Melanin and Melanin-Related Polymers as Materials with Biomedical and Biotechnological Applications-Cuttlefish Ink and Mussel Foot Proteins as Inspired Biomolecules.
International journal of molecular sciences
The huge development of bioengineering during the last years has boosted the search for new bioinspired materials, with tunable chemical, mechanical, and optoelectronic properties for the design of semiconductors, batteries, biosensors, imaging and therapy probes, adhesive hydrogels, tissue restoration, photoprotectors, etc. These new materials should complement or replace metallic or organic polymers that cause cytotoxicity and some adverse health effects. One of the most interesting biomaterials is melanin and synthetic melanin-related molecules. Melanin has a controversial molecular structure, dependent on the conditions of polymerization, and therefore tunable. It is found in animal hair and skin, although one of the common sources is cuttlefish (Sepia officinalis) ink. On the other hand, mussels synthesize adhesive proteins to anchor these marine animals to wet surfaces. Both melanin and mussel foot proteins contain a high number of catecholic residues, and their properties are related to these groups. Dopamine (DA) can easily polymerize to get polydopamine melanin (PDAM), that somehow shares properties with melanin and mussel proteins. Furthermore, PDAM can easily be conjugated with other components. This review accounts for the main aspects of melanin, as well as DA-based melanin-like materials, related to their biomedical and biotechnological applications.
10.3390/ijms18071561
Biosynthesis of catechol melanin from glycerol employing metabolically engineered Escherichia coli.
Mejía-Caballero Alejandra,de Anda Ramón,Hernández-Chávez Georgina,Rogg Simone,Martinez Alfredo,Bolívar Francisco,Castaño Victor M,Gosset Guillermo
Microbial cell factories
BACKGROUND:Melanins comprise a chemically-diverse group of polymeric pigments whose function is related to protection against physical and chemical stress factors. These polymers have current and potential applications in the chemical, medical, electronics and materials industries. The biotechnological production of melanins offers the possibility of obtaining these pigments in pure form and relatively low cost. In this study, Escherichia coli strains were engineered to evaluate the production of melanin from supplemented catechol or from glycerol-derived catechol produced by an Escherichia coli strain generated by metabolic engineering. RESULTS:It was determined that an improved mutant version of the tyrosinase from Rhizobium etli (MutmelA), could employ catechol as a substrate to generate melanin. Strain E. coli W3110 expressing MutmelA was grown in bioreactor batch cultures with catechol supplemented in the medium. Under these conditions, 0.29 g/L of catechol melanin were produced. A strain with the capacity to synthesize catechol melanin from a simple carbon source was generated by integrating the gene MutmelA into the chromosome of E. coli W3110 trpD9923, that has been modified to produce catechol by the expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, transketolase and anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. In batch cultures with this strain employing complex medium with 40 g/L glycerol as a carbon source, 1.21 g/L of catechol melanin were produced. The melanin was analysed by employing Fourier transform infrared spectroscopy, revealing the expected characteristics for a catechol-derived polymer. CONCLUSIONS:This constitutes the first report of an engineered E. coli strain and a fermentation process for producing a catechol melanin from a simple carbon source (glycerol) at gram level, opening the possibility of generating a large quantity of this polymer for its detailed characterization and the development of novel applications.
10.1186/s12934-016-0561-0
Metabolic engineering of Escherichia coli to optimize melanin synthesis from glucose.
Chávez-Béjar María I,Balderas-Hernandez Victor E,Gutiérrez-Alejandre Aída,Martinez Alfredo,Bolívar Francisco,Gosset Guillermo
Microbial cell factories
BACKGROUND:Natural aromatic polymers, mainly melanins, have potential and current applications in the cosmetic, pharmaceutical and chemical industries. The biotechnological production of this class of compounds is based on tyrosinase-dependent conversion of L-tyrosine and other aromatic substrates into melanins. The purpose of this work was to apply metabolic engineering for generating Escherichia coli strains with the capacity to synthesize an aromatic polymer from a simple carbon source. RESULTS:The strategy was based on the expression in E. coli of the MutmelA gene from Rhizobium etli, encoding an improved mutant tyrosinase. To direct the carbon flow from central metabolism into the common aromatic and the L-tyrosine biosynthetic pathways, feedback inhibition resistant versions of key enzymes were expressed in strains lacking the sugar phosphotransferase system and TyrR repressor. The expressed tyrosinase consumed intracellular L-tyrosine, thus causing growth impairment in the engineered strains. To avoid this issue, a two phase production process was devised, where tyrosinase activity was controlled by the delayed addition of the cofactor Cu. Following this procedure, 3.22 g/L of melanin were produced in 120 h with glucose as carbon source. Analysis of produced melanin by Fourier transform infrared spectroscopy revealed similar characteristics to a pure eumelanin standard. CONCLUSIONS:This is the first report of a process for producing melanin from a simple carbon source at grams level, having the potential for reducing production cost when compared to technologies employing L-tyrosine as raw material.
