Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA.
Jesse Brown VA Medical Center, Chicago, Illinois, USA.
Cell Microbiol. 2021 Apr;23(4):e13298. doi: 10.1111/cmi.13298. Epub 2020 Dec 22.
Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP-induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin-4 and E-cadherin. Therefore, we utilised confluent Caco-2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco-2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 10 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co-treated with CP + CQ as compared to CP or CQ alone, suggesting that CP-induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin-4 and E-cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP-induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.
自噬是一种降解和回收大分子和细胞器以维持细胞内环境稳定的过程，也已被证明有助于消除入侵的病原体。相反，各种病原体，包括寄生虫，已被证明可以调节/利用宿主自噬，促进其细胞内感染周期。在这方面，微小隐孢子虫（CP），一种小肠的原生动物寄生虫，正在成为一个主要的全球健康挑战。然而，隐孢子虫病的病理生理学大多是未知的。我们最近证明 CP 通过降低特定紧密连接（TJ）和黏附连接（AJ）蛋白的表达，如闭合蛋白、Claudin-4 和 E-钙黏蛋白，诱导上皮屏障破坏。因此，我们利用紧密连接的 Caco-2 细胞单层作为肠道上皮细胞（IEC）的体外模型，研究自噬在隐孢子虫病病理生理学中的潜在作用。自噬通过增加 LC3II（微管相关蛋白 1 轻链 3）与 LC3I 蛋白的比值和降低 p62/SQSTM1 蛋白水平来评估。CP 处理 Caco-2 细胞 24 小时诱导自噬，以 0.5×10 个卵囊/孔的最大效应观察到。CP 降低 mTOR（雷帕霉素的哺乳动物靶标，自噬的抑制剂）磷酸化，表明通过 mTOR 失活诱导自噬。利用溶酶体抑制剂氯喹（CQ）测量自噬通量显示，CP 共处理的细胞中 LC3II 水平的增加更为明显，与 CP 或 CQ 单独处理相比，这表明 CP 诱导的 LC3II 增加是由于增强的自噬体形成，而不是由于溶酶体清除受损。CP 感染不改变 ATG7，一种关键的自噬蛋白。然而，CP 降低 occludin、Claudin-4 和 E-钙黏蛋白的作用部分被 ATG7 的 siRNA 沉默所阻断，这表明自噬在 CP 诱导这些 TJ/AJ 蛋白减少中的作用。我们的结果提供了 CP 在宿主 IEC 中诱导自噬的新证据，这可能改变了对隐孢子虫病病理生理学有贡献的重要宿主细胞过程。
