An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms*

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and β-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.

Over the past three decades, insect immunity has been studied extensively with a variety of insects, including flies, mosquitoes, and moths. The insect immune system consists of two arms that operate upon the invasion of microorganisms into the hemocoel: (i) a humoral system resulting in the production of soluble antimicrobial peptides (1), melanin formation, and clotting via the activation of the prophenoloxidase cascade (2) and (ii) a cellular system based on hemocytes that is involved in defense reactions, including phagocytosis, nodulation, and encapsulation (3)(4)(5). These immune responses are triggered by the specific recognition of microorganisms by host proteins referred to as pattern recognition receptors (PRRs), 2 which are capable of binding to a variety of ligands, including lipopolysaccharides (LPS), lipoteichoic acid (LTA), peptidoglycan, and ␤-1,3-glucan (6 -9), which are cell wall components of three major groups of microorganisms: Gram-negative and Grampositive bacteria and fungi. These PRRs have principally been detected as soluble proteins in hemolymph and surface proteins on hemocytes.
The insect cellular responses primarily perform a function in the clearance of foreign invaders from hemolymph at the early phase of infection (10). Among three cellular immune reactions, phagocytosis is a highly conserved process that is known to occur in all metazoan phyla. This process is mediated by blood cells that either directly recognize the microorganisms or recognize targets that are coated in opsonic molecules. Thus far, insect phagocytosis has also generally been considered an essential immune reaction as it is in vertebrates (11). Nonetheless, phagocytosis is not currently as completely understood as other insect immune mechanisms. In particular, very little information has been discovered relevant to the phagocytic opsonins present in insect hemolymph, although several membrane receptors for phagocytosis have been identified.
In this study, we have purified a new protein with molecular mass of 9 kDa from the hemolymph of the Galleria mellonella larvae. In several respects, including the amino acid sequence homology, it was determined that the protein was quite similar to a cationic protein with mass of 9 kDa isolated from hemolymph of Manduca sexta (12). This Manduca protein was named cationic protein 8 (CP8) as its amino acid sequence was homologous to a small CP8 that had been found previously in Bombyx mori (GenBank accession number AY655143) and Bombyx mandarina (GenBank accession number EF126182). Therefore, our protein is referred to as GmCP8 (G. mellonella CP8) in this study. GmCP8 evidenced a marked binding activity to LPS, LTA, and ␤-1,3-glucan. As a multiligand receptor, the protein was shown to be capable of recognizing the three microorganisms selected in this study: Escherichia coli, Micrococcus luteus, and Candida albicans. Subsequently, we attempted to ascertain which immune responses were caused by the binding of the protein to microorganisms. According to the results of our in vitro and in vivo experiments, it was determined that GmCP8 had opsonin activity for the phagocytosis of the three microorganisms by hemocytes. A better understanding of this novel hemolymph protein, which was identified as both a multiligand receptor and a phagocytic opsonin, might provide us with crucial insights into a cellular defense system exploited by insects to deal with a broad variety of invaders.

EXPERIMENTAL PROCEDURES
Insects and Preparation of Cell-free Hemolymph and Hemocytes-G. mellonella larvae were cultivated on an artificial diet at 30°C in a dark incubator (13). Hemolymph was collected directly from the cut prolegs of the larvae into sterile tubes containing a few crystals of phenylthiourea. After the centrifugation of the sample at 10,000 ϫ g for 10 min at 4°C, the cellfree hemolymph, hereafter designated as plasma, was utilized immediately or stored at Ϫ70°C. To prepare the hemocytes, hemolymph was collected in a chilled tube containing 1 ml of anticoagulant buffer (93 mM NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM EDTA, and a few crystals of phenylthiourea, pH 4.6). The hemolymph was subsequently centrifuged at 180 ϫ g for 5 min at 4°C. The sediments containing the hemocytes were then washed twice in Grace's insect medium (GIM; Sigma). Isolated hemocytes were used immediately for experiments, although they were confirmed to be more than 90% viable at 4 h after breeding.
