A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice

Abstract

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.

Figure 1

ADAM10 contributes to lethal S. aureus pneumonia. (a) Survival curves for mice harboring conditional deletion of Adam10 in the respiratory epithelium (ADAM10^−/−) relative to non-deleted littermate controls following infection with S. aureus strain Newman. n = 14 mice, control and 15 mice, ADAM10^−/−. Hematoxylin and eosin stained lung tissues derived from control and ADAM10^−/− mice 18–24 hours post-infection with a sublethal (b) or lethal (c) inoculum of S. aureus. Scale bars = 40 μm (b) and 1 mm (c).

Figure 2

Hla induces ADAM10-dependent epithelial barrier disruption and E-cadherin cleavage. (a) Cell-associated metalloprotease activity measured in A549 cells transfected with irrelevant (Irr) or ADAM10 siRNA following treatment with 10 μg ml⁻¹ (300 nM) active Hla or the non-toxigenic mutant Hla_H35L. Activity was quantified by detection of a fluorescent substrate product. (b) Electrical cell substrate impedance sensing (ECIS) recordings of A549 monolayers treated with PBS (black), the Hla_H35L mutant (50 μg ml⁻¹, red), or irrelevant (Irr, blue) and ADAM10 (A10, green) siRNA transfectants treated with 50 μg ml⁻¹ Hla. (c) Immunoblot analysis of full-length E-cadherin (FL) and accumulation of the C-terminal cleavage fragment (CTF) following treatment of A549 cells with controls DMSO and ionomycin compared to 20 μg ml⁻¹ Hla_H35L or Hla over the time course indicated. (d) Concentration dependence of E-cadherin cleavage in A549 cells exposed to 1–50 μg ml⁻¹ (30 nM – 1.5 μM) Hla for 1 hour. (e) Immunofluorescence microscopy images demonstrating surface expression of E-cadherin (green) following treatment with Hla. Nuclei (blue) are stained with the fluorescent DNA stain DAPI. Mean pixel intensity scored for ≥75 cells, 20.5 ± 0.87 (PBS) and 16.5 ± 1.72 (Hla), P = 0.02. Scale bar = 20 μm. (f and g) Cellular metalloprotease activity and E-cadherin cleavage induced by treatment of A549 cells with 10 μg ml⁻¹ Hla, the monomeric Hla_H35L mutant, a pre-pore locked mutant (Hla_PPL) that is reverted to the wild-type toxin in the presence of dithiothreitol (Hla_PPL+DTT), or Hla in the presence of the pore-blocking methyl-β-cyclodextrin (MβCD) (f), or by Hla in the presence of media (F12K), PBS, Dulbecco’s PBS (DPBS) or PBS supplemented with 0.9 mM Ca²⁺, 0.493 mM Mg²⁺ or 2.67 mM K⁺ (g). Error bars represent SEM.

Figure 3

Hla is required for E-cadherin cleavage and disruption of epithelial barrier function in S. aureus pneumonia. Bronchoalveolar lavage (BAL) fluid analysis 6, 12, and 24 hours post-infection of C57BL/6J mice infected with S. aureus USA300 or its isogenic mutant harboring a disruption of the hla locus (Hla-) to assess E-cadherin cleavage, measured by immunoblotting for the released N-terminal extracellular fragment (NTF) (a). Simultaneous evaluations of barrier disruption were made assessing BAL for cell count (b) and protein concentration (c) in groups of 7 animals. (d) BAL fluid analysis from C57BL/6J mice that received an intranasal dose of 0.4 μg purified Hla or Hla_H35L, or control PBS, assessed as described in (a). (e) Histopathology of murine lung tissues 4 hours post-treatment with PBS or Hla. Tissues were analyzed by hematoxylin and eosin staining (upper panels) or E-cadherin immunohistochemistry (lower panels). Scale bars = 40 μm. Immunoblot analysis of cleaved E-cadherin NTF (f) and quantification of cell and protein content (g) present in BAL fluid from ADAM10^−/− mice relative to control littermates following treatment with 0.4 μg purified Hla delivered by intranasal route. E-cadherin cleavage (h) and cell/protein recovery (i) from BAL samples of C57BL/6J mice infected with either 3 × 10⁸ WT or Hla- S. aureus as compared to infection with 7 × 10⁸ Hla- S. aureus. Statistical analysis for panels b, c, g and i was performed using a two-tailed Student’s t-test, where * denotes P < 0.05 and ** denotes P < 0.02.

Figure 4

An ADAM10-specific metalloprotease inhibitor prevents Hla-mediated injury. (a) Toxin-induced (20 μg ml⁻¹) E-cadherin cleavage detected by immunoblot analysis of Hla treated A549 cells that were pretreated with the metalloprotease inhibitor GI254023X (20 μM) or DMSO vehicle (FL, full length; CTF, C-terminal fragment). (b) ECIS-based monitoring of A549 monolayer resistance following toxin treatment (20 μg ml⁻¹) of cells exposed to GI254023X (20 μM) or DMSO vehicle control. (c) Binding and oligomerization of radiolabelled, active Hla to A549 cells following treatment with GI254023X (Hla₇, oligomeric Hla; Hla, monomeric Hla). (d) Immunoblot analysis of cleaved E-cadherin NTF present in BAL fluid from GI254023X-treated mice relative to DMSO treated animals following intranasal instillation of purified Hla. (e) Mortality curves in mice treated with DMSO vehicle or GI254023X upon challenge with lethal inocula of strain Newman (upper panel, n = 14 mice, 5 × 10⁸ S. aureus per mouse, lower panel, n = 8 mice, 6.3 × 10⁸ S. aureus per mouse).