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 × 10⁶ 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.

中文翻译：

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小隐孢子虫感染诱导肠上皮细胞自噬


自噬是大分子和细胞器的降解和回收过程，以维持细胞稳态，也被证明有助于消除入侵的病原体。相反，包括寄生虫在内的各种病原体已被证明可以调节/利用宿主自噬，促进其细胞内感染周期。在这方面，小隐孢子虫（CP）这种小肠原生寄生虫正在成为全球健康的重大挑战。然而，隐孢子虫病的病理生理学大多未知。我们最近证明了 CP 通过降低特定紧密连接 (TJ) 和粘附连接 (AJ) 蛋白（例如 occludin、claudin-4 和 E-cadherin）的表达来诱导上皮屏障破坏。因此，我们利用汇合的 Caco-2 单层细胞作为肠上皮细胞 (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。通过 LC3II（微管相关蛋白 1 轻链 3）与 LC3I 蛋白比率的增加以及 p62/SQSTM1 蛋白水平的降低来评估自噬。 Caco-2 细胞 CP 处理 24 小时诱导自噬，用 0.5 × 10 ⁶卵囊/孔观察到最大效果。 CP 降低了 mTOR（雷帕霉素的哺乳动物靶标，自噬抑制剂）的磷酸化，表明通过 mTOR 失活诱导自噬。使用溶酶体抑制剂氯喹 (CQ) 测量自噬通量显示，与单独使用 CP 或 CQ 相比，CP + CQ 联合处理的细胞中 LC3II 水平增加更明显，表明 CP 诱导的 LC3II 增加是由于自噬体形成增强所致而不是溶酶体清除受损。 CP 感染不会改变 ATG7（一种关键的自噬蛋白）。 然而，在 siRNA 沉默 ATG7 后，CP 导致的 occludin、claudin-4 和 E-cadherin 的减少被部分阻断，这表明自噬在 CP 诱导的这些 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，这可能会改变导致隐孢子虫病病理生理学的重要宿主细胞过程。