Melanin, Radiation, and Energy Transduction in Fungi

ABSTRACT

Melanin pigments are found in many diverse fungal species, where they serve a variety of functions that promote fitness and cell survival. Melanotic fungi inhabit some of the most extreme habitats on earth such as the damaged nuclear reactor at Chernobyl and the highlands of Antarctica, both of which are high-radiation environments. Melanotic fungi migrate toward radioactive sources, which appear to enhance their growth. This phenomenon, combined with the known capacities of melanin to absorb a broad spectrum of electromagnetic radiation and transduce this radiation into other forms of energy, raises the possibility that melanin also functions in harvesting such energy for biological usage. The ability of melanotic fungi to harness electromagnetic radiation for physiological processes has enormous implications for biological energy flows in the biosphere and for exobiology, since it provides new mechanisms for survival in extraterrestrial conditions. Whereas some features of the way melanin-related energy transduction works can be discerned by linking various observations and circumstantial data, the mechanistic details remain to be discovered.

INTRODUCTION

Melanins are dark pigments that are made by diverse fungi (1, 2). Even fungi that produce white colonies, such as Candida albicans, have the ability to make melanins (3, 4). Melanins have elicited considerable interest in microbial pathogenesis because they are important virulence factors for many pathogenic microbes, and their presence is associated with reduced susceptibility to antifungal drugs (5, 6). Melanins are multifunctional molecules that give cells structural strength as well as reduced susceptibility to temperature extremes, heavy metals, and molecules produced by the immune system such as oxygen- and nitrogen-derived oxidants and microbicidal proteins (2, 7–10).

Despite the importance of melanins in biology, their structure remains largely unsolved (11). Melanins are composed of covalently polymerized indole- and phenol-type compounds resulting in a material that is insoluble and acid resistant. Melanin structures exhibit local order with global heterogeneity, thus resulting in an amorphous material. Insolubility combined with an amorphous nature means that the structure of melanin cannot be solved with currently available analytical techniques such as X-ray diffraction. A remarkable property of melanins is that they are stable free radicals and manifest a distinctive electron spin resonance signature that is used in their identification (12). Despite the difficulties involved in working with melanin, considerable progress has been made in eliciting its structure by combining results from several techniques including solid-state nuclear magnetic resonance (11, 13, 14).

Melanin production in fungi is a result of three pathways known as the polyketide, 3,4-dihydroxyphenylalanine (L-DOPA), and l-tyrosine degradation synthetic pathways, which produce two chemically different compounds with similar properties (2, 7, 15, 16). Melanin synthesis involves free radical reactions with toxic intermediates. Consequently, melanin synthesis in both animals and fungi occurs in specialized structures known as melanosomes (17). Melanin can be located internally or in and on the cell wall, where it is closely associated with other cellular components such as lipids, carbohydrates, and proteins, although the nature of these associations is poorly understood due to the inherent difficulties in studying melanin structure. For Cryptococcus neoformans, one of the fungi in which the process of melanization is best understood, melanin synthesis is catalyzed by a laccase, and the pigment is exclusively 3,4-dihydroxyphenylalanine-melanin. C. neoformans melanin is synthesized in vesicles that are exported to the extracellular space and are assembled in the cell membrane into concentric layers, where the pigment is closely associated with polysaccharide and other cellular structures such as chitin and its derivatives (11–14).

Among the remarkable properties of melanin is its capacity for energy transduction (18). Melanin is unique among biological compounds in that its absorption spectrum reveals absorption of all wavelengths in the UV-visible-infrared spectrum. The capacity of melanin to absorb all these wavelengths is presumably a function of its complex molecular structure, which allows it to interact with these frequencies of light. The ability of melanin to absorb electromagnetic radiation extends into the range of X rays and gamma rays, such that it has a shielding capacity that is approximately half that of lead and twice that of carbon (19). Mice given fungal melanin are capable of surviving lethal doses of gamma irradiation, presumably as a result of the pigment protecting the digestive tract and associated lymphatic tissue (20). The ability of melanin to absorb and convert electromagnetic energy is associated with a variety of biological effects that give melanotic fungi tremendous resilience, which translates into their ability to survive in hostile environments.

MELANOTIC FUNGI INHABIT RADIATION-EXTREME ENVIRONMENTS

Melanotic fungi inhabit some of the most extreme environments known, and it is generally believed that the presence of melanin contributes to survival through a variety of mechanisms. For example, Antarctic black rock fungi include a large number of diverse melanotic species that colonize the rocks of one of the most extreme environments on Earth (21). For the purposes of this article we review the literature reporting melanotic fungi in high-radiation environments. The remarkable finding that the damaged reactor at Chernobyl and surrounding soils host a large population of melanotic fungal species is perhaps the best-known example of fungi living in a high-radiation environment (22–24). The Antarctic highlands, where many black rock fungi thrive exposed on the surface and interior of rocks, is also a high-radiation environment (21), especially during the long austral summer and considering the recent historical weakening of the atmospheric protection in the southern hemisphere through ozone depletion. Notably, black fungi recovered from Antarctica can survive in simulated Mars conditions (25). The melanotic fungus Ulocladium chartarum was able to grow under space flight conditions, with a rate of growth that exceeded that achieved on Earth (26).

