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Recently, our group demonstrated that the bean bug, Riptortus pedestris, is a good experimental symbiosis model to study the molecular cross-talk between the host insect and the gut symbiont. The Burkholderia symbiont is orally acquired by host nymphs from the environment in every generation. However, it is still unclear how Riptortus specifically interacts with entomopathogens that are abundant in the environmental soil. In preliminary experiments, we observed that a potent entomopathogen, Serratia marcescens, can colonize the midgut of Riptortus insects and was recovered from the midgut when Serratia cells were orally administered, suggesting that this pathogenic bacterium can escape host immune defenses in the salivary fluid. We examined how orally fed Serratia cells can survive in the presence of antimicrobial substances of the Riptortus salivary fluid. In this study, a 15 kDa trialysin-like protein from the salivary gland of R. pedestris and a potent virulence factor of Serratia cells, a serralysin metalloprotease, from the culture medium of S. marcescens were successfully purified to homogeneity. When the purified Riptortus trialysin (rip-trialysin) was incubated with purified serralysin, rip-trialysin was specifically hydrolyzed by serralysin, leading to the loss of antimicrobial activity. These results clearly demonstrated that a potent virulent metalloprotease of S. marcescens functions as a key player in the escape from the salivary fluid-mediated host immune response, resulting in successful colonization of S. marcescens in the host midgut., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Valuable insect models have tremendously contributed to our understanding of innate immunity and symbiosis. Bean bug, Riptortus pedestris, is a useful insect symbiosis model due to harboring cultivable monospecific gut symbiont, genus Burkholderia. Bean bug is a hemimetabolous insect whose immunity is not well-understood. However, we recently identified three major antimicrobial peptides of Riptortus and examined the relationship between gut symbiosis and host immunity. We found that the presence of Burkholderia gut symbiont positively affects Riptortus immunity. From studying host regulation mechanisms of symbiont population, we revealed that the symbiotic Burkholderia cells are much more susceptible to Riptortus immune responses than the cultured cells. We further elucidated that the immune-susceptibility of the Burkholderia gut symbionts is due to the drastic change of bacterial cell envelope. Finally, we show that the immune-susceptible Burkholderia symbionts are able to prosper in host owing to the suppression of immune responses of the symbiotic midgut., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus is a Gram-positive bacterial pathogen that is decorated by glycopolymers, including wall teichoic acid (WTA), peptidoglycan, lipoteichoic acid, and capsular polysaccharides. These bacterial surface glycopolymers are recognized by serum antibodies and a variety of pattern recognition molecules, including mannose-binding lectin (MBL). Recently, we demonstrated that human serum MBL senses staphylococcal WTA. Whereas MBL in infants who have not yet fully developed adaptive immunity binds to S. aureus WTA and activates complement serum, MBL in adults who have fully developed adaptive immunity cannot bind to WTA because of an inhibitory effect of serum anti-WTA IgG. Furthermore, we showed that human anti-WTA IgGs purified from pooled adult serum IgGs triggered activation of classical complement-dependent opsonophagocytosis against S. aureus. Because the epitopes of WTA that are recognized by anti-WTA IgG and MBL have not been determined, we constructed several S. aureus mutants with altered WTA glycosylation. Our intensive biochemical studies provide evidence that the β-GlcNAc residues of WTA are required for the induction of anti-WTA IgG-mediated opsonophagocytosis and that both β- and α-GlcNAc residues are required for MBL-mediated complement activation. The molecular interactions of other S. aureus cell wall components and host recognition proteins are also discussed. In summary, in this review, we discuss the biological importance of S. aureus cell surface glycopolymers in complement activation and host defense responses., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Riptortus pedestris harboring Burkholderia symbiont is a useful symbiosis model to study the molecular interactions between insects and bacteria. We recently reported that the lipopolysaccharide O-antigen is absent in the Burkholderia symbionts isolated from Riptortus guts. Here, we investigated the symbiotic role of O-antigen comprehensively in the Riptortus-Burkholderia model. Firstly, Burkholderia mutant strains deficient of O-antigen biosynthesis genes were generated and confirmed for their different patterns of the lipopolysaccharide by electrophoretic analysis. The O-antigen-deficient mutant strains initially exhibited a reduction of infectivity, having significantly lower level of symbiont population at the second-instar stage. However, both the wild-type and O-antigen mutant symbionts exhibited a similar level of symbiont population from the third-instar stage, indicating that the O-antigen deficiency did not affect the bacterial persistence in the host midgut. Taken together, we showed that the lipopolysaccharide O-antigen of gut symbiont plays an exclusive role in the initial symbiotic association., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The relation between gut symbiosis and immunity has been reported in various animal model studies. Here, we corroborate the effect of gut symbiont to host immunity using the bean bug model. The bean bug, Riptortus pedestris, is a useful gut symbiosis model due to the monospecific gut symbiont, genus Burkholderia. To examine the effect of gut symbiosis to host immunity, we generated the gut symbiont-harboring (symbiotic) insect line and the gut symbiont-lacking (aposymbiotic) insect line. Upon bacterial challenges, the symbiotic Riptortus exhibited better survival than aposymbiotic Riptortus. When cellular immunity was inhibited, the symbiotic Riptortus still survived better than aposymbioic Riptortus, suggesting stronger humoral immunity. The molecular basis of the strong humoral immunity was further confirmed by the increase of hemolymph antimicrobial activity and antimicrobial peptide expression in the symbiotic insects. Taken together, our data clearly demonstrate that Burkhoderia gut symbiont positively affect the Riptortus systemic immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The cell envelopes of many Gram-positive bacteria contain wall teichoic acids (WTAs). Staphylococcus aureus WTAs are composed of ribitol phosphate (RboP) or glycerol phosphate (GroP) backbones substituted with D-alanine and N-acetyl-D-glucosamine (GlcNAc) or N-acetyl-D-galactosamine (GalNAc). Two WTA glycosyltransferases, TarM and TarS, are responsible for modifying the RboP WTA with α-GlcNAc and β-GlcNAc, respectively. We recently reported that purified human serum anti-WTA IgG specifically recognizes β-GlcNAc of the staphylococcal RboP WTA and then facilitates complement C3 deposition and opsonophagocytosis of S. aureus laboratory strains. This prompted us to examine whether anti-WTA IgG can induce C3 deposition on a diverse set of clinical S. aureus isolates. To this end, we compared anti-WTA IgG-mediated C3 deposition and opsonophagocytosis abilities using 13 different staphylococcal strains. Of note, the majority of S. aureus strains tested was recognized by anti-WTA IgG, resulting in C3 deposition and opsonophagocytosis. A minority of strains was not recognized by anti-WTA IgG, which correlated with either extensive capsule production or an alteration in the WTA glycosylation pattern. Our results demonstrate that the presence of WTAs with TarS-mediated glycosylation with β-GlcNAc in clinically isolated S. aureus strains is an important factor for induction of anti-WTA IgG-mediated C3 deposition and opsonophagocytosis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Because gut symbiotic bacteria affect host biology, host insects are expected to evolve some mechanisms for regulating symbiont population. The bean bug, Riptortus pedestris, harbors the Burkholderia genus as a gut symbiont in the midgut organ, designated as the M4 region. Recently, we demonstrated that the lysate of M4B, the region adjacent to M4, harbors potent antibacterial activity against symbiotic Burkholderia but not to cultured Burkholderia. However, the bona fide substance responsible for observed antibacterial activity was not identified in the previous study. Here, we report that cathepsin-L-like protease purified from the lysate of M4B showed strong antibacterial activity against symbiotic Burkholderia but not the cultured Burkholderia. To further confirm this activity, recombinant cathepsin-L-like protease expressed in Escherichia coli also showed antibacterial activity against symbiotic Burkholderia. These results suggest that cathepsin-L-like protease purified from the M4B region plays a critical role in controlling the population of the Burkholderia gut symbiont., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host-symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ's ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host's control and symbiont's motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.

