In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

Extracellular serine protease cascades mediate immune signaling and responses in insects. In the tobacco hornworm Manduca sexta, nearly 30 serine proteases (SPs) and their homologs (SPHs) are cloned from hemocytes and fat body. Some of them participate in prophenoloxidase (proPO) activation and proSpätzle processing. Here we report the cDNA cloning of hemolymph protease-1b (HP1b), which is 90% identical and 95% similar to HP1a (formerly HP1). The HP1a and HP1b mRNA levels in hemocytes was down- and up-regulated after an immune challenge, respectively. Quantitative real-time polymerase chain reactions revealed their tissue-specific and development-dependent expression, mostly in hemocytes of the feeding larvae. We isolated HP1 precursor (proHP1) from larval hemolymph and observed micro-heterogeneity caused by N-linked glycosylation. Supplementation of the purified proHP1 to plasma samples from naïve larvae or induced ones injected with bacteria caused a small PO activity increase, much lower than those elicited by recombinant proHP1a/b, but no proteolytic cleavage was detected in the zymogens. Incubation of proHP1a/b or their catalytic domains with a cationic detergent, cetylpyridinium chloride, induced an amidase activity that hydrolyzed LDLH-p-nitroanilide. Since LDLH corresponds to the P4–P1 region before the proteolytic activation site of proHP6, we propose that the active but uncleaved proHP1 may cut proHP6 to generate HP6 that in turn activates proPAP1 and proHP8. The catalytic domain of HP1a/b, which by itself does not activate purified proHP6 or hydrolyze LDLH-p-nitroanilide, somehow generated active HP6, HP8, PAP1 and PO in plasma. Together, these results indicate that proHP1 participates in the proPO activation system, although detailed mechanism needs further exploration.     

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal beta-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, an HP14 substrate similar to Drosophila snake. The recombinant proHP21 is a 51.1 kDa glycoprotein with an amino-terminal clip domain, a linker region, and a carboxyl-terminal serine proteinase domain. HP14, generated by incubating proHP14 with beta-1,3-glucan and beta-1,3-glucan recognition protein-2, activated proHP21 by limited proteolysis between Leu(152) and Ile(153). Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 to be Arg(355) and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in PO activity. HP21 cleaved proPO activating proteinase-2 precursor (proPAP-2) after Lys(153) and generated an amidase activity, which activated proPO in the presence of serine proteinase homolog-1 and 2. In summary, we have discovered and reconstituted a branch of the proPO activation cascade in vitro: beta-1,3-glucan recognition--proHP14 autoactivation--proHP21 cleavage--PAP-2 generation--proPO activation--melanin formation.     

A positive feedback mechanism in the Manduca sexta prophenoloxidase activation system

In Manduca sexta, pathogen recognition triggers a branched serine proteinase cascade which generates active phenoloxidase (PO) in the presence of a proPO-activating proteinase (PAP) and two noncatalytic serine proteinase homologs (SPHs). PO then catalyzes the production of reactive compounds for microbe killing, wound healing, and melanin formation. In this study, we discovered that a minute amount of PAP1 (a final component of the proteinase pathway) caused a remarkable increase in PO activity in plasma from na?ve larvae, which was significantly higher than that from the same amounts of PAP1, proPO and SPHs incubated in vitro. The enhanced proPO activation concurred with the proteolytic activation of HP6, HP8, PAP1, SPH1, SPH2 and PO precursors. PAP1 cleaved proSPH2 to yield bands with mobility identical to SPH2 generated in vivo. PAP1 partially hydrolyzed proHP6 and proHP8 at a bond amino-terminal to the one cut in the PAP1-added plasma. PAP1 did not directly activate proPAP1. These results suggest that a self-reinforcing mechanism is built into the proPO activation system and other plasma proteins are required for cleaving proHP6 and proHP8 at the correct site to strengthen the defense response, perhaps in the early stage of the pathway activation.     

