Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent

Abstract

Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10-8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.

Fig 1. Schiffer-Edmundson helical wheel representation of temporin-SHa and its analogs.

SHa, temporin-SHa; [K³]SHa, [K³]temporin-SHa; [A^2,6,9]SHa, [A^2,6,9]temporin-SHa; [A^2,6,9, K³]SHa, [A^2,6,9, K³]temporin-SHa. Apolar residues are represented in black and polar/basic residues in gray/white. Amino acid modifications are in bold. An amphipathic character, with two well-separated polar and apolar faces, is observed. Adapted from Heliquest.

Fig 2

Time-killing curves of SHa and its analog [K³]SHa against S. aureus ST1065 (A) and E. coli ML-35p (B). Bacteria (10⁶ cfu/ml) were incubated in phosphate-buffered saline (PBS) with temporins at concentrations 2-fold above the MIC obtained for S. aureus ST1065 (6 μM for [K³]SHa and 12 μM for SHa). The negative control corresponds to bacteria incubated in PBS without peptide (w/o peptide). The data are shown as the means ± SEM from a single experiment carried out in triplicate and are representative of three independent experiments.

Fig 3. Time-kill curves of temporins against L. infantum.

Parasites (2 x 10⁶ cells/ml) were incubated in HBSS with various concentrations (3, 6 and 12 μM) of synthetic SHa (A) and [K³]SHa (B). HBSS without peptide (w/o peptide) or containing 96 μM [A^2,6,9, K³]SHa was used as a negative control (C). The data are shown as the means ± SEM of one representative experiment obtained from three independent experiments carried out in duplicate.

Fig 4. Temporin-induced membrane permeabilization of E. coli ML-35p.

Bacteria were incubated with different concentrations of SHa (A) or [K³]SHa (B). The leakage kinetics were measured as the production of ONP at 405 nm resulting from hydrolysis of ONPG by the cytoplasmic bacterial β-galactosidase. C, comparison of the membrane leakage of temporins (SHa and [K³]SHa), dermaseptin B2 (B2) and melittin at the same concentration (10 μM). The negative control without peptide is also indicated (w/o peptide). D, no permeabilization was observed with [A^2,6,9]SHa (2, 4, 6, 8 and 10 μM). E, Extracellular release of cytoplasmic β-galactosidase after 60 min incubation of bacteria with 10 μM peptide followed by centrifugation (1,000 x g, 10 min, 4°C) to measure ONP production (A₄₀₅) in the supernatant. The results are expressed as the means ± SEM after subtraction of the negative control values (no peptide) from the test values and correspond to one representative experiment carried out in triplicate.

Fig 5

Temporin-induced SYTOX Green (SG) influx into the bacteria K. pneumoniae ATCC 13883 (A) and S. pyogenes ATCC 19615 (B), and the parasites L. infantum (C), L. braziliensis (D), L. major (E), and T. cruzi (F). Bacteria (10⁶ cfu/ml) and parasites (2.5 x 10⁶ cells/ml) were preincubated with 1 μM SG, and peptides (SHa or [K³]SHa) were added after fluorescence stabilization. Membrane alteration is correlated with the fluorescence of the DNA fluorescent probe (λ_excitation = 485 nm and λ_emission = 520 nm). For bacteria, the data are expressed as the means ± SEM after subtraction of the negative control values (w/o peptide) from the test values. For parasites, the results (mean ± SEM) were plotted as a percentage of the fluorescence relative to that of parasites fully permeabilized by 0.1% Triton X-100. The curves correspond to one experiment carried out in triplicate and are representative of two independent experiments.

Fig 6

Dose- and time-dependent propidium iodide (PI) staining (A) and luciferase release in the extracellular medium (B) of L. infantum parasites upon addition of temporins. L. infantum promastigotes (10⁶ cells/ml) were incubated with different concentrations (10, 20 and 40 μM) of SHa or [K³]SHa for different times. PI-positive cells were counted by flow cytometry after adding PI (1 μg/ml) to the parasites. The luciferase activity in the extracellular medium was determined after centrifugation of the parasites and measurement of the luminescence using the Steady-Glo® Luciferase Assay System (Promega). The data are expressed as the means ± SEM of two experiments carried out in triplicate.

