隐孢子虫通过宿主细胞EGFR通路调控自噬的胞内生存机制

Autophagy is an important cellular activity in helping the infected cells to remove pathogens, whereas the pathogens also possess the ability to escape autophagy. EGFR signaling pathway has been known to regulate autophagy. Pathogens have the ability to use cellular signal transduction pathway in regulating autophagy to promote pathogens’ intracellular survival ability, which is a major research topic of pathogenic mechanisms. Cryptosporidium is an intracellular zoonotic protozoon. However, it is still unclear how Cryptosporidium manipulate autophagy through cellular EGFR signaling pathway in order to adapt to the specific pathogenic mechanism of intracellular survival. Based on our previous work that sporozoan EGF like protein can recognize cellular epidermal growth factor receptor (EGFR), we propose here to systemically investigate how Cryptosporidium activate host cell EGFR signaling pathway; what Cryptosporidium molecule is used to activate EGFR signal pathway; how EGFR pathway regulate autophagy in pathogen infected cells; what are the relationships among EGFR pathway and the survival ability of intracellular pathogens. The proposed research will help to elucidate the mechanisms of how Cryptosporidium uses EGF-like to recognize and activate host cellular EGFR, which in turn regulate host cellular autophagy for pathogen escape and intracellular adaptation and survival. Furthermore, this could also lead to the discovery of key molecular targets for drug design.

自噬是细胞重要的生命活动，可促进感染细胞对病原的清除，病原也具有逃避自噬的能力。EGFR等信号通路调控自噬过程。病原具有利用细胞的信号通路来调控自噬利于其胞内生存的能力，这种能力是当今病原致病机制研究的主题之一。隐孢子虫是一种人兽共患的胞内原虫。但关于隐孢子虫通过EGFR信号通路调控自噬以适应胞内生存的具体致病机制还尚不明确。针对这一问题，拟从隐孢子虫激活宿主细胞EGFR通路作用及其分子基础、隐孢子虫对宿主细胞自噬的影响及其分子机制、EGFR通路在隐孢子虫介导细胞自噬中的调控作用、干预EGFR通路和自噬对虫体胞内生存的影响深入系统研究。以期揭示隐孢子虫如何通过EGF-like识别EGFR，并激活EGFR信号通路，最后逃避自噬以适应胞内生存的致病新机制。并力图找到防控隐孢子虫病药物设计的关键干预节点。

微小隐孢子虫是一种重要的人兽共患寄生虫，其感染造成的微小隐孢子虫病严重制约养殖业发展并危害人类健康。细胞自噬是宿主应对病原感染的重要机制，阐明细胞自噬对微小隐孢子虫感染的作用并分析其调控机制，有利于防治微小隐孢子虫病。本研究通过构建微小隐孢子虫感染HCT-8细胞模型，在不同刺激时间和不同刺激比例的条件下，检测EGFR-PI3K/Akt、MAPK通路、自噬信号以及miRNA的激活情况，并通过干预不同信号通路分析其调控机制同时阐明其对虫体增殖的作用，以探索微小隐孢子虫胞内生存机制、寻找干预靶点。结果显示，微小隐孢子虫感染HCT-8细胞后，能够激活EGFR、PI3K/Akt以及MAPK通路并诱导miR-30a和miR-26a的表达，同时发现虫体EGF-like与细胞EGFR存在相互作用；微小隐孢子虫感染诱导细胞完全自噬，自噬相关分子Beclin1和ATG7参与自噬形成；通过干预AKT、MAPK和miRNA的表达，发现AKT、miR-30a信号通过Beclin1和ATG7分子、ERK和p38 MAPK通过自身磷酸化抑制细胞自噬，而miR-26a促进细胞自噬；自噬能够抑制微小隐孢子虫增殖，而虫体可以通过激活EGFR-PI3K/Akt通路和MAPK通路、促进miR-30a表达和降低miR-26a表达来抑制宿主自噬，从而保证其胞内增殖。综上，本研究首次阐明微小隐孢子虫刺激HCT-8细胞后，通过诱导EGFR-PI3K/Akt信号、促进miR-30a表达、抑制miR-26a表达促进ERK和p38 MAPK蛋白磷酸化的三条途径抑制细胞自噬以实现胞内增殖，该研究为后续研发抗微小隐孢子虫的干预药物提供了靶标。