Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus alpha-hemolysin-mediated cellular injury

Abstract

Staphylococcus aureus alpha-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of staphylococcal diseases, including those caused by methicillin-resistant epidemic strains. Hla is secreted as a water-soluble monomer that undergoes a series of conformational changes to generate a heptameric, beta-barrel structure in host membranes. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor in the context of the cell membrane. It is reported here that a disintegrin and metalloprotease 10 (ADAM10) interacts with Hla and is required to initiate the sequence of events whereby the toxin is transformed into a cytolytic pore. Hla binding to the eukaryotic cell requires ADAM10 expression. Further, ADAM10 is required for Hla-mediated cytotoxicity, most notably when the toxin is present at low concentrations. These data thus implicate ADAM10 as the probable high-affinity toxin receptor. Upon Hla binding, ADAM10 relocalizes to caveolin 1-enriched lipid rafts that serve as a platform for the clustering of signaling molecules. It is demonstrated that the Hla-ADAM10 complex initiates intracellular signaling events that culminate in the disruption of focal adhesions.

Fig. 1.

Hla interacts with ADAM10 expressed on rabbit red blood cells. (A) Rabbit or human erythrocyte ghost preparations were precipitated with GST control or GST-Hla_H35L and visualized by silver stain analysis. **Purified GST protein; *purified GST-Hla_H35L fusion. (B) GST or GST-Hla_H35L precipitates were immunoblotted for human ADAM10 or GST. (C) Flow cytometric analysis of ADAM10 on rabbit and human erythrocytes and human A549 cells.

Fig. 2.

ADAM10 interacts with Hla and mediates toxin binding to human epithelial cells. (A) A549 alveolar epithelial cells were treated with nontoxigenic Hla_H35L or active Hla, then Hla immunoprecipitates from cell lysates prepared in Triton X-100 or RIPA lysis buffer were immunoblotted for ADAM10. (B) Hla induces the relocalization of ADAM10 to discrete sites on the cell membrane that colocalize with caveolin 1. Quantification of the number of ADAM10 punctae ≥0.05 μm in diameter averaged on a per-cell basis revealed 0.08 ± 0.14, PBS; 1.63 ± 0.48, Hla; 0.13 ± 0.14, Hla_H35L, with error values representing the SD. P = 8.7 × 10⁻⁶ PBS vs. Hla, P = 1.3 × 10⁻⁵ Hla vs. Hla_H35L. A minimum of 45 cells was counted for each treatment condition. Quantification of ADAM10–caveolin 1 colocalization was determined by the Manders’ coefficient assessed on 20 distinct images (0.15 ± 0.08, PBS; 0.28 ± 0.13, Hla; P = 0.0005) (Scale bars, 10 μm.) (C) Binding of [³⁵S]-methionine–labeled active Hla examined as a function of ADAM10 surface expression, quantified as the mean fluorescence intensity (MFI) shift by flow cytometric analysis in a panel of human epithelial cells. (D) Binding of [³⁵S]-methionine–labeled active Hla to A549 cells treated with irrelevant or ADAM10-specific siRNAs (designated ADAM10.1 and ADAM10.2). *P < 2 × 10⁻⁶. (E) Flow cytometric detection of surface expressed ADAM10 in A549 cells.

Fig. 3.

ADAM10 is required for Hla-mediated cytotoxicity. (A) Irrelevant or ADAM10 siRNA transfected A549 cells were cocultured with WT S. aureus, S. aureus deficient in production of Hla (Hla−), or S. aureus Hla− complemented with plasmid-encoded Hla (Hla− phla), and cell injury measured by lactate dehydrogenase (LDH) release. *P < 0.008 vs. irrelevant siRNA treated cells. Error bars delineate the SEM examined in six replicates from two independent experiments. (B) LDH release from A549 cells treated with the indicated concentrations of purified, active Hla. *P < 0.0002 vs. irrelevant siRNA treated cells. Error bars delineate the SD of six replicates. (C) Detection of radiolabeled, oligomeric Hla (Hla₇) bound to irrelevant or ADAM10 siRNA transfected A549 cells. (D) LDH release in a panel of epithelial cell lines cultured with purified active Hla examined as a function of ADAM10 surface expression, quantified as the mean fluorescence intensity (MFI) shift by flow cytometric analysis.

Fig. 4.

ADAM10 is required for Hla-mediated focal adhesion disruption. (A) Cell lysates from irrelevant or ADAM10 siRNA transfected A549 cells treated with active Hla were probed with an antibody specific for phospho-Tyr397 FAK (Upper) or anti-FAK control (Lower). (B) Lysates prepared as described in A were probed with an antibody specific for phospho-Tyr-416 Src (Upper) or anti-Src control (Lower). (C) Lysates prepared as described in A were probed with an antibody specific for phospho-Tyr-165 p130Cas (Upper) or anti-p130Cas control (Lower). (D) Lysates prepared as described in A were probed with an antibody specific for phospho-Tyr-118 paxillin (Upper) or antipaxillin control (Lower). (E) Irrelevant (Top) or ADAM10 (Middle, Bottom) siRNA transfected A549 cells were treated with PBS or active Hla and stained to detect vinculin (red) and filamentous actin (phalloidin, green). (Scale bars, 10 μm.)

Fig. 5.

Model depicting the ADAM10-Hla complex localized to caveolin 1-enriched membrane domains, causing cytolysis and focal adhesion disruption in toxin-sensitive cells.