Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila

The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. The Drosophila genome encodes three POs, which are primarily produced as zymogens or prophenoloxidases (PPO). Two of them, PPO1 and PPO2, are produced by crystal cells. Here we have generated flies carrying deletions in PPO1 and PPO2. By analyzing these mutations alone and in combination, we clarify the functions of both PPOs in humoral melanization. Our study shows that PPO1 and PPO2 are responsible for all the PO activity in the hemolymph. While PPO1 is involved in the rapid early delivery of PO activity, PPO2 is accumulated in the crystals of crystal cells and provides a storage form that can be deployed in a later phase. Our study also reveals an important role for PPO1 and PPO2 in the survival to infection with Gram-positive bacteria and fungi, underlining the importance of melanization in insect host defense.     

Effect of the insect phenoloxidase on the metabolism of l‐DOPA

Insect prophenoloxidase (PPO) induces melanization around pathogens. Before melanization, PPO is cleaved into phenoloxidase (PO) by serine proteases. Insect PPO can also be activated by exogenous proteases secreted by pathogens as well as by other compounds, such as ethanol and cetylpyridinium chloride (CPC). However, the effect of these activators on the activity of PO is unclear. In this study, the insect endogenous serine protease AMM1, α‐chymotrypsin, and ethanol were used to activate recombinant Drosophila PPO1 (rPPO1), and the PO activity differed depending on the activator applied. The PO‐induced intermediates during melanization also varied markedly in their numbers and abundances. Therefore, this study indicates that the mechanism of PPO activation influences PO activity. It also suggests that PO‐induced different intermediates may affect the antibacterial activity during melanization due to their toxicity.     

Molecular control of phenoloxidase-induced melanin synthesis in an insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Sp?tzle-processing enzyme is a key enzyme in the production of cleaved Sp?tzle from pro-Sp?tzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Sp?tzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.     

Regulation and function of the melanization reaction in Drosophila

The melanization reaction, involving the synthesis of melanin to encapsulate pathogens, is a prominent immune response in Drosophila, the mosquito, and other insects and arthropods. Biochemical studies with large insects have defined a basic model for how melanization is activated and regulated upon microbial infection. In this model, recognition of a microorganism triggers a serine protease cascade that activates phenol oxidase (PO), a key enzyme in the melanin biosynthetic pathway, and serpin-type protease inhibitors are involved in inhibiting the cascade. In the past few years, genetic studies in Drosophila have identified serine proteases and serpins that regulate activation of PO and melanization in vivo. These studies, along with molecular genetic analysis of melanization in the mosquito, have provided new insight into the role that melanization plays in fighting microbial infection.     

A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae.

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.     

A NOVEL TYPE OF HEMOCYTES LOCALIZING MELANIZATION WITH HIGH‐SPREADING BEHAVIOR IN MYTHIMNA SEPARATA

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)‐catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2–4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti‐PPO antibody. We were unable to detect PPO‐mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO‐mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.     

The Involvement of Hemocyte Prophenoloxidase in the Shell-Hardening Process of the Blue Crab,Callinectes sapidus

Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.     

An immune-responsive Serpin regulates the melanization cascade in Drosophila

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.     

A new factor in the Aedes aegypti immune response: CLSP2 modulates melanization

Microbial infections in the mosquito Aedes aegypti activate the newly identified CLSP1 and CLSP2 genes, which encode modular proteins composed of elastase-like serine protease and C-type lectin domains. These genes are predominantly regulated by the immune deficiency pathway, but also by the Toll pathway. Silencing of CLSP2, but not CLSP1, results in the activation of prophenoloxidase (PPO), the terminal enzyme in the melanization cascade, suggesting that CLSP2 is a negative modulator of this reaction. Haemolymph PPO activation is normally inhibited in the presence of Plasmodium parasites, but in CLSP2-depleted mosquitoes, the Plasmodium-induced block of melanization is reverted, and these mosquitoes are refractory to the parasite. Thus, CLSP2 is a new component of the mosquito immune response.     