Purification of GmCP8 and Production of Anti-GmCP8 Antibody-In our previous study, GmCP8 was isolated as one of three fungus-binding proteins from the plasma of G. mellonella larvae (14). GmCP8, which was referred to as fungus-binding protein 1 in the previous study, was injected into New Zealand White rabbits to generate an anti-GmCP8 Ab in accordance with standard protocols. In this study, the anti-GmCP8 Ab was used to trace GmCP8 in the purification procedures. The purification process, which consisted of a three-stage procedure, permitted us to acquire a sizable quantity of GmCP8 from the G. mellonella plasma. In brief, plasma (10 ml) was mixed with an equal volume of 10% acetic acid and agitated overnight at 4°C. After 30 min of centrifugation at 10,000 ϫ g, the supernatant was removed and loaded onto a Sephadex G-50 gel filtration column equilibrated with 5% acetic acid. Fractions were eluted at a flow rate of 6 ml/h and collected at 20-min intervals. Every third fraction was analyzed via SDS-PAGE and immunoblotted with anti-GmCP8 Ab. The GmCP8-containing fractions were then pooled and applied to a C 4 reversed-phase HPLC column (Vydac 214TP54). After washing the column with water containing 0.1% trifluoroacetic acid for 10 min, fractions were eluted with a linear gradient of 0 -60% acetonitrile in 0.1% trifluoroacetic acid for 60 min at a flow rate of 0.5 ml/min. The purified GmCP8 was then subjected to Tricine SDS-PAGE and acid-urea-PAGE analyses to confirm purity. The molecular mass of the purified GmCP8 was determined via matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry at the Korea Basic Science Institute.
Quantitative Reverse Transcription-PCR and Tissue Culture-To evaluate the tissue specificity of GmCP8 expression, the non-immunized or immunized larvae were dissected in accordance with a predetermined time schedule. For the immunization of insects, 5 ϫ 10 3 cells of log phase E. coli were injected into each of the larvae. The fat body, midgut, and integument were collected and rinsed repeatedly with insect Ringer's solution (IRS; 2.3 mM NaHCO 3 , 128 mM NaCl, 1.3 mM KCl, and 1.8 mM CaCl 2 , pH 6.2). In all cases, total RNA was isolated, and GmCP8 expression was analyzed immediately in the collected tissues. Total RNA samples were prepared with an RNA extrac-tion kit (SV Total RNA Isolation System, Promega, Madison, WI). Similarly, the fat body and the hemocyte RNA samples were also isolated at predetermined times following immunization. First strand cDNA was synthesized with total RNA (1-2 g), oligo(dT) 18 , and Moloney murine leukemia virus reverse transcriptase (200 units; Bioneer, Daejon, Korea) at 42°C for 1 h. G. mellonella actin transcripts were used as an internal standard for the normalization of the cDNA templates. Relative levels of GmCP8 cDNA in the samples were measured via semiquantitative PCR using two primers corresponding to the reverse complement of nucleotides (dashed-underlined arrows 42°C, 1 min; and 72°C, 1 min. The cycle numbers were chosen to yield comparable band intensities while preventing saturation. For the tissue cultures, the fat body, midgut, and integument were prepared in accordance with the same procedure as described above. The dissected tissues were rinsed twice in GIM, and ϳ250 mg of each tissue was transferred to separate wells of 12-well tissue culture plates (Nunclon), each of which contained 1 ml of the same medium supplemented with 100 units/ml penicillin G and 100 g/ml streptomycin (Sigma). In the case of the hemocyte sample, ϳ2 ϫ 10 6 cells from 10 larvae were utilized for each culture. The cultures were incubated for 16 h at 30°C with shaking at 100 rpm. The supernatants of each sample were collected via centrifugation and then dialyzed with a 1,000 molecular weight cutoff dialysis bag (Spectrum) in 50 mM Tris-HCl buffer, pH 7.0 for 16 h. Additionally, the protein extracts of tissues were prepared in SDS-PAGE sample buffer (1 M Tris, 10% SDS, and 21% glycerol, pH 8.8) and homogenized with a pestle or by pipetting. Following a brief centrifugation, all protein samples were analyzed via immunoblot assays using anti-GmCP8 Ab.
