CLIPB8 is part of the prophenoloxidase activation system in Anopheles gambiae mosquitoes

In insects and other arthropods the formation of eumelanin (melanization) is a broad spectrum and potent immune response that is used to encapsulate and kill invading pathogens. This immune response is regulated by the activation of prophenoxidase (proPO), which is controlled by proteinase cascades and its serpin inhibitors, together forming the proPO activation system. While the molecular composition of these protease cascades are well understood in insect model systems, major knowledge gaps remain in mosquitoes. Recently, a regulatory unit of melanization in Anopheles gambiae was documented, comprised of the inhibitory serpin-clip-serine proteinase, CLIPB9 and its inhibitor serpin-2 (SRPN2). Partial reversion of SRPN2 phenotypes in melanotic tumor formation and adult survival by SRPN2/CLIPB9 double knockdown suggested other target proteinases of SRPN2 in regulating melanization. Here we report that CLIPB8 supplements the SRPN2/CLIPB9 regulatory unit in controlling melanization in An. gambiae. As with CLIPB9, knockdown of CLIPB8 partially reversed the pleiotropic phenotype induced by SRPN2 silencing with regards to adult survival and melanotic tumor formation. Recombinant SRPN2 protein formed an SDS-stable protein complex with activated recombinant CLIPB8, however did not efficiently inhibit CLIPB8 activity in vitro. CLIPB8 did not directly activate proPO in vitro nor was it able to cleave and activate proCLIPB9. Nevertheless, epistasis analysis using RNAi placed CLIPB8 and CLIPB9 in the same pathway leading to melanization, suggesting that CLIPB8 either acts further upstream of CLIPB9 or is required for activation of a yet to be identified serine proteinase homolog. Taken together, this study identifies CLIPB8 as an additional player in proPO activation cascade and highlights the complexity of the proteinase network that regulates melanization in An. gambiae.     

A genetic module regulates the melanization response of Anopheles to Plasmodium

Two modes of refractoriness to Plasmodium, ookinete lysis and melanization, are known in the malaria vector, Anopheles gambiae. Melanization, a potent insect immune response, is manifested in a genetically selected refractory strain and in susceptible mosquitoes that are depleted of specific C-type lectins (CTLs). Here we use a systematic in vivo RNA interference-mediated reverse genetic screen and other recent results to define a melanization-regulating genetic module or network. It encompasses at least 14 genes, including those that encode five Easter-like clip domain serine proteases and four Masquerade-like serine protease homologues of the mosquito CLIPB and CLIPA subfamilies respectively. We show that several but not all CLIPB genes promote Plasmodium melanization, exhibiting partial functional overlap and synergy. We also report that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanization, while three other CLIPAs are novel synergistic inhibitors of this response. Importantly, the roles of certain CLIPAs and CLIPBs are strain specific, indicating that this network may differ between strains. Finally, we provide evidence that in susceptible mosquitoes melanization induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.     

Silencing the genes for dopa decarboxylase or dopachrome conversion enzyme reduces melanization of foreign targets in Anopheles gambiae

The production of melanin is a complex biochemical process in which several enzymes may play a role. Although phenoloxidase and serine proteases are clearly key components, the activity of other enzymes, including dopa decarboxylase and dopachrome conversion enzyme may also be required. We tested the effect of knockdown of gene expression for these two enzymes on melanization of abiotic targets in the mosquito, Anopheles gambiae. Knockdown of dopa decarboxylase and dopachrome conversion enzyme resulted in a significant reduction of melanization of Sephadex beads at 24 h after injection. Knockdown of a third enzyme, phenylalanine hydroxylase, which is involved in endogenous production of tyrosine, had no effect on bead melanization. Quantitative analysis of gene expression demonstrated significant upregulation of phenylalanine hydroxylase, but not the other two genes, following injection.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Caspase-1-Like Regulation of the proPO-System and Role of ppA and Caspase-1-Like Cleaved Peptides from proPO in Innate Immunity

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.     

Activation of prophenoloxidase and inhibition of melanization in the haemolymph of immune Galleria mellonella larvae

After immunization of Galleria larvae with bacterial lipopolysaccharide, inhibition of haemolymph melanization developed parallel with antibacterial immunity. Failure of melanization was correlated with significantly decreased amounts of active cell-associated phenoloxidase (PO). In normal, nonimmune haemolymph, cellular PO originated from plasma proPO activated by, and largely attached to, the haemocytes; activation was maximal by 150 min. With immune haemolymph, such cellular activation of plasma proPO did not occur. Immune plasma proPO was activated by homogenization, but not by freeze-thawing. Normal plasma proPO was activated by freeze-thawing, but only to a very slight extent by homogenization. Mixing immune plasma with the freeze-thaw activated PO in normal plasma (1:2, v:v) caused a 37%, average reduction in PO activity. The results suggest that inhibition of melanization in immune Galleria haemolymph is caused by plasma factor(s) inhibiting the activation of plasma proPO by haemocytes.     

