ISJ 3: 89-96, 2006 ISJ 3: 103-110, 2006 ISSN 1824-307X MINIREVIEW The multiple functions of the PGRP family in Drosophila immunity A Goto, S Kurata Graduate School of Pharmaceutical Sciences, Tohoku University, Japan Accepted November 13, 2006 Abstract The innate immune system discriminates between infectious non-self and self using germ-line- encoded pattern recognition receptors (PRRs) that are highly conserved from insects to mammals. Peptidoglycan recognition protein (PGRP) is one of the hallmark pattern recognition receptors responsible for detecting unique bacteria-derived peptidoglycans. The PGRP family comprises several members (13 in Drosophila, 7 in Anopheles, and 4 in mammals) and are differentially expressed on immune-responsive organs. Some PGRPs have amidase or bactericidal activities and function as immune modulators, whereas others have lost their enzymatic activity, but still have crucial roles in the activation of innate immune signaling. Evidence from recent Drosophila studies suggests that PGRPs have a role in a variety of immune reactions, such as in the activation of the prophenoloxidase cascade, the production of antimicrobial peptides through the activation of the Toll and Imd pathways, intracellular bacteria recognition, and phagocytosis. Key words: innate immunity; PGRP (peptidoglycan recognition protein); PRR (pattern recognition receptor); PAMP (pathogen-associated molecular pattern); amidase; antimicrobial peptide; phagocytosis __________________________________________________________________________________________ Introduction Insects are diverse group of organisms and comprise more than 80 % of the animal kingdom. To prevail as such a vast majority of species among other organisms, insects have developed excellent defense mechanisms against bacterial infection. In contrast to adaptive immunity, which requires highly specific receptors created by somatic gene rearrangement, the innate immune system is a defense mechanism that exploits germ-line encoded gene products. The principal mechanisms of innate immunity are difficult to study in vertebrates largely due to the effects of the adaptive immune response. Thus, insects have become a favorite model system because, as invertebrates, they depend solely on the innate immune system to fight off infection. The powerful genetic and molecular techniques with the complete genome sequencing make Drosophila an attractive model for deciphering the precise mechanisms of the innate immune response (Hoffmann, 2003; Hultmark, 2003). ___________________________________________________________________________ Corresponding author: Shoichiro Kurata Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan E-mail: kurata@mail.pharm.tohoku.ac.jp Although the innate immune system is composed of multiple complex processes including cellular and humoral responses, much of the attention in Drosophila has focused on humoral reactions. The primary response is the most immediate response (within minutes) against microorganism invasion and depends on constitutively present endogenous molecules in the hemolymph, such as coagulation factors, cell surface recognition receptors or prophenoloxidase (proPO) for wound healing, phagocytosis and melanization which confine microorganisms in small spaces, respectively (Theopold et al., 2003; Goto et al., 2003; Ashida, 2004; Kurata et al., 2006). Shortly after the primary response, the secondary response begins and ultimately leads to the production of antimicrobial peptides (AMPs) from the fat body, the functional equivalent of the mammalian liver. To date, there are seven known distinct antimicrobial peptides in Drosophila and they are basically small cationic molecules with specific membrane- attackable bactericidal activity against various types of microorganisms. The transcriptional regulation of AMP expression is under the control of two distinct pathways, the Toll and immune deficiency (Imd) pathways (Kurata, 2004; Kurata et al., 2006). The Toll pathway was originally identified as a 103 mailto:kurata@mail.pharm.tohoku.ac.jp signaling cascade involved in dorsoventral patterning in embryos (Morisato and Anderson, 1995; Roth, 2003). Subsequent genetic analysis serendipitously led to the discovery that it also controls the expression of the antifungal peptide Drosomycin, which is predominantly triggered by