Assays for Binding of GmCP8 to Microorganisms and Microbial Surface Molecules-1 ϫ 10 9 formaldehyde-fixed bacteria (E. coli and M. luteus) or C. albicans were resuspended in plasma diluted 3-fold with IRS. The mixture was incubated for 2 h with gentle shaking at 30°C and then centrifuged for 10 min at 2,000 ϫ g at 4°C. The supernatant was removed, and cells in the sediment were washed three times in phosphate-buffered saline (PBS; 1.47 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 2.7 mM KCl, and 137 mM NaCl, pH 7.4) containing 0.02% Tween 20. The bound proteins were subsequently eluted with 100 l of SDS-PAGE sample buffer. After 10 min of centrifugation at 2,000 ϫ g at 4°C, the supernatants were subjected to Tricine-SDS-PAGE analysis. Duplicate gels were used for immunoblot assays using anti-GmCP8 Ab. In an effort to assess binding activity to polysaccharides, 2 g of GmCP8 was incubated for 2 h with 2 mg of peptidoglycan (Fluka, 53243), curdlan (insoluble polymer of ␤-1,3-glucan; Sigma, C7821), or cellulose (insoluble polymer of ␤-1,4-glucan; Sigma, S5504) in 100 l of IRS containing 1% bovine serum albumin at 30°C. Each sample was then centrifuged for 10 min at 10,000 ϫ g at 4°C. The sediment was washed three times with PBS containing 0.02% Tween 20 and then diluted 3-fold with Tricine-SDS-PAGE sample buffer. After a brief centrifugation, the supernatants were analyzed via immunoblot assay. Additionally, the specific binding activity of GmCP8 to LPS (Sigma, L2630) or LTA (Sigma, L2515) was determined via a dansyl polymyxin (DPX) displacement assay (15). In this test, polymyxin B (Sigma, P0972) and apoli-pophorin-III (apoLp-III) that had been isolated previously from the plasma of G. mellonella (16) were used as positive controls. To further verify the specific binding of GmCP8 to the surfaces of the three microorganisms, we assessed the inhibitory effects of LPS, LTA, or laminarin (soluble polymer of ␤-1,3-glucan; Sigma, L9634) on the binding of GmCP8 to two bacteria and C. albicans. 2 g of GmCP8 was mixed with 100 g of each ligand in 50 l of IRS. After 1 h of incubation at 30°C, 50 l of IRS containing 1 ϫ 10 8 formaldehyde-fixed bacteria or C. albicans were added to each sample. As described above, the mixtures were incubated, and the protein detached from each microorganism was subjected to Western blotting analysis. Samples of GmCP8 that had not been preincubated with each of the ligands were used as positive controls.
Assays for Microbicidal Activity of Hemocytes-Each sample of two log phase bacteria (1 ϫ 10 8 cells) or C. albicans (1 ϫ 10 7 cells) was incubated for 2 h in 100 l of IRS containing GmCP8 (100 g) or plasma (10 l) as a positive control at 30°C in a shaking incubator. After two washes with IRS, 1 l of each sample was mixed with 99 l of GIM containing hemocytes (1 ϫ 10 6 cells) or no hemocytes. As a negative control, microorganisms treated neither with GmCP8 nor plasma were incubated with hemocytes under identical conditions. After 1.5 h of incubation in a rotary shaker at 30°C, 10 l of each sample was diluted with 990 l of GIM, and 10-l portions were then removed and plated onto tryptic soy broth or Sabouraud dextrose broth agar plates for bacteria and C. albicans, respectively. The resultant cultures were enumerated in colony-forming units (CFU) after 24 h of culture at 37°C. The microbicidal activities of hemocytes were expressed as the viable rate (in percent) of each microorganism calculated as follows: (number of CFU in test sample/number of CFU in the corresponding sample that was not treated with hemocytes) ϫ 100. To assess the effects of GmCP8 on nodulation against microorganisms, each GmCP8-coated microorganism was incubated with hemocytes as above and then visualized under light microscopy after staining with hematoxylin solution. Each microorganism was also coated with plasma or apoLp-III from G. mellonella and used as a control sample.