10.1186/1475-2859-12-108
Bioproduction, characterization, anticancer and antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories Streptomyces glaucescens NEAE-H.
El-Naggar Noura El-Ahmady,El-Ewasy Sara M
Scientific reports
In this present study, a newly isolated strain, Streptomyces sp. NEAE-H, capable of producing high amount of black extracellular melanin pigment on peptone-yeast extract iron agar and identified as Streptomyces glaucescens NEAE-H. Plackett-Burman statistical design was conducted for initial screening of 17 independent (assigned) variables for their significances on melanin pigment production by Streptomyces glaucescens NEAE-H. The most significant factors affecting melanin production are incubation period, protease-peptone and ferric ammonium citrate. The levels of these significant variables and their interaction effects were optimized by using face-centered central composite design. The maximum melanin production (31.650 μg/0.1 ml) and tyrosinase activity (6089.10 U/ml) were achieved in the central point runs under the conditions of incubation period (6 days), protease-peptone (5 g/L) and ferric ammonium citrate (0.5 g/L). Melanin pigment was recovered by acid-treatment. Higher absorption of the purified melanin pigment was observed in the UV region at 250 nm. It appeared to have defined small spheres by scanning electron microscopy imaging. The maximum melanin yield was 350 mg dry wt/L of production medium. In vitro anticancer activity of melanin pigment was assayed against skin cancer cell line using MTT assay. The IC value was 16.34 ± 1.31 μg/ml for melanin and 8.8 ± 0.5 μg/ml for standard 5-fluorouracil.
10.1038/srep42129
Melanin and fungi.
Gómez Beatriz L,Nosanchuk Joshua D
Current opinion in infectious diseases
PURPOSE OF REVIEW:Production of melanin has been associated with virulence in diverse microorganisms. Melanization of fungi has been noted for many years in predominantly subcutaneous infections such as chromoblastomycosis and more recently most extensively studied in a yeast causing systemic infection, Cryptococcus neoformans. Pigmented fungi are increasingly important human pathogens and currently available antifungals are often sub-optimal for serious infections. This review focuses on recent publications on melanin in fungi with particular reference to the role of melanin in virulence, protection against antifungal drugs, and promoting survival in the environment. RECENT FINDINGS:Inhibition of melanin production by C. neoformans can prolong survival of lethally infected mice. In contrast, melanin in C. neoformans and Histoplasma capsulatum yeast cells can bind amphotericin B and caspofungin, thereby reducing the fungicidal affects of these drugs. H. capsulatum and Paracoccidioides brasiliensis have only recently been shown to produce melanin in vitro and during infection. Additionally, melanin derived from melanized C. neoformans yeast and Aspergillus niger conidia can activate complement, which may modify immune responses to infection. Studies on C. neoformans laccase have revealed that the enzyme is located on the cell wall, which may allow for interactions with the host. Melanization reduces the susceptibility of C. neoformans to enzymatic degradation and toxicity from a heavy metal, which may afford protection to the fungus against similar insults in the environment. SUMMARY:Melanin has been referred to as 'fungal armor' due to the ability of the polymer to protect microorganisms against a broad range of toxic insults. Recent publications continue to reveal important contributions of melanin to survival of fungi in the environment and during infection.
10.1097/00001432-200304000-00005
Actinobacterial melanins: current status and perspective for the future.