Gram-positive or fungal infection (Lemaitre et al., 1996). Activation of the Toll pathway is initiated through the binding of the proteolytically cleaved cytokine Spaetzle to the Toll receptor, which then transduces its signal to the cells. In the cell, a Toll receptor-adaptor complex composed of dMyD88, Tube, Pelle, and other unidentified factors induces the phosphorylation and degradation of the ankyrin- repeat inhibitor protein Cactus. The nuclear factor NF-κB protein Dif, which is normally retained in the cytoplasm by binding to Cactus, is then translocated into the nucleus and triggers the expression of the antifungal peptide Drosomycin together with numerous effector molecules (Hoffman and Reichhart, 2002; Hoffmann, 2003). This discovery unambiguously led to the identification of the Toll- like receptor (TLR) family in mammals (Takeda et al., 2003; Royet et al., 2005). In contrast, the Imd pathway is predominantly activated by Gram-negative bacterial infection. While analyzing the expression of antibacterial genes in a phenoloxidase cascade mutant, imd was serendipitously discovered as the first recessive mutation that impairs the inducibility of all antibacterial peptides in the Drosophila immune response (Lemaitre et al., 1995). Six years later, the imd gene product was identified. It is a 25-kDa protein containing a death domain with significant sequence similarity to that of the mammalian tumor necrosis factor-receptor interacting protein RIP (Georgel et al., 2001). Activation of the Imd pathway triggers the kinase cascade of dTAK (a MAP 3 kinase homologue) and the IKK complex, and ultimately leads to phosphorylation of the Rel protein Relish (Leulier et al., 2002; Naitza et al., 2002; Vidal et al., 2001). Phosphorylated Relish is then cleaved by DREDD (a mammalian homolog of caspase-8), and its DNA binding domain leaving the I-κB domain in the cytoplasm is translocated into the nucleus and triggers AMP expression (Leulier et al., 2000; Stoven et al., 2003). Thus, over the past decade, our understanding of Drosophila innate immunity has dramatically centered on the field of intracellular signaling mechanisms. There remain crucial questions, however, about how the innate immune system recognizes microorganisms, and discriminates between self and non-self infections, and how the signals from outside the cells subsequently reach the cognate innate receptors to activate the Toll or Imd pathway. The discovery of pattern recognition receptors (PRR) such as peptidoglycan recognition proteins (PGRPs) that recognize unique bacterial cell component peptidoglycans (PGNs) led to our deeper understanding of the innate immune recognition concept (Medzhitov and Janeway, 2002; Royet, 2004). Here, we review recent advances in the understanding of the multifunctional PGRP family, a representative pattern recognition molecule, in Drosophila. Discovery of the PGRP family The name ”peptidoglycan recognition protein” was first introduced by Ashida’s group (Yoshida et al., 1996). They purified a 19-kDa protein from the hemolymph and cuticles of silkworm (Bombyx mori) and demonstrated that it binds to Gram-positive bacteria and PGN, and activates the prophenoloxidase cascade (Yoshida et al., 1996). Later, its corresponding cDNA was cloned (Ochiai et al., 1999). PGRP gene homologs from a moth (Trichoplusia ni) and subsequently from mouse and human were in turn cloned, indicating that the PGRP family is highly conserved from insects to mammals (Kang et al., 1998). Completion of various genome projects (e.g., Drosophila melanogaster, Anopheles gambiae, human, mouse, etc.) also boosted the discovery of diverse PGRP family. The Drosophila genome encodes at least 13 PGRP family members, which are subdivided into seven short transcripts (PGRP-S; ~200 amino acids long) and six long transcripts (PGRP-L; from 200 to 600 amino acids long). The short transcripts are predicted to be secreted molecules due to the presence of signal peptides and are induced in response to bacterial infection, whereas the long transcripts are constitutively present on the cell surface or integrated into the plasma membrane, except for PGRP-LB (Werner et al., 2000). Extracellular recognition by the PGRP family PGRP roles in the immune response A significant breakthrough with regard to the role of PGRP in the immune response came through a large-scale