Aβ抗真菌活性与聚集形态和细胞壁多糖的关系探究

Relationship Between the Antifungal Activity of Aβwith Aggregation Morphology and Cell Wall Polysaccharides

阿尔茨海默症(alzheimer’s disease,AD)的“微生物感染假说”表明,AD的发生发展与脑部真菌感染密切相关。β淀粉样蛋白(β-amyloid,Aβ)作为一种“抗菌肽”已被证明能够对多种病原微生物产生抗菌活性,然而其在抗真菌方面的机制仍不明确。本文主要从AD的“微生物感染假说”入手,探究几丁质等细胞壁多糖在Aβ的抗真菌活性中所发挥的作用。Aβ的聚集状态与其抗菌活性存在相关性,为探究不同聚集状态的Aβ与其抗菌活性的关系并尝试解析Aβ抗真菌的具体作用机制和靶点,选择病原真菌白色念珠菌作为研究对象,证实了Aβ中更容易发生淀粉样变性的具有42个氨基酸的Aβ42对白色念珠菌浮游菌和生物膜的代谢活力具有抑制作用,并且其抑菌效果与Aβ42浓度呈现明显的正相关。通过特异性标记真菌细胞壁的钙荧光白(calcofluor white,CFW)染色法验证了Aβ42对白色念珠菌菌丝生成具有抑制作用。体外制备获得了Aβ42的寡聚体及纤维状聚体,并通过透射电镜进行了形态验证,随后将不同聚集状态的Aβ42作用白色念珠菌。结果表明:不同聚集状态的Aβ42其抗真菌活性存在差异,当Aβ42寡聚体转变为纤维状聚体时其抗真菌活性显著降低。采用扫描电子显微镜观察到经过Aβ42处理后的白色念珠菌菌丝体减少、胞外基质丢失且细胞壁结构发生显著变化。为进一步揭示Aβ42抗真菌的相关作用机制,通过硫黄素T(ThT)荧光染色发现白色念珠菌细胞壁多糖中的几丁质和葡聚糖可能与Aβ42结合并影响其聚集,从而发挥其抗真菌的活性。此外,MTT法结果证实了细胞壁多糖的寡聚类似物片段能够通过与Aβ42的竞争性结合削弱Aβ42的抗菌效果。以上结果表明,Aβ可能通过与真菌细胞壁多糖结合并破坏细胞壁完整性发挥其抗菌活性。该研究为AD发病机制中的“微生物感染假说”提供了新的数据支持和理论依据。

The"microbial infection hypothesis"of Alzheimer’s Disease(AD)suggests that the development of AD is closely related to fungal infections in the brain.β-amyloid(Aβ),as an"antibacterial peptide",has been shown to have antifungal activity against a variety of pathogenic microorganisms,but its antifungal mechanism remains unclear.This paper mainly starts from the"microbial infection hypothesis"of AD,and explores the role of cell wall polysaccharides such as chitin in the antifungal activity of Aβ.There is a correlation between the aggregation status of Aβand its antifungal activity.Therefore,in order to explore the relationship between Aβin different aggregation status and its antifungal activity and try to analyze the specific mechanism and target of Aβantifungal,this study selected the pathogenic fungus Candida albicans as the research object.It was explored that Aβ42 with 42 amino acids in Aβ,which is more prone to amyloidosis,had inhibitory effect on the metabolic activity of Candida albicans planktonic cells and biofilm,and its fungistatic effect was significantly positively correlated with the concentration of Aβ42.The inhibitory effect of Aβ42on Candida albicans hyphae was verified by Calcofluor White(CFW)staining,which specifically labeled fungal cell walls.The oligomers and fibrillar aggregates of Aβ42 were prepared in vitro,and the morphology was verified by transmission electron microscopy.Then Aβ42 in different aggregation states was treated with Candida albicans.The results showed that the antifungal activities of Aβ42 in different aggregation states were different.The antifungal activity of Aβ42 was significantly reduced when the Aβ42 oligomers were converted into fibrillar polymers.Scanning electron microscopy showed that the mycelium of Candida albicans treated with Aβ42 decreased,the extracellular matrix was lost,and the cell wall structure changed significantly.In order to further reveal the antifungal mechanism of Aβ42,it was found by Thioflavin T(ThT)fluorescence staining that chitin and glucan in the cell wall polysaccharide of Candida albicans may bind to Aβ42 and affect its aggregation,thereby exerting its antifungal activity.In addition,the MTT assay results confirmed that the oligomeric analog fragments of cell wall polysaccharides can weaken the antifungal effect of Aβ42 through competitive binding with Aβ42.The above results suggest that Aβmay exert its antifungal activity by binding to fungal cell wall polysaccharides and destroying cell wall integrity.This study provides new data support and theoretical basis for the"microbial infection hypothesis"in AD pathogenesis.