Manivasagan Panchanathan,Venkatesan Jayachandran,Sivakumar Kannan,Kim Se-Kwon
World journal of microbiology & biotechnology
Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. Melanins are biological macromolecules with multiple important functions, yet their structures are not well understood. Melanins are frequently used in medicine, pharmacology, and cosmetics preparations. Melanins also have great application potential in agriculture industry. They have several biological functions including photoprotection, thermoregulation, action as free radical sinks, cation chelators, and antibiotics. Plants and insects incorporate melanins as cell wall and cuticle strengtheners, respectively. Actinobacteria are the most economically as well as biotechnologically valuable prokaryotes. However, the melanin properties are, in general, poorly understood. In this review an evaluation is made on the present state of research on actinobacterial melanins and its perspectives. The highlights include the production and biotechnological applications of melanins in agriculture, food, cosmetic and medicinal fields. With increasing advancement in science and technology, there would be greater demands in the future for melanins produced by actinobacteria from various sources.
10.1007/s11274-013-1352-y
Biosynthesis of fungal melanins and their importance for human pathogenic fungi.
Langfelder Kim,Streibel Martin,Jahn Bernhard,Haase Gerhard,Brakhage Axel A
Fungal genetics and biology : FG & B
For more than 40 years fungi have been known to produce pigments known as melanins. Predominantly these have been dihydroxyphenylalanine (DOPA)-melanin and dihydroxynaphthalene (DHN)-melanin. The biochemical and genetical analysis of the biosynthesis pathways have led to the identification of the genes and corresponding enzymes of the pathways. Only recently have both these types of melanin been linked to virulence in some human pathogenic and phytopathogenic fungi. The absence of melanin in human pathogenic and phytopathogenic fungi often leads to a decrease in virulence. In phytopathogenic fungi such as Magnaporthe grisea and Colletotrichum lagenarium, besides other possible functions in pathogenicity, DHN-melanin plays an essential role in generating turgor for plant appressoria to penetrate plant leaves. While the function of melanin in human pathogenic fungi such as Cryptococcus neoformans, Wangiella dermatitidis, Sporothrix schenckii, and Aspergillus fumigatus is less well defined, its role in protecting fungal cells has clearly been shown. Specifically, the ability of both DOPA- and DHN-melanins to quench free radicals is thought to be an important factor in virulence. In addition, in several fungi the production of fungal virulence factors, such as melanin, has been linked to a cAMP-dependent signaling pathway. Many of the components involved in the signaling pathway have been identified.
10.1016/s1087-1845(02)00526-1
Synthesis and assembly of fungal melanin.
Eisenman Helene C,Casadevall Arturo
Applied microbiology and biotechnology
Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from L-3,4-dihydroxyphenylalanine (L-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.
10.1007/s00253-011-3777-2
Bacterial melanin production by heterologous expression of 4‑hydroxyphenylpyruvate dioxygenase from Pseudomonas aeruginosa.
Bolognese Fabrizio,Scanferla Chiara,Caruso Enrico,Orlandi Viviana Teresa
International journal of biological macromolecules
Pyomelanin is a reddish-brown pigment produced by bacteria of different genera and plays a variety of physiological roles. Proposals have been regarding the use of pyomelanin in various environmental, industrial and, more recently, cosmetic applications. In Pseudomonas aeruginosa, the enzyme 4‑hydroxyphenylpiruvate dioxygenase (Hpd) converts 4-hydroxyphenylpiruvate into homogentisic acid, which represents the key intermediate for melanin biosynthesis. This work aimed to obtain Escherichia coli cells overexpressing hpd gene from the PAO1 strain to produce large amounts of pyomelanin for biotechnological purposes. The recombinant dioxygenase expression gave E. coli JM109 the ability to produce pyomelanin. A series of biotransformations led us to choose the best experimental conditions for pyomelanin production. Cells were grown at the mid-exponential phase in a mineral medium with added glucose 10 mM as carbon and energy sources and casamino acid 0.2% w/v as an amino acid source. The administration of tyrosine 1 mM after 30 min of exposure to arabinose 1% w/v made it possible to purify 213 mg/L of pyomelanin after 6 days of biotransformation. In addition to the interesting biotechnological outcomes, the resulting expression system supports the correlation between the hpd gene from P. aeruginosa PAO1 and pyomelanin synthesis.
10.1016/j.ijbiomac.2019.04.061
Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application.
Wang Zheng,Tschirhart Tanya,Schultzhaus Zachary,Kelly Erin E,Chen Amy,Oh Eunkeu,Nag Okhil,Glaser Evan R,Kim Eunkyoung,Lloyd Pamela F,Charles Paul T,Li Weiyao,Leary Dagmar,Compton Jaimee,Phillips Daniel A,Dhinojwala Ali,Payne Gregory F,Vora Gary J
Applied and environmental microbiology
Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium , one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from formed nanoparticles that were assembled as "melanin ghost" structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis. Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from -produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.
