fungal infection is independent of the semmelweis and osiris genes. A further genetic screen has identified the persephone gene, which encodes a hemolymph trypsin-like serine protease that mediates the fungal-dependent cleavage of Spatzle and the activation of Toll. Overexpression of the persephone gene is sufficient to lead to Spatzle-dependent induction of the Toll pathway in the absence of an immune challenge (Ligoxygakis et al., 2002a). Lately, Ferrandon and his colleagues have studied flies with mutant GNBP3, a protein encoded by the gene hades. They found that GNBP3 is a pattern-recognition receptor that is required for the detection of P-1,3-glucan, a fungal cell wall component. They also found a parallel pathway that acts jointly with GNBP3 (Gottar et al, 2006). Specifically, when Drosophila persephone protease is proteolytically matured by the secreted fungal virulence protease PR1, it activates the Toll pathway. Thus, the detection of fungal infections in Drosophila relies both on the recognition of P-1,3-glucan, an invariant microbial cell wall component, and on the effects of virulence factors such as PR1 protease (Figure 2).
As shown in Figure 2, the Drosophila Toll signaling pathway is divided into three steps: (1) extracellular recognition of invading Gram-positive bacteria or fungi by the PGRP-SA/GNBP1 complex or GNBP3, respectively, and signaling amplification step by several serine proteases, leading to the cleavage of pro-Spatzle; (2) activation via binding between a Toll ligand, processed Spatzle and a Toll receptor; and (3) intracellular activation and the expression of effector molecules. Specifically, upon recognition of invading Gram-positive bacteria or fungi, recognition signals are amplified by the serine protease proteolytic cascade, as in mammalian blood coagulation or complement activation cascades (Krem and Di Cera, 2002). It has been suggested that the activation of pro-Spatzle is achieved by a set of serine proteases distinct from those involved in Toll activation during embryonic development. Recently, Lemaitre and colleagues identified modular serine protease (ModSP), the most upstream serine protease of Drosophila Toll cascade (Buchon et al., 2009). They demonstrated that Drosophila ModSP integrates signals originating from the GNBP3 and PGRP-SA/GNBP1 complex, and connects them to the downstream serine proteases. The terminal serine protease that processes Spatzle has been identified, and named Drosophila Spatzle-processing enzyme (SPE) (Jang et al., 2006). Even though several Drosophila clip-domain-containing serine proteases, such as Grass and Spirit, which are suggested to act between ModSP and SPE, have been identified, the biochemical functional studies of these serine proteases are not yet complete (Kambris et al., 2006).
Upon binding of the cleaved Spatzle to the Toll receptor, the production of AMPs is induced from the fat body (Lemaitre and Hoffmann, 2007; Weber et al, 2003). The Spatzle—Toll complex recruits a set of downstream molecules. First, Toll/IL-1 Receptor (TIR) and/or the death-domain-containing adaptor molecules dMyD88 and Tube lead to the activation of the kinase Pelle. Pelle induces the degradation of Cactus, an inhibitor that maintains the cytoplasmic localization of Dif and Dorsal, two transactivators of the NF-kB family. The transloca-tion of these transcription factors induces the expression of several immune genes, including AMP genes, through binding to the kB DNA motifs of their promoter.
The IMD pathway is named after the first Dro-sophila mutant flies, called immune deficiency (IMD), which are susceptible to Gram-negative bacteria infection, but are more resistant to fungi and Gram-positive bacteria infection. The imd gene encodes a death-domain-containing protein similar to that of the receptor interacting protein (RIP) of the mammalian tumor necrosis factor receptor (TNFR) pathway (Georgel et al., 2001). In 2002, three groups simultaneously reported the identity of the upstream receptor molecule of the Drosophila IMD pathway: PGRP-LC, a putative transmembrane protein that is required for the activation of the Gram-negative-mediated IMD pathway (Choe et al., 2002; Gottar et al., 2002; Ramet et al., 2002). Compared to the IMD null mutant, loss-of-function mutants of PGRP-LC showed less severe phenotypes, suggesting that PGRP-LC is only one of several receptors that sense Gram-negative bacteria. Kurata and colleagues reported that Drosophila PGRP-LE, another member of the PGRP family, activates the IMD pathway when this gene is overexpressed (Takehana et al., 2002). Taken together, these studies showed that PGRP-LC and PGRP-LE function as receptor molecules for the activation of the Gram-negative bacteria-mediated IMD pathway (Figure 2).
