The cleavage of perylenequinones through photochemical oxidation acts as a detoxification mechanism for the producer

Abstract

Perylenequinones (PQs) belong to a class of photosensitizers, generated by some fungi for parasitization or combating invaders. However, PQs generate reactive oxygen when exposed to light irradiation and cause nonselective damage to the producer host. The mechanism underlying the self-resistance of the producer is less understood. By using high-performance liquid chromatography and UV-visible absorption spectroscopic analysis, we found that PQs from an endolichenic fungus Phaeosphaeria sp. were transformed into new derivatives when the culture was exposed to visible or ultraviolet light. This transformation was accompanied by the reduction of its antimicrobial activity. In order to unveil the underlying mechanism, the purified hypocrellin A and calphostin D were employed in the photochemical analysis. The obtained light cleaved products were found to be nontoxic to the tested microbes and this photo-driven detoxification could be taken as a self-resistant strategy for the producer.

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Lichens, symbiotic organisms consisting of fungi (called mycobionts) and chlorophytes or cyanophytes (called photobionts), are distributed worldwide and can survive under various harsh environmental conditions.¹ The photobiont provides photosynthate to the mycobiont while mycobionts produce secondary metabolites to protect lichens against harmful radiation, herbivores, and microbial infections.² In addition to the fungal mycobiont, lichens frequently harbor diverse endolichenic fungi, which live within the lichen thallus much the same way as endophytic fungi live within healthy plant tissues.³ The secondary metabolites generated by numerous endolichenic fungi diversified the structures of natural products and played important roles for the lichen host in adapting to variable environments.⁴ Our previous chemical investigation of endolichenic fungus Phaeosphaeria sp. from lichen Heterodermia obscurata (Nyl.) Trevis led to the discovery of a serial of perylenequinones (PQs), a class of photosensitizers.⁵ Investigation of PQs revealed that they are toxic to tumor cells⁵ and that light can enhance the toxicity of PQs against the tumor cells and microbes for the high yields of reactive oxygen species (ROS), including superoxide anion radical (O₂˙⁻), hydroxyl radical (OH˙⁻), and singlet oxygen (¹O₂).^5,6 Lichen utilizes these phototoxins produced by endolichenic fungus to prevent pathogenic invasions.⁵ PQs are also produced by some pathogenic fungi and involved in parasitizing plants.⁷ The light induced ROS production by PQs are recognized to help lichens or pathogens to destroy the membranes of host plants to obtain nutrients.^5,8 However, continuous generation of toxic oxygen species would cause damage to the producer. The underlying self-resistant mechanism of the toxin-producer from photodynamic inactivation remains unclear. Here, we reported that light-driven cleavage of PQs into inactive forms could be taken as a detoxification strategy for the toxin-producer.

The PQ pigments were secreted by our previously investigated endolichenic fungus Phaeosphaeria sp. and coagulated into red granules revealed by confocal laser scanning microscopy (CLSM) observation (Fig. 1). The crude PQs were obtained by extracting the fermentation culture of Phaeosphaeria sp. using EtOAc. PQs extracts which were irradiated by UV (350 nm) for 5 h or incubated in the darkness followed by an antifungal test against pathogenic fungus Candida albicans. We found that the extracts changed from dark red to orange red when irradiated by UV (Fig. S1†). Furthermore, the fungicidal activity of irradiated extracts was greatly reduced compared to that of untreated ones (Fig. 2a). This significant change of bioactivity often resulted from the structure variation of the PQs. High-performance liquid chromatography (HPLC) was then employed to detect the chemical variations. The HPLC chromatogram of the dark incubated group showed a series of PQs indicated by peaks P1–P4 (Fig. 2b), which gave the characteristic absorption bands of the UV-visible spectrum at 220, 267, 341, 464, 540, and 581 nm (Fig. 2c). In the HPLC chromatogram of the UV (350 nm) light irradiation group, the amounts of original PQs components were greatly decreased and new peaks (P5–P8) were generated (Fig. 2b). Visible light (4 days irradiation) could also drive the photochemical transformation, although this process was slower than that of UV irradiation (5 h). The generated peaks showed a common UV-visible absorption (Fig. 2d) different from the spectrum of original PQs (Fig. 2c). The disappearance of the absorption bands at 540 and 581 nm of the light-induced products (Fig. 2d) implied that the 4,9-dihydroxy-3,10-perylene-quinonoid chromophore in PQs was destroyed. These observations demonstrated that the structural cleavage of PQ pigments resulted in altered phototoxicity because the presence of the 4,9-dihydroxy-3,10-perylene-quinonoid skeleton was an essential requirement for the production of active oxygen species by PQs.^6e

Fig. 1 Confocal microscopic view of fermentation products.

