Exoenzyme Y induces extracellular active caspase-7 accumulation independent from apoptosis: modulation of transmissible cytotoxicity

Abstract

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 <i>Pseudomonas aeruginosa</i> (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 Δ<i>exoUexoT</i>::Tc pUCP<i>exoY</i>) 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.