We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Piceatannol (PCT), an anti-colitic natural product, undergoes extensive Phase II hepatic metabolism, resulting in very low bioavailability. We investigated whether colon-targeted delivery of PCT could enhance anti-colitic effects and how therapeutic enhancement occurred at the molecular level. Molecular effects of PCT were examined in human colon carcinoma cells and inflamed colons. The anti-colitic effects of PCT in a colon-targeted capsule (colon-targeted PCT) were compared with PCT in a gelatin capsule (conventional PCT) in a trinitrobenzene sulfonic acid-induced rat colitis model. Colon-targeted PCT elicited greatly enhanced recovery of the colonic inflammation. In HCT116 cells, PCT inhibited nuclear factor kappaB while activating anti-colitic transcription factors, nuclear factor-erythroid 2 (NF-E2) p45-related factor 2, and hypoxia-inducible factor-1. Colon-targeted PCT, but not conventional PCT, modulated production of the target gene products of the transcription factors in the inflamed colonic tissues. Rectal administration of PCT, which simulates the therapeutic action of colon-targeted PCT, also ameliorated rat colitis and reproduced the molecular effects in the inflamed colonic tissues. Colon-targeted delivery increased therapeutic efficacy of PCT against colitis, likely resulting from multitargeted effects exerted by colon-targeted PCT. The drug delivery technique may be useful for therapeutic optimization of anti-colitic lead compounds including natural products.

