S aureus lta

Fluorescence polarization measurements were conducted on a CLARIOstar (BMG Labtech) spectrophotometer in a Greiner 364-well flat bottom black microplate. Data were acquired at 23 °C in water using the FITC fluorescence of FITC-pseudonajide and 35 flashes in standard mode. FITC-pseudonajide concentration was set to 5 μM and volume was adjusted to 20 μL in all wells. S. aureus LTA was purchased from Sigma. Titration series was set in triplicate using LTA monomer concentrations ranging from 2.5 μM to 5 mM. Data were recorded within one hour, analyzed using Kaleidagraph (Synergy Software) and fitted using a 1:1 model and the following equation: P_obs = P_free + [[(P_max-P_free)/2C₀] * [(K_d + L₀ + C₀)-((K_d + L₀ + C₀)^{^2}-4C₀L₀)^{^0.5}]] with P = polarization, C₀ = pseudonajide concentration, L₀ = total LTA concentration.

The LPS and LTA binding assay was performed to investigate the interaction between PN5 and E. coli O111:B4 LPS and S. aureus LTA (Sigma-Aldrich) by CD spectroscopy. E. coli LPS and S. aureus LTA were diluted in 10 mM sodium phosphate buffer (pH 7.4) to 1 mg mL⁻¹. The peptide (50 μM) was added to E. coli LPS and S. aureus LTA. The temperature was regulated by a PTC-423S controller and was set to 20°C. The spectra were measured from 190 to 250 nm using a CD spectropolarimeter (Jasco) operating at room temperature (25°C) (61 (link)).

BODIPY-TR-cadaverine (Life Technologies) was prepared in 10 mM NaCl or 5 mM HEPES (pH 7.5) and pre-warmed at 37°C. Purified P. aeruginosa LPS and S. aureus LTA (Sigma-Aldrich) were prepared in distilled water. Colistin and meropenem (Santa Cruz Biotechnology) were used as positive and negative controls respectively (Ouberai et al., 2011 (link)). A final 100 μl mixture of 5 μM BODIPY-TR-cadaverine (in 10 mM NaCl or 5 mM HEPES, pH 7.5), 15 μg/ml LPS or LTA, and peptides or antibiotics in a concentration ranged from 0.02 to 272 μM was added to a 96-well black assay plate. The fluorescence at 580/620 nm was measured at 37°C with a Synergy 4 fluorescence microplate reader (BioTek). Displacement percentage of KAMP relative to colistin and meropenem was determined by: fluorescence of (probe/endotoxin/KAMP mixture - probe/endotoxin/meropenem mixture) / fluorescence of (probe/endotoxin/colistin mixture - probe/endotoxin/meropenem mixture) × 100%.

S. aureus LTA and SPB were supplied by Sigma-Aldrich (St Louis, MO, USA). All antibodies used in Western blots were purchased from Beyotime Institute of Biotechnology (Haimen, China). All other chemicals were reagent grade.