Melanin-like Compounds Research Articles

Heterologous production and characterization of a pyomelanin of Antarctic Pseudomonas sp. ANT_H4: a metabolite protecting against UV and free radicals, interacting with iron from minerals and exhibiting priming properties toward plant hairy roots

BackgroundAntarctica has one of the most extreme environments in the world. This region is inhabited by specifically adapted microorganisms that produce various unique secondary metabolites (e.g. pigments) enabling their survival under the harsh environmental conditions. It was already shown that these natural, biologically active molecules may find application in various fields of biotechnology.ResultsIn this study, a cold-active brown-pigment-producing Pseudomonas sp. ANT_H4 strain was characterized. In-depth genomic analysis combined with the application of a fosmid expression system revealed two different pathways of melanin-like compounds biosynthesis by the ANT_H4 strain. The chromatographic behavior and Fourier-transform infrared spectroscopic analyses allowed for the identification of the extracted melanin-like compound as a pyomelanin. Furthermore, optimization of the production and thorough functional analyses of the pyomelanin were performed to test its usability in biotechnology. It was confirmed that ANT_H4-derived pyomelanin increases the sun protection factor, enables scavenging of free radicals, and interacts with the iron from minerals. Moreover, it was shown for the first time that pyomelanin exhibits priming properties toward Calendula officinalis hairy roots in in vitro cultures.ConclusionsResults of the study indicate the significant biotechnological potential of ANT_H4-derived pyomelanin and open opportunities for future applications. Taking into account protective features of analyzed pyomelanin it may be potentially used in medical biotechnology and cosmetology. Especially interesting was showing that pyomelanin exhibits priming properties toward hairy roots, which creates a perspective for its usage for the development of novel and sustainable agrotechnical solutions.

Excitation-Dependent Fluorescence Helps to Indicate Fungal Contamination of Aquatic Environments and to Differentiate Filamentous Fungi

Fungal contamination of aquatic environments can lead to an adverse impact on the environment and human health. (1) The search for fast, inexpensive and appropriate methods for detection of fungi is very moving rapidly due to their significant impact on ecosystem functions and human health. (2) We focused on examination of fluorescence proxies able to distinguish chromophoric matter occurring in different fungi. Spectroscopic studies were performed on five strains of filamentous fungi: Trichoderma harzianum, Fusarium solani, Alternaria alternata, Cladosporium cladosporioides and Aspergillus terreus. (3) The results showed that most of the fungal autofluorescence was emitted by amino acids, melanin-like compounds, NAD(P)H and flavins. The spectra of five fungal species cultivated as planktonic or surface-associated forms turned out to be different. Protein fluorescence can be used to detect general microbial contamination. Presence of excitation wavelength dependent mode and the “blue shift” of fluorescence (emission bands 400–500 nm) can be suggested as specific feature of fluorescence of fungal melanin-containing samples. (4) The determination based on fluorescence spectra obtained at a certain excitation/emission wavelengths pair and at whole excitation-emission matrices (EEMs) coupled to principal component analysis (PCA) algorithms as a tool of improving detection capabilities can be suggested to enable fast and inexpensive monitoring of fungal contamination of aquatic environments.

Two-Electron Oxidation of Dopamine Controlled by Surface Modification of Few-Layer Graphene

Oxidation of dopamine (DA) at classical electrodes is suggested to go through three steps and then form melanin-like compounds, which would inhibit electron transfer and result in the fouling of electrode. In addition, the selective oxidation of DA in the presence of ascorbate (VC) should also be considered for the design of electrode. Here, graphenes modified with different surface groups are used to investigate the oxidation behaviour of DA. Compared with classical electrodes, negatively charged few-layer graphene (AA-GN) shows a good performance in selective oxidation of DA and can also control the oxidation of DA in the first step, which would further prevent the fouling of electrode. The rate constant and number of electron transfer are calculated to be about 7.38s−1 and 2, respectively, which further indicates that the oxidation of DA was controlled in the first step. Furthermore, AA-GN-modified electrode is used to selectively detect DA with a detection limit of 10nM in the prescence of excess VC. At last, AA-GN-modified macro-electrode is used to monitor DA release from living cells for the first time.

