Table 1.
List of novel USCLs used in this study.
Table 2.
Minimum inhibitory concentration (MICa, in μg/mL) of USCLs against reference bacterial strains.
Table 3.
Minimum inhibitory concentration (MICa, in μg/mL) of selected USCLs against a panel of Gram-positive bacterial strains.
Table 4.
Minimum inhibitory concentration (MICa, in μg/mL) of selected USCLs against a panel of Gram-negative bacteria and C. albicans strains.
Fig 1.
Growth kinetics of S. aureus ATCC 25923 in presence of different concentrations of USCLs.
Bacterial suspensions (1×106 cells/mL) was grown up to 4h in presence of indicated concentrations of Lp-I (a) and Lp-IRR (b), and the OD620 was measured every 10 min. The results are the mean ± SEM of three independent experiments. *p < 0.05 vs untreated cells (ctrl) at 4h incubation time; **p < 0.0001 vs untreated cells (ctrl) at 4 h incubation time (Student-Newman-Keuls Multiple Comparisons Test, ANOVA).
Fig 2.
Effects of Lp-I and Lp-IRR on human epidermal keratinocytes HaCaT cells and the human epidermal model EpiDerm.
The cell viability is expressed as a percentage of the OD measured on untreated cells (ctrl) assumed as 100% viability. Cytotoxic activity on HaCaT cells was evaluated after 1h (a) or 24h (b) incubation with indicated concentrations of USCLs. (c) The cytotoxicity with EpiDerm test was evaluated after 1h incubation with 100 μg/mL of both USCLs; 5% SDS was used as positive control. Each value represents the mean ± SEM of 3 experiments performed in triplicate. *p < 0.05 vs untreated cells (ctrl); **p < 0.0001 vs untreated cells (ctrl) (Student-Newman-Keuls Multiple Comparisons Test, ANOVA).
Fig 3.
Binding sensorgrams for USCLs onto immobilized LUVs.
Solutions with increasing concentrations (1.5, 3, 6 and 12 μg/mL) of Lp-I (a, c) and Lp-IRR (b, d) were injected onto L1 sensor chip surface with immobilized LUVs (DPPC/DPPG 4:1 v/v). The baseline was adjusted to zero at each injection.
Fig 4.
Evaluation of membrane-damaging activity of Lp-I and Lp-IRR on S. aureus ATCC 25923 by PI-uptake assay and scanning electron microscopy.
The permeabilization assay with Lp-I (a) and Lp-IRR (b) on S. aureus cells has been performed in MHB. Bacterial cells (1×106 CFU/mL) were incubated, for different incubation times, with USCLs at the concentration equal to their MIC, ½ MIC or 2×MIC. % PI-positive: percentage of propidium iodide positive cells. The background level of permeabilized cells, obtained with untreated samples, was always lower than 2% and was subtracted to the corresponding USCL-treated sample. Data are a mean ± SEM of four independent experiments. Scanning electron microscopy of 107 CFU/mL S. aureus cells untreated (c) or after 60 min incubation at 37°C with 30 μg/mL of Lp-I (d) or Lp-IRR (e).