Fig 7. Changes in the membrane potential of bacteria and parasites upon addition of temporins.

S. aureus ATCC 25923 (A), L. infantum (B), L. amazonensis (C) and T. cruzi (D) were equilibrated with DiSC₃(5) (1 μM for S. aureus and 2.5 μM for parasites). SHa or [K³]SHa was then added (t = 0) at a concentration of 5 μM (bacteria) or 50 μM (parasites), and changes in the fluorescence were monitored for 15 min (bacteria) or 20 min (parasites) at λ_excitation = 622 nm and λ_emission = 670 nm. The curves correspond to a single experiment representative of three independent experiments.

Fig 8

AFM and FEG-SEM visualization of the effect of temporins-SH on P. aeruginosa bacteria (A–C) and parasites (L. infantum promastigotes and T. cruzi epimastigotes; D–I). A–C, AFM imaging of P. aeruginosa: A, untreated control bacteria; B, bacteria after 1 h incubation with 50 μM SHa; C, bacteria treated for 1 h with 6 μM [K³]SHa. Bacteria were severely damaged by temporins compared to the control. D–G, AFM imaging of L. infantum promastigotes and T. cruzi epimastigotes: D and E, L. infantum untreated (D) or treated with 5 μM [K³]SHa (E); F and G, T. cruzi without peptide (F) or with 5 μM [K³]SHa (G). H and I, FEG-SEM visualization of L. infantum promastigotes without peptide (H) or with 5 μM [K³]SHa. Morphological changes were observed for parasites that were incubated with peptides (E, G and I).

Fig 9. Kinetics of mitochondrial membrane depolarization of L. infantum promastigotes.

Parasites were incubated for 3 h at 26°C with different concentrations of peptide (3 and 6 μM, final concentrations). Mitochondrial membrane potential was monitored by flow cytometry using the fluorescence probe TMRE. A, index of variation for SHa. B, index of variation for [K³]SHa. Negative and positive controls were assayed without peptides or with 500 μM CCCP, respectively. The index of variation is expressed in arbitrary units (a.u.). The curves were obtained from a single experiment representative of three independent experiments.

Fig 10

DNA fragmentation (A) and cell cycle analysis (B–D) of L. infantum promastigotes. Parasites were treated with different concentrations (12.5, 25 and 50 μM, final concentrations) of SHa or [K³]SHa. Miltefosine (hexadecylphosphocholine, 50 μM), a drug used for the treatment of leishmaniasis that is known to induce apoptosis, and [A^2,6,9, K³]SHa (50 μM) were used as positive and negative controls, respectively. A, DNA fragmentation was assessed by TUNEL assay, and fluorescence values were corrected (subtraction of negative control fluorescence value) and converted into a histogram that represents the percentage of FITC-positive cells. Parametric data were analyzed by a one-way ANOVA and Dunnett’s post-test using GraphPad Prism 5.0. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B-D, L. infantum promastigotes were stained with propidium iodide and analyzed by flow cytometry. Flow cytograms are shown: B, parasites untreated or treated with 50 μM of [A^2,6,9, K³]SHa or miltefosine; C, parasites treated with SHa (12.5, 25 μM or 50 μM); D, parasites treated with [K³]SHa (12.5, 25 μM or 50 μM). The sub-G1 peak is shown with an arrow. Flow cytograms correspond to a single experiment representative of three independent experiments and were obtained using FlowJo vX.0.7 software.

Fig 11. Multipassage resistance selection.

