EPR spectroscopy of C. neoformans cells

All melanins contain a small population of semiquinone radicals within their structure and exhibit a characteristic EPR signal. Melanized C. neoformans cells collected from cultures grown in the presence of L-DOPA are black and exhibit an apparent singlet X-band EPR signal (77 K) at g = 2.0030 with a linewidth (peak to trough) of 5 Gauss (Fig. 2) similar to that of synthetic eumelanin [17], [28]. Comparison of the signal intensity to that from a suspension of melanized-cell ghosts suggests a melanin concentration of ∼3.0 mg/mL in typical cell samples. The radical concentration in melanins is known to respond to effectors including pH, temperature, and light, which alter the equilibrium between reduced and oxidized quinoid species [15], [16]. To investigate the properties of melanin in the cells and for later experiments in which the effects of γ-irradiation were monitored, the EPR signal from cell suspensions was recorded before and after illumination with high intensity light from a Xe lamp. This approach provides a probe of the integrity of melanin subunit structure, as it depends on the formation of new semiquinone radicals different from the intrinsic radicals.

Illumination of cells at room temperature caused an increase in EPR signal intensity (measured at 77 K) in a dose-dependent manner, reaching a maximum of 2.5-fold after ∼60 min (Fig. 3A). Illumination of frozen cell suspensions produced a 10-fold maximum increase in intensity after ∼60 min (Fig. 3B). Samples stored in the dark at 77 K retained the increased intensity for at least several days after illumination (not shown). In general, the response to light is consistent with the formation of semiquinones that persist briefly after illumination at room temperature, but which are trapped at 77 K. A small broadening of the signal (less than 1 Gauss) was also noted as previously reported [29] suggesting that the new radical sites are structurally distinct from the intrinsic radical sites. Non-melanized cell samples, which were pale in color, did not exhibit an EPR signal related to the typical melanin signal before or after Xe-lamp illumination (not shown).

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larger image TIFF original image Download: Figure 3. EPR spectra (77 K) of melanized C. neoformans cells after Xe-lamp illumination. A) cells were illuminated at room temperature for the time periods indicated and frozen immediately after removal from the light source; B) cells were frozen after collection from cultures and were illuminated at 77 K for the time periods indicated. https://doi.org/10.1371/journal.pone.0025092.g003

In addition to the melanin radicals produced photochemically, illumination at 77 K produced other dilute paramagnetic species (arrows, Fig. 3B) in samples of both melanized and non-melanized cells. These features arise from photo-induced cellular radicals and paramagnetic centers in illuminated quartz (shoulder at g ∼1.999) [23]. No difference was observed between melanized and non-melanized cells in these background signals.

The above survey of melanin radical behavior in whole C. neoformans cells provided the basis for interpretation of the effects of γ-irradiation. Cell suspensions were irradiated with two different doses of γ rays (10 and 30 min, ∼120 and 360 Gy total, capable of killing about 50 and 80% of cells, respectively [9]) followed by freezing in liquid nitrogen within 1 min of removal from the beam. These radiation doses are in a range known from prior work to demonstrate significant protection of melanized C. neoformans cells relative to non-melanized cells [9]. Here, the γ-irradiation caused a 30% increase in melanin radical signal intensity measured at 77 K for the higher dose (Fig. 4A). This gain in intensity, which could arise from a number of different processes, was reversed upon thawing and incubating the samples at room temperature (not shown) and was not explored further. In non-melanized cell samples, no EPR signal related to the typical melanin signal was detected after γ-irradiation (not shown).

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larger image TIFF original image Download: Figure 4. EPR spectra (77 K) of γ-irradiated and Xe-lamp illuminated C. neoformans cells. A) melanized cells γ-irradiated at room temperature (11.94 Gy/min), then frozen in liquid nitrogen; B) γ-irradiated melanized cells from (A) illuminated for the indicated time periods after freezing at 77 K; C) γ-irradiated (11.94 Gy/min) frozen (77 K) melanized cells, non-melanized cells, and PBS; D) γ-irradiated melanized cells from (C) stored frozen for 2 weeks then illuminated at 77 K for the indicated time periods. https://doi.org/10.1371/journal.pone.0025092.g004

To probe for changes in the photoresponse of melanin, γ-irradiated melanized cell samples were subjected to illumination at 77 K as above. The EPR signal intensity again increased approximately 10-fold (Fig. 4B) compared to the intensity increase before irradiation (Fig. 3B) suggesting that no loss in melanin integrity had occurred.

To test for rapidly reversible changes occurring at room temperature that escaped detection in the EPR samples described above (which were frozen after removal from the radiation beam), melanized cells were γ-irradiated at 77 K. The observations here were complicated by new EPR signals that overlapped with and nearly completely masked the melanin radical signal, which is indicated by the arrow in Fig. 4C. The broad new signals arise from hydroxyl and hydroperoxyl radicals produced in γ-irradiated ice, consistent with water radiolysis as their principal source [30]–[33]. These signals, which slowly decay by radical recombination [32] upon storage at 77 K or quickly decay at room temperature, were also seen with nearly equal intensities for γ-irradiated non-melanized cell suspensions and frozen PBS. Any of these new radicals for which EPR signals were detected, as well as other short-lived species produced during radiolysis of water such as e− aq , CO 2 −•, O 2 −•, H• [13], [31] could contribute to damage to cells and melanin in our experiments and in environments where ionizing radiation is present.

In order to examine melanin radical signals in the samples that had been γ-irradiated, the non-melanin signals were allowed to decay by thawing the sample for 1 hr. The EPR spectrum after refreezing showed an intensity of the melanin radical signal was approximately 80% of its value before γ-irradiation (not shown). To extend this analysis, Xe-lamp illumination was applied; a lower photoresponse was observed for the two γ-irradiated samples, compared with the 10-fold increase for the non-irradiated sample (Fig. 4D). The small losses in the intrinsic radical population and in the photoresponse suggest that some damage had occurred to melanin, which may also have occurred in the room temperature protocol but was not detected by EPR. Therefore, a search for small molecule products was undertaken.