Flow Cytometry Analysis and Microscopic Observation for Phagocytosis-Each of the heat-killed microorganisms was labeled with FITC in accordance with the procedure described previously (17). FITC-labeled bacteria (1 ϫ 10 8 cells) or yeast (1 ϫ 10 7 cells) were coated with GmCP8 according to the same procedure described above. FITC-labeled bacteria (1 ϫ 10 7 ) or yeast (1 ϫ 10 6 ) coated with GmCP8 were mixed with hemocytes (2 ϫ 10 5 ) in a total volume of 100 l of GIM in each well of 96-well tissue culture plates (Nunclon). After 1 h of incubation at 30°C, each mixture was transferred to a 1.5-ml centrifuge tube and washed twice in 100 l of GIM. Then, to quench the fluorescence of any non-internalized particles, the samples were treated with 100 l of 0.4% trypan blue solution (Fluka) for 10 min. After washing with GIM, each sample was visualized via light and fluorescence microscopy (Leica DMLB). Additionally, the phagocytosis of each FITC-labeled microorganism by hemocytes was evaluated via flow cytometry analysis with a FACS flow cytometer (BD Biosciences) with computer-assisted evaluation of data (FACScan software). For FACS analysis, all hemocyte samples were fixed in 2% paraformaldehyde in IRS at 4°C, and ϳ10,000 -20,000 hemocytes of each sample were counted. Alternatively, to evaluate the inhibitory effects of each ligand on the phagocytic opsonin activity of GmCP8, 100 g of GmCP8 was preincubated in 50 l of IRS containing 1 mg of LPS, LTA, or laminarin for 1 h at 30°C and utilized for FACS analysis as above. Additionally, the phagocytosis of non-coated microorganisms by hemocytes was measured as a control in accordance with the same procedures described above.
Confocal Microscopic Observation-2 l of IRS containing each of the FITC-labeled microorganisms (10 7 CFU for each bacterium and 10 6 CFU for C. albicans) was injected into the hemocoels of live G. mellonella larva. After 1 h of incubation at room temperature, the hemocytes were extracted from the larvae in accordance with the procedure described above. The hemocytes were then placed on the slide glass and incubated for 20 min at 30°C. After washing with PBS, the hemocytes were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The slides were then washed with PBS and exposed for 10 min to 0.4% Triton X-100 in PBS followed by two additional washes in PBS. After 1 h of blocking with 5% bovine serum albumin in PBS, the slides were incubated with anti-GmCP8 Ab diluted to 1:500 in 1% bovine serum albumin in PBS for 1.5 h at 30°C. The samples were washed three times in PBS and incubated for 1 h with a fluorescein rhodamine-conjugated goat anti-rabbit IgG Ab (Alexa Flour 546 goat anti-rabbit, Invitrogen) diluted 1:300 in 1% bovine serum albumin in PBS. After treatment with the secondary Ab, the samples were again washed three times in PBS and stained with TO-PRO-3 iodide (Invitrogen) to visualize the nuclei of the hemocytes. After the final wash, the slides were mounted with an aqueous mounting medium and visualized under an FV500 laser-scanning confocal microscope (Olympus). For each of the samples, 10 sequential optical sections of 1 m each were collected, and only one section is shown.

RESULTS
Purification of GmCP8 and Its Amino Acid Sequence-Via a three-step procedure consisting of acid extraction, gel permeation chromatography, and reverse phase HPLC, GmCP8 was purified from the plasma of G. mellonella larvae. As shown in Fig. 1A, GmCP8 was eluted in a symmetric HPLC peak. Fig. 1, B and C, show the purified GmCP8 on the Tricine-SDS-PAGE and acid-urea-PAGE gels, respectively. As the protein appeared in two discrete bands on the acid-urea-PAGE gel, it was assumed that GmCP8 might occur in two isoforms, namely an amidated and an acidic C-terminal form. The results of MALDI analysis showed two mass peaks with a difference of 58 Da, which corresponds to the mass of glycine (Fig. 1D). The fulllength cDNA encoding GmCP8 was cloned from the larval fat body using a combination of reverse transcription-PCR and 5Ј-rapid amplification of cDNA ends-PCR (supplemental Fig. S1). The observed cDNA structure indicated that it comprised 87 amino acids with a C-terminal glycine, which has been observed frequently in the cDNA structures of other proteins harboring an amidated C terminus (18). It was also noted that GmCP8 harbored six intradisulfide bonds because it contained 12 cysteine residues (Fig. 1E). Overall, the calculated New Insect Opsonin for Phagocytosis of Microorganisms AUGUST 13, 2010 • VOLUME 285 • NUMBER 33

JOURNAL OF BIOLOGICAL CHEMISTRY 25245
average masses of the two isoforms of GmCP8 were estimated at 9,169 and 9,226 Da, which are almost identical to the measured masses. Therefore, it was concluded that GmCP8 was a small compact protein that was not conjugated with any other molecule and that one of two GmCP8 isoforms harbored an amidated C-terminal serine as Gly 87 participated in the formation of a C-terminal amide. Additionally, it was determined that the amino acid sequence of GmCP8 exhibits a high degree of similarity with the inducible serine protease inhibitor-1 (ISPI-1; Swiss-Prot accession number P81905), the fungal protease inhibitor-1 (AmFPI-1; Swiss-Prot accession number B0JFB8), and CP8 (GenBank accession number EF467286) from three different lepidopteran insects, G. mellonella (13), Antheraea mylitta (19), and M. sexta (12), respectively (supplemental Fig. S2).