Nitric oxide (NO)-releasing nanoparticles (NPs) have emerged as a wound healing enhancer and a novel antibacterial agent that can circumvent antibiotic resistance. However, the NO release from NPs over extended periods of time is still inadequate for clinical application. In this study, we developed NO-releasing poly(lactic-co-glycolic acid)-polyethylenimine (PEI) NPs (NO/PPNPs) composed of poly(lactic-co-glycolic acid) and PEI/diazeniumdiolate (PEI/NONOate) for prolonged NO release, antibacterial efficacy, and wound healing activity. Successful preparation of PEI/NONOate was confirmed by proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet/visible spectrophotometry. NO/PPNPs were characterized by particle size, surface charge, and NO loading. The NO/PPNPs showed a prolonged NO release profile over 6 days without any burst release. The NO/PPNPs exhibited potent bactericidal efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa concentration-dependently and showed the ability to bind on the surface of the bacteria. We also found that the NO released from the NO/PPNPs mediates bactericidal efficacy and is not toxic to healthy fibroblast cells. Furthermore, NO/PPNPs accelerated wound healing and epithelialization in a mouse model of a MRSA-infected wound. Therefore, our results suggest that the NO/PPNPs presented in this study could be a suitable approach for treating wounds and various skin infections.

Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella.

Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca2(+) release from intracellular Ca2(+) stores and anti-PGN-IgG dependent Ca2(+) mobilization is controlled via a phospholipase Cγ-2-mediated pathway.

In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE-activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs., (© 2014 John Wiley & Sons Ltd.)

