Department of Pathogenic Biology, Jilin Medical University, Jilin, China.
Department of Clinical Laboratory, Jilin City Hospital of Chemical Industry, Jilin, China.
Parasit Vectors. 2024 Aug 19;17(1):347. doi: 10.1186/s13071-024-06436-w.
The encystation of Acanthamoeba castellanii has important ecological and medical significance. Blocking encystation is the key to preventing transmission and curing infections caused by A. castellanii. The formation of autophagosomes is one of the most important changes that occur during the encystation of Acanthamoeba. Our previous studies have shown that the heat shock protein 20 of A. castellanii (Ac-HSP20) is involved in its encystation. This study aimed to determine the role and mechanism of Ac-HSP20 in regulating autophagy involved in the encystation of A. castellanii.
Immunofluorescence assay, western blotting and transmission electron microscopy were used to analyze the dynamic changes in autophagy during the initiation and continuation of encystation. The knockdown of Ac-HSP20 was performed to clarify its regulation of encystation and autophagy and to elucidate the molecular mechanism by which Ac-HSP20 participates in autophagy to promote cyst maturation.
The encystation rates and autophagosomes were significantly decreased by treatment with the autophagy inhibitor 3-MA. The autophagy marker LC3B and autophagic lysosomes increased with the induced duration of encystation and reached the maximum at 48 h. The encystation rate, LC3B expression and autophagosomes decreased when Ac-HSP20 was knocked down by siRNA transfection. In addition, the expression levels of Ac-HSP20 and LC3B increased and the expressions of p-AKT and p-mTOR decreased after 48 h of encystation without knockdown. However, the expressions of p-AKT and p-mTOR increased while the expression of LC3B decreased under the knockdown of Ac-HSP20. Furthermore, the protein expression of LC3B increased when the PI3K/AKT/mTOR signaling pathway was inhibited but decreased when the pathway was activated.
The results demonstrated that autophagy is positively correlated with the encystation of A. castellanii, and Ac-HSP20 regulates autophagy to maintain the homeostasis of A. castellanii by inhibiting the PI3K /AKT /mTOR signaling pathway, thus promoting the maturation and stability of encystation.
棘阿米巴包囊形成具有重要的生态和医学意义。阻止包囊形成是预防棘阿米巴传播和感染的关键。自噬体的形成是棘阿米巴包囊形成过程中最重要的变化之一。我们之前的研究表明，棘阿米巴热休克蛋白 20（Ac-HSP20）参与了棘阿米巴的包囊形成。本研究旨在确定 Ac-HSP20 在调节参与棘阿米巴包囊形成的自噬中的作用和机制。
通过免疫荧光分析、western blot 和透射电镜分析自噬在包囊起始和持续过程中的动态变化。通过 Ac-HSP20 的 siRNA 转染敲低来阐明其对包囊形成和自噬的调节作用，并阐明 Ac-HSP20 参与自噬以促进囊泡成熟的分子机制。
自噬抑制剂 3-MA 处理后，包囊形成率和自噬体明显减少。自噬标记物 LC3B 和自噬溶酶体随着包囊诱导时间的延长而增加，在 48 h 时达到最大值。Ac-HSP20 通过 siRNA 转染敲低后，包囊形成率、LC3B 表达和自噬体减少。此外，在没有敲低的情况下，在 48 h 的包囊形成后，Ac-HSP20 和 LC3B 的表达水平增加，p-AKT 和 p-mTOR 的表达水平降低。然而，在 Ac-HSP20 敲低的情况下，p-AKT 和 p-mTOR 的表达水平增加，而 LC3B 的表达水平降低。此外，当抑制 PI3K/AKT/mTOR 信号通路时，LC3B 的蛋白表达增加，而当激活该通路时，LC3B 的蛋白表达减少。
结果表明，自噬与棘阿米巴的包囊形成呈正相关，Ac-HSP20 通过抑制 PI3K/AKT/mTOR 信号通路来调节自噬，从而维持棘阿米巴的内稳态，促进包囊的成熟和稳定。