Production of GmCP8 in Insect Tissues-The sites of GmCP8 synthesis were identified via immunoblotting and quantitative reverse transcription-PCR analyses. Fig. 2A shows an SDS-PAGE analysis for proteins extracted from the hemocytes, fat body, midgut, and integument (left panel). In the immunoblotting assay, the GmCP8 band was detected only in the fat body extract (right panel). However, GmCP8 was detected in the culture supernatants of three tissues: fat body, midgut, and integument (Fig. 2B). Therefore, it was determined that GmCP8 was synthesized in these three tissues and secreted into the hemo-lymph. It is worth noting that GmCP8 was not generated in the hemocytes. Consistent with these results, reverse transcription-PCR analysis showed that the GmCP8-encoded gene was expressed in three tissues but not in the hemocytes (Fig. 2C). Additionally, we attempted to determine whether GmCP8 gene expression was up-regulated in the fat body or induced in the hemocytes in response to bacterial infection. Our results showed that GmCP8 mRNA was present at a constant level in the fat body but was not detected in the hemocytes during the entire test period (Fig. 2D). Therefore, it was concluded that GmCP8 was constitutively produced in the fat body but not in the hemocytes even under bacterial infection conditions.
Recognition of Microorganisms by GmCP8-Plasma was mixed with three types of microorganisms fixed with formaldehyde, and proteins detached from the microbial surfaces were analyzed via Tricine-SDS-PAGE under reducing conditions. GmCP8 was then detected via an immunoblotting assay using anti-GmCP8 Ab. As demonstrated in Fig. 3A, GmCP8 bound strongly to the surfaces of all three microorganisms. In the subsequent experiments, we attempted to determine whether GmCP8 bound specifically to the cell wall components of three microorganisms, such as LPS, LTA, peptidoglycan, and glucan. In the DPX displacement assays, it was determined that the  binding of DPX to LPS or LTA was inhibited by GmCP8 in a dose-dependent fashion (Fig. 3B). The inhibitory effects of GmCP8 on the binding of DPX to LPS and LTA were similar to or stronger than those of apoLp-III, which was shown previously to bind to LPS and LTA (20,21). It was therefore concluded that GmCP8 could recognize E. coli and M. luteus via specific binding to LPS or LTA. Additionally, the specific binding of GmCP8 to fungal cell wall components was assessed using insoluble polymers of different polysaccharides (Fig. 3C). As a result, GmCP8 was shown to bind to curdlan (polymer of ␤-1,3-glucan) but not to peptidoglycan or cellulose (polymer of ␤-1,4-glucan). It was also concluded that the recognition of C. albicans by GmCP8 was mediated by its specific binding to ␤-1,3-glucan. Additionally, the specific microbial recognition of GmCP8 was confirmed by determining whether or not GmCP8 was capable of binding to the microbial surface after its incubation with each of three cell wall components. As a result, it was determined that each ligand substantially inhibited the binding of GmCP8 to the microbial surface (Fig. 3D).
Antimicrobial Effect of G. mellonella Hemocytes Mediated by GmCP8- Fig. 4A shows the microbicidal effects of hemocytes against three microorganisms under different conditions. When hemocytes were incubated directly with three microorganisms for 1.5 h, no antimicrobial activity of hemocytes was detected. In contrast, the hemocytes evidenced marked microbicidal activity against three microorganisms that had been pretreated with plasma. These results showed that G. mellonella hemocytes could not mount their defense reactions against invading microorganisms without the aid of plasma components. It was also determined that hemocytes exerted their antimicrobial effects against three microorganisms coated with purified GmCP8, thereby indicating that GmCP8 performed a critical function in the cellular defense reactions of G. mellonella. In the following experiment, we attempted to determine whether or not GmCP8 was involved in nodule formation via an in vitro hemocyte aggregation assay.
Our results showed that, in contrast to the two control samples prepared with plasma and apoLp-III, hemocyte aggregation was not observed when the hemocytes were incubated with microorganisms pretreated with GmCP8 (Fig. 4B).

