Screening of Microbial Metabolites Affecting Cell Membrane Function (An Attempt to Effectuation of Ineffective Inhibitors for Fungal Growth by Permeability Changes by the Metabolites)

据报道，一些酵母菌在弱暴露于聚乙二醇胺和两性霉素B等破坏细胞膜的药物时，会对各种抗生素和抑制剂变得敏感。在本研究中，我们试图寻找能够特异性作用于酵母细胞膜屏障的微生物代谢物，使不起作用的不渗透抑制剂能够进入细胞并显示其活性。用NTG处理酿酒酵母IFO 0203，得到对精氨酸类似物刀豆氨酸和尿嘧啶类似物5-氟尿嘧啶不敏感的Arg-p3(精氨酸渗透酶缺陷突变株)和ura-p17(尿嘧啶渗透酶缺陷突变株)。此外，(3)白色念珠菌IFO 1061对尿嘧啶缺乏敏感性。对约3100种培养滤液进行了抗(1)-(3)活性筛选，只有在有刀豆碱或5-氟尿嘧啶存在的情况下才有抗酵母活性。结果共分离到4株微生物(一株丝状真菌青霉、一株放线菌和一株链霉菌)被发现是此类活动的有前途的生产者。从它们的发酵液中分离得到4种活性成分(影响细胞膜的代谢产物)，分别鉴定为金丝桃素、脱水黄腐酸、链菌素和四霉素A。除刀豆碱和5-氟尿嘧啶外，与这些影响细胞膜的代谢产物联合使用时，对真菌生长也有抑制作用。大黄素抑制酵母细胞亚麻酸和亚油酸的生物合成。由此可见，多烯脂肪酸在细胞膜对药物的通透性屏障中起着重要作用。需要进一步的研究来阐明其他筛选的代谢物的作用方式。

It has been reported that some yeasts become hypersensitive to various antibiotics and inhibitors when they are weakly exposed to cell membrane-damaging agents such as polygodial and amphotericin B. In this study we tried to search for microbial metabolites which can act specifically on the membrane barrier of yeast cells, so that impermeable inhibitors showing no effect would be able to enter the cells and show their activities.1. Yeasts for screening test were prepared : Cells of Saccharomyces cerevisiae IFO 0203 were treated with NTG to give (1) arg-p3 (arginine permease-deficient mutant) and (2) ura-p17 (uracil permease-deficient mutant) which are insensitive to the arginine analogue canavanine and the uracil analogue 5-fluorouracil, respectively. Also, (3) Candida albicans IFO 1061 was found to be deficient in uracil permease.2. About 3100 kinds of culture filtrates were subjected to the screening whether they showed anti-yeast activity against (1) - (3) only when they were tested in the presence of canavanine or 5-fluorouracil. As a result, 4 microorganisms (an unidentified filamentous fungus, Penicillium sp., an unidentified actinomycetes and Streptomyces sp.) were found to be promising producers of such activities.3. From their culture broths, 4 active principles (cell membrane-affecting metabolites) were isolated and identified as chrysodin, anhydrofulvic acid, chainin and tetramycin A, respectively.4. Besides canavanine and 5-fluorouracil, some other ineffective inhibitors for fungal growth were effectuated when combined with these cell membrane-affecting metabolites.5. Chrysodin inhibited the biosynthesis of linolenic acid and linoleic acid in yeast cells. Thus, it seems that polyenoic fatty acids play an important role in the permeability barrier of cell membrane to drugs. Further studies are needed to clarify the mode of action of other screened metabolites.