Detection of DOPA-Melanin in the Dimorphic Fungal Pathogen Penicillium marneffei and Its Effect on Macrophage Phagocytosis In Vitro

The fungal pathogen Penicillium marneffei produces melanin-like pigment in vitro. The synthetic pathway of melanin and its possible influence in the protective yeast cells surviving within macrophage cells are not known. In this work, P. marneffei produced brown black pigment in the presence of L-DOPA and black particles were extracted from yeast cells treated with proteolytic enzymes, denaturant and concentrated hot acid. Kojic acid inhibited the brown-black pigment production of P. marneffei yeast grown on brain heart infusion agar. Transmitting electron microscopy showed spherical granular electron-dense particles with an average diameter of 100 nm in a beaded arrangement in the innermost cell wall. Electron-paramagnetic resonance revealed that the black particles contain a stable free radical compound. The UV-visible and Fourier transform infrared spectra of particles extracted from P. marneffei and synthetic DOPA-melanin showed a high degree of similarity. Melanized yeast cells decreased phagocytosis by macrophage cells and increased resistance to intracellular digestion in vitro. These results indicate that P. marneffei can synthesize DOPA-melanin or melanin-like compounds in vitro and suggest that the DOPA-melanin pathway is associated with cell wall structure and enhances the resistance to phagocytosis by macrophages.

Dopamine-Mediated Sclerotization of Regenerated Chitin in Ionic Liquid.

Chitin is a promising structural material for biomedical applications, due to its many advantageous properties and abundance in nature. However, its usage and development in the biomedical field have been stagnant, because of chitin’s poor mechanical properties in wet conditions and the difficulties in transforming it into an applicable form. To overcome these challenges, we created a novel biomimetic chitin composite. This regenerated chitin, prepared with ionic liquid, showed improved mechanical properties in wet conditions by mimicking insect cuticle and squid beak sclerotization, i.e., catechol-meditated cross-linking. By ionic liquid-based heat treatment, dopamine oxidation produced melanin-like compounds and dopamine-meditated cross-links without any solvent evaporation and oxidant utilization. The dopamine-meditated sclerotization increased the ultimate tensile strength (UTS) of the regenerated chitin by 2.52-fold, measured after six weeks of phosphate-buffered saline (PBS) submersion. In addition, the linear swelling ratio (LSR) of the chitin film was reduced by about 22%. This strategy raises a possibility of using regenerated chitin as an artificial hard tissue in wet conditions.

The role of L-DOPA on melanization and mycelial production in Malassezia furfur.

Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.

Detection of Antibodies against Paracoccidioides brasiliensis Melanin inIn VitroandIn VivoStudies during Infection

Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

The oxidation metabolites of endomorphin 1 and its fragments induced by free radicals

Endomorphin 1 (EM1), an endogenous micro-opioid receptor agonist, acts as a free radical scavenger in vitro and an antioxidant in vivo. The modification of EM1 by ROS and the properties of the OM attracted our attention. In vitro assays were performed via RP-HPLC, spectrophotometric measurements, EPR and amino acid analysis, Schmorl's reaction to define the formation of melanin-like compounds transformed from EM1, collectively named EM1-melanin and by solubility assay, radioligand-binding assay, NADH oxidation, superoxide anion scavenging assay to study some physical and chemical properties of EM1-melanin. Possible pathways of the formation of EM1-melanin were proposed.

Coccidioides posadasii produces melanin in vitro and during infection

Using techniques developed to study melanization in other fungi, we demonstrate that Coccidioides posadasii arthroconidia, spherules, and endospores produce melanin or melanin-like compounds in vitro and tissue forms synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, it may affect the pathogenesis of coccidioidomycosis.

Production of melanin by Aspergillus fumigatus.

