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.     

Involvement of pro-phenoloxidase 3 in lamellocyte-mediated spontaneous melanization in Drosophila

Phenoloxidase (PO), a melanin-forming enzyme around the foreign bodies, is an important component of the host defense system in invertebrates. Pro-PO is the enzymatically inactive zymogen form of PO. In the Drosophila genome, three Pro-PO isoforms have been identified to date. These include Pro-PO1 and 2, which are primarily expressed in crystal cells, and Pro-PO3, which is predominantly found in the lamellocytes. In this study, we demonstrated that Drosophila Pro-PO3, but not Pro-PO1 or 2, is enzymatically active in its zymogen form. These findings were evidenced by spectacular melanin forming capacities of various cells and tissues that overexpressed these proenzymes. Furthermore, the melanization phenotype observed in the lamellocyte-enriched hop(Tum-1) mutant was drastically reduced in the absence of PPO3, indicating that PPO3 plays a major role in the lamellocyte-mediated spontaneous melanization process. Taken together, these findings indicate that the biochemical properties, activation mode and in vivo role of Pro-PO3 are likely distinct from those of the other two Pro-PO enzymes involved in Drosophila physiology.     

Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin

Melanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint, our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level, our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors.     

Growth conditions influence melanization of Brazilian clinical Sporothrix schenckii isolates

Sporothrix schenckii is known to produce DHN melanin on both conidial and yeast cells, however little information is available regarding the factors inducing fungal melanization. We evaluated whether culture conditions influenced melanization of 25 Brazilian S. schenckii strains and one control strain (ATCC 10212). Tested conditions included different media, pH, temperature, incubation time, glucose concentrations, and presence or absence of tricyclazole or L-DOPA. Melanization was reduced on Sabouraud compared to defined chemical medium. The majority of strains produced small amounts of melanin at 37 °C and none melanized at basic pH. Increased glucose concentrations did not inhibit melanization, rather increasing glucose enhanced pigment production in 27% of strains. Melanin synthesis was also enhanced by the addition of L-DOPA and its addition to medium with tricyclazole, an inhibitor of melanin synthesis, resulted in fungal melanization, including hyphal melanin production. Our results suggest that different S. schenckii strains have distinct control of melanization and that this fungus can use phenolic compounds to enhance melanization in vitro.     

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.     

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.     

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.     

Catechol Formation and Melanization by Na+ -Dependent Azotobacter chroococcum: a Protective Mechanism for Aeroadaptation?

Aeroadaptive microaerophilic Azotobacter chroococcum 184 produced a cell-associated black pigment when grown at high aeration rates under nitrogen-fixing conditions. This pigment was shown to be a catechol melanin. Polyphenol oxidase activity was detected in cell extracts of cells grown for 72 h. Melanin formation was optimal in the later stages of growth, and there was no correlation between nitrogenase activity and melanization. Nitrogenase activity in strain 184 was optimal at 10% O₂, and melanin formation was suppressed by O₂ limitation. In the presence of charcoal, an adsorbent of toxic oxygen intermediates, and benzoic acid, a scavenger of hydroxyl radicals, melanization was inhibited. However, in the presence of copper, the intensity of pigment color increased and melanization was accelerated. Copper also eliminated catalase and peroxidase activities of the organism but still permitted aerobic growth. In the presence of low levels of iron, melanization was accelerated under high aeration rates, and under low rates of aeration, melanization was observed only at higher levels of iron. Hydroxamate-siderophore production was detectable in the presence of soluble iron under high rates of aeration but was repressed by the same levels of iron under low aeration rates. Unlike melanization and hydroxamate formation, catechol formation was observed under both low and high rates of aeration under nitrogen-fixing conditions. Catechol formation and melanization were repressed by 14 mM NH₄⁺, at which level nitrogenase activity was also repressed. Copper reversed the repressive effect of NH₄⁺. A role for catechol formation and melanization in aeroadaptation is proposed.     

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.     

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.     

Melanization and morphological effects on antifungal susceptibility of Penicillium marneffei

The biosynthesis of melanin has been linked with virulence in diverse pathogenic fungi. Penicillium marneffei, a dimorphic fungus, is capable of melanization in both mycelial and yeast phases, and the pigment may be produced during infection to protect the fungus from the host immune system. To investigate the impact of yeast morphological transformation on antifungal susceptibility, P. marneffei was cultured on various media including minimal medium, 1 % tryptone, brain heart infusion broth, and malt extract broth by using the standardized susceptibility protocol (the M27-A protocol, RPMI medium) for yeasts. We also investigated whether P. marneffei melanization affected its susceptibility to antifungal drugs by adding l-DOPA into culture broths. There were no differences in the minimum inhibitory concentrations of P. marneffei yeast cells previously grown in various culture broths with or without l-DOPA using the M27A protocol (into which no melanin substrate can be added due to a rapid colour change of the RPMI medium to black) for testing amphotericin B, clotrimazole, fluconazole, itraconazole and ketoconazole. However, both melanized and non-melanized P. marneffei displayed increased resistance to antifungal drugs when l-DOPA was added into a selected assay medium, 0.17 % yeast nitrogen base, 2 % glucose, and 1.5 % agar. Hence, active melanin formation appears to protect P. marneffei by enhancing its resistance to antifungal drugs.     

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.     

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.     

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.     

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.     

Cryptococcus neoformans Can Utilize the Bacterial Melanin Precursor Homogentisic Acid for Fungal Melanogenesis

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin “ghost”-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.     

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.     

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D ¹³C-¹³C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.