Oxidation mechanism of Penicillium digitatum spores through neutral oxygen radicals

The use of non-equilibrium cold plasma has recently attracted much attention in biological fields, such as medicine, agriculture, and fisheries, because of its many advantages including low-temperature processing and short processing time. There have been many reports on the inactivation of microorganisms using non-equilibrium atmospheric-pressure plasma (NEAPP).^1,2) Various factors, such as ultraviolet (UV) light, neutral and charged species, and electric fields, are simultaneously produced by NEAPP, and these may synergistically cause the inactivation of microorganisms. In particular, there have been many reports indicating that reactive oxygen species (ROS), including ground-state atomic oxygen [O(³P_j)], hydroxyl radicals (OH), singlet oxygen molecules [O₂(¹Δ_g)], superoxide anions (O₂⁻), and ozone (O₃), are effective at inactivating microorganisms, such as Escherichia coli and Bacillus spp.^3–24) However, in all of the studies, multiple ROS including charged species, in addition to UV radiation, were involved, and no lifetimes or inactivation rates were determined for individual species. It is therefore indispensable to systematically discuss inactivation effects of individual species on the basis of quantitative measurements.

In previous works, we have inactivated Penicillium digitatum spores using plasma treatments and investigated inactivation mechanisms by considering intracellular changes.^25–27) P. digitatum spores cause the formation of green mold on citrus fruits, which is a difficult-to-inactivate postharvest disease. The spores, which are classified as fungi, differ from the bacteria that are typically employed when researching the plasma inactivation in that they have a much more resistant structure, a different composition, and different cell membrane functions. Various biological responses causing inactivation by plasma treatment, namely, (1) lethal oxidation of biomembranes, (2) electrostatic disruption of cell membranes, and (3) biological responses such as apoptosis, are considered. Plasma can trigger a complex sequence of biological responses in tissues and cells; therefore, the inactivation mechanism with plasma treatment is complicated. In many cases of plasma treatment, the morphology of bacteria is changed by effects such as etching, cracking, leakage, and size reduction.^{9,11,14,16,17,19,28,29)} On the other hand, in the case of cancer cells, plasma can induce apoptosis due to a signaling pathway.^30–33) Joshi et al. reported that oxidative stress leads to oxidative DNA damage and membrane lipid peroxidation in E. coli treated with a dielectric barrier discharge plasma.³⁴⁾ We observed the change in spores treated with NEAPP by a fluorescent microscopy and compared it with those treated with UV light or moist heat. The intracellular organelles as well as the cell membranes in the spores treated with the plasma were stained with the dye without a major morphological change of the membranes, while the organelles were not stained in the spores treated with UV light or moist heat. Another dye revealed that organelles were oxidized by plasma treatment, in contrast to UV light or moist heat treatments. Moreover, we found an electron-spin-resonance (ESR) signal from the P. digitatum spores by real-time in-situ ESR measurements. The signal was identified to be semiquinone and disappeared with the oxygen plasma treatment.²⁷⁾ These results suggest that only plasma treatment induces a minor structural change or functional inhibition of cell membranes, which leads to the oxidation of the intracellular organelles without major deformation of the membranes through the penetration of reactive oxygen species generated by the plasma into the cell. In these studies using the NEAPP, multiple ROS including charged species, in addition to UV radiation, were involved, and no inactivation rates were determined for individual species. It is, therefore, indispensable to discuss inactivation effects of individual species.

In order to clarify the inactivation effects of neutral oxygen species, we developed an atmospheric-pressure oxygen radical source that supplies only neutral oxygen species.^35,36) We measured the densities of O(³P_j), O₂(¹Δ_g), and O₃, produced by the radical source using vacuum ultraviolet absorption spectroscopy (VUVAS), and showed that the inactivation rate corresponded to the O(³P_j) density against the O₂/(Ar + O₂) gas flow ratio and exposure distance, while inactivation efficiencies of O₂(¹Δ_g) and O₃ are negligible.³⁷⁾ We concluded that O(³P_j) is an effective species for inactivating P. digitatum spores.^37,38)