10.1128/AEM.02749-19
DOPA and DHN pathway orchestrate melanin synthesis in Aspergillus species.
Pal Anuradha K,Gajjar Devarshi U,Vasavada Abhay R
Medical mycology
Melanins are high molecular weight hydrophobic pigments that have been studied for their role in the virulence of fungal pathogens. We investigated the amount and type of melanin in 20 isolates of Aspergillus spp.; A. niger (n = 3), A. flavus (n = 5), A. tamarii (n = 3), A. terreus (n = 3), A. tubingensis (n = 3), A. sydowii (n = 3). Aspergillus spp. were identified by sequencing the internal transcribed spacer (ITS) region. Extraction of melanin from culture filtrate and fungal biomass was done and followed by qualitative and quantitative analysis of melanin pigment. Ultraviolet (UV), Fourier transformed infrared (FT-IR), and electron paramagnetic resonance (EPR) spectra analyses confirmed the presence of melanin. The melanin pathway was studied by analyzing the effects of inhibitors; kojic acid, tropolone, phthalide, and tricyclazole. The results indicate that in A. niger and A. tubingensis melanin was found in both culture filtrate and fungal biomass. For A. tamarii and A. flavus melanin was extracted from biomass only, whereas melanin was found only in culture filtrate for A. terreus. A negligible amount of melanin was found in A. sydowii. The maximum amount of melanin from culture filtrate and fungal biomass was found in A. niger and A. tamarrii, respectively. The DOPA (3,4-dihydroxyphenylalanine) pathway produces melanin in A. niger, A. tamarii and A. flavus, whereas the DHN (1,8-dihydroxynaphthalene) pathway produces melanin in A. tubingensis and A. terreus. It can be concluded that the amount and type of melanin in aspergilli largely differ from species to species.
10.3109/13693786.2013.826879
Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins.
Barek Hanine,Sugumaran Manickam,Ito Shosuke,Wakamatsu Kazumasa
Pigment cell & melanoma research
Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6-dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.
10.1111/pcmr.12672
Scalable Biosynthesis of Melanin by the Basidiomycete Armillaria cepistipes.
Ribera Javier,Panzarasa Guido,Stobbe Annika,Osypova Alina,Rupper Patrick,Klose Daniel,Schwarze Francis W M R
Journal of agricultural and food chemistry
Natural melanin features many interesting properties, including the ability to shield electromagnetic radiation, the ability to act as scavenger for radical and reactive oxygen species and the capacity to chelate different metal ions. For these reasons, melanin is becoming increasingly relevant for the development of functional materials with potential applications in cosmetics, drug delivery, and water purification. However, the extraction and purification of melanin from conventional sources (e.g., sepia ink, hair, and wool) is inefficient and not easily scalable, hence diverting its technological applications. Some fungal species, especially wood-decay basidiomycetes, can be regarded as promising sources of melanin. In the present study, we screened different fungi in regard to their melanin-biosynthesis abilities using l-tyrosine as a precursor, and we found that an Armillaria cepistipes strain (Empa 655) produced the highest yield of melanin (27.98 g L). Physicochemical characterization of the obtained fungal melanin revealed a typical eumelanin structure. The method for the biosynthesis of fungal melanin we propose is efficient, scalable, and sustainable and has the potential to provide support for further technological exploitation.
10.1021/acs.jafc.8b05071
The melanization road more traveled by: Precursor substrate effects on melanin synthesis in cell-free and fungal cell systems.
The Journal of biological chemistry
Natural brown-black eumelanin pigments confer structural coloration in animals and potently block ionizing radiation and antifungal drugs. These functions also make them attractive for bioinspired materials design, including coating materials for drug-delivery vehicles, strengthening agents for adhesive hydrogel materials, and free-radical scavengers for soil remediation. Nonetheless, the molecular determinants of the melanin "developmental road traveled" and the resulting architectural features have remained uncertain because of the insoluble, heterogeneous, and amorphous characteristics of these complex polymeric assemblies. Here, we used 2D solid-state NMR, EPR, and dynamic nuclear polarization spectroscopic techniques, assisted in some instances by the use of isotopically enriched precursors, to address several open questions regarding the molecular structures and associated functions of eumelanin. Our findings uncovered: 1) that the identity of the available catecholamine precursor alters the structure of melanin pigments produced either in fungal cells or under cell-free conditions; 2) that the identity of the available precursor alters the scaffold organization and membrane lipid content of melanized fungal cells; 3) that the fungal cells are melanized preferentially by an l-DOPA precursor; and 4) that the macromolecular carbon- and nitrogen-based architecture of cell-free and fungal eumelanins includes indole, pyrrole, indolequinone, and open-chain building blocks that develop depending on reaction time. In conclusion, the availability of catecholamine precursors plays an important role in eumelanin development by affecting the efficacy of pigment formation, the melanin molecular structure, and its underlying scaffold in fungal systems.
