ADAM10 mediates vascular injury induced by Staphylococcus aureus α-hemolysin

Abstract

Staphylococcus aureus is a leading cause of bacteremia and sepsis. The interaction of S. aureus with the endothelium is central to bloodstream infection pathophysiology yet remains ill-understood. We show herein that staphylococcal α-hemolysin, a pore-forming cytotoxin, is required for full virulence in a murine sepsis model. The α-hemolysin binding to its receptor A-disintegrin and metalloprotease 10 (ADAM10) upregulates the receptor's metalloprotease activity on endothelial cells, causing vascular endothelial-cadherin cleavage and concomitant loss of endothelial barrier function. These cellular injuries and sepsis severity can be mitigated by ADAM10 inhibition. This study therefore provides mechanistic insight into toxin-mediated endothelial injury and suggests new therapeutic approaches for staphylococcal sepsis.

Figure 1.

Role of Staphylococcus aureus α-hemolysin in lethal infection and endothelial barrier disruption. A, BALB/c mice (n = 10) were inoculated with wild-type (WT) or toxin-deficient (Hla-) S. aureus Newman (1 × 10⁸ colony-forming units [CFUs], upper panel) and USA300/LAC (2.5 × 10⁷ CFUs, lower panel) via retroorbital intravenous route and observed for acute lethal disease. B, Human pulmonary artery endothelial cells (HPAECs) transfected with either irrelevant (irr) or ADAM10-specific siRNA (A10) were treated with 150 nM purified Hla or Hla_H35L, and cell-associated metalloprotease activity was measured using a fluorogenic substrate assay. C, HPAECs were treated with the indicated concentrations of purified Hla, and barrier resistance was continuously measured using an electric cell-substrate impedance sensing (ECIS) system. D, Immunoprecipitation and immunoblot analysis of full length (FL) and C-terminal cleavage fragment (CTF) of vascular endothelial (VE)–cadherin in HPAEC treated with 225 nM Hla or the negative control dimethyl sulfoxide (DMSO) and positive control ionomycin. E, Immunofluorescence microscopy of VE-cadherin (green) in HPAEC treated with 150 nM Hla. Nuclei were visualized with DAPI (blue). F, Miles assay in BALB/c mice demonstrating Evans blue dye extravasation from the vasculature into the skin following subcutaneous injection of purified toxin (1 μg per mouse). Abbreviations: IgG, immunoglobulin G; PBS, phosphate-buffered saline.

Figure 2.

ADAM10 inhibition prevents endothelial barrier disruption by Staphylococcus aureus α-hemolysin. Aand B, HPAECs treated with 20 μM GI254023X, an ADAM10 inhibitor, for 16–18 h. Vascular endothelial (VE)–cadherin cleavage following Hla administration observed by Western blot analysis (A) and immunofluorescence microscopy (B) of VE-cadherin (green) and nuclei (blue). FL, full-length VE-cadherin; CTF, C-terminal fragment. C and D, BALB/c mice treated with 200 mg/kg GI254023X/day or dimethyl sulfoxide (DMSO) control were examined in a Miles assay following subcutaneous injection of active Hla, demonstrating dye extravasation into the skin (C) and following intravenous injection of S. aureus strain Newman to evaluate lethal disease progression (D) (n = 18). Abbreviation: IgG, immunoglobulin G.