Melanins, or melanin-like compounds, may play a role in the pathogenesis of a number of human fungal infections. This study investigated the production of melanin by the important opportunistic pathogen Aspergillus fumigatus. Conidia from A. fumigatus were harvested and treated with proteolytic enzymes, denaturant and hot, concentrated acid; this yielded dark particles which were similar in size and shape to the original propagules. Electron spin resonance spectroscopy revealed that the conidial-derived particles were stable free radicals consistent with an identification as melanin. Melanin particles were used to immunize BALB/c mice in order to produce a total of five anti-melanin monoclonal antibodies (mAbs). The latter mAbs were strongly reactive both with intact conidia and with extracted melanin particles by ELISA and immunofluorescence reactivity. Immunofluorescence labelling with the novel mAbs was used to examine the temporal expression of melanin during in vitro culture of A. fumigatus--melanization was confined to conidial structures and was absent from hyphae. SDS-PAGE L-3,4-dihydroxyphenylalanine (L-DOPA) substrate analysis confirmed the presence of a laccase-type activity in conidial extracts, but not in hyphae. Melanin-binding mAbs were used to detect the presence of melanized conidia in three patients with nasal aspergilloma, indicating that in vivo melanization may occur during infection.

Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection.

Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.

Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection.

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogen Histoplasma capsulatum var. capsulatum produced melanin or melanin-like compounds in vitro and during infection. Growth of H. capsulatum mycelia in chemically defined minimal medium produced pigmented conidia. Growth of H. capsulatum yeast in chemically defined minimal medium with L-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected with H. capsulatum and from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance to H. capsulatum yeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that H. capsulatum conidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection.

Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-dihydroxyphenylalanine (L-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in L-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

Melanization decreases the susceptibility of Cryptococcus neoformans to enzymatic degradation.

Cryptococcus neoformans is a free-living fungus that is primarily found in soils contaminated with avian excreta. Recent studies have shown that C. neoformans can synthesize melanins or melanin-like compounds in avian excreta. Melanization has been associated with protection of C. neoformans against harsh environmental conditions, such as ultraviolet radiation and extremes of temperature. In this study we examined whether melanization can protect C. neoformans against enzymatic degradation. Our results demonstrated that in vitro melanization decreases the susceptibility of C. neoformans to hydrolytic enzymes. This suggests a role for melanin in protection of C. neoformans against enzymatic degradation by antagonistic microbes in the environment.

Melanization of Cryptococcus neoformans in murine infection.

Cryptococcus neoformans is a fungus that is pathogenic in humans and that can produce melanin in vitro. Melanization is associated with virulence, but there is no evidence that melanin is made during infection. Melanins are difficult to study because they are amorphous and insoluble. Melanin-binding peptides from a phage display library were used to demonstrate that C. neoformans makes melanin-like compounds in tissue. Melanin-binding peptides were characterized by a high proportion of positively charged and aromatic residues. Two other methods, demonstration of an antibody response to melanin in mice infected with C. neoformans and analysis of yeast cell walls in infected tissue by light microscopy, were used to support these findings. The demonstration that C. neoformans melanizes in tissue has important implications for pathogenesis and drug discovery.

The presence of melanin inSynchytrium endobioticum

Synchytrium endobioticum is a quarantined pathogen of potatoes, which persists as a resting spore in soil for decades. We tested resting spores for the presence of melanin, which in other fungi is as- sociated with protection from light and microbial degradation. The pigmented materials were extract- ed and oxidized by the potassium permanganate method. Data on native and treated extracts and a melanin standard were obtained from high pressure liquid chromatography, ultraviolet-visible and infra- red spectra, and indicate that melanin or melaninlike compounds are present in the resting spore.

The Presence of Melanin in Synchytrium endobioticum

Synchytrium endobioticum is a quarantined pathogen of potatoes, which persists as a resting spore in soil for decades. We tested resting spores for the presence of melanin, which in other fungi is associated with protection from light and microbial degradation. The pigmented materials were extracted and oxidized by the potassium permanganate method. Data on native and treated extracts and a melanin standard were obtained from high pressure liquid chromatography, ultraviolet-visible and infrared spectra, and indicate that melanin or melaninlike compounds are present in the resting spore.