ethylmethane sulfonate mutagenesis screen from Drosophila. Michel et al isolated a mutation called semmelweis (seml), named after the Hungarian physician Ignaz Phillipp Semmelweis who is a pioneer of antiseptic treatments (Celine, 1999; Michel et al., 2001). The PGRP-SAseml mutants lead to defects in AMP expression and render flies more susceptible to several Gram-positive bacterial infections. The mutated region was found to be on a highly conserved cysteine residue that is changed to a tyrosine in PGRP-SA. Hemolymph transfer experiments revealed that PGRP-SA is secreted into the hemolymph. Surprisingly, activation of the Toll pathway by fungi and of the Imd pathway by Gram-negative bacteria is normal compared to wild- type, raising the possibility that there are other PRRs specific for fungi and Gram-negative bacteria (Michel et al., 2001). Consistent with this hypothesis, three independent groups identified PGRP-LC mutations (ird7 or totem) (Choe et al., 2002; Gottar et al., 2002; Ramet et al., 2002). PGRP-LC mutant flies are highly susceptible to some Gram-negative bacterial infections and do not express Imd-mediated AMP genes (such as Diptericin and Attacin). Epistatic analysis indicates that PGRP-LC encoding a single transmembrane protein acts upstream of Imd, suggesting that PGRP-LC functions in the activation of the Imd pathway in response to Gram-negative bacterial infection. PGRP-SA binds to lysine (Lys)-type PGN 104 from Gram-positive bacteria, whereas PGRP-LC binds to meso-diaminopimelic (DAP)-type PGN from Gram-negative bacteria, reinforcing the structural basis for the recognition mechanisms (Leulier et al., 2003; Kaneko et al., 2004). Cooperative roles of PGRPs for detecting pathogens Curiously, the PGRP-LC mutant phenotypes are not as severe as that of IKK� or Relish null mutants in terms of both AMP expression and survival rates upon some Gram-negative bacterial infections (Gottar et al., 2002). Accordingly, some Gram-positive bacteria such as Staphylococcus saprophyticus, Staphylococcus aureus, or Enterococcus faecalis induce normal activation of the Toll pathway in a PGRP-SA mutant background, but this activation is completely blocked in Dif mutants (Bischoff et al., 2004). Those findings led to the observation that the one to one PRR/ pathogen associated molecular pattern (PAMP) concept is inappropriate for innate immune sensing mechanisms. This interpretation was soon validated by the discovery of multi-functional PGRP-LE, which contains a unique acidic domain, functions in the activation of the Imd pathway and also triggers the proPO cascade when it is ectopically expressed (Takehana et al., 2002). More importantly, PGRP- LC/PGRP-LE double mutants are more susceptible against Gram-negative bacteria (E. coli) infection than either single mutant alone, suggesting that these two proteins cooperate to recognize Gram- negative bacteria and lead to the activation of the Imd pathway (Takehana et al., 2004). Similarly, in the case of Gram-positive bacteria sensing, other cooperative receptor complexes are required upstream of Toll. Gram-negative bacteria binding protein (GNBP), which binds Gram-negative bacteria, was first identified from silkworm (Lee et al., 1996). Gobert et al (2003) subsequently reported that GNBP1 mutants are not resistant against some Gram-positive bacterial infections, showing a similar immune defective phenotype as the PGRP-SA mutant. The finding that GNBP1 physically interacts with PGRP-SA in the circulation to activate the Toll pathway supports this observation (Gobert et al., 2003; Pili-Floury et al., 2004; Wang et al., 2006). Moreover, the generation of another PGRP family member mutant, the PGRP- SD loss-of-function mutant, has been reported (Bischoff et al., 2004). PGRP-SD mutants are highly susceptible to some types of Gram-positive bacterial infection, and this mutation exacerbates the PGRP- SA and GNBP1 mutant phenotypes, suggesting that PGRP-SD is another Gram-positive bacteria recognition molecule involved in the activation of the Toll pathway. Taken together, there are four known PGRP family members, PGRP-SA, -SD, -LC, and –LE, that are crucial for microbe recognition and for activating the innate immune signaling pathways (Toll, Imd , and proPO cascades) (Fig. 1). Fig. 1 Extracellular recognition by PGRP family. In hemolymph, meso-diaminopimelic (DAP)-type peptidoglycans (PGNs) from Gram-negative and some Gram-positive bacteria (Bacillus species) are recognized by PGRP-LC or PGRP-LE and activate the Imd pathway to trigger Diptericin or Attacin expression. In the case of lysine (Lys)-type Gram-positive bacterial infection, either PGRP-SC1a, -SD, -SA, GNBP1 alone or in combination is responsible for the activation of the Toll pathway through binding of the endogenous ligand spaetzle (Spz). 