Involvement of Manduca sexta peptidoglycan recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase system

Although the importance of peptidoglycan recognition proteins (PGRPs) in detecting bacteria and promoting immunity is well recognized in Drosophila melanogaster and other insect species, such a role has not yet been experimentally established for PGRPs in the tobacco hornworm, Manduca sexta. In this study, we purified M. sexta PGRP1 from the baculovirus-insect cell expression system, tested its association with peptidoglycans and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 is not an amidase and lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid-peptidoglycans (DAP-PGs). M. sexta PGRP1 gene was constitutively expressed at a low level in fat body, and the mRNA concentration became much higher after an injection of Escherichia coli. Consistently, the protein concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of Staphylococcus aureus. In addition, this recognition protein completely bound to insoluble PGs from Micrococcus luteus, Bacillus megaterium and Bacillus subtilis, whereas its association with the bacterial cells was low even though their peptidoglycans are exposed on the cell surface. After PGRP1 had been added to plasma of naïve larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with peptidoglyans or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. Taken together, these results indicated that PGRP1 is a member of the M. sexta prophenoloxidase activation system, which recognizes peptidoglycans from certain bacteria and initiates the host defense response. The unexplained difference between the purified PGs and intact bacteria clearly reflects our general lack of understanding of PGRP1-mediated recognition and how it leads to proPO activation.     

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta

Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R³⁶⁶ at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R⁴¹⁰ as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg³⁶⁶ by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg⁴¹⁰ for trypsin-like proteases, Gly⁴⁰⁶ and Ala⁴⁰⁹ for the elastase and Thr⁴⁰⁴ for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0–50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade

Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of approximately 32 and approximately 37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.     

Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6

Insect immune responses include prophenoloxidase (proPO) activation and Toll pathway initiation, which are mediated by serine proteinase cascades and regulated by serpins. Manduca sexta hemolymph proteinase-6 (HP6) is a component of both pathways. It cleaves and activates proPO activating proteinase 1 (PAP1) and hemolymph proteinase-8 (HP8), which activates proSpätzle. Inhibitors of HP6 could have the capability of regulating both of these innate immune proteinase cascade pathways. Covalent complexes of HP6 with serpin-4 and serpin-5 were previously isolated from M. sexta plasma using immunoaffinity chromatography with serpin antibodies. We investigated the inhibition of purified, recombinant HP6 by serpin-4 and serpin-5. Both serpin-4 and serpin-5 formed SDS-stable complexes with HP6 in vitro, and they inhibited the activation of proHP8 and proPAP1. Serpin-5 inhibited HP6 more efficiently than did serpin-4. Injection of serpin-5 into larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced the bacteria-induced expression of attacin, cecropin and hemolin genes in fat body. Injection of serpin-4 had a weaker effect on antimicrobial peptide expression. These results indicate that serpin-5 may regulate the activity of HP6 to modulate proPO activation and antimicrobial peptide production during immune responses of M. sexta.     

Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism,mediates immune responses

Tissue damage or pathogen invasion triggers the auto-proteolysis of an initiating serine protease (SP), rapidly leading to sequential cleavage activation of other cascade members to set off innate immune responses in insects. Recently, we presented evidence that Manduca sexta hemolymph protease-1 zymogen (proHP1) is a member of the SP system in this species, and may activate proHP6. HP6 stimulates melanization and induces antimicrobial peptide synthesis. Here we report that proHP1 adopts an active conformation (*) to carry out its function, without a requirement for proteolytic activation. Affinity chromatography using HP1 antibodies isolated from induced hemolymph the 48 kDa proHP1 and also a 90 kDa band (detected by SDS-PAGE under reducing conditions) containing proHP1 and several serpins, as revealed by mass spectrometric analysis. Identification of tryptic peptides from these 90 kDa complexes included peptides from the amino-terminal regulatory part of proHP1, indicating that proHP1* was not cleaved, and that it had formed a complex with the serpins. As suicide inhibitors, serpins form SDS-stable, acyl-complexes when they are attacked by active proteases, indicating that proHP1* was catalytically active. Detection of M. sexta serpin-1, 4, 9, 13 and smaller amounts of serpin-3, 5, 6 in the complexes suggests that it is regulated by multiple serpins in hemolymph. We produced site-directed mutants of proHP1b for cleavage by bovine blood coagulation factor Xa at the designed proteolytic activation site, to generate a form of proHP1b that could be activated by Factor Xa. However, proHP1b cut by Factor Xa failed to activate proHP6 and, via HP6, proHP8 or proPAP1. This negative result is consistent with the suggestion that proHP1* is a physiological mediator of immune responses. Further research is needed to investigate the conformational change that results in conversion of proHP1 to active proHP1*.     

