Woo J-H, Kamei Y
Marine and Highland Bioscience Center, Saga University, Saga 847-0021 Karatsu, Japan.
Appl Microbiol Biotechnol. 2003 Sep;62(4):407-13. doi: 10.1007/s00253-003-1235-5. Epub 2003 May 23.
Previously we reported an antifungal protein specific to Pythium porphyrae, a causative agent of red rot disease afflicting seaweed Porphyra spp. This study was carried out to identify the antifungal mechanism of the antifungal protein to P. porphyrae. When we first examined the effect of an anti- Pythium protein (SAP) on the P. porphyrae cell walls, SAP did not decompose the six structural polysaccharides in Pythium cell walls. However, hyphal growth was significantly inhibited in Pythium cells treated with 50 microg/ml of SAP by MTT assay. Protoplasmic leakage was observed in P. porphyrae hyphae treated with SAP for 1 h, followed by hyphal swelling and disintegration, using SYTOX Green, and SAP permeabilized the membrane of P. porphyrae in a dose-dependent manner. Treating P. porphyrae cells with SAP in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a membrane-depolarizing agent, significantly reduced the membrane permeability to SYTOX Green. Moreover, a similar effect was observed when the P. porphyrae cells were treated with SAP in the presence of MgCl2. In contrast, identical treatment in the presence of KCl significantly increased the membrane permeability to SYTOX Green. These results suggested that anti- Pythium mechanism of SAP was related to alteration of the membrane permeability in P. porphyrae.
此前我们报道了一种对紫菜疫霉特异的抗真菌蛋白，紫菜疫霉是侵害紫菜属海藻的红腐病病原体。本研究旨在确定该抗真菌蛋白对紫菜疫霉的抗真菌机制。当我们首次检测抗腐霉菌蛋白（SAP）对紫菜疫霉细胞壁的影响时，SAP并未分解腐霉菌细胞壁中的六种结构多糖。然而，通过MTT分析，用50微克/毫升的SAP处理的腐霉菌细胞中，菌丝生长受到显著抑制。用SYTOX Green检测发现，用SAP处理1小时的紫菜疫霉菌丝中出现原生质泄漏，随后菌丝肿胀并解体，且SAP以剂量依赖的方式使紫菜疫霉的膜通透性增加。在存在膜去极化剂羰基氰化物间氯苯腙（CCCP）的情况下，用SAP处理紫菜疫霉细胞，显著降低了对SYTOX Green的膜通透性。此外，在存在MgCl2的情况下用SAP处理紫菜疫霉细胞时也观察到类似效果。相反，在存在KCl的情况下进行相同处理，显著增加了对SYTOX Green的膜通透性。这些结果表明，SAP的抗腐霉菌机制与紫菜疫霉膜通透性的改变有关。