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Riptortus-Burkholderia symbiotic system is an experimental model system for studying the molecular mechanisms of an insect-microbe gut symbiosis. When the symbiotic midgut of Riptortus pedestris was investigated by light and transmission electron microscopy, the lumens of the midgut crypts that harbor colonizing Burkholderia symbionts were occupied by an extracellular matrix consisting of polysaccharides. This observation prompted us to search for symbiont genes involved in the induction of biofilm formation and to examine whether the biofilms are necessary for the symbiont to establish a successful symbiotic association with the host. To answer these questions, we focused on purN and purT, which independently catalyze the same step of bacterial purine biosynthesis. When we disrupted purN and purT in the Burkholderia symbiont, the ΔpurN and ΔpurT mutants grew normally, and only the ΔpurT mutant failed to form biofilms. Notably, the ΔpurT mutant exhibited a significantly lower level of cyclic-di-GMP (c-di-GMP) than the wild type and the ΔpurN mutant, suggesting involvement of the secondary messenger c-di-GMP in the defect of biofilm formation in the ΔpurT mutant, which might operate via impaired purine biosynthesis. The host insects infected with the ΔpurT mutant exhibited a lower infection density, slower growth, and lighter body weight than the host insects infected with the wild type and the ΔpurN mutant. These results show that the function of purT of the gut symbiont is important for the persistence of the insect gut symbiont, suggesting the intricate biological relevance of purine biosynthesis, biofilm formation, and symbiosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The majority of insects possess symbiotic bacteria. Since symbiont titers can affect host phenotypes of biological importance, host insects are expected to evolve some mechanisms for regulating symbiont population. Here we report that, in the Riptortus-Burkholderia gut symbiosis, titers of the beneficial symbiont transiently decrease at the pre-molt stages in host development. This molting-associated suppression of the symbiont population is coincident with the increase of antimicrobial activity in the symbiotic midgut, which is observed in both symbiotic and aposymbiotic insects. Two genes, pyrrhocoricin-like antimicrobial peptide and c-type lysozyme, exhibit significantly increased expression in the symbiotic midgut at the pre-molt stages. These results suggest that the molting-associated up-regulation of antimicrobial activity in the symbiotic midgut represents a physiological mechanism of the host insect to regulate symbiosis, which is presumably for defending molting insects against injury and infection and/or for allocating symbiont-derived energy and resources to host molting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5'-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3'. The 'helix-short helix-helix' motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Many insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the host's control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bug Riptortus pedestris harbors a betaproteobacterial extracellular symbiont of the genus Burkholderia in the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harbors Burkholderia symbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against the Burkholderia symbiont but not the cultured Burkholderia and other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region of R. pedestris that plays a critical role in controlling the population of the Burkholderia gut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (Imd) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, Tenebrio molitor. The cDNA sequence is comprised of 495 bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type beta-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram (-) bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, Listeria monocytogenes suggesting putative function in innate immunity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic "Cys-Cys-Gly" motif and "Cys188" residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

We investigated the mode of action underlying the anti-mycoplasma activity of cationic antimicrobial peptides (AMPs) using four known AMPs and Mycoplasma pulmonis as a model mycoplasma. Scanning electron microscopy revealed that the integrity of the M. pulmonis membrane was significantly damaged within 30 min of AMPs exposure, which was confirmed by measuring the uptake of propidium iodine into the mycoplasma cells. The anti-mycoplasma activity of AMPs was found to depend on the binding affinity for phosphatidylcholine, which was incorporated into the mycoplasma membrane from the growth medium and preferentially distributed in the outer leaflet of the lipid bilayer., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Many bacteria accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which confer resistance to nutritional depletion and other environmental stresses. Here, we report an unexpected involvement of the bacterial endocellular storage polymer, PHA, in an insect-bacterium symbiotic association. The bean bug Riptortus pedestris harbors a beneficial and specific gut symbiont of the β-proteobacterial genus Burkholderia, which is orally acquired by host nymphs from the environment every generation and easily cultivable and genetically manipulatable. Biochemical and cytological comparisons between symbiotic and cultured Burkholderia detected more PHA granules consisting of poly-3-hydroxybutyrate and associated phasin (PhaP) protein in the symbiotic Burkholderia. Among major PHA synthesis genes, phaB and phaC were disrupted by homologous recombination together with the phaP gene, whereby ΔphaB, ΔphaC, and ΔphaP mutants were generated. Both in culture and in symbiosis, accumulation of PHA granules was strongly suppressed in ΔphaB and ΔphaC, but only moderately in ΔphaP. In symbiosis, the host insects infected with ΔphaB and ΔphaC exhibited significantly lower symbiont densities and smaller body sizes. These deficient phenotypes associated with ΔphaB and ΔphaC were restored by complementation of the mutants with plasmids encoding a functional phaB/phaC gene. Retention analysis of the plasmids revealed positive selection acting on the functional phaB/phaC in symbiosis. These results indicate that the PHA synthesis genes of the Burkholderia symbiont are required for normal symbiotic association with the Riptortus host. In vitro culturing analyses confirmed vulnerability of the PHA gene mutants to environmental stresses, suggesting that PHA may play a role in resisting stress under symbiotic conditions.