Spectroscopic features of native and bleached opio-melanins

Opioid peptides can be converted by tyrosinase into melanin-like compounds, in which the peptide moiety is retained. Such pigments, named opio-melanins, exhibit a characteristic absorption spectrum with a maximum at about 330 nm and a different solubility behaviour with respect to dopa-melanin, being completely soluble in hydrophylic solvents at neutral and basic pH. Opio-melanins precipitate in aqueous solutions below pH 5.0, and show apparent p K a values of 3.1, 3.6 and 4.4 for Tyr-Gly-melanin, Tyr-Gly-Gly-melanin and leuenk-melanin, respectively. The concomitant oxidation of dopa and opioid peptides by tyrosinase produces mixed polymers, showing the distinctive absorption peak at 330 nm. In the dark, in the pH range 5.5–7.0 the pigments are completely stable, whereas H 2O 2 addition provokes a slight degradation. At higher pH values or under simulated solar illumination with or without hydrogen peroxide, bleaching occurs more rapidly than in dopa-melanin. Upon photoirradiation the absorption spectrum of opio-melanins undergoes a marked variation, the peak at 330 nm being replaced by a broad shoulder in the range 280–350 nm. The absorption spectra of native and bleached pigments and the extent of opio-melanins degradation by bleaching agents, confirm the hypothesis that the different initial structure of the precursors accounts for a final diverse polymeric architecture of these pigments with respect to dopa-melanin.

Biological Control of Blue-Stain Fungi in Wood

Biological control of blue-stain fungi, such as Ophiostoma spp., that are detrimental to the wood products industry, was demonstrated in laboratory and field trials by a colorless strain of O. piliferum. Cartapip-97. This strain lacks melaninlike compounds responsible for the discoloration of sapwood. Inoculation of logs with Cartapip in the laboratory 2 wk before challenging with other fungi resulted in 58-68% colonization for Cartapip in isolated wood chips, while O. piliferum. O. piceae, O. minus, Phanerochaete gigantea, or Trichoderma harzianum colonized 0, 0, 0, 0, and 61%, respectively. Inoculation of logs with Cartapip 4 wk before other fungi resulted in similar trends with strong inhibition of blue-stain fungi. Simultaneous inoculation of logs with Cartapip and other fungi resulted in decreased colonization by both Cartapip and Ophiostoma species. When blue-stain fungi, P. gigantea or T. harzianum were inoculated 2 wk before Cartapip, colonization for these fungi ranged from 19 to 64% in cultured wood chips, whereas Cartapip ranged from 0 to 45% among the different treatments. Inoculation of O. piliferum and O. piceae prior to Cartapip resulted in inhibition of Cartapip. Two field trials demonstrated the exclusion of blue-stain fungi with prior colonization of the sapwood by Cartapip. Four weeks after inoculation of logs in the field, 92-100% of cultured wood chips were colonized by Cartapip in both trials, while blue-stain fungi colonized only 0-8%. In contrast, blue-stain fungi colonized 63% of the cultured wood chips in untreated control logs during the first field trial, and 29 and 71% for untreated control and antitranspirant treatments, respectively, during the second field trial. Results from both laboratory and field trials show the effectiveness of Cartapip for protecting freshly cut wood from blue-stain fungi

Some biochemical properties of melanins from opioid peptides

Opioid peptides are converted by mushroom tyrosinase into melanin-like compounds retaining the peptide moiety (opio-melanins). Opio-melanins, owing to the presence of the linked aminoacids and in contrast with DOPA-melanin, are soluble compound. The enkephalin-generated melanins are cleaved by carboxypeptidase A and pronase whereas aminopeptidase M cannot remove aminoacids from the pigment. Enkephalins, as well as other opioid peptides, (α-endorphin, kyotorphin, esorphins) if oxidized in presence of DOPA and tyrosinase are readily incorporated into DOPA-melanin. The resulting mixed-melanins (opio-melanin + DOPA-melanin) can be solubilized in hydrophylic solvents. Melanin from leuenkephalin exhibits paramagnetism as evidenced by an EPR spectrum identical to that of DOPA-melanin, but unlike the latter pigment, it does not appear to oxidize NADH, probably for the presence of the peptide moiety that exerts a hampering effect on the oxidizing capacity.