Document Type

Article

Publication Title

American Journal of Physiology-Lung Cellular and Molecular Physiology

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 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.

First Page

L380

Last Page

L390

DOI

10.1152/ajplung.00508.2019

Publication Date

Summer 8-2020

Department

College of Medicine

Comments

Copyright © 2020 the American Physiological Society

Please visit the publisher's website for any inquires about this work or to access any downloable tables or figures at the following link:

https://doi.org/10.1152/ajplung.00508.2019

Supplemental materials, along with copyright, usage permissions, and citations can be accessed at the following links:

Supplemental Video S1; available online at https://doi.org/10.6084/m9.figshare.12132699.v1

Supplemental Video S2; available online at https://doi.org/10.6084/m9.figshare.12137427.v1

Fig. 1B and Supplemental Figure S1; https://doi.org/10.6084/m9.figshare.12137511

Fig. 2 and Supplemental Fig. S2; available online at https://doi.org/10.6084/m9.figshare.12137529

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