参与真菌/细菌互作的新型真菌凝集素蛋白的抗菌功能研究

Antibiotic resistance is one of the top threats to global health. Due to their antimicrobial activities, lectins have received more attention in recent years and are recognized as one of the important sources for antimicrobial compounds. The filamentous fungi are rich sources in lectin genes. However, little knowledge has gained on their antimicrobial activity and the related mechanisms. On the other hand, most fungal lectin genes remain silent under pure culture conditions, which limits the analysis of their antimicrobial activities and the related mechanisms. In comparison, fungal cocultivation is a promising way to active the fungal silent genes. Coprinopsis cinerea contains 7 lectin genes in the genome. Previously, our results showed that 5 lectin genes from C. cinerea were triggered to overexpress when cocultured with other microbes and may be involved in the recognition and antimicrobial processes during cocultivation. As a result, this project plans to identify and characterize the five lectins from co-culture systems that every lectin is most significantly up-regulated. The structure, characterization and antimicrobial mechanisms of these lectins on microbes will be evaluated. Our research will lay a foundation for the development and application of novel fungal lectins with antimicrobial activities.

凝集素所展现出的抑菌活性使其成为潜在抗菌剂的重要来源。丝状真菌的基因组富含大量凝集素编码基因。然而，其抗菌活性鲜有报道，其抗菌作用及机制研究匮乏。另一方面，大量真菌凝集素基因在纯培养条件下处于沉默状态，限制了对其蛋白功能及机制研究。模拟自然条件，通过微生物共培养策略可激活大量沉默基因的表达。灰盖鬼伞（Coprinopsis cinerea）基因组中包含7个凝集素编码基因。前期我们发现，C. cinerea在与不同微生物共培养时，有5个凝集素基因显著上调表达，可能差异性的调控了C. cinerea与其他微生物的识别和抗菌过程。在此基础上，本课题拟选择各凝集素上调最显著的相互作用体系，分离纯化获得这5个凝集素蛋白，对其进行结构和性质表征，并进一步探讨其抗菌机制，为具有抗菌活性的新型凝集素的开发及应用奠定基础。

丝状真菌凝集素结构新颖、性质稳定，是潜在的抗菌剂和抗癌剂。然而，大量真菌凝集素基因在纯培养条件下处于沉默状态，限制了对其蛋白功能及应用的开发。前期，我们分别建立了灰盖鬼伞菌（Coprinopsis cinerea）与几种细菌或真菌的相互作用体系，本项目通过扩大相互作用菌的范围，拟获取可能参与菌间识别和抗性过程的凝集素，并制备后进行抗菌和抗癌功能研究。我们的转录组学数据显示，在与不同真菌或细菌共培养时，C. cinerea的多种抗性蛋白参与了互作过程，其中有五个凝集素差异性上调表达；进一步，通过选择合适的异源表达体系和融合标签，制备获得各凝集素；结构分析显示其分别为半乳凝集素和β-三叶草型凝集素；五个凝集素均有较好的热稳定性和凝集活性，其中半乳凝集素的最适活性均为中性偏碱条件；融合标签的加入改变了半乳凝集素CGL1和CGL2的聚集状态和糖结合特异性，增加了其体外抗菌特性；进一步研究表明，具有费氏埃希菌抗性的三个凝集素与细胞壁成分并无直接相互作用，但可导致细胞膜破裂；凝集素CGL1、CGL2和8598对结肠癌细胞具有很好的抑制作用，分别以凋亡和坏死途径诱导癌细胞死亡；动物模型结果显示，CGL1和CGL2的体内抗结肠癌效果差异显著。我们的结果不仅为具有抗菌或抗癌活性的新型凝集素的开发奠定基础，也可为其机制研究提供模型。