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor onto bacterial cell membranes. These proteins play important roles in a wide variety of bacterial physiological processes, including virulence, and induce innate immune reactions by functioning as ligands of the mammalian Toll-like receptor 2. We review recent advances in our understanding of bacterial lipoprotein structure, biosynthesis and structure-function relationships between bacterial lipoproteins and Toll-like receptor 2. Notably, 40 years after the first report of the triacyl structure of Braun's lipoprotein in Escherichia coli, recent intensive MS-based analyses have led to the discovery of three new lipidated structures of lipoproteins in monoderm bacteria: the lyso, N-acetyl and peptidyl forms. Moreover, the bacterial lipoprotein structure is considered to be constant in each bacterium; however, lipoprotein structures in Staphylococcus aureus vary between the diacyl and triacyl forms depending on the environmental conditions. Thus, the lipidation state of bacterial lipoproteins, particularly in monoderm bacteria, is more complex than previously assumed., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Bacterial lipoproteins/lipopeptides inducing host innate immune responses are sensed by mammalian Toll-like receptor 2 (TLR2). These bacterial lipoproteins are structurally divided into two groups, diacylated or triacylated lipoproteins, by the absence or presence of an amide-linked fatty acid. The presence of diacylated lipoproteins has been predicted in low-GC content gram-positive bacteria and mycoplasmas based on the absence of one modification enzyme in their genomes; however, we recently determined triacylated structures in low-GC gram-positive Staphylococcus aureus, raising questions about the actual lipoprotein structure in other low-GC content gram-positive bacteria. Here, through intensive MS analyses, we identified a novel and unique bacterial lipoprotein structure containing an N-acyl-S-monoacyl-glyceryl-cysteine (named the lyso structure) from low-GC gram-positive Enterococcus faecalis, Bacillus cereus, Streptococcus sanguinis, and Lactobacillus bulgaricus. Two of the purified native lyso-form lipoproteins induced proinflammatory cytokine production from mice macrophages in a TLR2-dependent and TLR1-independent manner but with a different dependence on TLR6. Additionally, two other new lipoprotein structures were identified. One is the "N-acetyl" lipoprotein structure containing N-acetyl-S-diacyl-glyceryl-cysteine, which was found in five gram-positive bacteria, including Bacillus subtilis. The N-acetyl lipoproteins induced the proinflammatory cytokines through the TLR2/6 heterodimer. The other was identified in a mycoplasma strain and is an unusual diacyl lipoprotein structure containing two amino acids before the lipid-modified cysteine residue. Taken together, our results suggest the existence of novel TLR2-stimulating lyso and N-acetyl forms of lipoproteins that are conserved in low-GC content gram-positive bacteria and provide clear evidence for the presence of yet to be identified key enzymes involved in the bacterial lipoprotein biosynthesis.

