This explains our finding that no measurable MIC (minimal inhibitory concentration) could be measured even if high

concentrations of peptides were tested (up to 128 μg/mL for pre-elafin/trappin-2 and elafin and up to 256 μg/mL for cementoin). Fluorescein-labeled pre-elafin/trappin-2 CP-690550 mouse incubated with P. aeruginosa accumulates within the cytosol and both elafin and pre-elafin/trappin-2 AZD0156 clinical trial bind DNA in vitro Weak membrane depolarization and leakage of liposome-entrapped calcein, while indicating little membrane disruption, does not exclude that transient pores may form upon incubation of P. aeruginosa with pre-elafin/trappin-2 and derived peptides, as suggested by SEM examination. Formation of transient pores could lead to the translocation of the peptides across membranes.

We previously reported that fluorescein-labeled pre-elafin/trappin-2 heavily decorated P. aeruginosa cells as assessed by fluorescence microscopy [27]. Here we used confocal microscopy to examine the fate of fluorescein-labeled pre-elafin/trappin-2 upon a 1 h incubation with selleck inhibitor P. aeruginosa. As shown in Fig. 4, the whole bacterial cell was fluorescent in all consecutive 0.2 μm sections. This is taken as evidence that pre-elafin/trappin-2 not only binds the surface, but also accumulates within the bacterial cytosol. Figure 4 Confocal microscopy of P. aeruginosa incubated with fluorescein-labeled pre-elafin/trappin-2. Mid-logarithmic phase cultures of P. aeruginosa were incubated for 1 h at 37°C with fluorescein-labeled pre-elafin/trappin-2 and observed by confocal microscopy at 400 × magnification. From left to right, consecutive 0.2 μm sections of a fluorescent bacterial cell. Given the polycationic character

of pre-elafin/trappin-2 and derived peptides and the apparent ability of pre-elafin/trappin-2 to traverse lipid bilayers, we considered the possibility that they could interact with nucleic acids. To test this hypothesis, we evaluated whether any of the pre-elafin/trappin-2 and derived peptides could induce an electrophoretic mobility shift (EMSA) of DNA. As shown in Fig. 5, the EMSA assay revealed that pre-elafin/trappin-2 binds to DNA in vitro at a peptide:DNA ratio of 5:1 about and greater. Similar results were also obtained with the elafin domain. In contrast, no DNA shift was observed for the cementoin peptide up to a 100:1 ratio. Hence, despite the fact that the cementoin peptide has a greater positive charge (+4) than elafin (+3), the structure of the elafin domain appears necessary and sufficient for binding to DNA in vitro. Figure 5 Electrophoretic mobility shift assay of plasmid DNA incubated in the absence or presence of pre-elafin/trappin-2, elafin and cementoin. Plasmid pRS426 (100 ng) was incubated with the indicated ratios of peptide/DNA (w/w) for 1 h and then analyzed by agarose gel electrophoresis followed by staining with ethidium bromide. Above are representative gels from an experiment performed in triplicata.