Fig. 2 The alteration of compositions and bioactivities for the light-treated fermentation extracts of Phaeosphaeria sp. (a) Comparison of the antifungal activity against C. albicans ATCC10231 for the fermentation extracts with UV light treatment or not. Asterisk** indicates p < 0.01. (b) Analysis of the compositions of fermentation extracts irradiated by visible light, UV light or incubated in the darkness using HPLC (60–100% MeOH–H₂O; 0.8 mL min⁻¹; 280 nm). (c) The UV-visible absorption spectra for peaks of P1–P4. (d) The UV-visible absorption spectra for peaks of P5–P8.

Considering the structures of PQs, we used hypocrellin A (HA) and calphostin D, the two main components from Phaeosphaeria sp. to testify this detoxification process with the alteration of structures. HA, as a phototoxic agent, has potent antifungal, antibacterial and antivirus activities.^6d,g,9 Combined experimental approaches including HPLC, UV-visible spectroscopy absorption, mass spectrum (MS), nuclear magnetic resonance (NMR), and antimicrobial activity assay were utilized. In response to visible light or UV light irradiation, HA was partially converted into compound 1 (Scheme 1). Analysis of aliquots of light treated HA samples using HPLC showed that this transformation was time dependent (Fig. 3a and b). 1 was found to be formed in the initial 2 h and reached the maximum after 48 h when HA was irradiated with visible light, and UV irradiation accelerated the photo-transformation. The structure of 1 was established by comparing its 1D NMR data with that reported (Table S1 and Fig. S6 and S7†),¹⁰ and the double bond in the middle ring of HA was oxidated into two carbonyl groups. Bioactivity assays showed that compound 1 completely lost antimicrobial action even when the concentration used reached up to 16 mg L⁻¹. On the other hand, HA maintained antimicrobial activity against C. albicans, Gram positive strains Staphylococcus aureus and Bacillus subtilis (Fig. 3c–e).

Scheme 1 UV-visible light-induced conversion of HA or calphostin D to compounds 1–3.

Fig. 3 Light caused the structure and bioactivity alteration of HA. (a and b) HPLC chromatograms (85% MeOH–H₂O, 0.8 mL min⁻¹ (a); 90% MeOH–H₂O, 1.8 mL min⁻¹ (b); 280 nm) obtained for the irradiation of HA in methanol solution at different time intervals using visible light (a) or UV light (350 nm) (b); (c–e) comparison of the activities of HA and its photoderivative compound 1 against C. albicans ATCC10231 (c), S. aureus ATCC6538 (d) and B. subtilis ATCC9372 (e).

Exposure of calphostin D to UV light irradiation generated two photoderivatives (Fig. S2†). After purification with HPLC and using various spectroscopic techniques, including ESI-MS and NMR (Table S2 and Fig. S8–S11†), the structures of the two products were determined to be 2 and 3 (Scheme 1) as depicted,¹¹ suggesting the double bond in the middle ring of calphostin D was cleaved by oxidation into two carbonyl groups, respectively. This suggests that PQs could be specifically cleaved by light, although calphostin D and two photoderivatives 2 and 3 did not display any phototoxic activities in our tested conditions (data not shown).

Conclusions

Our findings demonstrate that both visible and UV light resulted in the oxygenation cleavage of PQs. The formed photoderivatives with disrupted structures lost the ability to generate photo-induced reactive oxygen. Toxin producers were reported to harbor multiple self-protective strategies either through enzyme degradation of the toxins, encoding specific resistance genes or target mutation.¹² This transformation uncovers an alternative detoxification mechanism for the phototoxin producer. Whether this type of photochemical conversion has general applicability for detoxification in other organisms needs to be determined in further investigation.

Acknowledgements

This work was financially supported by the National Natural Science Foundation (nos 81273383, 81172956, 81402804), the China Postdoctoral Science Foundation (2014M551925) and the Fundamental Research Funds of Shandong University (2014GN032).

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Footnotes

† Electronic supplementary information (ESI) available: General experimental procedures, bioassays, 1D and 2D NMR, and UV-visible absorption spectra. See DOI: 10.1039/c5ra02238j

‡ These authors contributed equally to this work.

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