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. 2007 May 23;2(5):e457.
doi: 10.1371/journal.pone.0000457.

Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi

Ekaterina Dadachova  1 , Ruth A Bryan, Xianchun Huang, Tiffany Moadel, Andrew D Schweitzer, Philip Aisen, Joshua D Nosanchuk, Arturo Casadevall
Affiliations

Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi

Ekaterina Dadachova et al. PLoS One. .

Abstract

Background: Melanin pigments are ubiquitous in nature. Melanized microorganisms are often the dominating species in certain extreme environments, such as soils contaminated with radionuclides, suggesting that the presence of melanin is beneficial in their life cycle. We hypothesized that ionizing radiation could change the electronic properties of melanin and might enhance the growth of melanized microorganisms.

Methodology/principal findings: Ionizing irradiation changed the electron spin resonance (ESR) signal of melanin, consistent with changes in electronic structure. Irradiated melanin manifested a 4-fold increase in its capacity to reduce NADH relative to non-irradiated melanin. HPLC analysis of melanin from fungi grown on different substrates revealed chemical complexity, dependence of melanin composition on the growth substrate and possible influence of melanin composition on its interaction with ionizing radiation. XTT/MTT assays showed increased metabolic activity of melanized C. neoformans cells relative to non-melanized cells, and exposure to ionizing radiation enhanced the electron-transfer properties of melanin in melanized cells. Melanized Wangiella dermatitidis and Cryptococcus neoformans cells exposed to ionizing radiation approximately 500 times higher than background grew significantly faster as indicated by higher CFUs, more dry weight biomass and 3-fold greater incorporation of (14)C-acetate than non-irradiated melanized cells or irradiated albino mutants. In addition, radiation enhanced the growth of melanized Cladosporium sphaerospermum cells under limited nutrients conditions.

Conclusions/significance: Exposure of melanin to ionizing radiation, and possibly other forms of electromagnetic radiation, changes its electronic properties. Melanized fungal cells manifested increased growth relative to non-melanized cells after exposure to ionizing radiation, raising intriguing questions about a potential role for melanin in energy capture and utilization.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Microscopic images of melanized fungal cells:
a) light microscopy image of C. neoformans melanin “ghosts”; (b–e) TEM images of C. sphaerospermum “ghosts” derived from cells grown on nutrient rich or nutrient-deficient media: b) potato dextrose agar; c) Sabaroud dextrose agar; d) water agar with casein; e) water agar with dextrose. Original magnification: light microscopy image – X 1,000; TEM images – X 13,000.
Figure 2
Figure 2. Chemical composition and paramagnetic properties of melanin:
a) structure of eumelanin oligomer; b) structure of pheomelanin oligomer; (c – f) HPLC of permanganate-oxidized melanins: c) chromatogram of background solution; d) C. neoformans; e) C. sphaerospermum grown on Sabaroud dextrose agar; f) W. dermatitidis isolate 8656 (wild type); (g–i) ESR spectra: g) W. dermatitidis isolate 8656; h) C. neoformans before irradiation; i) C. neoformans after irradiation with 0.3 kGy dose. PDCA - pyrrole-2,3-dicarboxylic acid; PTCA - pyrrole-2,3,5-tricarboxylic acid; TTCA - 1,3-thiazole-2,4,5-tricarboxylic acid; TDCA - 1,3-thiazole-4,5-dicarboxylic acid. Absorption was monitored at 255 nm and displayed on a linear scale. ESR spectra were obtained by suspending “ghosts” in water except for (h) which was performed in dry state. Ordinate in g–i is the derivative of the ESR absorption in arbitrary units.
Figure 3
Figure 3. HPLC of melanin derived from C. sphaerospermum grown on different substrates: a) potato dextrose agar; b) Sabaroud dextrose agar; c) water agar with casein; d) water agar with dextrose.
PDCA - pyrrole-2,3-dicarboxylic acid; PTCA - pyrrole-2,3,5-tricarboxylic acid; TTCA - 1,3-thiazole-2,4,5-tricarboxylic acid; TDCA - 1,3-thiazole-4,5-dicarboxylic acid. Absorption was monitored at 255 nm and displayed on a linear scale. Cs - C. sphaerospermum.
Figure 4
Figure 4. ESR spectra of melanin derived from C. sphaerospermum grown on different substrates: a) potato dextrose agar; b) Sabaroud dextrose agar; c) potato dextrose agar impregnated with 25 µg/mL tricyclazole.
Differences in C. sphaerospermum ESR spectra in comparison with C. neoformans are marked with arrows. ESR spectra were obtained by suspending “ghosts” in water. Ordinate is the derivative of the ESR absorption in arbitrary units. Cs - C. sphaerospermum.
Figure 5
Figure 5. The influence of ionizing radiation or heat on the metabolic activity of melanized and non-melanized C. neoformans cells.
a, b) irradiated and non-irradiated cells: a) XTT; b) MTT. c) XTT of cells grown at room temperature (22°C) or at 30°C. The cells were kept in the dark while being exposed to ionizing radiation or different temperatures. Following the exposure, XTT or MTT reagents were added to the samples and absorbance was measured at 492 or 550 nm for XTT and MTT, respectively.
Figure 6
Figure 6. Growth and incorporation of 14C-acetate by melanized C. neoformans H99 cells and non-melanized Lac(-) H99 cells lacking the laccase enzyme under conditions of limited nutrients supply in a radiation field of 0.05 mGy/hr or at background radiation level.
a) growth of melanized H99 cells; b) growth of non-melanized Lac(-) H99 cells; c) incorporation of 14C-acetate into melanized H99 cells; d) incorporation of 14C-acetate into non-melanized Lac(-) H99 cells; e) ratio of irradiated to non-irradiated cells CFUs and cpms ratios (normalized CFUs and cpms) for melanized H99 and non-melanized Lac(-) H99 cells.
Figure 7
Figure 7. Survival of non-melanized and melanized C. sphaerospermum cells following exposure to external gamma rays: average volume (left side plates) and radial growth rate (right side plates) of melanized and melanin-deficient C. sphaerospermum colonies grown on agar plates with (top panels) or without (bottom panels) sucrose in a radiation field of 0.05 mGy/hr or at background radiation level (control).
rad - irradiated, ctrl - control, mel - melanized, suc- sucrose added. The volume of half-sphere was calculated as V/2 = π/12 d3. Radial linear growth rate of C. sphaerospermum colonies was calculated as K = (Rt−Ro)/(t−to), where K is radial linear growth rate, mm/hr; Rt and Ro - colony radii at time t and time to, respectively.
Figure 8
Figure 8. Growth of W. dermatitidis.
a) wild type 8656; b) albino mutant wdpks1Δ-1 with a disrupted polyketide synthase gene; c) a strain complemented with wild type gene wdpks1. The cells were grown under conditions of limited nutrients in a radiation field of 0.05 mGy/hr or at background radiation level. The cells were exposed to radiation for various times and plated for CFUs on YPD.
See this image and copyright information in PMC

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