放線菌や真菌は，多くの有用な化合物を提供してきた．しかしながらその遺伝子は２割程度しか働いておらず，新たな新規天然物を生産するであろう生合成遺伝子が眠ったままの休眠遺伝子であることがわかっている．我々は近年，病原放線菌と動物細胞の共培養法を開発し，休眠遺伝子活性化に成功している．この新規手法は，病原微生物が動物に感染する際の状況を再現し，疑似感染状態を模倣したものである．本研究では，本共培養法を病原真菌にも応用し，新たな共培養特異的化合物を見いだし，その生産機構に迫ることを目的とした．
Aspergillus terreus と，単球細胞 THP-1 との共培養により得られた共培養特異的化合物を，大量培養の後，単離・構造決定し，butyrolactone Iaと命名した．大変興味深いことに，butyrolactone Iaは，免疫抑制活性を示し，NO生産を濃度依存的に減少させた．さらに，NOを産生する酵素iNOSの発現量も激減し，マクロファージが産生するサイトカインであるIL-1βも減少した．このように，病原菌が免疫細胞の刺激により，免疫抑制化合物を産生したことは，大変，興味深い結果である．
Aspergillus luchuensis とマクロファージ様 J774.1 細胞との共培養から，2種のイタコン 酸誘導体の単離に成功している．どうやって麹菌が細胞から刺激され，化合物を生産するようになるのか，その産生メカニズムを検討した．J774.1細胞の培養上清を菌に加えるだけでイタコン酸誘導体が産生された．培養上清を有機溶媒と水で分配し菌に加えたところ，水層に反応して化合物が産生された．また，上清をオートクレーブしたところ，化合物は生産されなかったことから，熱に強く極性の高い，化合物（ペプチド）等に反応していることが示唆された．現在，さらにメカニズムを検討中である．
Actinomycetes and fungi have provided many useful compounds. However, about 80% of the biosynthetic genes are remain silent, therefore such cryptic (silent) genes will produce new natural products. In recent years, we have developed a co-culture method of pathogenic actinomycetes and animal cells, and have succeeded in activating cryptic genes. This new method reproduces the situation when pathogenic microorganisms infect animals and imitates pseudo-infected conditions. The purpose of this study was to apply this co-culture method to pathogenic fungi, discover new co-culture-specific compounds, and investigate their production mechanisms.
A coculture-specific compound obtained by coculturing Aspergillus terreus and monocytic THP-1 was isolated, structurally determined, and designated as butyrolactone Ia. Interestingly, butyrolactone Ia exhibited immunosuppressive activity and decreased NO production in a dose-dependent manner. In addition, the expression of iNOS, an enzyme that produces NO, decreased sharply, and IL-1b, a cytokine produced by macrophages, also decreased. Thus, it is very interesting that pathogenic bacteria produced immunosuppressive compounds by stimulating immune cells.
We have successfully isolated two itaconic acid derivatives from the co-culture of Aspergillus luchuensis and macrophage-like J774.1 cells. We investigated the production mechanism of how koji mold is stimulated by cells to produce compounds. Itaconic acid derivatives were produced only by adding the culture supernatant of J774.1 cells to the mold. When the culture supernatant was divided into an organic solvent and water and added to the mold, compounds were produced in response to the aqueous layer. In addition, when the supernatant was autoclaved, no compound was produced, suggesting that it reacts with compounds (peptides) that are resistant to heat and have high polarity. We are currently investigating the mechanism further.
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