105 Intracellular recognition by PGRP-LE Large-scale gain-of-function genetic screening using AMP reporters led to the identification of PGRP-LE. PGRP-LE overexpression leads to constitutive activation of the Imd pathway as well as the proPO cascade, which ultimately leads to the formation of large melanotic tumors in the hemolymph (Takehana et al., 2002). PGRP-LE loss- of-function mutants were isolated in subsequent studies 2 years after the identification of PGRP-LE (Takehana et al., 2004). The phenotypes of a single PGRP-LE gene mutation are fairly weak in terms of their survival rates against E. coli infection and AMP expression. However, when combined with the PGRP-LC mutation, the PGRP-LE/PGRP-LC double mutants exhibit high susceptibility to E. coli and to other DAP-type PGN-containing bacteria (Takehana et al., 2004). This finding clearly suggests that PGRP-LE synergistically or redundantly acts with PGRP-LC to activate the Imd pathway. To gain a better understanding of the cooperative recognition mechanisms between PGRP-LC and PGRP-LE, two independent groups recently conducted very exciting experiments using monomeric DAP-type PGN tracheal cytotoxin (TCT) (Kaneko et al., 2006; Lim et al., 2006). TCT is a disaccharide-tetrapeptide fragment of PGN that contains a 1,6-anhydro-arranged muramic acid and a DAP residue at the third position of the stem peptides (Cookson et al., 1989), and is continuously released from Gram-negative bacteria as a turnover product of the PGN in the cell wall (Park, 2001). The presence of such a small diffusible monomeric PGN is an ideal signature for the innate immune recognition system by PRRs, as the PGN layer of Gram-negative bacteria is basically hidden under the outer lipopolysaccharide (LPS) layers. In vitro cell culture studies demonstrated that TCT potently activates the Imd pathway via two different PGRP- LC isoforms: PGRP-LCa and PGRP-LCx (Kaneko et al., 2004). Deletion mutant studies demonstrated that the RHIM-like motif of PGRP-LC is responsible for the signaling, but is dispensable for the interaction with Imd (Kaneko et al., 2006; Choe et al., 2005). Whereas a single PGRP-LC or PGRP-LE mutant responds normally to TCT, E. coli PGN, or live E. coli, the response of PGRP-LC/PGRP-LE double mutants is completely abolished. Clonal expression studies demonstrated that PGRP-LE acts in a cell-autonomous manner on some immune responsive tissues, such as malpighian tubes. Direct delivery of TCT into the cytosol by calcium phosphate transfection triggers an enhanced AMP expression that is dependent on PGRP-LE, suggesting that PGRP-LE is a second receptor for TCT (Kaneko et al., 2006). This idea is supported by analysis of the crystal structure of PGRP-LEpg (PGRP domain of PGRP-LE)/TCT complex, which indicates that TCT strongly binds to PGRP-LEpg with an apparent Kd of 27 nM, and induces PGRP-LE multimerization through head-to-tail dimer formation (Lim et al., 2006). Collectively, these results indicate that PGRP- LE potentially has dual functions depending on its localization; on the outside of the cells, it cooperates with PGRP-LC through its PGRP domain and triggers the proPO cascade, and inside the cells, it acts as an intracellular recognition receptor for activation of the Imd pathway (Fig. 2). Fig. 2 Recognition mechanisms of PGRP-LE. Monomeric DAP-type PGNs (TCT) binds to the PGRP domain of PGRP-LE. In the hemolymph, this complex triggers the proPO cascade. TCT can also bind to PGRP-LCx to form three potential types of receptor complexes (LEpg/TCT/LC, LCx/TCT/LCa or LCx/PGN/LCx) on the cell surface. The RHIM-like motif of PGRP-LCx and LCa is responsible for the activation of the Imd pathway. The PGRP-LE/TCT complex also functions in intracellular recognition. Immune modulator activities of the PGRP family A representative feature of the PGRP family is that all PGRPs in insects and mammals have in common an approximately 160 amino