The biochemical characterization of novel antimicrobial peptides (AMPs) and the determination of ligand molecules that induce AMP production are essential for understanding the host innate immune response in insects. Here, we purified a new 14-kDa AMP, named tenecin 4, from the larval hemolymph of the beetle Tenebrio molitor. Tenecin 4 contains 14% glycine residues and has moderate similarities both to the C-terminal region of Drosophila attacin and to silk-moth gloverin proteins. Purified tenecin 4 showed bactericidal activity against Gram-negative Escherichia coli but not against Gram-positive Bacillus subtilis or the fungus Candida albicans. Tenecin 4 production was induced by Toll cascade-activating ligands, such as β-1,3-glucan, lysine-type peptidoglycan and active Spätzle, and by the probable Imd pathway-activating ligand monomeric meso-diaminopimelic acid-type peptidoglycan. Taken together, these data show that tenecin 4 is a defense protein against Gram-negative pathogens and is induced by multiple ligands in Tenebrio larvae., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We recently reported that D-alanylation of Staphylococcus aureus wall teichoic acid (WTA) mitigates an induction of the Toll-mediated humoral response in Drosophila by interfering with peptidoglycan (PG) recognition by PG recognition protein-SA (PGRP-SA). Here, we investigated the mode of this interference by using an in vitro cell free system from larvae of the coleoptran insect Tenebrio molitor. WTA modification on PG had a potent inhibitory effect on PGRP-SA-mediated Toll proteolytic cascade activation, and the D-alanylation of WTA enhanced its inhibitory effect. Purified D-alanylated WTA released from PG lost its inhibitory action on both Toll cascade activation and PGRP-SA binding to insoluble PG. The inhibition of PGRP-SA binding to PG by D-alanylated WTA took place not only on polymeric PG but also on WTA-attached disaccharide units of monomeric PG. These results suggest that D-alanylation-mediated evasion requires the covalent bonding of D-alanylated WTA to PG, but not net-like cross-linking structure of PG., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bacterial lipoproteins are known to be diacylated or triacylated and activate mammalian immune cells via Toll-like receptor 2/6 or 2/1 heterodimer. Because the genomes of low G+C content gram-positive bacteria, such as Staphylococcus aureus, do not contain Escherichia coli-type apolipoprotein N-acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of S. aureus are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from S. aureus RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from S. aureus SA113 strain was diacylated. The determination of exact lipidated structures of S. aureus lipoproteins is thus crucial for elucidating the molecular basis of host-microorganism interactions. Toward this purpose, we intensively used MS-based analyses. Here, we demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipoprotein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resistant fatty acid. Further MS/MS analysis of the lipoprotein lipase digest revealed that the lipoprotein lipase-resistant fatty acid was acylated to α-amino group of the N-terminal cysteine residue of SitC. Triacylated forms of SitC with various length fatty acids were also confirmed in cell lysate of the RN4220 and Triton X-114 phase in three other S. aureus strains, including SA113 strain and one Staphylococcus epidermidis strain. Moreover, four other major lipoproteins including SA2202 in S. aureus strains were identified as N-acylated. These results strongly suggest that lipoproteins of S. aureus are mainly in the N-acylated triacyl form., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30kDa protein from hemocytes of the freshwater crayfish, Pacifastacus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type peptidoglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPHs are involved in this activation possibly by forming a complex with LGBP and without a PGRP., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Bacteria and other potential pathogens are cleared rapidly from the body fluids of invertebrates by the immediate response of the innate immune system. Proteolytic cascades, following their initiation by pattern recognition proteins, control several such reactions, notably coagulation, melanisation, activation of the Toll receptor and complement-like reactions. However, there is considerable variation among invertebrates and these cascades, although widespread, are not present in all phyla. In recent years, significant progress has been made in identifying and characterizing these cascades in insects. Notably, recent work has identified several connections and shared principles among the different pathways, suggesting that cross-talk between them may be common., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Genetic studies have elegantly characterized the innate immune response in Drosophila melanogaster. However, these studies have a limited ability to reveal the biochemical mechanisms underlying the innate immune response. To investigate the biochemical basis of how insects recognize invading microbes and how these recognition signals activate the innate immune response, it is necessary to use insects, from which larger amounts of hemolymph can be extracted. Using the larvae from two species of beetle, Tenebrio molitor and Holotrichia diomphalia, we elucidated the mechanisms underlying pathogenic microbe recognition. In addition, we studied the mechanism of host defense molecule amplification. In particular, we identified several pattern recognition proteins, serine proteases, serpins and antimicrobial peptides and examined how these molecules affect innate immunity.

The Drosophila Toll receptor does not interact directly with microbial determinants, but is instead activated by a cleaved form of the cytokine-like molecule Spätzle. During the immune response, Spätzle is processed by complex cascades of serine proteases, which are activated by secreted pattern-recognition receptors. Here, we demonstrate the essential role of ModSP, a modular serine protease, in the activation of the Toll pathway by gram-positive bacteria and fungi. Our analysis shows that ModSP integrates signals originating from the circulating recognition molecules GNBP3 and PGRP-SA and connects them to the Grass-SPE-Spätzle extracellular pathway upstream of the Toll receptor. It also reveals the conserved role of modular serine proteases in the activation of insect immune reactions.