MELANOTIC FUNGI RESPONSES TO RADIATION

Three lines of evidence indicate that melanotic fungi respond to radiation: kinetic attraction toward radioactive sources, faster growth, and metabolic changes. Although these lines of evidence are not completely independent (e.g., hyphal growth toward a radioactive source and enhanced colony growth both reflect cell growth, which in turn is associated with metabolic changes), they are sufficiently distinct to discuss separately.

Radiotropism

The term “radiotropism” refers to the ability of some fungi to grow toward radiation sources (27, 28). This phenomenon was first reported among fungal species colonizing the damaged reactor at Chernobyl and involved the migration toward and degradation of carbon containing “hot particles” (28). Radiotropism was triggered primarily by gamma radiation, but it is possible that both alpha and beta radiation could be “sensed” by the fungi as well. Although the mechanism and purpose of fungal cell migration toward a radioactive source remain unknown, the careful work documenting this phenomenon indicates that fungi respond to radiation and suggest intentional migration and attraction to this energy source.

Enhanced Growth

In 2007 our group reported differential growth effects on melanotic and nonmelanotic strains of C. neoformans and Exophiala (Wangiella) dermatitidis exposed to gamma radiation such that the melanin-containing strains grew faster than albino strains (29). This phenomenon was subsequently confirmed and expanded (see below) by another group (30). Enhanced fungal growth in melanotic fungi in response to high-energy electromagnetic radiation has also been reported for other fungal species. Large increases in colony-forming units (>100-fold) were observed for Alternaria alternata exposed to gamma radiation (31). Exposure of Aspergillus versicolor to radioactive nuclides increased hyphal length and spore germination, phenomena that were attributed to enhanced growth by radiation (24). Hence, enhanced growth of melanotic fungi in high-radiation environments has been observed for several fungal species by at least four independent groups. Analysis of the energy levels that triggered radiation-related fungal growth revealed that whereas low levels of radiation (150 peak kilovoltage [kVp]) enhanced the growth of both melanized and nonmelanized C. neoformans, higher levels (320 kVp) triggered enhanced growth only in the melanized cells (32).

Metabolic Changes

Analysis of wild-type and albino mutant wdpks1 E. dermatitidis strains’ gene expression after exposure to high-energy radiation revealed differential expression of about 3,000 genes involving multiple pathways, such that genes for cell cycles of amino acid synthesis were downregulated, while stress response genes were upregulated (30). Of particular interest was the observation that the wild-type (melanotic) but not the albino wdpks1 strain manifested ribosomal biogenesis genes’ upregulation to radiation exposure, leading the authors to suggest the possibility that melanin-derived energy was being used for protein synthesis (30). An experiment with three melanotic fungal species in a simulated Mars environment that included exposure to 200 nm UV radiation revealed some initial changes in protein expression followed by adaptation with resumption of normal expression, leading the investigators to conclude that these organisms could survive in that environment (33). An analysis of the response of Hormoconis resinae to chronic radiation resulting in cumulative doses of 2 to 3 Gy found that radiation was associated with enhanced synthesis of melanin and antioxidant enzymes (34).

INTERACTIONS OF MELANIN WITH HIGH-ENERGY ELECTROMAGNETIC RADIATION

For melanin to capture electromagnetic radiation in a manner that is suitable for conversion to biological energy, it must interact with it. Whereas melanin is known to absorb all UV, visible, and infrared frequencies, the phenomena of radiotropism and enhanced growth upon exposure to gamma rays depend on the ability of the pigment to interact with much higher-energy radiation. Such interactions have now been demonstrated by several methodologies. Irradiation of melanin with gamma rays resulted in changed electronic properties as measured by electron spin resonance spectra (20). A demonstration of the capacity of melanin to serve as an energy transduction molecule for high-energy electromagnetic radiation came from the observation that an electric current was produced by a melanin electrode placed in a gamma-ray beam (35).