Phenoloxidase in larvae of Plodia interpunctella (Lepidoptera: Pyralidae): molecular cloning of the proenzyme cDNA and enzyme activity in larvae paralyzed and parasitized by Habrobracon hebetor (Hymenoptera: Braconidae)

Phenoloxidase (PO) is a major component of the insect immune system. The enzyme is involved in encapsulation and melanization processes as well as wound healing and cuticle sclerotization. PO is present as an inactive proenzyme, prophenoloxidase (PPO), which is activated via a protease cascade. In this study, we have cloned a full-length PPO1 cDNA and a partial PPO2 cDNA from the Indianmeal moth, Plodia interpunctella (Hubner) (Lepidoptera: Pyralidae) and documented changes in PO activity in larvae paralyzed and parasitized by the ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae). The cDNA for PPO1 is 2,748 bp and encodes a protein of 681 amino acids with a calculated molecular weight of 78,328 and pI of 6.41 containing a conserved proteolytic cleavage site found in other PPOs. P. interpunctella PPO1 ranges from 71-78% identical to other known lepidopteran PPO-1 sequences. Percent identity decreases as comparisons are made to PPO-1 of more divergent species in the orders Diptera (Aa-48; As-49; and Sb-60%) and Coleoptera (Tm-58; Hd-50%). Paralyzation of host larvae of P. interpunctella by the idiobiont H. hebetor results in an increase in phenoloxidase activity in host hemolymph, a process that may protect the host from microbial infection during self-provisioning by this wasp. Subsequent parasitization by H. hebetor larvae causes a decrease in hemolymph PO activity, which suggests that the larval parasitoid may be secreting an immunosuppressant into the host larva during feeding.     

Two proteases defining a melanization cascade in the immune system of Drosophila

The melanization reaction is used as an immune mechanism in arthropods to encapsulate and kill microbial pathogens. In Drosophila, the serpin Spn27A regulates melanization apparently by inhibiting the protease that activates phenoloxidase, the key enzyme in melanin synthesis. Here, we have described the genetic characterization of two immune inducible serine proteases, MP1 and MP2, which act in a melanization cascade regulated by Spn27A. MP1 is required to activate melanization in response to both bacterial and fungal infection, whereas MP2 is mainly involved during fungal infection. Pathogenic bacteria and fungi may therefore trigger two different melanization cascades that use MP1 as a common downstream protease to activate phenoloxidase. We have also shown that the melanization reaction activated by MP1 and MP2 plays an important role in augmenting the effectiveness of other immune reactions, thereby promoting resistance of Drosophila to microbial infection.     

Serpin-5 regulates prophenoloxidase activation and antimicrobial peptide pathways in the silkworm,Bombyx mori

The prophenoloxidase (PPO) activation pathway and Toll pathway are two critical insect immune responses against microbial infection. Activation of these pathways is mediated by an extracellular serine protease cascade, which is negatively regulated by serpins. In this study, we found that the mRNA abundance of silkworm serpin-5 (BmSpn-5) increased dramatically in the fat body after bacterial infection. The expression level of antimicrobial peptides (AMPs), gloverin-3, cecropin-D and -E decreased in the silkworm larvae injected with recombinant BmSpn-5 protein. Meanwhile, the inhibition of beads melanization, systemic melanization and PPO activation by BmSpn-5 was also observed. By means of immunoaffinity purification and analysis by mass spectrometry, we identified that the silkworm clip domain serine proteases BmHP6 and BmSP21 form a complex with BmSpn-5, which suggests that BmHP6 and SP21 are the cognate proteases of BmSpn-5 and are essential in the serine protease cascade that activates the Toll and PPO pathways. Our study provides a comprehensive characterization of BmSpn-5 and sheds light on the multiple pathways leading to PPO activation and their regulation by serpins.     