The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections

The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity.     

MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.     

Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation

In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.     

Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta

Manduca sexta microbe binding protein (MBP) is a member of the β-1,3-glucanase-related protein superfamily that includes Gram-negative bacteria-binding proteins (GNBPs), β-1,3-glucan recognition proteins (βGRPs), and β-1,3-glucanases. Our previous and current studies showed that the purified MBP from baculovirus-infected insect cells had stimulated prophenoloxidase (proPO) activation in the hemolymph of naïve and immune challenged larvae and that supplementation of the exogenous MBP and peptidoglycans (PGs) had caused synergistic increases in PO activity. To explore the underlying mechanism, we separated by SDS-PAGE naïve and induced larval plasma treated with buffer or MBP and detected on immunoblots changes in intensity and/or mobility of hemolymph (serine) proteases [HP14, HP21, HP6, HP8, proPO-activating proteases (PAPs) 1–3] and their homologs (SPH1, SPH2). In a nickel pull-down assay, we observed association of MBP with proHP14 (slightly), βGRP2, PG recognition protein-1 (PGRP1, indirectly), SPH1, SPH2, and proPO2. Further experiments indicated that diaminopimelic acid (DAP) or Lys PG, MBP, PGRP1, and proHP14 together trigger the proPO activation system in a Ca²⁺-dependent manner. Injection of the recombinant MBP into the 5th instar naïve larvae significantly induced the expression of several antimicrobial peptide genes, revealing a possible link between HP14 and immune signal transduction. Together, these results suggest that the recognition of Gram-negative or -positive bacteria via their PGs induces the melanization and Toll pathways in M. sexta.     

The cost of immunity in the yellow fever mosquito, Aedes aegypti depends on immune activation

Although host immunity offers the obvious benefit of reducing parasite infection, it is often traded-off with other fitness components. We investigated whether the cost of an immune response in the yellow fever mosquito, Aedes aegypti, is modulated by the antigen that activates the melanization immune response. Thus, one of three different novel antigens were injected into the mosquito's thorax--either a glass bead, a negatively charged (C-25) Sephadex bead, or a neutral (G-25) Sephadex bead--and fecundity and bead melanization were observed. Glass beads are immunologically inert and were therefore used as an inoculation control. The fecundity of mosquitoes inoculated with these beads did not differ from the fecundity of mosquitoes that did not melanize negatively charged or neutral beads. The ability of A. aegypti to melanize negatively charged Sephadex beads was associated with reduced fecundity, showing a clear cost of immunity. In contrast, melanization of the neutral beads was quite strong but had no effect on fecundity. Thus, the cost of what appeared to be the same immune response--melanization of a bead--depended on the type of bead that stimulated the immune system. Such differences might help to explain variation of immune efficacy against different parasites in natural populations.     

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.     

Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.     

The extended loop of the C-terminal carbohydrate-recognition domain of Manduca sexta immulectin-2 is important for ligand binding and functions

Our previous research showed that immulectin-2 (IML-2), a C-type lectin from the tobacco hornworn, Manduca sexta, is a pattern recognition receptor (PRR) that can bind to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PG) and β-1,3-glucan, and IML-2 plays an important role in cellular encapsulation, melanization, phagocytosis, and prophenoloxidase (proPO) activation. Unlike most mammalian C-type lectins that contain a single carbohydrate-recognition domain (CRD), IML-2 is composed of tandem CRDs, and the C-terminal CRD2 contains an extended loop, which is not present in most C-type CRDs. We hypothesize that the extended loop may participate in ligand binding, encapsulation, melanization, phagocytosis and/or proPO activation in M. sexta. To test this hypothesis, two deletion mutant proteins (IML-2Δ220-244 and IML-2Δ220-257), in which the extended loop of the CRD2 was partially or completely deleted, were expressed and purified. By comparing the characteristics of recombinant IML-2, IML-2Δ220-244 and IML-2Δ220-257, we found that deletion of the extended loop in CRD2 impaired the ability of IML-2 to bind microbial PAMPs and to stimulate proPO activation, indicating that the extended loop of IML-2 plays an important role in ligand binding and biological functions.     

Anopheles gambiae immune responses to Sephadex beads: involvement of anti-Plasmodium factors in regulating melanization

We have performed a global genome expression analysis of mosquito responses to CM-25 Sephadex beads and identified 27 regulated immune genes, including several anti-Plasmodium factors and other components with likely roles in melanization. Silencing of two bead injection responsive genes, TEP1 and LRIM1, which encode proteins known to mediate Plasmodium killing, significantly compromised the ability to melanize the beads. In contrast, silencing of two Plasmodium protective c-type lectins, CTL4 and CTLMA2, did not affect bead melanization. This data suggest that the anti-Plasmodium factors have dual functions, as determinants of both Plasmodium killing and melanization of the parasite and other foreign bodies, while the Plasmodium protective factors are specifically utilized by the parasite for evasion of mosquito defense mechanisms.     

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.     

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.