acid-long PGRP domain with similarity to the bacteriophageT7 lysozyme, a zinc-dependent N-acetyl-muramyl-L- alanine amidase (Steiner, 2004; Royet et al., 2005). Among the 13 PGRP family members in Drosophila, the first group is categorized as non-catalytic PGRPs, which lack the zinc binding residues required for amidase activity, but retain the binding capacities to PGN (PGRP-SA, -SD, -LA, -LC, LD, - LE, -LF). The second group is the catalytic PGRPs, which have zinc-dependent amidase activity that either reduces or eliminates the biological activities of the PGNs (PGRP-SC1a, -SC1b, -LB, -SB1, - SC2). The first striking discovery of the potential role of catalytic PGRPs in the Drosophila immune response was reported for PGRP-SC1b (Mellroth et al., 2003). In vitro studies performed by Mellroth et al. (2003) demonstrated that recombinant SC1b hydrolyzes the lactylamide bond between the glycan strand and the stem peptides of PGN, and the degraded PGN has less immune stimulatory activity compared with undigested PGN, indicating that 106 PGRP-SC1b has scavenger activity. Unexpectedly, Chang et al. (2004), in the course of crystal structure analysis, demonstrated that recombinant PGRP-SA has L,D-carboxypeptidase activity that cleaves the DAP-type muropeptide, but not the Lys- type compound. Moreover, three independent groups reported the roles of catalytic PGRPs: PGRP-SC1/2 (Bischoff et al., 2006), PGRP-LB (Zaidman-Rémy et al., 2006), and PGRP-SB1 (Mellroth and Steiner, 2006). First, Bischoff et al. (2006) used RNA interference techniques to generate PGRP-SC1/2 loss of function mutants and reported that the mutants overactivate the Imd pathway after bacterial infection, and feeding-induced infection by Erwinia carotovora carotovora is enhanced compared to controls. Thus, PGRP-SC1/SC2 might modulate the activation of the Imd pathway in the gut, which is constantly threatened by bacterial infection (Bischoff et al., 2006). The effects of phenotypes of recently isolated PGRP-SC1a mutants (picky) on the Toll pathway are confusing with respect to the complex immune modulator mechanisms (Garver et al., 2006). Second, biochemical analysis indicates that PGRP-LB has specific amidase activity for DAP- type PGN, including TCT. Similar to the RNA- interference generated PGRP-SC1/2 mutants, the analysis of the time course of AMP expression induced by Gram-negative bacteria or TCT infection revealed a strong immune response by the RNA- interference generated PGRP-LB mutants compared to wild-type controls (Zaidman-Rémy et al., 2006). Therefore, they concluded that PGRP-LB negatively regulates the Imd pathway and functions as a scavenger receptor for Gram-negative bacterial PGN. Finally, recent biochemical analysis demonstrated that PGRP-SB1 is an N- acetylmuramoyl L-alanine amidase that preferentially hydrolyzes DAP-type PGN. In contrast to PGRP-LB, PGRP-SB1 lacks enzymatic activity against TCT. In addition, this report first demonstrated that PGRP-SB1 possesses bactericidal activity against B. megaterium (Mellroth and Steiner, 2006). Taken together, these findings suggest that non-catalytic PGRPs preferentially function in the detection of PGN, and catalytic PGRPs serve as scavenger receptors to detoxify harmful PGN and modulate the signaling pathways, although catalytic PGRPs that specifically cleave Lys-type PGN have yet to be identified. Roles of PGRPs in phagocytosis Phagocytosis is a phenomenon whereby invading microbes or altered self-like apoptotic or infected cells are engulfed and, eventually, digested by host cells called phagocytes (Aderem and Underhill, 1999). In Drosophila, most humoral immune responses leading to Toll and Imd pathway activation are mainly mediated by the fat body, whereas cellular responses such as phagocytosis, encapsulation, or melanization are mediated by blood cells called hemocytes (Hoffmann and Reichhart, 2002). There are three known hemocyte types in Drosophila. Plasmatocytes account for approximately 90 % of hemocytes and are the main executors of phagocytosis. Crystal cells, approximately 5% of the hemocyte population, contain a set of substrates and enzymes responsible for proPO-mediated humoral melanization. Lamellocytes, the third hemocyte type, show a large flattened morphology. Under non-infected conditions, lamellocytes are absent, but once large invaders, such as wasps, are infected, lamellocytes are differentiated from the lymph glands, the main hemocyte-producing organs in larvae (Meister and Lagueux, 2003). In addition to several phagocytic receptors, such as Croquemort (“catcher of death” ) (Franc et al., 1996, 1999; Stuart et al., 2005), scavenger receptor (SR) -CI (Ramet et al., 2001; Ulvila et al., 2006), Peste (Philips et al., 2005), Eater (Kocks et al., 2005), and Dscam (Watson et al., 2005), PGRP family members are also involved in phagocytosis. PGRP-LC was the first PGRP family member discovered to be involved in phagocytosis. A double-stranded (ds)RNA interference-based screen of the Drosophila macrophage cell line S2 identified 34 genes, including PGRP-LC, that are essential for phagocytosis. Fluorescence Activated Cell Sorting analysis indicated that S2 cells with PGRP-LC knock-down had substantially decreased phagocytic activity towards Gram-negative bacteria E. coli, but not Gram-positive bacteria S. aureus. They also demonstrated that long transcripts of the same types of PGRP family, PGRP-LA or PGRP– LD, are not involved in phagocytosis (Ramet et al., 2002). Conversely, another group recently reported that PGRP-SC1a mutation (picky) impairs phagocytic ability S. aureus, but not E. coli or S. cerevisiae (yeast), based on the results of a forward genetic ethylmethane sulfonate screen. Moreover, activation of the Toll pathway is significantly impaired in PGRP-SC1a mutants. Transgenic rescue experiments indicated that the amidase activity of PGRP-SC1a does contribute to Toll pathway activation, but is essential for the uptake of S. aureus (Garver et al., 2006). Conclusion Over the past 5 years, the concept of the PRR/PAMP theory originally introduced by Charles Janeway has been verified experimentally using versatile molecular and genetic strategies (Janeway and Medzhitov, 2002). In mammals, functional characterization of Toll-like receptor family has considerably deepened our understanding of the recognition mechanisms of innate immunity (Takeda and Akira, 2005). In contrast, it seems that insect Tolls are not involved in immune functions as pattern recognition receptors, but are more likely to have other functions. Drosophila Toll is a kind of cytokine receptor that is activated by the endogenous proteolytically cleaved ligand spaetzle (Imler and Hoffmann, 2002). Now, the PGRP family members are coming to the forefront as molecular sensors against microbial infection in insects. Recent functional characterization of the PGRP family members in Drosophila has enabled us broaden the partial picture of pattern recognition mechanisms: 1) Specific types of PGN (Lys-type or DAP-type) can be detected by different PGRPs; 2) 107 Catalytic amidase PGRPs act as immune modulators; 3) Microbe detection by PGRPs triggers the activation of innate immune signaling pathways; and 4) PGRPs possess receptor functions for phagocytosis. Still, a fundamental question remains as to how the signals are transmitted into the cells to kill microbes after PGNs are recognized by PGRPs. Further studies will address this issue to complete the picture of the PRR/ PAMP mechanisms. Acknowledgement This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Japan Society for the Promotion of Science; the Program for the Promotion of Basic Research Activities for Innovative Biosciences; and the Naito Foundation. References Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17: 593-623, 1999. Ashida M. Activation of prophenoloxidase cascade with peptidoglycan. Tanpakushitsu Kakusan Koso 49: 1168-1173, 2004. Bischoff V, Vignal C, Boneca IG, Michel T, Hoffmann JA, Royet J. Function of the Drosophila pattern-recognition receptor PGRP- SD in the detection of Gram-positive bacteria. Nat. Immunol. 5: 1175-1180, 2004. Bischoff V, Vignal C, Duvic B, Boneca IG, Hoffmann JA, Royet J. Downregulation of the Drosophila innate immune response by peptidoglycan recognition proteins SC1 and SC2. PLoS Pathog. 2: e14, 2006. Celine LF. Semmelweis. L'imaginaire. Gallimard, Paris, 1999. Chang CI, Pili-Floury S, Herve M, Parquet C, Chelliah Y, Lemaitre B, et al. A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D- carboxypeptidase activity. PLoS Biol.2: E277, 2004. Choe KM, Lee H, Anderson KV. Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a single-transducing innate immune receptor. Proc. Natl. Acad. Sci. USA 102: 1122- 1126, 2005. Choe KM, Werner T, Stoven S, Hultmark D, Anderson KV. Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296: 359-362, 2002. Cookson BT, Tyler AN, Goldman WE. Primary structure of the peptidoglycan-derived tracheal cytotoxin of Bordetella pertussis. Biochemistry 28: 1744-1749, 1989. Franc NC, Dimarcq JL, Lagueux M, Hoffmann J, Ezekowitz RA. Croquemort, A novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 4: 431-443, 1996. Franc NC, Heitzler P, Ezekowitz RA, White K. Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284: 1991- 1994, 1999. Garver LS, Wu J, Wu LP. The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila. Proc. Natl. Acad. Sci. USA 103: 660-665, 2006. Georgel P, Naitza S, Kappler C, Ferrandon D, Zachary D, Swimmer C, et al. Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. Dev. Cell 1: 503-514, 2001. Gobert V, Gottar M, Matskevich AA, Rutschmann S, Royet J, Belvin M, et al. Dual activation of the Drosophila toll pathway by two pattern recognition receptors. Science 302: 2126-2130, 2003. Goto A, Kadowaki T, Kitagawa Y. Drosophila hemolectin gene is expressed in embryonic and larval hemocytes and its knock down causes bleeding defects. Dev. Biol. 264: 582-591, 2003. Gottar M, Gobert V, Michel T, Belvin M, Duyk G, Hoffmann JA, et al. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416: 640-644, 2002. Hoffmann JA, Reichhart JM. Drosophila innate immunity: an evolutionary perspective. Nat. Immunol. 3: 121-126, 2002. Hoffmann JA. The immune response of Drosophila. Nature 426: 33-38, 2003. Hultmark D. Drosophila immunity: paths and patterns. Curr. Opin. Immunol. 15: 12-19, 2003. Imler JL, Hoffmann JA. Toll receptors in Drosophila: a family of molecules regulating development and immunity. Curr. Top. Microbiol. Immunol. 270: 63-79, 2002. Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216, 2002. Kaneko T, Goldman WE, Mellroth P, Steiner H, Fukase K, Kusumoto S, et al. Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 20: 637-649, 2004. Kaneko T, Yano T, Aggarwal K, Lim JH, Ueda K, Oshima Y, et al. 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, 2006. Kang D, Liu G, Lundstrom A, Gelius E, Steiner H. A peptidoglycan recognition protein in innate immunity conserved from insects to humans. Proc. Natl. Acad. Sci. USA 95: 10078-10082, 1998. Kocks C, Cho JH, Nehme N, Ulvila J, Pearson AM, Meister M, et al. Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123: 335-346, 2005. Kurata S, Ariki S, Kawabata S. Recognition of pathogens and activation of immune responses in Drosophila and horse crab innate immunity. Immunobiology 211: 237-249, 2006. Kurata S. Recognition of infectious non-self and activation of immune responses by peptidoglycan recognition protein (PGRP)-family in Drosophila. Dev. Comp. Immunol. 28: 89-95, 2004. 108 Lee WJ, Lee JD, Kravchenko VV, Ulevitch RJ, Brey PT. Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori. Proc. Natl. Acad. Sci. USA 93: 7888-7893, 1996. Lemaitre B, Kromer-Metzger E, Michaut L, Nicolas E, Meister M, Georgel P, et al. A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense. Proc. Natl. Acad. Sci. USA 92: 9465- 9469, 1995. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86: 973-983, 1996. Leulier F, Parquet C, Pili-Floury S, Ryu JH, Caroff M, Lee WJ, et al. The Drosophila immune system detects bacteria through specific peptidoglycan recognition. Nat. Immunol. 4: 478-484, 2003. Leulier F, Rodriguez A, Khush RS, Abrams JM, Lemaitre B. The Drosophila caspase Dredd is required to resist gram-negative bacterial infection. EMBO Rep. 1: 353-358, 2000. Leulier F, Vidal S, Saigo K, Ueda R, Lemaitre B. Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults. Curr. Biol. 12: 996-100, 2002. Lim JH, Kim MS, Kim HE, Yano T, Oshima Y, Aggarwal K, et al. Structural basis for preferential recognition of diaminopimelic acid-type peptidoglycan by a subset of peptidoglycan recognition proteins. J. Biol. Chem. 281: 8286- 8295, 2006. Medzhitov R, Janeway CA Jr. Decoding the patterns of self and nonself by the innate immune system. Science 296: 298-300, 2002. Meister M, Lagueux M. Drosophila blood cells. Cell Microbiol. 5: 573-580, 2003. Mellroth P, Karlsson J, Steiner H. A scavenger function for a Drosophila peptidoglycan recognition protein. J. Biol. Chem. 278: 7059- 7064, 2003. Mellroth P, Steiner H. PGRP-SB1: An N- acetylmuramoyl l-alanine amidase with antibacterial activity. Biochem. Biophys. Res. Commun. 350: 994-999, 2006. Michel T, Reichhart JM, Hoffmann JA, Royet J. Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 414: 756-759, 2001. Morisato D, Anderson KV. Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo. Annu. Rev. Genet. 29: 371- 399, 1995. Naitza S, Rosse C, Kappler C, Georgel P, Belvin M, Gubb D, et al. The Drosophila immune defense against gram-negative infection requires the death protein dFADD. Immunity 17: 575-581, 2002. Ochiai M, Ashida M. A pattern recognition for peptidoglycan. Cloning the cDNA and the gene of the silkworm, Bombyx mori. J. Biol. Chem. 274: 11854-11858, 1999. Park JT. Identification of a dedicated recycling pathway for anhydro-N-acetylmuramic acid and N-acetylglucosamine derived from Escherichia coli cell wall murein. J. Bacteriol. 183: 3842- 3847, 2001. Philips JA, Rubin EJ, Perrimon N. Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309: 1251-1253, 2005. Pili-Floury S, Leulier F, Takahashi K, Saigo K, Samain E, Ueda R, et al. In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults. J. Biol. Chem. 279: 12848-12853, 2004. Ramet M, Manfruelli P, Pearson A, Mathey-Prevot B, Ezekowitz RA. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416: 644-648, 2002. Ramet M, Pearson A, Manfruelli P, Li X, Kozeil H, Gobel V, et al. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 15: 1027-1038, 2001. Roth S. The origin of dorsoventral polarity in Drosophila. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358: 1317-1329, 2003. Royet J, Reichhart JM, Hoffmann JA. Sensing and signaling during infections in Drosophila. Curr. Opin. Immunol. 17: 11-17, 2005. Royet J. Infectious non-self recognition in invertebrates: lessons from Drosophila and other insect models. Mol. Immunol. 41: 1063-1075, 2004. Steiner H. Peptidoglycan recognition proteins: on and off switches for innate immunity. Immunol. Rev. 198: 83-96, 2004. Stoven S, Silverman N, Junell A, Hedengren-Olcott M, Erturk D, Engstrom Y, et al. Caspase- mediated processing of the Drosophila NF-κB factor Relish. Proc. Natl. Acad. Sci. USA 100: 5991-5996, 2003. Stuart LM, Deng J, Silver JM, Takahashi K, Tseng AA, Hennessy EJ, et al. Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170: 477-485, 2005. Takeda K, Akira S. Toll-like receptors in innate immunity. Int. Immunol. 17: 1-14, 2005. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu. Rev. Immunol. 21: 335-376, 2003. Takehana A, Katsuyama T, Yano T, Oshima Y, Takada H, Aigaki T, et al. Overexpression of a pattern- recognition receptor, peptidoglycan-recognition protein-LE, activates imd/relish-mediated antibacterial defense and the prophenoloxidase cascade in Drosophila larvae. Proc. Natl. Acad. Sci. USA 99: 13705-13710, 2002. Takehana A, Yano T, Mita S, Kotani A, Oshima Y, Kurata S. Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity. EMBO J. 23: 4690-4700, 2004. Theopold U, Schmidt O, Soderhall K, Dushay MS. Coagulation in arthropods: wound closure and healing. Trends Immunol. 25: 289-294, 2003. 109 Ulvila J, Parikka M, Kleino A, Sormunen R, Ezekowitz RA, Kocks C, et al. Double-stranded RNA is internalized by scavenger receptor- mediated endocytosis in Drosophila S2 cells. J. Biol. Chem. 281: 14370-14375, 2006. Vidal S, Khush RS, Lulier F, Tzou P, Nakamura M, Lemaitre B. Mutations in the Drosophila dTAK1 reveal a conserved function for MAPKKKs in the control of rel/NF-�B-dependent innate immune responses. Genes Dev. 15: 1900-1912, 2001. Wang L, Weber AN, Atilano ML, Filipe SR, Gay NJ, Ligoxygakis P. Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP- SA. EMBO J. 25: 5005-5014, 2006. Watson FL, Puttmann-Holgado R, Thomas F, Lamar DL, Hughes M, Kondo M, et al. Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309: 1874- 1878, 2005. Werner T, Liu G, Kang D, Ekengren S, Steiner H, Hultmark D. A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 97: 13772-13777, 2000. Yoshida H, Kinoshita, Ashida M. Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J. Biol. Chem. 271: 13854-13860, 1996. Zaidman-Rémy A, Herve M, Poidevin M, Pili-Floury S, Kim MS, Blanot D, et al. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24: 463-473, 2006. 110 The multiple functions of the PGRP family in Drosophila immunity A Goto, S Kurata Discovery of the PGRP family References