背景:卡氏棘阿米巴的包育具有重要的生态学和医学意义。阻断包膜是防止传播和治疗由A引起的感染的关键。自噬体的形成是棘阿米巴囊化过程中发生的最重要的变化之一。我们以前的研究表明,A.castellanii(Ac-HSP20)的热休克蛋白20参与其囊封。本研究旨在确定Ac-HSP20在调节自噬中的作用和机制。
方法:免疫荧光分析,采用western印迹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。对Ac-HSP20进行敲除以阐明其对包膜和自噬的调控,并阐明Ac-HSP20参与自噬促进囊肿成熟的分子机制。
结果:用自噬抑制剂3-MA治疗可显著降低包膜率和自噬体。自噬标记LC3B和自噬溶酶体随诱导包膜持续时间的增加而增加,并在48h时达到最大值。当通过siRNA转染敲低Ac-HSP20时,LC3B表达和自噬体降低。此外,未敲除48h后,Ac-HSP20和LC3B的表达水平升高,p-AKT和p-mTOR的表达降低。然而,在Ac-HSP20敲低的情况下,p-AKT和p-mTOR的表达增加,而LC3B的表达减少。此外,当PI3K/AKT/mTOR信号通路被抑制时,LC3B的蛋白表达增加,而当通路被激活时,LC3B的蛋白表达减少。
结论:结果表明,自噬与A.castellanii的包膜呈正相关,和Ac-HSP20通过抑制PI3K/AKT/mTOR信号通路调节自噬以维持卡氏A的稳态,从而促进封闭的成熟和稳定。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.