In this study, we observed the changes in spores of P. digitatum treated with an oxygen radical source by in-situ fluorescent confocal-laser microscopy in order to investigate the biological responses causing the inactivation through neutral oxygen species. A confocal-laser microscope has a high resolution, especially in the depth direction. From fluorescent cross-sectional images with a z-axis resolution of approximately 0.2 µm, we can analyze the intracellular changes of the spores treated with oxygen radicals in detail. We performed two fluorescent assays to investigate the effects of ROS. One assay involves the use of 1,1'-dioctadecyl-3,3,Y,3'-tetramethylindocarbocyanine perchlorate (DiI) as a probe for a vital fluorescence membrane dye. DiI belongs to a family of cyanine dyes that have been used to investigate the structure and dynamics of cell membranes and artificial lipid bilayers.^39–44) The octadecyl group of DiI binds to the acyl groups of phospholipids in the cell membrane owing to the long carbon chain, resulting in the staining of cell membranes. Therefore, we can analyze the changes in the function and morphology of cell membranes. The other assay involves the use of diphenyl-1-pyrenylphosphine (DPPP). DPPP reacts with lipid peroxide in biomembranes and produces DPPP oxide.^45,46) Therefore, DPPP assay is used to prove the oxidation of microorganisms.

Figure 1 shows a schematic diagram of the in-situ fluorescent confocal-laser microscope system with an oxygen radical source. The oxygen radical source (Fuji Machine Tough Plasma) was described in detail in Refs. 35 and 36. The radical source was based on atmospheric-pressure high-density O₂/Ar plasma, which generates electrons with a high density of about 10¹⁶ cm⁻³. Charged species and optical radiation from the O₂/Ar plasma were blocked by electrodes and the exit aperture of the radical source, so that only neutral species are supplied to the samples. The exit configuration of the radical source used in this study was a slit aperture of 0.5 × 16 mm². The treatment area was purged with the gases from the radical source using a plastic cover to eliminate the influence of atmospheric gases. The flows of Ar and O₂ gases were sufficient to purge the ambient gas from the area surrounded by the plastic cover. A P. digitatum spore suspension of 10 mg/ml was prepared from distilled water, 1% surfactant (Tween20), and spores. Then, 1 µl of the suspension was spotted on a 35-mm-ϕ dish and dried.

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Fluorescent images were obtained using an inverted confocal-laser microscope (Olympus FV1000-D) equipped with a 100× oil immersion objective lens (Olympus UPLSAPO100×O). Two fluorescent dyes were used to investigate the effects of oxygen radicals. A staining solution consisted of a mixture of 1 µl of DiI aqueous solution (Lonza Walkersville Vybrant^® DiI Cell-Labeling Solution, 1 mM) and 99 µl of 1% Tween20. The irradiation of DiI with light from a mercury lamp filtered at a wavelength of 549 nm, where the DiI is bound to the acyl groups of phospholipids in various membranes, produces green fluorescence at 565 nm. Cell membranes and intracellular organelles, such as nuclei, mitochondria, endoplasmic reticula, and Golgi bodies, have membrane structures that are made of phospholipids. Thus, we can analyze the physical destruction of cell membranes or the disappearance of their function. To observe the fluorescence, an excitation filter (500–550 nm), a barrier filter (565–625 nm), and a dichroic mirror (long pass; 560 nm) were used in the microscope.

Another staining solution consisted of a mixture of 10 mg of DPPP aqueous solution (Dojindo Laboratories) and 999 ml of dimethylsulfoxide. DPPP reacts with lipid peroxide and forms DPPP oxide. Lipid peroxide is produced via the oxidation of lipids in biomembranes, which also exist in the intracellular organelles. Thus, DPPP assay can be used to prove the oxidation of P. digitatum by radical treatment. The irradiation of DPPP oxide with light from a mercury lamp filtered at a wavelength of 352 nm produces blue fluorescence at 380 nm. An excitation filter (340–390 nm), a barrier filter (long pass; 400 nm), and a dichroic mirror (long pass; 400 nm) were used for DPPP observation.