A, plot of MICs against E. coli lineages adapted to increasing concentrations of temporins or ampicillin. B, control: MICs against lineages grown in the same conditions without antimicrobial agents (MilliQ water). The following temporins were tested: SHa, D-SHa (SHa with all residues in D-configuration), and [K³]SHa. The conventional antibiotic ampicillin was also used for comparison. E. coli ATCC 25922 was continuously re-cultured in the presence of doubling concentrations of antimicrobial agents from 1/16 of the MIC until adaptation to the MIC (50 passages, 10 bacterial lineages with 1/16 MIC, 1/8 MIC, 1/4 MIC, 1/2 MIC, and MIC) (see Materials and Methods). The MIC of the antimicrobial agent was determined against the adapted E. coli lineages originating from different last passages: passage 5 (E. coli with no antimicrobial agent), 15 (E. coli with a concentration of antimicrobial agent equal to 1/16 MIC), 25 (1/8 MIC), 35 (1/4 MIC), 45(1/2 MIC), and 55 (MIC). MIC values were obtained in triplicate and represent the average of three independent experiments. Curves representing the MIC as a function of the passage number were obtained from the means ± SEM of MIC values of at least three independent experiments.

Fig 12. CD and NMR investigation of temporins.

A, CD spectra of SHa, [K³]SHa and [A^2,6,9]SHa (30 μM) in DMPC:DMPG 3:1 (mol:mol) LUVs (1 mg/ml in PBS). No ordered structure was found in PBS. CD measurements are reported as the dichroic increment (Δε) per residue. The relative helix content was deduced as the percent of helix = [Δε₂₂₂ x –10], where Δε_{222 nm} is the dichroic increment at 222 nm. B, NMR chemical shift deviations (CSDs) of Hα protons of SHa and [K³]SHa in 50 mM DHPC/25 mM DMPG bicelles. C, Residual peak volume after addition of 2% 1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3-phosphocholine (12-doxylPC) paramagnetic probe. For each residue, 1 to 3 cross-peaks corresponding to HN-Hα and HN-Hβ NOE correlations were integrated. The HN protons of residues 1 and 2 were not detected. The standard deviation of peak volumes integrated for each residue is indicated.

Fig 13. Surface plasmon resonance (SPR) analysis of temporin binding to negatively charged DMPC/DMPG 3:1 (mol:mol) LUVs.

A, binding of temporins directly to the L1 sensor chip surface. SHa and [K³]SHa injected at a concentration of 5 μM (20 μl during 1 min) interact with the carboxymethylated dextran containing covalently attached alkyl chains, as indicated by the significant amount of temporin non-specific binding (SHa: 197 RU, [K³]SHa: 240 RU) remaining on the sensor chip surface after the end of peptide injection. B and C, binding of SHa (B) and [K³]SHa (C) after injection of BSA. In contrast, no peptide interaction was observed after binding of 0.2 mg/ml BSA (15 μl injected during 3 min) to the sensor chip surface followed by injection of SHa or [K³]SHa (5 μM). D, complete SPR cycle used for the binding of temporins. In the example, 0.2 mg/ml BSA was first injected (15 μl during 3 min) on the L1 surface to prevent non-specific binding of temporins and was followed by an injection (2 μl during 2 min) of 0.2 mg/ml DMPC/DMPG LUVs and then of peptide (300 nM of SHa in the example; 20 μl during 1 min). Complete regeneration of the surface was obtained using 40 mM of the detergent n-octyl-β-D-glucopyranoside (OG) (30 μl injected during 1 min). E and F, determination of the binding affinity of temporins SHa (E) and [K³]SHa (F). Peptides diluted in HBS-N buffer were tested at different concentrations (0 to 300 nM) for their binding to DMPC/DMPG LUVs. The baseline corresponds to HBS-N alone. The following K_D values were calculated by BIAevaluation software analysis: K_D (SHa) = 1.3 ± 0.4 x 10⁻⁷ M, χ² = 3.7 ± 1.3 (n = 3); K_D ([K³]SHa) = 3.1 ± 0.7 x 10⁻⁸ M, χ² = 3.2 ± 0.7 (n = 3). Chi² (χ²) values below 10 indicate a good fit of the Langmuir (1:1) binding model. G and H, selective SPR binding of temporins SHa (G) and [K³]SHa (H) toward anionic model membranes. Negatively charged DMPG or zwitterionic DMPC LUVs were injected onto the L1 sensor chip precoated with BSA (0.2 mg/ml). Temporins (500 nM) were then injected, and binding to the DMPG (solid line) and DMPC (dashed line) LUVs was monitored. RU: resonance units; SI: start of injection; EI: end of injection. The curves correspond to a single experiment representative of three different experiments.