Elsinochrome C

Background: Fungal perylenequinones (PQs) are a class of photoactivated polyketide mycotoxins produced by plant-associated fungi. Hypocrellins, the effective anticancer photodynamic therapy (PDT) agents are main bioactive PQs isolated from a bambusicolous Shiraia fruiting bodies. We found previously that bacterial communities inhabiting fungal fruiting bodies are diverse, but with unknown functions. Bacillus is the most dominant genus inside Shiraia fruiting body. To understand the regulation role of the dominant Bacillus isolates on host fungus, we continued our work on co-culture of the dominant bacterium B. cereus No.1 with host fungus Shiraia sp. S9 to elucidate bacterial regulation on fungal hypocrellin production. Results: Results from "donut" plate tests indicated that the bacterial culture could promote significantly fungal PQ production including hypocrellin A (HA), HC and elsinochrome A-C through bacterial volatiles. After analysis by gas chromatograph/mass spectrometer and confirmation with commercial pure compounds, the volatiles produced by the bacterium were characterized. The eliciting roles of bacterial volatile organic compounds (VOCs) on HA production via transcriptional regulation of host Shiraia fungus were confirmed. In the established submerged bacterial volatile co-culture, bacterial volatiles could not only promote HA production in the mycelium culture, but also facilitate the release of HA into the medium. The total production of HA was reached to 225.9 mg/L, about 1.87 times that of the fungal mono-culture. In contrast, the live bacterium suppressed markedly fungal PQ production in both confrontation plates and mycelium cultures by direct contact. The live bacterium not only down-regulated the transcript levels of HA biosynthetic genes, but also degraded extracellular HA quickly to its reductive product. Conclusion: Our results indicated that bacterial volatile release could be a long-distance signal to elicit fungal PQ production. Biodegradation and inhibition by direct contact on fungal PQs were induced by the dominate Bacillus to protect themselves in the fruiting bodies. This is the first report on the regulation of Bacillus volatiles on fungal PQ production. These findings could be helpful for both understanding the intimate fungal-bacterial interactions in a fruiting body and establishing novel cultures for the enhanced production of bioactive PQs.

Biofilm-associated Candida albicans infections, the leading cause of invasive candidiasis, can cause high mortality rates in immunocompromised patients. Photodynamic antimicrobial chemotherapy (PACT) is a promising approach for controlling infections caused by biofilm-associated C. albicans. This study shows the effect of Elsinochrome A (EA) against different stages of C. albicans biofilms in vitro by XTT reduction assay and crystal violet staining. The mechanism of action of EA on C. albicans biofilm was analyzed with flow cytometry, confocal laser microscopy, and the Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). EA-mediated PACT significantly reduced the viability of C. albicans, with an inhibition rate on biofilm of 89.38% under a concentration of 32 μg/mL EA. We found that EA could not only inhibit the adhesion of C. albicans in the early stage of biofilm formation, but that it also had good effects on pre-formed mature biofilms with a clearance rate of 35.16%. It was observed that EA-mediated PACT promotes the production of a large amount of reactive oxygen species (ROS) in C. albicans and down-regulates the intracellular expression of oxidative-stress-related genes, which further disrupted the permeability of cell membranes, leading to mitochondrial and nuclear damage. These results indicate that EA has good photodynamic antagonizing activity against the C. albicans biofilm, and potential clinical value.

Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.