We have recently isolated an invertebrate cytokine from a freshwater crayfish, which we named astakine 1. Interestingly this protein is expressed exclusively in hemocytes and hematopoietic tissue and is essential for the release of new hemocytes into the open circulatory system of these animals. This astakine has a prokineticin (PK) domain but lacks the N-terminal AVIT amino acids and hence receptor binding may differ from vertebrate PKs. Accordingly, here we report that a receptor for astakine 1 on hematopoietic tissue (Hpt) cells is identical to the beta-subunit of F1ATP synthase. In this study we have used several different methods to clearly demonstrate that ATP-synthase is located on the plasma membrane of a subpopulation of Hpt cells and there may function as a receptor for astakine, whereas mature blood cells (hemocytes) do not have any ATP-synthase on the outside of their plasma membranes. Our results clearly show that ATP synthase beta subunits are present on the cell surface of Hpt cells and highlight the need for more detailed studies on intracellular traffic connections between mitochondria and other membrane compartments.

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus Delta ltaS mutant could grow at 30 degrees C but not at 37 degrees C. Even at the permissive temperature, Delta ltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of Delta ltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the Delta ltaS mutant at 37 degrees C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the Delta ltaS mutation was found to be synthetically lethal with the Delta tagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30 degrees C, suggesting that LTA and WTA compensate for one another in an essential function.

Mannose-binding lectin (MBL) exists in the serum as a complex with MBL-associated serine protease (MASP). A recent paper described how MASP-free recombinant rat MBL stimulates the phagocytosis of Escherichia coli and Staphylococcus aureus by rat Kupffer cells through an increase in the level of a phagocytosis receptor. We have examined the effect of human MBL on the phagocytic action of human macrophages. Purified recombinant human MBL stimulated the phagocytosis of E. coli by THP-1 macrophages, leaving that of latex beads, apoptotic human cells, zymosan particles or S. aureus unchanged. This stimulatory effect was observed when either phagocytes or targets were preincubated with MBL. Furthermore, MBL bound to THP-1 macrophages as well as to E. coli, but not to S. aureus, through lipid A. These results indicated that human MBL in the absence of MASP stimulates macrophage phagocytosis of E. coli by bridging targets and phagocytes.

Melanisation is an important immune response in many invertebrates. Recent evidence also strongly implies that the melanisation (prophenoloxidase activating) cascade is intimately associated with the appearance of factors stimulating cellular defence by aiding phagocytosis and encapsulation reactions. However, some controversy exists in the field, and at least in flies and mosquitoes, the successful combat of some pathogens does not seem to be dependent on phenoloxidase activity. This may be because of redundancy among separate immune mechanisms, inappropriate testing, species differences or a combination thereof. Recently, by using RNA interference against phenoloxidase or in specific host-pathogen interactions where the pathogen prevents melanin production by the host, convincing data have confirmed the importance of this cascade in invertebrate innate immunity.

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic alpha- helical barrel domain and a membrane-embedded beta-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membraneembedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family can fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor. Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.

Apoptotic cell phagocytosis is initiated through the specific interaction between markers for phagocytosis present at the surface of targets and their receptors of phagocytes. Although many molecules have been proposed to be phagocytosis markers and receptors in mammals, information as to the identity of those molecules is limited for invertebrate animals. Calreticulin, a molecular chaperone that functions in the lumen of the endoplasmic reticulum, was recently reported to be the second general marker, the membrane phospholipid phosphatidylserine being the first, for mammalian apoptotic cells to be recognized by phagocytes. We here asked whether or not calreticulin serves as a marker for phagocytosis in Drosophila. Phagocytosis of apoptotic S2 cells by Drosophila hemocyte-derived l(2)mbn cells, which we previously showed to occur independent of phosphatidylserine, was inhibited by the addition of anti-calreticulin antibody. This inhibition was observed when the target cells, but not phagocytes, were pre-incubated with the antibody. In addition, RNA interference-mediated reduction of calreticulin expression in apoptotic S2 cells, but not in l(2)mbn cells, reduced the level of phagocytosis. An immunocytochemical analysis revealed that calreticulin is widely distributed at the surface of viable S2 cells. After the induction of apoptosis, cell surface calreticulin seemed to form aggregates, with no change in its amount. Furthermore, in embryos of a mutant Drosophila strain that expresses calreticulin at a reduced level, the level of phagocytosis of apoptotic cells was about a half of that observed in embryos of a wild-type strain. These results collectively indicate that calreticulin is the first molecule to be identified as a marker for phagocytosis of apoptotic cells by Drosophila phagocytes.