Role of GmCP8 in Phagocytosis of Microorganisms by G. mellonella
Hemocytes-As GmCP8 was determined not to be involved in nodule formation, which was one of two cellular immune responses that might occur in response to the invasion of microorganisms into the with each of three insoluble polysaccharides, GmCP8 binding was analyzed by immunoblot assay using anti-GmCP8 Ab. GmCP8 was detected only in the curdlan sample. CP8, GmCP8; PGN, peptidoglycan; Cur, curdlan; Cel, cellulose. D, binding of the GmCP8 to three microorganisms was inhibited by soluble components of the microbial cell wall. In all cases, it was determined that GmCP8 pretreated with each ligand did not bind to the microbial surface. Symbols ϩ and Ϫ indicate addition and no addition of corresponding ligand, respectively. CP8, 1 g of GmCP8.  Ͻ 0.001). B, each of the microorganisms was coated with GmCP8, apoLp-III (100 g), or plasma as described under "Experimental Procedures." After washing with IRS, opsonized microorganisms were mixed with hemocytes and incubated in a rotary shaker for 1.5 h at 30°C prior to examination under a light microscope. For negative controls, non-coated microorganisms were incubated with hemocytes under the same condition. hemocoel, we evaluated its possible role in the phagocytosis of microorganisms by hemocytes. First, we attempted to determine whether or not the binding of GmCP8 to three types of microorganisms could opsonize them for phagocytosis. Fig. 5 shows the fluorescence microscopic and flow cytometric analyses for the phagocytosis of the three microorganisms by G. mellonella hemocytes. The results showed that the GmCP8treated microorganisms were internalized into the hemocytes to a substantially greater extent than were the untreated microorganisms. To further assess the function of GmCP8 as a molecular bridge between microorganisms and hemocytes, we evaluated the inhibitory effects of three soluble ligands (LPS, LTA, and laminarin) on the opsonophagocytic activity of GmCP8. As a consequence, the treatment of microorganisms with GmCP8 preincubated with each ligand did not augment the uptake of the corresponding microorganism by hemocytes. As shown in all FACS data in Fig. 5, peak c in the three graphs overlapped almost completely with peak a for the control samples, which were not treated with GmCP8. These results clearly revealed that the opsonin activity of GmCP8 was attributable to its specific binding to the surfaces of microorganisms. Furthermore, the GmCP8-mediated phagocytosis noted in the hemo-cytes was examined in greater detail under in vivo conditions. Each of three FITC-labeled microorganisms was injected into the hemocoel of live G. mellonella larvae, and then the hemocytes collected from the insects were subjected directly to confocal microscopic analysis. As shown in Fig. 6, each microorganism was observed around the nucleus of hemocytes, and its FITC fluorescence was shown to overlap with the rhodamine fluorescence of the secondary Ab attached to anti-GmCP8 Ab in the case of E. coli or enclosed by rhodamine fluorescence in the cases of M. luteus and C. albicans. Considering that GmCP8 was not generated in the hemocytes, it became apparent that GmCP8 was internalized into the hemocytes from the hemolymph while bound to the surfaces of microorganisms. Collectively, our results demonstrated that invading microorganisms were specifically bound to GmCP8 occurring in the hemolymph and were then subjected to GmCP8-mediated phagocytosis by the hemocytes.

DISCUSSION
Insects have become a preferred system for the study of certain aspects of innate immunity as insects can defend themselves against microbial pathogens without an adaptive immune system. Whereas the fruit fly and the mosquito are useful and genetically tractable models that permit the rapid identification of novel immune factors, lepidopteran insects are frequently utilized because their large size makes them suitable for biochemical experiments for the analysis of immune mechanisms. Because insects lack the specificity of microbial recognition achievable with an adaptive system, they generate a variety of PRRs to cope with a very diverse range of microbial pathogens and also utilize a combination of several PRRs, thereby expanding their ability to detect invaders (22,23). In this study, we have purified a new multiligand receptor from the plasma of G. mellonella larvae and have also demonstrated its immunological role in host-pathogen interactions using the purified protein. GmCP8 evidenced potent microbial binding and phagocytosis-promoting activities, clearly indicating this protein as an opsonin for the phagocytosis of the three microorganisms selected in this study.
