Lysostaphin

Gentamicin, vancomycin, linezolid, and lysostaphin were from Sigma-Aldrich (St. Louis, MO, USA). FBS, DMEM, RPMI 1640, calcium and magnesium free phosphate-buffered saline (PBS without Ca²⁺ and Mg²⁺), and penicillin-streptomycin (PEST) were obtained from Gibco (Life Technologies, Paisley, UK). CytoTox96 non-radioactive cytotoxicity assay kit was obtained from Promega (Promega, Madison, WI, USA). Sytox green and LIVE/DEAD BacLight Bacterial Viability kit were purchased from ThermoFisher Scientific (Invitrogen, ThermoFisher Scientific, Eugene, OR, USA).

Bacterial DNA was isolated from 1 ml of a fresh over-night culture of S. aureus 6850, cellular DNA was isolated from 2 × 10⁷ A549 cells. Cells were pelleted for 5 min at 3200× g and 4 °C. The pellet was resuspended in 493 μl TE buffer [10 mM Tris/HCl, 1 mM EDTA, pH 8.0]. For lysis, cells were incubated with 4 μg ml⁻¹ lysostaphin (Sigma, Munich, Germany) for 10 min at 37 °C. Subsequently, 2.4 μg ml⁻¹ Proteinase K (Sigma) was added and the suspension was incubated for further 10 min at 37 °C. Lysis was stopped by incubation for 5 min at 95 °C. One volume of phenol/chloroform/isoamyl alcohol (Roth, Karlsruhe, Germany) was added and samples were centrifuged for 10 min at 20800× g. The upper aqueous phase was used to repeat the phenol/chloroform/isoamyl alcohol extraction step. The aqueous phase was transferred to a clean tube and mixed with 1/10 volume of 5 M NaCl and 6/10 volume of isopropanol. DNA was precipitated for 0.5 h at 20800× g, 4 °C. The pellet was washed once with 70% ethanol and centrifuged for 15 min at 20800× g and 4 °C. Finally, the pellet was air dried and dissolved in water. Bacterial DNA concentration and purity were determined by NanoDrop (PEQLAB Biotechnology, Erlangen, Germany) measurement.

Preparation of a S. aureus ΔvraGΔhemB whole cell extract was done as previously described with some modifications [28 (link)]. Briefly, overnight bacterial cultures were diluted 1/1000 in fresh BHI broth, and then incubated at 35°C (225 rpm) until an A_600nm of ~ 0.8 was reached. Bacterial cells were centrifuged, and pellets were washed twice in ice-cold PBS and resuspended in a ratio of 5 ml of PBS per ml of pellet. Bacterial suspensions were then treated with 100 μg of lysostaphin (Sigma-Aldrich, Oakville, ON, Canada) per ml of pellet for 1 h at 37°C, and then 3 μg of protease inhibitor cocktail (Sigma-Aldrich), 8 μg of RNAse A (Sigma-Aldrich) and 8 μg of DNAse (Qiagen, Toronto, ON, Canada) per ml of pellet were added to the suspension. After 30 min at room temperature, cells were mechanically disrupted by 3 to 4 passages in a SLM Aminco French Pressure cell disrupter, and then centrifuged at 12,000 × g at 4°C for 10 min to remove unbroken cells. The supernatant was collected and used as the whole cell extract. Total protein concentration was determined by the bicinchoninic acid method (BCA) Protein Assay Kit (Thermo Fisher Scientific, Ottawa, Canada).

Western ligand affinity blotting was performed as previously described [4 (link)], with modifications. Briefly, overnight cultures were diluted 1:50 in TSB medium and grown at 37°C with shaking for 4 hours. Cells were washed and surface-associated protein extracts were prepared through resuspension in PBS media containing 20 μg/mL lysostaphin (Sigma), 20 μg/mL DNAse (New England Biolabs), 1 mM phenylmethanesulfonylfluoride (PMSF, Thermo Scientific), and 1:100 dilution of a protease inhibitor cocktail (Sigma, P2714). Cell extracts were incubated at 37°C for 30 minutes and spun at 12,000 x g for 1 minute at 4°C. The protein concentration in the supernatant was determined with a BCA Protein Assay Kit (Pierce), and 20 μg of each sample was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). FnBPs were detected by incubation for 1 hour with biotinylated human fibronectin (50 μg/mL) in PBS containing 0.1% Tween 20 (PBST). An EZ-Link sulfo-N-hydroxysuccinimide-LC biotinylation kit (Pierce) was used to biotinylate human fibronectin (Sigma). After washing with PBST, membranes were incubated for 1 hour with streptavidin-peroxidase conjugate (Roche; 1:3000 dilution). Finally, membranes were developed with the ECL Western blotting system (Pierce). A S. aureus fnbA fnbB double mutant [19 (link)] was used as a negative control.

TLR9 murine agonist CpG (ODN1826), TLR9 murine antagonist CpG (ODN2088), monoclonal anti-mTLR9 antibody, mouse monoclonal anti-hemagglutinin-tag (HA) Ab, pUNO-mTLR9-HA, pUNO-mcs purchased from Invivogen (San Diego, CA). Avian Myeloblastosis Virus-Reverse Transcriptase (AMV-RT) enzyme, SYBR Green Jump Start ready mix, Lysostaphin, actinomycin-D, and Tricarbonyl-dichloro-ruthenium (II) dimer (CORM2) were obtained from Sigma Aldrich (St. Louis, MO). The Micro-Bicinchoninic Acid assay (BCA) protein assay kit was purchased from Pierce Biotechnology (Rockford, IL). The Cobalt Protoporphyrin-IX (CoPP), Zinc Protoporphyrin-IX (ZnPP), Biliverdin was from Frontier Scientific, Logan, UT. The RNA isolation kit was acquired from Qiagen (Valencia, CA). Murine PMCs were isolated from HO-1^+/+ and HO-1^-/- mice and cultures were established as reported earlier [6 (link)].