10.1074/jbc.RA118.005791
Bacterial Enzymes Catalyzing the Synthesis of 1,8-Dihydroxynaphthalene, a Key Precursor of Dihydroxynaphthalene Melanin, from Sorangium cellulosum.
Sone Yusuke,Nakamura Shuto,Sasaki Makoto,Hasebe Fumihito,Kim Seung-Young,Funa Nobutaka
Applied and environmental microbiology
1,8-Dihydroxynaphthalene (1,8-DHN) is a key intermediate in the biosynthesis of DHN melanin, which is specific to fungi. In this study, we characterized the enzymatic properties of the gene products of an operon consisting of , , and from the Gram-negative bacterium Heterologous expression of , , and in caused secretion of a dark-brown pigment into the broth. High-performance liquid chromatography (HPLC) analysis of the broth revealed that the recombinant strain produced 1,8-DHN, indicating that the operon encoded a novel enzymatic system for the synthesis of 1,8-DHN. Simultaneous incubation of the recombinant SoceCHS1, BdsA, and BdsB with malonyl-coenzyme A (malonyl-CoA) and NADPH resulted in the synthesis of 1,8-DHN. SoceCHS1, a type III polyketide synthase (PKS), catalyzed the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN) THN was in turn converted to 1,8-DHN by successive steps of reduction and dehydration, which were catalyzed by BdsA and BdsB. BdsA, which is a member of the aldo-keto reductase (AKR) superfamily, catalyzed the reduction of THN and 1,3,8-tetrahydroxynaphthalene (THN) to scytalone and vermelone, respectively. The stereoselectivity of THN reduction by BdsA occurred on the -face to give ()-scytalone with more than 99% optical purity. BdsB, a SnoaL2-like protein, catalyzed the dehydration of scytalone and vermelone to THN and 1,8-DHN, respectively. The fungal pathway for the synthesis of 1,8-DHN is composed of a type I PKS, naphthol reductases of the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (SD). These findings demonstrated 1,8-DHN synthesis by novel enzymes of bacterial origin. Although the DHN biosynthetic pathway was thought to be specific to fungi, we discovered novel DHN synthesis enzymes of bacterial origin. The biosynthesis of bacterial DHN utilized a type III PKS for polyketide synthesis, an AKR superfamily for reduction, and a SnoaL2-like NTF2 superfamily for dehydration, whereas the biosynthesis of fungal DHN utilized a type I PKS, SDR superfamily enzyme, and SD-like NTF2 superfamily. Surprisingly, the enzyme systems comprising the pathway were significantly different from each other, suggesting independent, parallel evolution leading to the same biosynthesis. DHN melanin plays roles in host invasion and adaptation to stress in pathogenic fungi and is therefore important to study. However, it is unclear whether DHN biosynthesis occurs in bacteria. Importantly, we did find that bacterial DHN biosynthetic enzymes were conserved among pathogenic bacteria.
10.1128/AEM.00258-18
Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans.