The functions of hemocytes in innate immune response are reviewed with emphasized on their roles in coagulation, melanization and opsonization. Also the ways in which hemocytes are produced in and released from hematopoietic tissue are discussed.

Invertebrate animals, which lack adaptive immune systems, have developed other systems of biological host defense, so called innate immunity, that respond to common antigens on the cell surfaces of potential pathogens. During the past two decades, the molecular structures and functions of various defense components that participated in innate immune systems have been established in Arthropoda, such as, insects, the horseshoe crab, freshwater crayfish, and the protochordata ascidian. These defense molecules include phenoloxidases, clotting factors, complement factors, lectins, protease inhibitors, antimicrobial peptides, Toll receptors, and other humoral factors found mainly in hemolymph plasma and hemocytes. These components, which together compose the innate immune system, defend invertebrate from invading bacterial, fungal, and viral pathogens. This review describes the present status of our knowledge concerning such defensive molecules in invertebrates.

The prophenoloxidase (proPO) cascade is a major innate immune response in invertebrates, which is triggered into its active form by elicitors, such as lipopolysaccharide, peptidoglycan, and 1,3-beta-D-glucan. A key question of the proPO system is how pattern recognition proteins recognize pathogenic microbes and subsequently activate the system. To investigate the biological function of 1,3-beta-D-glucan pattern recognition protein in the proPO cascade system, we isolated eight different 1,3-beta-D-glucan-binding proteins from the hemolymph of large beetle (Holotrichia diomphalia) larvae by using 1,3-beta-D-glucan immobilized column. Among them, a 20- and 17-kDa protein (referred to as Hd-PGRP-1 and Hd-PGRP-2) show high sequence identity with the short forms of peptidoglycan recognition proteins (PGRPs-S) from human and Drosophila melanogaster. To be able to characterize the biochemical properties of these two proteins, we expressed them in Drosophila S2 cells. Hd-PGRP-1 and Hd-PGRP-2 were found to specifically bind both 1,3-beta-D-glucan and peptidoglycan. By BIAcore analysis, the minimal 1,3-beta-D-glucan structure required for binding to Hd-PGRP-1 was found to be laminaritetraose. Hd-PGRP-1 increased serine protease activity upon binding to 1,3-beta-D-glucan and subsequently induced the phenoloxidase activity in the presence of both 1,3-beta-D-glucan and Ca(2+), but no phenoloxidase activity was elicited under the same conditions in the presence of peptidoglycan and Ca(2+). These results demonstrate that Hd-PGRP-1 can serve as a receptor for 1,3-beta-D-glucan in the insect proPO activation system.

Although many different pattern recognition receptors recognizing peptidoglycan and 1,3-beta-D-glucan have been identified in vertebrates and insects, the molecular mechanism of these molecules in the pattern recognition and subsequent signaling is largely unknown. To gain insights into the action mechanism of 1,3-beta-D-glucan pattern recognition protein in the insect prophenoloxidase (proPO) activation system, we purified a 53-kDa 1,3-beta-D-glucan recognition protein (Tm-GRP) to homogeneity from the hemolymph of the mealworm, Tenebrio molitor, by using a 1,3-beta-d-glucan affinity column. The purified protein specifically bound to 1,3-beta-D-glucan but not to peptidoglycan. Subsequent molecular cloning revealed that Tm-GRP contains a region with close sequence similarity to bacterial glucanases. Strikingly, two catalytically important residues in glucanases are replaced with other nonhomologous amino acids in Tm-GRP. The finding suggests that Tm-GRP has evolved from an ancestral gene of glucanases but retained only the ability to recognize 1,3-beta-D-glucan. A Western blot analysis of the protein level of endogenous Tm-GRP showed that the protein was specifically degraded following the activation of proPO with 1,3-beta-D-glucan and calcium ion. The degradation was significantly retarded by the addition of serine protease inhibitors but not by cysteine or acidic protease inhibitor. These results suggest that 1,3-beta-D-glucan pattern recognition protein is specifically degraded by serine protease(s) during proPO activation, and we propose that this degradation is an important regulatory mechanism of the activation of the proPO system.

To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+. This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+, a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and beta-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor.