Antimicrobial Photodynamic therapy enhanced by the peptide aurein 1.2

Abstract

In the past few years, the World Health Organization has been warning that the post-antibiotic era is an increasingly real threat. The rising and disseminated resistance to antibiotics made mandatory the search for new drugs and/or alternative therapies that are able to eliminate resistant microorganisms and impair the development of new forms of resistance. In this context, antimicrobial photodynamic therapy (aPDT) and helical cationic antimicrobial peptides (AMP) are highlighted for the treatment of localized infections. This study aimed to combine the AMP aurein 1.2 to aPDT using Enterococcus faecalis as a model strain. Our results demonstrate that the combination of aPDT with aurein 1.2 proved to be a feasible alternative capable of completely eliminating E. faecalis employing low concentrations of both PS and AMP, in comparison with the individual therapies. Aurein 1.2 is capable of enhancing the aPDT activity whenever mediated by methylene blue or chlorin-e6, but not by curcumin, revealing a PS-dependent mechanism. The combined treatment was also effective against different strains; noteworthy, it completely eliminated a vancomycin-resistant strain of Enterococcus faecium. Our results suggest that this combined protocol must be exploited for clinical applications in localized infections as an alternative to antibiotics.

Figure 1

Aurein 1.2 and Photosensitizers structures. (a) Aurein 1.2 helical wheel diagram showing the disposition of each residue when the peptide assumes its α-helical conformation, evidencing the spatial “separation” of hydrophobic and hydrophilic residues, an important feature for the peptide mechanism of action. (b) Molecular structures of the photosensitizers methylene blue (top), chlorin-e6 (middle) and curcumin (bottom).

Figure 2

Aurein 1.2 minimal bactericidal concentration (MBC). Standardized suspensions of Enterococcus faecalis were submitted to incubation with aurein 1.2 for different time periods (a) and with different concentrations of the peptide for 5 minutes (b). The symbols and the columns represent the average of three independent assays (n = 9) and the bars represent the standard deviation. Two-way ANOVA (a) or One-way ANOVA (b); p < 0.0001. Asterisks indicate where there is a significant difference in comparison with the control (no aurein 1.2–0 µM).

Figure 3

Combination treatment. Standardized suspensions of Enterococcus faecalis were submitted to sub-inhibitory photodynamic therapy combined or not to the peptide aurein 1.2. Columns represent the average of at least three independent assays performed in triplicates, and bars represent the standard deviation. The asterisks indicate where there is a significant difference in comparison with the control (no treatment). One-way ANOVA with Tukey’s post hoc. (*p < 0.05, **p < 0.01, ***p < 0.001). MB: methylene blue; Ce6: chlorin-e6; CUR: curcumin; AU: aurein 1.2.

Figure 4

Photosensitizer uptake. Standardized suspensions of Enterococcus faecalis were incubated with methylene blue (156 µM; (a) chlorin-e6 (84 µM; (b) or curcumin (68 µM; (c) with or without aurein 1.2 (16 µM) for 5 minutes in the dark. The line inside the boxes represent the medians; boxes represent the minimum and maximum values, and whiskers represent the 10–90 percentiles. The y-axis represents the concentration of PS internalized by the totality of cells. Four independent assays (n = 12). The asterisk indicates where there is a significant difference between the groups (**p = 0.0022). Mann-Whitney test. MB: methylene blue; Ce6: chlorin-e6; CUR: curcumin; AU: aurein 1.2.

Figure 5

Reactive oxygen species (ROS) detection. Cell-free solutions of methylene blue (156 µM; (a) or chlorin-e6 (84 µM; (b) were combined or not with aurein 1.2 (16 µM) and incubated with ROS-detecting fluorescent probes to detect either hydroxyl radicals (^•OH) or singlet oxygen (¹O₂) upon irradiation. (c) Standardized suspensions of Enterococcus faecalis were incubated with APF, washed, and then treated with Ce6-PDT or Ce6-PDT + AU. Statistical analysis (t test) revealed no significant difference between groups (p > 0.05 for all tests; n = 6). MB: methylene blue; Ce6: chlorin-e6; AU: aurein 1.2; a.u.: arbitrary unities.