For scanning electron microscopy (SEM) observation, spores were fixed in 3% glutaraldehyde solution for 1 h. The suspension was centrifuged at 1,000 × g for 5 min, washed twice with 1% Tween20 solution, and adjusted to a concentration of 0.6 mg/ml. Then, 10 µl of the suspension was spotted on a Nano-percolator (JEOL), air-dried, and coated with gold particles for 20 s.

Fluorescent and transparent microscopic images of P. digitatum spores stained with DiI are shown in Fig. 2 [(a) control (transparent), (b) control (fluorescent), (c) oxygen radical treatment (transparent), and (d) oxygen radical treatment (fluorescent)]. The O₂/(Ar + O₂) flow rate ratio and total flow rate were 0.6% and 5 slm, respectively. The spore samples were exposed to radicals 10 mm downstream from the radical head for 1.5 min. The spores were recovered with 300 µl of 1% Tween20, mixed with 3 µl of DiI solution, and incubated at 30 °C for 30 min. Then, 1 µl of the mixture was spotted on a glass-bottom dish (Matsunami) and dried. Figure 2(b) shows that the cell membranes of the spores were stained with DiI. This result shows that the cell membranes had a function of protecting the cell from being infiltrated with DiI, and only the phospholipids in the outer membranes were stained. Namely, the cell membranes in intact spores of P. digitatum are impermeable to DiI because they have selective permeability. The organelles inside the cells treated with oxygen radicals also emitted fluorescent light, as shown in Fig. 2(d). The fluorescent light was observed mainly from nuclear membranes and intracellular organelles, which consist of membrane structures including phospholipids. These results indicate that the neutral oxygen species cause the disappearance of the cell membrane's function, and no major morphological variation was observed in the spore cell membranes. Previously, we showed that the function of the cell membrane was not impaired by other inactivation methods, such as UV radiation and moist heat treatment.²⁶⁾ On the other hand, Cerioni et al. reported that the permeability of the cell membrane of P. digitatum spores increased with oxidative stress, such as by the application of hydrogen peroxide, and that the intracellular organelles became completely disorganized according to the results of transmission electron microscopy (TEM) observation.⁴⁷⁾ Therefore, oxidative stress leads to inhibition of the selective permeability of the cell membrane and the intracellular structure of spores.

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Figure 3 shows the time evolution of DiI staining of P. digitatum spores treated with oxygen radicals. Before oxygen radical treatment, the spores were stained with DiI, then the DiI solution containing the spores was treated. The O₂/(Ar + O₂) flow rate ratio and total flow rate were 0.6% and 5 slm, respectively. At 0 min treatment (before radical treatment), the fluorescence arising from not only the cell membrane, as shown in Fig. 2(b), but also the DiI solution around the spores, as a background, was detected. The fluorescence of the DiI solution disappeared, in contrast with that of the cell membrane, because the DiI dye easily interacts with oxygen radicals, resulting in photobleaching. The organelles inside the cell emitted fluorescent light after 0.5 min oxygen radical treatment. Then, the fluorescence gradually faded after 20 min treatment. This result indicates that the neutral oxygen radicals react with the surface of the cell membrane at the same time when the neutral oxygen radicals are exposed, which leads to the inhibition of the function of cell membranes, resulting in the penetration of ROS and dye into the spores. After 20 min treatment, the fluorescence of the intracellular organelles faded, which is attributed to the oxidation of bonding sites in phospholipids upon their reaction with DiI or to the oxidative degradation of DiI dye due to excess ROS.