The intracellular signaling mechanism of the IMD pathway, mostly determined by genetic studies, is summarized as follows (Lemaitre and Hoffmann, 2007) (Figure 2): upon Gram-negative bacterial infection, PGRP-LC recruits the IMD protein, which interacts with the adaptor molecule dFADD via death-domain interaction. The dFADD then recruits the Dredd caspase, which is proposed to associate with Relish. After cleavage of the Relish protein, the Relish transactivator domain translocates to the nucleus, while the inhibitory domain of Relish remains in the cytoplasm. Relish that was phos-phorylated by the inhibitory KB-kinase signaling complex is proposed to be activated by Tak1 and its adaptor TAB2 in the IMD pathway. The molecular mechanisms behind the dimerization of PGRP-LC and the activation of the downstream signal pathway by the ligand-recep-tor complex have not yet been determined.
The serpins belong to a superfamily of serine protease inhibitors that act as suicide substrates by binding cova-lently to their target proteases. Serpins are known to regulate various physiological processes and defense reactions in mammals and invetrebrates (Reichhart, 2005; Silverman et al., 2010; Whisstock et al, 2010). To date, four Drosophila serpins related to innate immunity (SPN43Ac, SPN27A, SPN77Ba, and SPN28D) have been analyzed in detail by genetic approaches.
Figure 2 Three major Drosophila signaling pathways that regulate systemic immune responses against bacterial, fungal and viral infection. (A) The Toll pathway is activated by the Lys-type peptidoglycan of Gram-positive bacteria and p-1,3-glucan of yeast and some fungi through two different pattern recognition proteins, such as PGRP-SA/GNBP1 and GNBP3, respectively. These recognition signals initiate the activation of the proteolytic serine protease cascade via ModSP, Grass, and Spatzle-processing enzyme (SPE), which are clip-domains containing serine protease, leading to cleavage of pro-Spatzle to the processed Spatzle by activated SPE. Upon binding processed Spatzle to Toll, dimerized Toll recruits dMyD88, Tube, and Pelle, resulting in the phosphorylation and proteosomal degradation of Catus. Catus degradation induces the translocation of Rel transcriptional factors, Dif and Dosal, to the nucleus. These factors bind to NF-kB-response elements and induce activation of transcription of AMP genes, such as Drosomycin. The virulence factor of fungi, such as PR1 protease, is also suggested to activate pro-SPE to activated SPE, leading to the cleavage of pro-Spatzle to the processed Spatzle. (B) The polymeric and monomeric DAP-type peptidoglycans of Gram-negative bacteria are recognized by PGRP-LE and PGRP-LCs. These recognition signals are transferred to the IMD, which is localized in cytoplasm. Upon binding of monomeric DAP-type peptidoglycan to PGRP-Lcx/Lca heterodimer or polymeric DAP-type peptidoglycan to PGRP-Lcx homodimer, IMD is recruited by the intracellular domain of PGRP-LCs. IMD then recruits dFADD and caspase Dredd, which cleave the phosphorylated Relish. leading to translocation of Rel domain to nucleus, where the Rel domain binds to NF-kB response elements and activates the transcription of AMP genes, such as Diptericin. The phosphorylation of Relish is suggested to be mediated by an inhibitory kB (IKK) complex (containing IRD5 and Kenny). dTAKI activation is induced by adaptor dTAB2 nd DIAP2. (C) Upon binding of cytokine Upd3, which is secreted from hemocytes into hemolymph by viral and wasp-parasitoid infection, to the dimerized Domelss receptors, JAK (Hopscotch) is activated. The JAK recruits the STATs (Start92E), which are phosphorylated and dimerized, leading to translocation to the nucleus to activate transcription of target genes, such as Vir-1.
SPN43Ac mutant flies accumulated cleaved Spatzle, resulting in constitutive activation of the Toll pathway and the expression of AMPs (Levashina et al., 1999). SPN27A and SPN28D are known to regulate the Toll pathway during early development (Hashimoto et al., 2003; Ligoxygakis et al., 2003; Scherfer et al., 2008), and are also involved in the melanin biosynthesis reaction (De Gregorio et al.,2002; Ligoxygakis et al., 2002b). However, the molecular identities of the serpin target serine proteases and the biochemical regulatory mechanisms of these serpins have not been clearly demonstrated, leading to a lack of molecular understanding of the roles of serpins in the Toll signaling cascade.
