Cryptosporidium parvum infection induces autophagy in intestinal epithelial cells.
Cryptosporidium parvum infection induces autophagy in intestinal epithelial cells.
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DOI：
10.1111/cmi.13298
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发表时间：
2021-04
影响因子：
3.4
通讯作者：
中科院分区：
文献类型：
--
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自噬是一种大分子和细胞器的降解和循环过程，以维持细胞的动态平衡，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调节/利用宿主自噬促进其细胞内感染循环。在这方面，微小隐孢子虫(Cryptosporidium parvum，CP)是一种寄生于小肠的原虫，正在成为全球主要的健康挑战。然而，隐孢子虫病的病理生理机制大多尚不清楚。我们最近证实，CP通过减少特异性紧密连接(TJ)和黏附连接(AJ)蛋白的表达，如occludin、claudin-4和E-cadherin，来破坏上皮屏障。因此，我们利用融合的Caco-2细胞单层作为肠上皮细胞(IECS)的体外模型，探讨自噬在隐孢子虫病病理生理学中的潜在作用。通过微管相关蛋白1轻链3与微管相关蛋白1轻链3的比值增加和p62/SQSTM1蛋白水平的降低来评价自噬。CP处理Caco-2细胞24小时可诱导自噬，0.5×106个卵囊/孔的自噬效果最好。CP降低了mTOR(雷帕霉素的哺乳动物靶标，一种自噬抑制因子)的磷酸化，提示通过mTOR失活来诱导自噬。利用溶酶体抑制剂氯喹(CQ)对自噬通量的测量表明，与CP或CQ单独处理相比，CP+CQ共同处理的细胞的LC 3II水平更显著地增加，这表明CP诱导的LC 3II水平的增加是由于促进了自噬小体的形成，而不是损害了溶酶体的清除。CP感染并没有改变ATG7，这是一种关键的自噬蛋白。然而，在siRNA沉默ATG7后，CP对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蛋白减少中起到了作用。我们的结果为CP诱导宿主IECS自噬提供了新的证据，这种自噬可能改变有助于隐孢子虫病病理生理的重要宿主细胞过程。
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 utilized 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 24h induced autophagy with a maximum effect observed with 0.5 x 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.
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