Intracellular Staphylococcus aureus triggers pyroptosis and contributes to inhibition of healing due to perforin-2 suppression
Irena Pastar, … , Hadar Lev-Tov, Marjana Tomic-Canic
Irena Pastar, … , Hadar Lev-Tov, Marjana Tomic-Canic
Published November 2, 2021
Citation Information: J Clin Invest. 2021;131(24):e133727. https://doi.org/10.1172/JCI133727.
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Intracellular Staphylococcus aureus triggers pyroptosis and contributes to inhibition of healing due to perforin-2 suppression

Abstract

Impaired wound healing associated with recurrent Staphylococcus aureus infection and unresolved inflammation are hallmarks of nonhealing diabetic foot ulcers (DFUs). Perforin-2, an innate immunity molecule against intracellular bacteria, limits cutaneous infection and dissemination of S. aureus in mice. Here, we report the intracellular accumulation of S. aureus in the epidermis of DFUs with no clinical signs of infection due to marked suppression of perforin-2. S. aureus residing within the epidermis of DFUs triggers AIM2 inflammasome activation and pyroptosis. These findings were corroborated in mice lacking perforin-2. The effects of pyroptosis on DFU clinical outcomes were further elucidated in a 4-week longitudinal clinical study in patients with DFUs receiving standard care. Increased AIM2 inflammasome and ASC-pyroptosome coupled with induction of IL-1β were found in nonhealing DFUs compared with healing DFUs. Our findings revealed that perforin-2 suppression, intracellular S. aureus accumulation, and associated induction of pyroptosis contribute to healing inhibition and prolonged inflammation in patients with DFUs.

Authors

Irena Pastar, Andrew P. Sawaya, Jelena Marjanovic, Jamie L. Burgess, Natasa Strbo, Katelyn E. Rivas, Tongyu C. Wikramanayake, Cheyanne R. Head, Rivka C. Stone, Ivan Jozic, Olivera Stojadinovic, Eran Y. Kornfeld, Robert S. Kirsner, Hadar Lev-Tov, Marjana Tomic-Canic

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Figure 2

Intracellular presence of S. aureus induces ASC pyroptosome assembly in DFUs.

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Intracellular presence of S. aureus induces ASC pyroptosome assembly in ...
(A) H&E staining of healthy foot skin (FS) and diabetic foot ulcers (DFUs) revealing presence of hole-like structures indicative of cell lysis, suggesting presence of pyroptosis. (B) Western blot of ASC pyroptosome showing ASC oligomerization in DFUs (n = 3 per group), demonstrating induction of pyroptosis in DFUs; Ctrl= human keratinocytes treated with poly dA/dT to induce pyroptosis. (C) Western blot of ASC pyroptosome demonstrated lack of ASC oligomerization during shorter infection allowing for bacterial adhesion (left blot) and induction of pyroptosis during longer infection allowing for MRSA internalization (right blot). Primary human keratinocytes were infected with MRSA at MOI of 100 for 15, 30, and 60 minutes; washed with PBS; and collected at 15-minute time increments (white triangles); or infected for 60, 120, and 180 minutes; washed; and collected at 30, 60, and 120 minutes (gray triangles) to allow bacteria to be internalized (20). Ctrl = uninfected cells. (D) Representative confocal imaging, corresponding to Western blots, of primary human keratinocytes infected with MRSA USA300 (green); DAPI (blue) = nuclei; rhodamine-conjugated phalloidin (orange) used to visualize actin; Ctrl = uninfected cells. Upper panel shows MRSA attached to keratinocytes during shorter infection time; bottom panel confirms intracellular localization of MRSA (white arrows); scale bar: 10 μm. Results depict representative experiment from 3 biological replicates.