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Figure 4 shows the ratio of the number of spores intracellularly stained with DiI as a function of treatment time. The ratio increases to a maximum value of 87% until 3 min at an exposure distance of 10 mm, and then gradually decreases. On the other hand, at an exposure distance of 20 mm, the ratio increases to a maximum value of 85% until 7 min. The decimal reduction time (D-value), which corresponds to the time required to inactivate 90% of the spores, increases from 1.2 to 19.8 min with increasing exposure distance from 10 to 20 mm, and more than 99.99% of the spores are inactivated after the treatment for 7 min. From the results of the density measurement of O₂(¹Δ_g), O(³P_j), and O₃ by VUVAS, the O(³P_j) density drastically decreases from 2.3 × 10¹⁴ to 1.3 × 10¹³ cm⁻³ as the exposure distance is changed from 10 to 20 mm. On the other hand, the O₂(¹Δ_g) density is almost constant at 8.0 × 10¹⁴ cm⁻³ and the O₃ density is below the detection limit of about 2.5 × 10¹³ cm⁻³. These results indicate that O(³P_j) is the crucial species responsible for the inactivation of P. digitatum spores, with only minor roles being played by O₂(¹Δ_g) and O₃.³⁷⁾ The rate of increase of the ratio at 10 mm is larger than that at 20 mm until 5 min as shown in Fig. 4. This is because the dose of O(³P_j) to the spores at 10 mm is larger than that at 20 mm. The decrease in the ratio of the number of intracellularly stained spores after 3 min at 10 mm is considered to be caused by excess ROS, which peroxidizes acyl-group sites in phospholipids to react with DiI. A similar behavior should occur at 20 mm. From these results, it is concluded that the treatment with neutral oxygen radicals introduces molecules, such as dye molecules and ROS, into spore cells.

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Fluorescent and transparent microscopic images of P. digitatum spores stained with DPPP are shown in Fig. 5 [(a) control (transparent), (b) control (fluorescent), (c) oxygen radical treatment (transparent), and (d) oxygen radical treatment (fluorescent)]. The O₂/(Ar + O₂) flow rate ratio and total flow rate were 0.6% and 5 slm, respectively. Spores were exposed to radicals 10 mm downstream from the radical head for 7 min. After the spores were recovered with 300 µl of 1% Tween20, 60 µl of the suspension was mixed with 6 µl of 1 mg/ml DPPP solution (Dojindo Laboratories) and incubated at 60 °C for 60 min. Then, 1 µl of the mixture was spotted on a glass-bottom dish and dried. Figure 5(d) shows that fluorescent light was emitted from intracellular organelles treated with oxygen radicals. DPPP reacts with lipid peroxide and forms DPPP oxide. Lipid peroxide is produced via the oxidation of lipids in biomembranes, which also exist in the intracellular organelles. This result indicates that the cell was oxidized by oxygen radical treatment.

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Figures 6(a) and 6(b) show SEM images of control spores and spores after 7 min oxygen radical treatment, respectively. The images indicate that no major morphological variation occurs even in spores treated with neutral oxygen radicals for 7 min.

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From the results of DiI staining, the function of the spore membrane was inhibited by oxygen radicals, which led to the penetration of molecules including DiI and ROS into the spores. The results of DPPP staining suggest that the production of lipid peroxide (LOOH) in the spores was induced by neutral oxygen species. In general, the radical-initiated autoxidation of lipids, which produces LOOH, occurs naturally in vivo without the induction of ROS.⁴⁸⁾ The increase in the amount of LOOH by oxygen radical treatment enhances DNA damage and enzyme inactivation, leading to the rapid inactivation of spores. In this study, we found that neutral oxygen species, especially O(³P_j), cause not only a minor structural change or functional inhibition without a major morphological change of the cell membranes but also the oxidation of intracellular organelles, resulting in the inactivation of spores.

To investigate the inactivation process of Penicillium digitatum spores by neutral oxygen species, the spores were treated with an atmospheric-pressure oxygen radical source, which eliminates the effects of radiation and charged species. The treated spores were stained with two fluorescent dyes and observed in-situ using a fluorescent confocal-laser microscope. The intracellular organelles as well as the cell membranes in the spores treated with the oxygen radical source were stained with DiI without a major morphological change of the membranes. Moreover, DPPP staining revealed that the organelles were oxidized by oxygen radical treatment. The experiment using oxygen radical source showed that the dose of atomic oxygen strongly affected the oxidation of the intracellular organelles and the inactivation rate, whereas the effects of O₂(¹Δ_g) and O₃ are small. From these results, it was found that neutral oxygen species, especially atomic oxygen causes a minor structural change or functional inhibition of cell membranes, which leads to the oxidation of the intracellular organelles through the penetration of reactive oxygen species into the cell.

This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas, "Frontier Science of Interactions between Plasmas and Nano-interfaces" (No. 21110006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.