Caspase-3 and -7 are executioner caspases whose enzymatic activity is necessary to complete apoptotic cell death. Here, we questioned whether endothelial cell infection leads to caspase-3/7-mediated cell death. Pulmonary microvascular endothelial cells (PMVECs) were infected with Pseudomonas aeruginosa (PA103). PA103 caused cell swelling with a granular appearance, paralleled by intracellular caspase-3/7 activation and cell death. In contrast, PMVEC infection with ExoY⁺ (PA103 ΔexoUexoT::Tc pUCPexoY) caused cell rounding, but it did not activate intracellular caspase-3/7 and it did not cause cell death. However, ExoY⁺ led to a time-dependent accumulation of active caspase-7, but not caspase-3, in the supernatant, independent of apoptosis. To study the function of extracellular caspase-7, caspase-7- and caspase-3-deficient PMVECs were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. Caspase-7 activity was significantly reduced in supernatants from infected caspase-7-deficient cells but was unchanged in supernatants from infected caspase-3 deficient cells, indicating an uncoupling in the mechanism of activation of these two enzymes. Because ExoY⁺ leads to the release of heat stable amyloid cytotoxins that are responsible for transmissible cytotoxicity, we next questioned whether caspase-7 contributes to the severity of this process. Supernatants obtained from infected caspase-7-deficient cells displayed significantly reduced transmissible cytotoxicity when compared with supernatants from infected wild-type controls, illustrating an essential role for caspase-7 in promoting the potency of transmissible cytotoxicity. Thus, we report a mechanism whereby ExoY⁺ infection induces active caspase-7 accumulation in the extracellular space, independent of both caspase-3 and cell death, where it modulates ExoY⁺-induced transmissible cytotoxicity.

中文翻译：

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外酶 Y 诱导细胞外活性 caspase-7 积累，不依赖于细胞凋亡：调节可传播的细胞毒性。

Caspase-3 和 -7 是刽子手 caspase，其酶活性是完成凋亡细胞死亡所必需的。在这里，我们质疑内皮细胞感染是否会导致 caspase-3/7 介导的细胞死亡。肺微血管内皮细胞 (PMVECs) 被铜绿假单胞菌(PA103)感染。PA103 引起细胞肿胀，呈颗粒状，同时伴有细胞内 caspase-3/7 活化和细胞死亡。相比之下，用 ExoY ⁺ (PA103 Δ exoUexoT ::Tc pUCP exoY ) 感染 PMVEC 会导致细胞变圆，但它不会激活细胞内 caspase-3/7，也不会导致细胞死亡。然而，ExoY ⁺导致上清液中活性 caspase-7 而非 caspase-3 的时间依赖性积累，与细胞凋亡无关。为了研究细胞外 caspase-7 的功能，caspase-7 和 caspase-3 缺陷型 PMVEC 使用成簇的规则间隔短回文重复序列 (CRISPR)/Cas9 技术生成。Caspase-7 活性在感染的 caspase-7 缺陷细胞的上清液中显着降低，但在感染的 caspase-3 缺陷细胞的上清液中没有变化，表明这两种酶的活化机制存在解偶联。因为 ExoY ⁺导致释放热稳定的淀粉样蛋白细胞毒素，这些毒素负责可传播的细胞毒性，接下来我们质疑 caspase-7 是否有助于这一过程的严重性。与来自受感染的野生型对照的上清液相比，从感染的 caspase-7 缺陷细胞中获得的上清液显示出显着降低的可传播细胞毒性，说明 caspase-7 在促进可传播细胞毒性效力方面的重要作用。因此，我们报告了一种机制，即 ExoY ⁺感染诱导细胞外空间中活性 caspase-7 的积累，独立于 caspase-3 和细胞死亡，其中它调节 ExoY ⁺诱导的可传播细胞毒性。