Lee Dongpil,Jang Eun-Ha,Lee Minjae,Kim Sun-Woo,Lee Yeonseon,Lee Kyung-Tae,Bahn Yong-Sun
mBio
Melanin is an antioxidant polyphenol pigment required for the pathogenicity of many fungal pathogens, but comprehensive regulatory mechanisms remain unidentified. In this study, we systematically analyzed melanin-regulating signaling pathways in and identified four melanin-regulating core transcription factors (TFs), Bzp4, Usv101, Mbs1, and Hob1, required for induction of the laccase gene (). Bzp4, Usv101, and Mbs1 independently regulate induction, whereas Hob1 controls Bzp4 and Usv101 expression. Both Bzp4 and Usv101 are localized in the cytoplasm under nutrient-rich conditions (i.e., in the presence of yeast extract-peptone-dextrose [YPD] medium) but translocate into the nucleus upon nutrient starvation (i.e., in the presence of yeast nitrogen base [YNB] medium without glucose), and Mbs1 is constitutively localized in the nucleus. Notably, the cAMP pathway is not involved in regulation of the four TFs, but the high-osmolarity glycerol response (HOG) pathway negatively regulates induction of and Next, we searched for potential kinases upstream of the core TFs and identified nine core kinases; their deletion led to defective melanin production and induction. Deletion of or abolished induction of and and perturbed nuclear translocation of Bzp4. Notably, Gsk3 also regulated expression of , , and , indicating that it is a critical melanin-regulating kinase. Finally, an RNA sequencing-based transcriptome analysis of the wild-type strain and of Δ, Δ, Δ, and Δ strains under nutrient-rich and nutrient-starved conditions revealed that the melanin-regulating core TFs govern redundant and distinct classes of genes involved in a variety of biological processes. Melanins are dark green, brown, or black pigments that serve as antioxidant, reactive oxygen species (ROS) scavengers that protect fungal pathogens from radiation and host immune responses. , the major etiological agent of fungal meningoencephalitis, also utilizes melanin as a key virulence factor. In this basidiomycete pathogen, melanin production is regulated by the cAMP and high-osmolarity glycerol response (HOG) pathways, and yet its complex signaling networks remain poorly described. In this study, we uncovered novel melanin synthesis regulatory networks consisting of core transcription factors (TFs), including Bzp4, Usv101, Hob1, and Mbs1, and core kinases Gsk3 and Kic1. These networks were identified through coupling systematic analyses of the expression and epistatic relationships of TF and kinase mutant libraries in the presence of diverse melanin substrates with transcriptome profiling of the core TF mutants. Thus, this report provides comprehensive insight into the melanin-regulating pathways in and other fungal pathogens.
10.1128/mBio.02267-19
The structural unit of melanin in the cell wall of the fungal pathogen .
The Journal of biological chemistry
Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.
10.1074/jbc.RA119.008684
Identification of a tyrosinase gene and its functional analysis in melanin synthesis of Pteria penguin.
Yu Feifei,Pan Zhenni,Qu Bingliang,Yu Xiangyong,Xu Kaihang,Deng Yuewen,Liang Feilong
Gene
Tyrosinase is a key rate-limiting enzyme in melanin synthesis. In this study, a new tyrosinase gene (Tyr) was identified from Pteria penguin and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpTyr was 1728 bp long, containing a 5'untranslated region (UTR) of 11 bp, a 3'UTR of 295 bp, and an open reading fragment of 1422 bp encoding 473 amino acids. Amino acid alignment showed PpTyr had the highest (50%) identity to tyrosinase-like protein 1 from Pinctada fucata. Phylogenetic tree analysis classified PpTyr into α-subclass of type-3 copper protein. Tissue expression analysis indicated that PpTyr was highly expressed in mantle, a nacre formation related tissue. After PpTyr RNA interference, PpTyr mRNA was significantly inhibited by 71.0% (P < 0.05). For other melanin-related genes, PpCreb2 and PpPax3 expression showed no significant change, but PpBcl2 was obviously increased. By liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis, the total content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2,3,5-tricarboxylic acid), as main markers for eumelanin, was sharply decreased by 66.6% after PpTyr RNAi (P < 0.05). The percentage of PDCA was also obviously decreased from 20.1% to 13.9%. This indicated that tyrosinase played a key role in melanin synthesis and color formation of P. penguin.
10.1016/j.gene.2018.02.060
Gene Regulated Melanin Synthesis by Tyrosinase Pathway in .
Yu Feifei,Qu Bingliang,Lin Dandan,Deng Yuewen,Huang Ronglian,Zhong Zhiming
International journal of molecular sciences
The paired-box 3 () is a transcription factor and it plays an important part in melanin synthesis. In this study, a new gene was identified from (, ) () by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed shared the highest (69.2%) identity with of . Tissue expression profile showed that had the highest expression in mantle, a nacre-formation related tissue. The silencing significantly inhibited the expression of , , and , genes involved in -mediated melanin synthesis, but had no effect on and an increase effect on . Furthermore, the knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase () knockdown. These data indicated that played an important regulating role in melanin synthesis by pathway in .
10.3390/ijms19123700