Figure 6

Absorbance spectrum and photobleaching of photosensitizers. (a) Normalized UV-VIS spectra of MB (; 156 µM), AU (; 16 µM) and MB combined with AU (), in aqueous solutions. (b) Photobleaching kinetics assessment of MB alone and in the presence of AU irradiated with a total energy dose of 45 J/cm². Statistical analyses revealed no significant difference between the two slopes (p = 0.2581). (c) Normalized UV-VIS spectra of Ce6 (; 84 µM), AU (; 16 µM) and Ce6 combined with AU (), in aqueous solutions. (d) Photobleaching kinetics assessment of Ce6 alone and in the presence of AU irradiated with a total energy dose of 30 J/cm². Statistical analyses revealed a significant difference between the two slopes (p < 0.0001). (e) Normalized UV-VIS spectra of CUR (; 68 µM), AU (; 16 µM) and CUR combined with AU (), in aqueous solutions. (f) Photobleaching kinetics assessment of CUR alone and in the presence of AU irradiated with a total energy dose of 12.5 J/cm². Statistical analyses revealed a significant difference between the two slopes (p = 0.0244). All assays were executed with Synergy H1M (Synergy H1 Multi-Mode Reader, BioTek, Winooski, VT, USA). CUR: curcumin; Ce6: chlorin-e6; MB: methylene blue; AU: aurein 1.2.

Figure 7

Circular dichroism of the AU. CD spectra of AU (16 µM) were recorded in aqueous solution (phosphate buffer 0.1 M pH 7.2) prior and after the addition of LPC (5 mM), MB (156 µM; (a), Ce6 (84 µM; (b), or CUR (68 µM; (c). CDs are an average of ten recorded spectra converted to mean-residue ellipticity [θ] (in deg cm² dmol⁻¹×10³) for each sample. (d) Mean residue ellipticity and helicity relative to Aurein 1.2 in different environments. CUR: curcumin; Ce6: chlorin-e6; MB: methylene blue; AU: aurein 1.2; LPC: lysophosphatidylcholine.

Figure 8

Chlorin-e6 and aurein 1.2 effects over the cell wall. (a) zeta potential: Standardized suspensions of Enterococcus faecalis were incubated with AU (16 µM), Ce6 (84 µM) or Ce6 + AU for 5 minutes in the dark. Particle size and the zeta potential of the bacterial cells were determined for each treatment and compared with the control (no treatment). The columns represent the average of 30 readings from three independent assays, bars represent the standard deviation. Different letters indicate a significant difference between groups. One-way ANOVA with Tukey’s posthoc. Particle size: p < 0.0001; zeta potential: p = 0.0282. (b) propidium iodide uptake: Standardized suspensions of Enterococcus faecalis were incubated with isopropanol 70%, AU (16 µM), Ce6 (84 µM) or Ce6 + AU for 5 minutes in the dark. Cells were washed to remove treatment and incubated with PI for 5 min. After removing excess PI, fluorescence was read at 535/617 nm. The columns represent the average of three independent assays performed in triplicates, bars represent the standard deviation. Different letters indicate a significant difference between groups. One-way ANOVA with Tukey’s posthoc, p < 0.0001. (−) CTRL: negative control (no treatment); (+) CTRL: positive control (isopropanol 70%); Ce6: chlorin-e6; AU: aurein 1.2.

Figure 9

Combined treatment against clinically relevant strains. Standardized suspensions of Staphylococcus aureus (a), Acinetobacter baumannii (b), Escherichia coli (c) or Enterococcus faecium VRE (d) were submitted to photodynamic therapy combined or not to the peptide aurein 1.2. MB-PDT: 156 µM of MB + 45 J/cm²; Ce6-PDT: 84 µM of Ce6 + 30 J/cm². Columns represent the average of at least three independent assays performed in triplicates, and bars represent the standard deviation. The asterisks indicate where there is a significant difference in comparison with the control (no treatment). One-way ANOVA with Tukey’s post hoc. (*p < 0.05, **p < 0.01, ***p < 0.001). MB: methylene blue; Ce6: chlorin-e6; CUR: curcumin; AU: aurein 1.2.