A pattern recognition serine proteinase triggers the prophenoloxidase activation cascade in the tobacco hornworm, Manduca sexta

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.     

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway.Innate immune systems of mammals and arthropods include extracellular serine proteinase cascade pathways, which rapidly amplify responses to infection and stimulate killing of pathogens. These proteinase-driven processes include the complement system of vertebrates (1, 2) and pathways in arthropods involving proteinases containing amino-terminal clip domains (3). Clip domain proteinases function in blood coagulation (4, 5), activation of prophenoloxidase (proPO) that leads to melanin synthesis (6–9), and stimulation of the Toll pathway to promote synthesis of antimicrobial peptides/proteins (AMPs)² secreted into the hemolymph (10, 11).The serine proteinase systems best characterized in arthropods are the horseshoe crab hemolymph coagulation pathway and the cascade leading to activation of the Toll pathway in dorsal-ventral development in Drosophila (12–14). Recent research also has led to better characterization of the proPO activation pathway in Manduca sexta (7, 15, 16) and the Toll-signaling pathway in the Drosophila immune response (17, 18) and to both the proPO and Toll pathways in the beetle Tenebrio molitor (11, 19).In the proPO activation pathway, soluble pattern recognition proteins initially recognize pathogen-associated molecular patterns such as bacterial peptidoglycan or fungal β-1,3-glucan (20–22). This interaction stimulates the sequential activation of a series of serine proteinases in hemolymph, leading to the activation of proPO-activating proteinase (PAP), also known as proPO activating enzyme (7, 23). Activated PAP converts inactive proPO to PO. PO catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to quinones that are involved in microbial killing, melanin synthesis, sequestration of parasites or pathogens, and wound healing (24, 25). Other proteins required for proPO activation are clip-domain serine proteinase homologs (SPHs), whose catalytic serine is replaced with glycine and, therefore, lack proteolytic activity (26, 27). Serine proteinase inhibitors, including members of the serpin superfamily, regulate the activation of proPO by inhibiting the activating proteinases (28, 29).Drosophila clip-domain serine proteinases Persephone, Grass, Spirit, and spätzle-processing enzyme (SPE) participate in the activation of Toll pathway, stimulating synthesis of antimicrobial peptides as an innate immune response (18, 30–32). Although genetic evidence indicates that Persephone and Spirit are upstream of SPE in the cascade, the substrate(s) of Persephone and Spirit have not been identified, and which proteinase directly activates SPE is unknown. Neither is it clear whether these enzymes may be related to the melanization pathway, which involves clip-domain proteinases MP2 and MP1 (33).Here we report the functional characterization of M. sexta HP6 and HP8, probable orthologs of Drosophila Persephone and SPE, respectively. We developed methods to activate purified recombinant proHP6 and proHP8 and discovered that HP6 participates in proPO activation by activating proPAP1 and that both HP6 and HP8 function in a pathway that stimulates the synthesis of AMPs in M. sexta.     

饲料添加β-1,3-葡聚糖对凡纳滨对虾生长性能、血清代谢、免疫相关基因表达和抗亚硝酸氮应激能力的影响

实验旨在研究β-1,3-葡聚糖的不同投喂方式对凡纳滨对虾(Litopenaeus vannamei)生长、血清代谢和抗亚硝酸氮应激能力的影响。选用480尾初体重(0.43±0.01) g的凡纳滨对虾,随机分为4组,即G0(全程投喂基础饲料)、G1组(全程投喂0.1%β-1,3-葡聚糖饲料)、G2组(0.1%β-1,3-葡聚糖饲料7d+基础饲料7d循环)和G3组(0.1%β-1,3-葡聚糖饲料14d+基础饲料14d循环)。在养殖84d后,应用亚硝酸钠进行120h亚硝酸氮应激实验。结果显示,各实验组凡纳滨对虾生长性能和全虾营养成分没有显著性差异。在养殖84d后, G2和G3组凡纳滨对虾肝胰腺脂肪酶活性显著高于G0和G1组(P<0.05), G1、G2和G3组凡纳滨对虾血清胆固醇和甘油三酯含量显著高于G0组(P<0.05), G3组凡纳滨对虾肌肉脂多糖/?-1,3-葡聚糖结合蛋白(LGBP)、酚氧化物酶原(proPO)和超氧化物歧化酶(SOD)mRNA表达显著高于G0和G1组(P<0.05)。亚硝酸氮应激120h, G1、G2和G3组凡纳滨对虾累计死亡率显著低于G0组(P&...     

Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a β-1,3-glucanase-related protein in Manduca sexta larval plasma

Detection of pathogenic invaders is the essential first step of a successful defense response in multicellular organisms. In this study, we have identified a new member of the ??-1,3-glucanase-related protein superfamily from the tobacco hornworm Manduca sexta. This protein, designated microbe binding protein (MBP), is 61% identical in sequence to Bombyx mori Gram-negative bacteria binding protein, but only 34-36% identical to M. sexta ??-1,3-glucan recognition protein-1 and 2. Its mRNA levels were strongly up-regulated in hemocytes and fat body of immune challenged larvae, along with an increase in concentration of the plasma protein. We expressed M. sexta MBP in a baculovirus-insect cell system. The purified protein associated with intact bacteria and fungi. It specifically bound to lipoteichoic acid, lipopolysaccharide, diaminopimelic acid-type peptidoglycans (DAP-PGs) from Escherichia coli and Bacillus subtilis, but less so to laminarin or Lys-type PG from Staphylococcus aureus. The complex binding pattern was influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP had been incubated with larval plasma on ice, a concentration-dependent increase in phenoloxidase (PO) activity occurred in the absence of any microbial elicitor. The activity increase was also observed in the mixture of plasma and a bacterial or fungal cell wall component. The prophenoloxidase (proPO) activation became more prominent when DAP-PGs, Micrococcus luteus Lys-PG, or lipoteichoic acid was included in the mixture of MBP and plasma. Statistic analysis suggested that a synergistic enhancement of proPO activation was caused by an interaction between MBP and these elicitors, but not S. aureus Lys-PG, lipopolysaccharide, curdlan, or laminarin. These data indicate that M. sexta MBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and serine proteinases, triggers the proPO activation system.     

Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm,Manduca sexta

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection.     

Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect

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.     

Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition

Toll-like receptor 2 (TLR2) has been shown to recognize several classes of pathogen-associated molecular patterns including peptidoglycan (PG). However, studies linking PG with TLR2 recognition have relied mainly on the use of commercial Staphylococcus aureus PG and have not addressed TLR2 recognition of other PG types. Using highly purified PGs from eight bacteria (Escherichia coli, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Helicobacter pylori, Bacillus subtilis, Listeria monocytogenes, Streptococcus pneumoniae and S. aureus), we show that these PGs are not sensed through TLR2, TLR2/1 or TLR2/6. PG sensing is lost after removal of lipoproteins or lipoteichoic acids (LTAs) from Gram-negative and Gram-positive cell walls, respectively. Accordingly, purified LTAs are sensed synergistically through TLR2/1. Finally, we show that elicited peritoneal murine macrophages do not produce tumour necrosis factor-alpha or interleukin-6 in response to purified PGs, suggesting that PG detection is more likely to occur intracellularly (through Nod1/Nod2) rather than from the extracellular compartment.     

First Documentation of Cultural Transmission of Predator Recognition by Larval Amphibians

Predation is a pervasive selective agent shaping a prey's behaviour, morphology and life history. To survive, prey animals have to respond adaptively to predation threats and this can be achieved through learned predator recognition. Cultural transmission of predator recognition is likely a widespread means of learning in social animals, including mammals, birds and fishes. However, no studies have investigated the cultural transmission of predator recognition in amphibians. In our study, we examined whether naïve woodfrog (Rana sylvatica) tadpoles can acquire the recognition of the odour of a predatory tiger salamander (Ambystoma tigrinum) from experienced conspecifics. After conditioning some tutors to recognize salamander odour, we paired naïve observer tadpoles with either a salamander‐naïve or salamander‐experienced tutor and exposed the pairs to either salamander odour or a water control. Observers were subsequently tested alone for a response to salamander odour. We found that when given salamander odour, observer tadpoles that were paired with a salamander‐experienced tutor successfully learned to recognize the salamander odour as a threat, whereas the observers paired with salamander‐naïve tutors did not. Likewise, tadpoles exposed to the water control did not learn to recognize the salamander regardless of whether they were paired with a naïve or experienced tutor. This is the first study demonstrating cultural transmission of predator recognition in an amphibian species.