The regulatory mechanisms of the Drosophila IMD pathway have also been studied; four negative regulators of the IMD pathway have been identified and characterized.
PGRP-LF, a membrane-bound non-catalytic PGRP containing two PGRP domains, was demonstrated to be a key negative regulator of the PGRP-LC-mediated IMD signaling pathway (Maillet et al., 2008). However, the inhibition of the IMD pathway by PGRP-LF was induced even in the absence of infection, allowing the prevention of aberrant activation of the IMD pathway by residual PGN fragments that are ingested from food or released by indigenous microbes. Pirk (poor IMD response upon knock-in), a protein interacting with PGRP-LC, is also known as Rutra (a new regulator of the IMD pathway); PIMS (PGRP-LC-interacting inhibitor of IMD signaling) is another negative regulator of the IMD pathway. The biological functions of these proteins are involved in the precise control of IMD pathway induction, but the exact biological functions are still not clear (Aggarwal et al., 2008; Kleino et al., 2008; Lho-cine et al., 2008). Furthermore, catalytic PGRPs such as PGRP-SC1 and PGRP-LB, which show amidase activity against PGN, were reported as another family of negative regulators of the IMD pathway. These PGRPs function as scavengers by cleaving the stem-peptide of PGN, thereby eliminating the immune-stimulating activity of PGN and leading to shutdown of the PGRP-LC-mediated IMD signaling pathway (Bischoff et al., 2006).
Although we have observed how Drosophila Toll and IMD pathways are activated through microbe-recognition proteins and their extracellular and intracellu-lar adaptor molecules after recognition of Gram-positive, Gram-negative bacteria or fungi, it is necessary to answer the question of which PAMP molecules of these microbes can activate these signaling pathways. In 2003, Lemai-tre and his colleagues first reported the determination of ligand molecules of the Drosophila Toll and IMD pathways, demonstrating that DAP-type PGN of Gram-negative bacteria and certain bacilli species function as the most potent activators of the IMD pathway, while the Toll pathway is predominantly activated by Lys-type PGN of Gram-positive bacteria (Leulier et al., 2003). These results clearly demonstrated that the discrimination between Gram-positive and Gram-negative bacteria in Drosophila relies on the recognition of specific forms of PGN but not other bacterial cell wall components, such as LPS, wall teichoic acid, lipoteichoic acid, and lipoprotein.
Activation and regulation of Tenebrio Toll signaling pathway As shown above, Drosophila genetics are very powerful tools for characterizing and ordering the components in the Drosophila Toll and IMD signaling pathways. However, this system is still limited in terms of determining the biochemical mechanisms involved in regulating the proteolytic Toll cascade. Since Drosophila has several alternative routes to the Toll pathway, used at various developmental stages and infection protocols, it is difficult to determine the clear activation mechanism of the Toll signaling cascade. For instance, Drosophila persephone is another serine protease linked to the Toll pathway and antifungal immunity, yet the biological functions of this molecule have only been partially characterized by Drosophila genetic studies. The proper identification of downstream factor(s) of persephone still awaits further investigation. To provide compelling biochemical data on how the Lys-type PGN and |-1,3-glucan recognition signals can be sequentially transferred to Spatzle during the Toll signaling pathway, it is necessary to use a larger insect that enables us to collect larger amounts of hemolymph. The coleopteran larvae T. molitor was used for intensive biochemical studies, resulting in the purification of nine proteins, which are involved in the activation of the Tenebrio Toll signaling pathway (Park et al., 2007; Kim et al., 2008; Roh et al., 2009) (Figure 3). The nine molecules include three pattern-recognition proteins (Tenebrio PGRP-SA, GNBP-1, GNBP-3), three serine protease zymogens (Tenebrio modular serine protease (MSP); Spatzle processing enzyme (SPE), and SPE-activating enzyme (SAE)), and recombinant pro-Spatzle and recombinant Toll-ecto domain-containing proteins, which were purified to homogeneity. The activation mechanism of the Tenebrio Toll signaling pathway was then studied biochemically using in vitro reconstitution experiments. We proposed that the Tenebrio PGRP-SA/GNBP1/MSP/SAE/SPE/Spatzle cascade is an essential unit that triggers the Lys-type PGN recognition signaling pathway in response to Gram-positive bacterial infection in the Tenebrio system (Kim et al, 2008).
The 1,3-glucan recognition signal is also transferred via the sequential activation of the three Tenebrio serine proteases, MSP, SAE, and SPE, which leads to the processing of pro-Spatzle to its mature form Spatzle, demonstrating that a three-step proteolytic cascade is essential for Toll pathway activation by fungal |-1,3-glucan in Tenebrio larvae. This cascade is shared with Lys-type PGN-induced Toll pathway activation (Roh et al., 2009). Furthermore, we demonstrated that |-1,3-glucan and Lys-type PGN activate the Toll signaling cascade using the same three-step proteolytic cascade that results in the production of two Tenebrio AMPs, tenecin 1 and tenecin 2. The amino acid sequence of tenecin 1 and its disulfide bond arrangement were found to be very similar to Drosophila defensin, while tenecin 2 showed high sequence identity (65% and 36%) with coleoptericin and holotricin-2, respectively. Holotri-cin-2, previously identified by our group, is also an inducible antibacterial peptide purified from coleopteran H. diomphalia larvae (Lee et al., 1994). The molecular mechanisms behind the recognition of Gram-positive bacteria or fungi by Tenebrio larvae in vivo, the activation of the Toll pathway, and the type of AMPs induced after the activation of the bacteria- or fungi-mediated Toll signaling cascade are clearly determined. Finally, the upstream pathogen recognition features of the Tenebrio Toll cascade are reminiscent of the complement activation by the lectin pathway in mammals in which the recognition of carbohydrates by mannose-binding lectin (MBL) leads to the auto-activation of MBL-associated serine proteases (MASPs) (Matsushita and Fujita, 1992). The domain organization of MASPs is similar to those of insect MSPs.
Figure 3 Comparison of Tenebrio and Manduca extracellular proteolytic Toll signaling pathways. (A) The Tenebrio Toll signaling pathway shares a three-step proteolytic cascade, which consists of three serine proteases, such as modular serine protease (MSP), Spatzle-processing enzyme (SPE), and SPE activating enzyme (SAE). Lys-type peptidoglycan and p-1,3-glucan are recognized by the Tenebrio PGRP-SA/GNBP1 complex and GNBP3, as in the Drosophila system. The recognition signals induce three serine proteases downstream. The activated SPE leads to the cleavage of pro-Spatzle to processed Spatzle, leading to the activation of the Toll cascade and, subsequently, production of the Tenebrio AMPs, tenecin 1 and 2. Three serpins (SPN 40, SPN 55, and SPN 45) make specific three serpin-serine protease complexes, and inhibit the processing of pro-Spatzle and phenoloxidase-mediated melanin synthesis. Polymeric DAP-type peptidoglycan of Gram-negative bacteria in Tenebrio system also uses the same cascade as that of the polymeric Lys-type peptidoglycan-mediated Toll signaling pathway. (B) The production of AMP and melanin synthesis in Manduca system is separated by two branches. Manduca AMP production begins with hemolymph protease 6 (HP6). The activated HP6 activates proHP8 to the active form of HP8, which can cleave pro-Spatzle to Spatzle, resulting in the induction of Manduca AMPs. The Michael Kanost group of Kansas State University proposed that activated HP6 can also activate prophenoloxidase activating proteinases-1 (PAP-1), which can cleave Manduca prophenoloxidase to phenoloxidase, leading to melanin synthesis in the presence of serine protease homolog 2. Also, Manduca prophenoloxidase is activated by a p-1,3-glucan recognition protein (pGRP)-mediated three-step proteolytic cascade (HP14/ HP21/PAP2&3), leading to melanin synthesis in the presence of SPH1.
Recently, the recognition mechanism of DAP-type PGN in the Tenebrio system has also been proposed (Yu et al., 2010), although the Drosophila IMD pathway responds to polymeric and monomeric DAP-type PGNs of Gram-negative bacteria and certain Gram-positive Bacillus species through PGRP-LC or PGRP-LE receptors. Unexpectedly, polymeric (but not monomeric) DAP-type PGN formed a complex with Tenebrio PGRP-SA, and this complex activated the three-step proteolytic cascade to produce processed Spatzle, leading to the induction of tenecin 1 in Tenebrio larvae, as in the Lys-type PGN-mediated Toll pathway (Figure 3). In addition, both polymeric and monomeric DAP-type PGNs induced the expression of Tenebrio PGRP-SC2, which is a DAP-type PGN-selective N-acetylmuramyl-L-alanine amidase that functions as a DAP-type PGN scavenger. PGRP-SC2 appears to function as a negative regulator of DAP-type PGN signaling by cleaving DAP-type PGN, rendering it incapable of inducing AMPs in Tenebrio larvae. These results demonstrated that the molecular recognition mechanism for polymeric DAP-type PGN differs between Tenebrio larvae and Drosophila adults, providing biochemical evidence of biological diversity in the innate immune responses of insects.
Because three serine proteases that are directly involved in the activation of the Tenebrio Toll cascade were identified, Tenebrio larvae were assumed to be a useful system for identifying and characterizing novel target serpins that directly regulate the Toll proteolytic cascade. As described above, because the molecular identities of the Drosoph-ila target serine proteases of the four Drosophila serpins and the biochemical regulatory mechanisms of these ser-pins were not clearly demonstrated, we tried to understand the molecular regulation mechanism biochemically using the Tenebrio system. Three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor larvae were purified (Jiang et al., 2009). These ser-pins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases (MSP, SAE, and SPE), and cooperatively blocked the Toll signaling cascade and |-1,3-glucan-mediated melanin biosynthesis. In addition, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of the processed Spatzle into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. In addition, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like SAE and SPE serine proteases. These cleavage patterns are also highly similar to those of the unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. It had been thought that the Drosophila Toll cascade was regulated by the activity of a single "bottle-neck" protease inhibitor, but our data presented the first indication that each individual protease in a cascade may be regulated by a specific serpin (Figure 3).
Manduca Toll signaling pathway The Toll cascade of another large insect was also studied biochemically. The Kanost group characterized more than 20 clip-domain-containing serine proteases in the hemolymph of the tobacco hornworm, M. sexta (Jiang and Kanost, 2000). Recently, they reported the function of two Manduca serine proteases, hemolymph proteases 6 and 8 (HP6 and HP8; An et al., 2009; see also Figure 3). HP6 and HP8 are each composed of an N-terminal clip domain and a C-terminal serine protease domain. HP6 was an apparent ortholog of Drosophila persephone, whereas HP8 was most similar to Drosophila and Tenebrio SPE, all of which activate the Toll pathway. Recombinant HP6 was found to activate prophenoloxidase-activating proteinase (proPAP1) in vitro and induce prophenoloxidase activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced the expression of AMPs such as attacin, cecropin, and lysozyme. These results suggest that proHP6 becomes activated in response to microbial infection, and participates in two immune pathways: activation of PAP1, which leads to prophenoloxidase activation and melanin synthesis, and activation of HP8, which stimulates the Manduca Toll-like signaling pathway. However, the most upstream receptors of the Toll pathway, such as Manduca PGRP-SA and GNBP1, have not yet been characterized in the Manduca system.
The Kanost group has done pioneering work in the biochemical characterization of insect serpins. They first described the M. sexta serpin-1, which has 12 different copies of exon 9 that undergo mutually exclusive alternative splicing to produce 12 putative protein isoforms. These isoforms differ in their carboxyl-terminal 39-46 residues, including the P1 residue, and inhibit serine proteases with different specificities (Kanost and Jiang, 1997). Recently, they also reported the biological function of the Manduca serpins: for example, serpin-1 isoforms can inhibit HP8, which activates pro-Spatzle, suggesting that serpin-1 isoforms may be involved in regulation of the Manduca Toll cascade (Ragan et al., 2010). The proposed model of activation and regulation of the Manduca Toll cascades is shown in Figure 3.
Lectin Induction
Lectins are defined as a protein family capable of recognizing specific oligosaccharides, which were initially purified from various plant seeds. Similar proteins have also been isolated from a number of organs in a wide range of vertebrates and invertebrates (Barondes, 1984). Because the diverse biological roles of purified insect lectins cannot all be mentioned in this topic, we will focus on the insect lectins that mainly participate in insect immunology. The pioneering work was performed by Natori and his colleagues using S. peregrina flesh fly larvae (Natori et al., 1999). Sarcophaga lectins functioning as defense proteins are synthesized by the fat body and secreted into the hemolymph when the larval body wall is pricked with a hypodermic needle (Okada and Natori, 1983). This 260-aa lectin is a typical C-type lectin that requires calcium ions for agglutinating activity. C-type lectins, a superfamily of calcium-dependent carbohydrate-binding proteins, are known to function in pathogen recognition, cell-cell interactions, and innate immunity in mammals (Weis et al., 1998). This lectin is rapidly induced when sheep red cells are injected, but is not present in the hemolymph of naive larvae. Elegant biochemical studies performed by the Natori group demonstrated that this lectin has dual functions in defense and development, and provided evidence that the Sarcophaga lectin activates insect hemocytes in some way, resulting in the activation of hemocyte-mediated digestion of non-self foreign cells (Nakajima et al., 1982; Komano et al., 1983). They showed that this lectin was also needed for the differentiation of ima-ginal discs in the pupal stage. When antibodies against Sarcophaga lectin were added to the culture medium of Sarcophaga imaginal discs, differentiation was strongly inhibited; none of the discs reached the stage of terminal differentiation (Kawaguchi et al., 1991). These results suggest that Sarcophaga lectin is not simply a defense molecule that is needed for the elimination of the invading non-self cells, but also a regulatory molecule in the development of imaginal discs.
Kanost and his colleagues also purified four soluble C-type lectins with two carbohydrate-recognition domains from the tobacco hornworm M. sexta, and named them immulectin-1 through -4 (Yu et al., 1999). Expression of all four immulectins is upregulated in the fat body upon bacterial challenge. Immulectin-1 and -4 agglutinate Gram-negative and Gram-positive bacteria and yeast. However, immulectin-2 binds to a wide range of microbial cell wall components such as lipoteichoic acid, laminarin (branched P-1,3-glucan), mannose, and LPS. Furthermore, knockdown of the immulectin-2 gene at the level of both mRNA and protein by RNA interference (RNAi) markedly decreased the ability of M. sexta to defend bacterial infection when exposed to either species of the insect pathogen Photorhabdus, suggesting that the Manduca lectin is not the only one that recognizes Pho-torhabdus; the lectin-mediated insect immune system also plays an essential role in defending bacterial infection (Eleftherianos et al., 2006). Similar tandem-domain C-type lectins are identified in other lepidopterans, and are involved in bacterial binding and hemocyte aggregation (Koizumi et al., 1999). However, further studies are necessary to determine the structure of the ligand molecules and the biological functions of these C-type insect lectins during activation of insect innate immunity.
Insect galectin homologs, which have the ability to bind P-galactoside sugars, are identified from two insects, D. melanogaster (Pace et al., 2002) and A. gambiae (Dimo-poulos et al., 1997). Fourteen galectins have been identified in mammals (Rabinovich et al., 2002). However, the identification of precise biological functions for these mammalian galectins is difficult because of the redundancy in tissue expression and the complexity of recognition mechanisms in the target cells. A benefit of working with insect systems such as Drosophila and Anopheles is the ease of genetic manipulation and the rapid generation time. In addition, there are relatively small numbers of putative galectins in the Drosophila and Anopheles genomes; lower organisms such as insects are therefore useful in deciphering the precise biological functions of galectins. The Kafa-tos group demonstrated that a putative galectin homolog was upregulated in the salivary and gut of A. gambiae when mosquitoes were infected with malaria and bacteria. The Anopheles galectin was suggested to function as a pattern-recognition protein by binding saccharide ligands on the microbial cell wall surface to trigger a host innate immune response (Dimopoulos et al., 2001). However, Drosophila galectin was expressed in naive hemocytes, but not by the fat body or larval hemolymph (Pace et al., 2002). Mammalian galectins were suggested to participate in the innate immune response by facilitating microbial recognition and/or lectin-mediated phagocytosis (Fradin et al., 2000; Rahnamaeian et al., 2009). The elucidation of Toll receptor-mediated immune response in insects has led to rapid progress in the understanding of innate immune function in mammals; the determination of biological functions of insect galectins may similarly provide insight into the novel biological functions of mammalian galectins.
