Figure 3

Bactericidal activity of MK against S. aureus is impaired by AL but not SA. (A) MK was compared with the classic HDP LL-37 in the viable counts assay using the S. aureus strain 5120 as target. Bacteria were incubated with increasing MK concentrations for 1 h and then plated, and CFUs were enumerated after an overnight incubation at 37°C. MK was the more potent antibacterial agent at lower concentrations, killing approximately 90% of the bacteria at 0.1 µmol/L. (B) MK antibacterial activity has potent bactericidal activity against the S. aureus strains Newman and 8325-4. (C) Scanning electron microscopy showing intact S. aureus after incubation in buffer alone for 1 h at 37°C (i) and disrupted bacteria with leakage of intracellular contents after incubation with MK (1 µmol/L) (ii). (D) MK was incubated with AL and SA for 3 and 18 h. Samples were separated by SDS-PAGE and the fragmentation pattern is visualized by Coomassie staining. AL generated bands of approximately 5 and 6 kDa, respectively, whereas SA generated two bands of similar sizes. (E) AL and SA degradation products of MK show differences in bactericidal activity. After incubation of MK with AL, only 25% killing was retained at a MK concentration corresponding to 1 µmol/L of the holoprotein. SA-generated fragments retained bactericidal activity in the order of the MK holoprotein. (F) Incubation of MK with culture supernatant from S. aureus 8325-4 wt and sarA (a strain overexpressing and releasing proteases) resulted in degradation of MK. A faint band is seen with supernatant from wt, and a distinct band is seen with sarA (arrow). In addition, a smaller band is observed in the latter case (*). The bands correspond to the molecular sizes generated with recombinant AL.