IMPLICATIONS OF MELANIN-MEDIATED ENERGY TRANSDUCTION

The phenomenon of radiation-induced growth in melanotic fungi was called radiosynthesis in analogy to photosynthesis, by which plants convert light energy into chemical energy that they can utilize for biological processes (29). Supporting this designation was the observation that irradiated melanin was able to reduce NAD to NADH, thus providing a critical link for the conversion of electromagnetic energy into chemical energy that was immediately biologically useful. However, the analogy of radiosynthesis to photosynthesis is only partial, for the latter is understood to include a series of complex reactions that involve the fixing of carbon to synthesize new biologically useful molecules. For fungi, there are some suggestions that they can utilize CO₂ for synthesizing organic molecules, but this topic has not been thoroughly explored. Studies of black rock fungi from Antarctica using ¹⁴C suggested that they could directly utilize CO₂ and incorporate carbon into organic molecules (36). Earlier studies of the mold Zygorrhynchus moelleri provided evidence for the direct nonphotosynthetic incorporation of CO₂ into pyruvate (37). Hence, one can imagine a situation in an extreme environment such as Antarctic mountains where the combination of radiation harvesting by melanin combined with some capacity for utilizing inorganic carbon could produce a process akin to radiosynthesis.

MAJOR QUESTIONS AND FUTURE RESEARCH DIRECTIONS

At this time there is experimental evidence for the following observations: (i) melanin is an energy-transducing molecule with the capacity to absorb a broad spectrum of electromagnetic radiation; (ii) melanotic fungi exposed to high-energy radiation grow faster than exposed nonmelanotic mutants and unexposed fungi, both melanotic and amelanotic; (iii) melanin can interact with high-energy electromagnetic radiation and transduce it to chemical and electrical energy. In aggregate, these observations suggest that melanotic fungi have the capacity to utilize high-energy electromagnetic radiation to sustain some biological processes. This, combined with the possibility that fungi have some capacity to fix carbon, raises the possibility of radiosynthesis. However, we lack information about the detailed processes by which electromagnetic energy is harvested and converted into biologically useful energy. The ability of irradiated melanin to convert NAD to NADH in vitro suggests that it may contribute to energy utilization through simple oxidation-reduction reactions that harvest electrons for biological use. However, there is also a possibility that melanin-related energy utilization involves a sophisticated molecular apparatus such as an antennae complex to shuttle electrons from extracellular cell wall-associated melanin to the cell interior.

The biological advantages of melanin-mediated energy transduction are readily apparent. For organisms in extreme environments such as black rock fungi of Antarctica, the ability to harvest some light energy for biological processes could provide a tremendous survival advantage relative to nonmelanotic organisms. However, the exact contribution of this mechanism relative to the energy available from conventional nutritional processes available to fungi from other sources such as degradation of plant matter is unknown. Establishing the importance of this mechanism in fungi is a much more difficult undertaking than showing the importance of photosynthesis in plants, because the latter are totally dependent on sunlight for their survival, whereas fungi can access nutrients from other sources. In this regard, it is noteworthy that experiments showing that electromagnetic radiation enhances growth have relied on measuring growth increments relative to nonirradiated conditions or albino mutant controls rather than establishing an absolute requirement for growth, since the latter criterion has been experimentally difficult. Consequently, establishing the importance of melanin energy capture is an open question that will probably require innovative experimental design.

IMPLICATIONS FOR BIOLOGY AND EXOBIOLOGY

Given the widespread abundance of melanotic fungi in the biosphere, any amount of conversion of electromagnetic energy into biologically useful energy is likely to have a major impact on estimates of planetary energy flows. For example, photosynthesis is estimated to convert approximately 130 terawatts of sunlight into biologically useful energy, an amount that could increase significantly with melanin-related energy conversion. The ability of melanotic fungi to harvest energy would make them autotrophs and place them alongside plants as important contributors to the conversion of solar and physical electromagnetic energy sources into biologically useful energy. Because the energy of the ionizing radiation is several orders of magnitude higher than the energy of white light, even a very inefficient mechanism of its harvesting will still produce energy transfer. The fact that melanin pigments are easy to synthesize and are found in all biological kingdoms raises the tantalizing possibility that melanin-related energy transduction is ancient, predating photosynthesis, and served as a significant energy-harvesting mechanism for early life on Earth, which emerged in conditions of much higher radiation exposure.

Melanin-related energy transduction has immediate implications for exobiology since it implies that this pigment not only confers the capacity for survival in extreme environments but also provides a means for harvesting electromagnetic energy. The survival of melanotic fungi in simulated Mars-like conditions suggests that the resiliency conferred by melanin already provides some Earth microbes with the capacity to survive on other worlds, thus extending the limits of terrestrial life.

FIGURE 1.

Melanized Cryptococcus neoformans and melanin “ghosts.” (A) Melanized C. neoformans cell in India Ink suspension to illustrate the cellular location of melanin. The cell wall appears dark because of the presence of melanin. Note that melanin is external to the body of the cell. (B) Melanin ghosts prepared from melanized cryptococcal cells by strong acid digestion (38), which removes cellular components except for the cell wall-associated melanin. (C) Scanning electron microscopy of a melanin ghost showing its granulated surface structure, porosity, and thickness.