CLIP proteases and Plasmodium melanization in Anopheles gambiae

Melanization is a potent immune response mediated by phenoloxidase (PO). Multiple Clip-domain serine proteases (CLIP) regulate PO activation as part of a complex cascade of proteases that are cleaved sequentially. The role of several CLIP as key activators or suppressors of the melanization responses of Anopheles gambiae to Plasmodium berghei (murine malaria) has been established recently using a genome-wide reverse genetics approach. Important differences in regulation of PO activation between An. gambiae strains were also identified. This review summarizes these findings and discusses our current understanding of the An. gambiae melanization responses to Plasmodium.     

Regulation of melanization by glutathione in the moth Pseudoplusia includens

The phenoloxidase (PO) cascade regulates the melanization of hemolymph, which serves as a conserved humoral immune response in insects and other arthropods. The reductant glutathione (GSH) has long been used to inhibit melanization of hemolymph from insects but whether GSH levels in hemolymph are sufficient to play a physiological role in regulating melanization is unknown. Here, we characterized the abundance and effects of GSH on the melanization of plasma from larval stage Pseudoplusia includens (Lepidoptera: Noctuidae). GSH concentration in newly collected plasma from day two fifth instars ranged from 50 to 115 μM, while the titer of tyrosine, a substrate for the PO cascade, was 141 μM. GSH titers rapidly declined in plasma after collection from larvae, but no melanin formation occurred until GSH levels fell below 20 μM. Added GSH dose-dependently blocked melanization while PO substrates overrode GSH inhibition. Experiments conducted in the absence of oxygen and presence of PO cascade inhibitors further suggested that depletion of GSH from plasma was primarily due to formation of reactive intermediates produced by activated PO. Additional studies identified hemocytes as a potential source of plasma GSH. Hemocyte lysates recycled oxidized glutathione (GSSG) into GSH using NADPH, while intact hemocytes released GSH into the medium. These results suggest that in addition to protease cascade-releated mechanisms that regulate phenoloxidase, GSH exerts another level of control on melanization of insect hemolymph.     

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.     

PGE(2) induces oenocytoid cell lysis via a G protein-coupled receptor in the beet armyworm, Spodoptera exigua

Eicosanoids mediate cellular and humoral immune responses in the beet armyworm, Spodoptera exigua, including activation of prophenoloxidase (PPO). PPO activation begins with release of its inactive zymogen, PPO, from oenocytoids in response to prostaglandins (PGs). Based on the biomedical literature, we hypothesized that PGs exert their actions via specific G protein-coupled receptor(s) in S. exigua. This study reports a G protein-coupled receptor (Se-hcPGGPCR1) gene, which is expressed in the hemocytes of S. exigua. The Se-hcPGGPCR1 consists of 420 amino acids and belongs to rhodopsin-type GPCRs. The high content of hydrophobic amino acid residues within the Se-hcPGGPCR1 protein is explained by prediction of seven-transmembrane domains that are characteristic of these GPCRs. Except for the eggs, Se-hcPGGPCR1 was expressed in all life stages. During the larval stage, it was expressed in hemocytes and gut, but not in fat body nor in epidermis. Real time quantitative RT-PCR showed that bacterial challenge induced more than 20-fold increases in its expression level. Fluorescence in situ hybridization showed that Se-hcPGGPCR1 was expressed in a specific hemocyte type, the oenocytoids. A specific eicosanoid, PGE₂, significantly induced oenocytoid lysis and increased PO activity in the plasma. In contrast, when Se-hcPGGPCR1 expression was suppressed by RNA interference (RNAi), the oenocytoid lysis and the PO activation in response to PGE₂ were not elevated above basal levels. A binding assay using intracellular calcium mobilization showed that the RNAi-treated hemocytes were significantly less responsive to PGE₂ than the control hemocytes. These results support our hypothesis with the specific finding that PGE₂ acts through Se-hcPGGPCR1 to activate PPO by lysing oenocytoids.     

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD₅₀ in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72 h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72 h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.