As is the case in higher vertebrates, phagocytosis is believed to be an essential cellular process in insect immune responses In FACS analyses, all samples treated with GmCP8 evidenced substantially enhanced uptake of the microorganisms into hemocytes (peak b) as compared with control samples that were treated with no GmCP8 (peak a) or GmCP8 preincubated with each of soluble ligands (LPS, LTA, or laminarin) (peak c). against invading microorganisms. Insect phagocytosis is also mediated by the binding of PRRs to the surfaces of microorganisms (10,11). Thus far, four main classes of PRRs associated with insect phagocytosis have been detected in the form of soluble proteins secreted from immune cells and membrane proteins attached to hemocytes, which interact with their cognate ligands expressed on the surfaces of Gram-negative and Grampositive bacteria as well as fungi (11,24). (i) A group of thioester-containing proteins (TEPs) are the best characterized phagocytic opsonins that have been detected in Drosophila melanogaster and Anopheles gambiae (25,26). It has also been noted that a variety of TEPs appear to be involved in the recognition and phagocytosis of different microorganisms: TEPII for E. coli, TEPIII for Staphylococcus aureus, and TEPVI for C. albicans (27). (ii) The scavenger receptor families comprise structurally unrelated transmembrane proteins that participate in the recognition of polyanionic ligands. They have been recognized as PRRs crucial for phagocytosis in many species, including mammals and insects (28). The insect scavenger receptor families are expanded to cope with a broad variety of microorganisms as each member has only a limited repertoire for microbial recognition (29,30). (iii) Down syndrome cell adhesion molecule (Dscam) is the immunoglobulin superfamily receptor previously identified as a potential phagocytic membrane receptor and opsonin in D. melanogaster and A. gambiae this is reminiscent of mammalian immunoglobulin (31,32). Therefore, it seems plausible that Dscam is an insect phagocytic PRR with the required repertoire to cope with a broad range of pathogens. (iv) Two novel phagocytosis-mediated transmembrane receptors, Eater and Nimrod C1 (33,34), have been previously identified in D. melanogaster. Whereas Eater was well defined as a phagocytic receptor that promotes bacteria binding and uptake, it remains to be clarified whether Nimrod C1 can recognize bacteria directly, although it has been demonstrated that the protein was capable of contributing to the phagocytosis of E. coli (34).
As described under "Results," the amino acid sequence of GmCP8 was shown to be homologous with those of ISPI-1 from G. mellonella, CP8 from M. sexta, and AmFPI-1 from A. mylitta (supplemental Fig. S2). Regarding their physiological functions, it has been reported that ISPI-1 and AmFPI-1 are involved in an insect defense mechanism as an inhibitor to regulate the proteolytic activity of a variety of proteases, including fungal protease (13,19), and Manduca CP8 with no inhibitor activity contributed to the activation of prophenoloxidase cascade in the hemolymph (12). However, it remains to be determined whether those three proteins also exhibit the same microbial binding and/or phagocytic activity as GmCP8. On the other hand, it was postulated that GmCP8 might be functionally similar to the CP8 from M. sexta rather than the other two proteins (ISPI-1 and AmFPI-1) in that it evidenced no activity as a protease inhibitor (data not shown), and its production was not inducible upon microbial infection as shown in Fig. 2D. Additionally, as in the case of Manduca CP8, GmCP8 was not generated in the hemocytes, which allowed us to confirm its opsonin activity via the direct injection of microorganisms into the hemocoels of live insects.
Thus far, there has been only minimal information regarding insect phagocytic opsonin as compared with other immune fac-tors. Furthermore, to the best of our knowledge, no phagocytic membrane receptors, such as the scavenger receptor family, have been reported in lepidopteran insects, thus suggesting that they may utilize an opsonin rather than a membrane receptor for the phagocytosis of microbial pathogens. Therefore, we assumed that GmCP8 might be involved in phagocytosis when it was found to exhibit marked microbial binding activity and also to mediate the microbicidal activity of hemocytes (Fig. 4A), but it was not shown to induce hemocyte aggregation upon incubation with microorganisms (Fig. 4B) nor was it implicated in other immune reactions such as prophenoloxidase activation (data not shown). Finally, according to the results of our in vitro and in vivo experiments, we can define GmCP8 as a new opsonin requisite for the phagocytosis of three different types of microorganisms, although its receptor on G. mellonella hemocytes, such as the Fc receptor or the complement receptor on mammalian phagocytes, has yet to be identified. Overall, the results presented herein provide us with new insights into some as yet incompletely characterized insect immune mechanisms specifically with regard to phagocytosis.