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

Abstract

Caspases-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 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. Since ExoY+ leads to 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. Supernatant obtained from infected caspase-7 deficient cells displayed significantly reduced transmissible cytotoxicity when compared to supernatant 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 from both caspase-3 and cell death, where it modulates ExoY+-induced transmissible cytotoxicity.

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