基于诱导细胞凋亡的低浓度芽孢杆菌脂肽抗真菌作用机制

Apoptosis is an important type of programmed cell death. Recent studies showed that PCD played an important role in the development, reproduction and aging of different filamentous fungi. Bacillus and its lipopeptides were proved to be a powerful weapon towards harmful fungi. And the known antifungal action of lipopeptide surfactin is to elicit pores on fungal cell membranes. But this action was based on the high concentration (1000µg/mL). However, as we all known, the sufficient amount of lipopeptide for perforation was difficult to produce by Bacillus sp. in the natural ecological conditions. So in this case, how to play the antifungal activity? Maybe some unknown action was exist in the natural environment. During our preliminary studies, the perforation cannot be finished by the low concentration (100µg/mL) of lipopeptide, but a surprise that surfactin with low concentration induced fungi PCD of was occurred. Therefore, we hypothesized that the antifungal action of surfactin with low concentration was induce fungi PCD. Thus, based on our preliminary studies, Fluorescence microscopy imaging, Flow Cytometry, TEM and other techniques was employed in this project, PCD markers induced by surfactin would be totally detected, the pathways and key regulatory factors of PCD would be discovered, and the role of PCD in the antifungal activity of lipopeptide would be analyzed. Our research would be perfect the antifungal action of Bacillus lipopeptide, would be provide new action targets and guidance for the development of new biopreservative and biopesticide, and would be provide new ideas and reference for the control practice against these harmful fungi.

凋亡是一种程序性细胞死亡类型，研究发现PCD也广泛存在于丝状真菌。芽孢杆菌及其脂肽被证明具有良好的抗真菌活性，其中脂肽Surfactin公认的抗真菌作用是高浓度脂肽参与的攻膜作用。而在自然环境中芽孢杆菌很难合成出足量脂肽，那么此情况下脂肽抗真菌活性将如何发挥？我们发现低浓度脂肽作用下真菌细胞膜并未出现穿孔，但出现有Hoechst33258染色阳性、染色质浓缩明显、ROS积累等PCD特征。我们推断低浓度脂肽很有可能是通过诱导PCD来发挥其作用的。项目将采用荧光显微技术、流式细胞技术、电镜技术等技术方法，考察低浓度脂肽诱导的禾谷镰刀菌PCD特征，解析PCD途径和关键调节因子，揭示诱导PCD在低浓度脂肽抗真菌活性中的作用。项目研究将丰富芽孢杆菌脂肽抗真菌作用机制，将为生物防腐剂和生物农药的开发提供新的作用靶点和指导，将为有害真菌防控实践提供新的思路和参考。

丝状真菌大多是农作物病原菌或者农产品腐败菌，丝状真菌及其毒素污染已经成为危害农产品质量和食品安全的重大问题，例如禾谷镰刀菌引起的小麦赤霉病及其毒素污染正严重影响小麦产量、品质及其食用安全性。芽孢杆菌脂肽Surfactin最初被发现抗病毒、抗肿瘤、抗支原体和表面活性等，关于其抗真菌活性近年才有报道。基于前期研究，我们推断脂肽Surfactin更可能是通过诱导真菌发生细胞凋亡来实现其抗真菌作用的。项目具体检测了脂肽Surfactin作用下禾谷镰刀菌是否出现细胞凋亡的典型特征，进而解析脂肽诱导的禾谷镰刀菌细胞凋亡的途径、关键因子及其在脂肽抗真菌活性中的作用。主要结果如下，在脂肽Surfactin作用下，禾谷镰刀菌出现Hoechst 33342染色分析阳性、Hoechst 33342/PI双染分析阳性、ROS累积检测阳性、磷脂酞丝氨酸外翻检测阳性、Metacaspase酶活检测阳性、线粒体膜电位检测阳性等细胞凋亡典型特征，确定脂肽Surfactin可以诱导禾谷镰刀菌发生细胞凋亡；揭示了脂肽Surfactin诱导的禾谷镰刀菌细胞凋亡依赖于ROS累积介导，明确了脂肽Surfactin抗真菌能力与其所诱发的ROS累积程度成正相关，ROS是脂肽Surfactin诱导的禾谷镰刀菌细胞凋亡的关键因子；阐明了脂肽Surfactin诱导的禾谷镰刀菌细胞凋亡属于内源性线粒体途径、metacaspase依赖通路。项目研究明确了低浓度脂肽可以诱导禾谷镰刀菌发生细胞凋亡，丰富了芽孢杆菌脂肽的抗真菌作用机理。更重要的是，自然条件下芽胞杆菌并不能合成足够多可以直接导致真菌细胞膜穿孔死亡的脂肽，低浓度脂肽诱导真菌细胞凋亡很有可能是芽孢杆菌在自然界中拮抗真菌的真正作用机理。项目研究将为有害真菌防控实践提供新的思路和参考。