Figure 6.

Mirolysin degrades LL-37 in a concentration- and time-dependent manner, abolishing its antimicrobial activity. A, B) LL-37 was incubated with mature mirolysin (mMir) at different substrate:enzyme molar ratios (concentration-dependent proteolysis) for 1 h (A) or at a constant 100 000:1 substrate:enzyme molar ratio for different time intervals (time-dependent proteolysis) (B) in 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl₂, 0.02% NaN₃, pH 7.6, at 37°C. The reaction was terminated by boiling with SDS-PAGE sample buffer, and samples were subjected to SDS-PAGE. LL-37 incubated with Mircat^E225A served as a negative control. C) LL-37 was incubated alone or with mirolysin (mMir) at a molar ratio of 100 000:1 for 10 and 60 min, then mixed with bacteria (E. coli ATTCC 25922), incubated for 2 h at 37°C, and plated onto agar plates to determine the numbers of CFU. Percentage survival was calculated and compared to bacteria grown without antimicrobial peptide LL-37 (100% survival). D) Bacteria were mixed with mMir at concentrations of 0.01, 0.1, and 1 nM or Mircat^E225A (control), then LL-37 was added, and after incubation for 2 h at 37°C the mixtures were plated onto agar plates to determine the numbers of CFU. Percentage survival was calculated and compared to bacteria treated with Mircat^E225A (no survival). E) LL-37, intact or cleaved by mMir, was mixed and then incubated for 5 min at room temperature with endotoxin from E. coli 0111:B4. Intact LL-37 incubated with Mircat^E225A was used as a negative control. The level of the Limulus test reactive endotoxin was determined and compared to endotoxin without LL-37 (100%). *p<0.05; **p<0.01; ***p<0.001.