Antimicrobial activity of native L- and D-PLNC8 αβ

We have recently shown that PLNC8 αβ is a potent antimicrobial agent against the Gram-negative bacteria P. gingivalis13,14. The purpose of this study was to determine the antimicrobial activity of PLNC8 αβ against susceptible and antibiotic-resistant strains of staphylococci.

Peptides are constantly subjected to proteolytic degradation in biological systems. In order to investigate the stability of PLNC8 αβ enantiomers against proteolytic degradation, the peptides were treated with trypsin for 16 h. The molecular weight of the peptides, before and after trypsin treatment, was determined using matrix assisted laser dissociation and ionization time of flight mass spectrometry (MALDI-ToF MS). Both PLNC8 α and β of the L-form, but not the D-form, were efficiently degraded by trypsin (Supplementary Fig. S3). Among the degradation products of L-PLNC8 β, the amino acid sequences of 1–21, 1–22 and 1–26 were found to be accumulated. Interestingly, these fragments of PLNC8 β were also shown to exhibit inhibitory effects on their own (see below).

The membrane activity of PLNC8 α and β, alone and when combined, was evaluated using liposomal model systems with different ratios of the zwitterionic lipid POPC and the negatively charged lipids POPS and POPG, with and without cholesterol. In liposomes comprised of POPC and POPS (95:5), 0.12 μM PLNC8 β triggered 50% release of encapsulated carboxyfluorescein (CF) after 30 min whereas only limited release was seen in the presence of PLNC8 α (Fig. 2A,B). The combination of PLNC8 α and β in a molar ratio of 1:1 showed efficient permeabilization of the liposomes and both the L- and the D-enantiomers of the peptides exerted similar activity, resulting in 50% release of CF at 0.08 and 0.06 µM for L- and D-PLNC8 αβ, respectively. To investigate the effect of the lipid composition, various ratios of POPC and POPG were evaluated. Whereas POPS can be used to mimic the negative net charge of bacterial membranes, POPG is a more common lipid in bacterial membranes22. At a ratio of POPC:POPG (95:5) the lipid membrane was slightly more resilient to perturbation by L-PLNC8 αβ compared to POPS containing lipid membranes, requiring a peptide concentration of 0.7 µM to reach 50% CF-release (Fig. 2C). This is likely a result of the slightly higher negative net charge of POPS. Exposing the liposomes to the individual peptides resulted in a similar trend. Peptide concentrations of 0.12 and 5.7 µM for PLNC8 β and PLNC8 α, respectively, were required in order to produce a 50% CF-release (Fig. S4A,B). Increasing the ratio of POPG further to 25–50% made the liposomes even more susceptible to the peptides, corresponding to a 100–1000 fold increase in efficiency (Fig. 2C). The individual peptides displayed a similar trend (Fig. S4A,B), although the effect on lipid composition was not as pronounced for PLNC8 α as for PLNC8 β. Liposome net charge is consequently a central aspect in regulating the membrane activity of the peptides. To investigate the potential influence of cholesterol on the permeabilizing effect of PLNC8, a model system comprising POPC:POPS:Cholesterol (65:5:30) was used. In the presence of cholesterol, a 100-fold higher concentration of PLNC8 αβ was required to trigger a similar CF release as in the absence of cholesterol (Fig. 2D). The same trend was observed for the individual peptides were the membrane activity of PLNC8 α was almost abolished in the presence of cholesterol (Fig. S4A,B). Cholesterol can thus protect eukaryotic cells from the lipid membrane perturbating effects of PLNC8.

Figure 2 Permeabilization of model lipid membranes by PLNC8 α and β. Release of (6)-carboxyfluorescein (CF) was recorded after exposure of liposomes composed of POPC:POPS (95:5) with (A) L-PLNC8 α, β and αβ or (B) D-PLNC8 α, β and αβ. CF-release from model systems containing 5, 25 or 50% POPG (C) or containing cholesterol (D) due to exposure to L-PLNC8 αβ. Full size image

The antimicrobial effects of both PLNC8 αβ enantiomers were rapid as indicated by the enhanced uptake of Sytox Green already after 2 min of treatment (Fig. 3A). When scrambling the peptide sequence, no bacterial membrane permeabilization was obtained, which indicate that the membrane interactions are likely folding dependent. The rapid lytic effects of PLNC8 αβ was further demonstrated on liposomes (POPC:POPS (95:5)), where PLNC8 β and PLNC8 αβ (1:1), but not PLNC8 α, caused complete lysis after 2 min (Supplementary Fig. S4). The same concentration dependent rapid permeabilization was observed for all liposomal model systems investigated (data not shown). Bacterial permeabilization by PLNC8 αβ was further verified by measuring aggregation and ATP release (Fig. 3B). At low concentrations (≤6.25 µM), PLNC8 αβ caused bacterial aggregation with minor ATP release. Increasing concentrations of PLNC8 αβ resulted in less bacterial aggregation while permeabilization was considerably enhanced, as revealed by a rapid (seconds) release of extracellular ATP.

Figure 3 Rapid bacterial permeabilization by PLNC8 αβ. (A) Uptake of Sytox Green by S. epidermidis ATCC 12228 after exposure to 5 µM of L-PLNC8 αβ, D-PLNC8 αβ or scrambled-PLNC8 αβ for 2 min, scale bar = 300 µm. (B) Aggregation (dotted line) and ATP release (solid line) were recorded to determine bacterial permeabilization by PLNC8 αβ. Low PLNC8 αβ concentration caused bacterial aggregation with minor lysis, while higher PLNC8 αβ concentration caused rapid and efficient permeabilization with no aggregation. Data from three independent experiments are presented as mean with standard error of the mean (SEM). Full size image

Antimicrobial peptides tend to adopt a more defined secondary structure when interacting with lipid membranes. The structural changes of PLNC8 αβ when interacting with a lipid model system composed of POPC:POPS (95:5) was determined using circular dichroism (CD) spectroscopy. CD spectra of both enantiomers of PLNC8 α and β, separately and when combined, showed induced structural changes when combined with the liposome model system (Supplementary Fig. S6). The enantiomers did, as expected, show mirrored spectra because of the opposite chirality of the peptides. Enantiomers of PLNC8 α only displayed minor structural changes while PLNC8 β showed a more pronounced change in structure, from random coil to a helical structure, when interacting with the liposomes. Interestingly, previous studies have indicated that PLNC8 β can adopt a β-sheet structure when interacting with liposomes with the same lipid composition but with both constituents at higher concentrations14, illustrating a structurally highly dynamic system. When combining both peptides (PLNC8 αβ), a large structural rearrangement was seen, which due to the spectral contributions from both peptides, is difficult to qualitatively and quantitatively define. In addition, PLNC8 αβ also showed tendencies to cause aggregation of the liposomal model system seen as a slight decrease in the CD intensity.

Both L- and D-enantiomers of PLNC8 αβ were tested for their antimicrobial activity on S. epidermidis. The individual peptides showed generally poor activity on S. epidermidis, while the combination of α and β resulted in a pronounced antimicrobial effect (data not shown). The inhibitory (MIC) and bactericidal (MBC) concentrations of the peptides on S. epidermidis were 6.25 and 12.5 µM, respectively, for the L-enantiomer, and 12.5 µM for the D-enantiomer.

PLNC8 αβ belongs to class IIb bacteriocins that consist of two separate peptides. Optimal antimicrobial activity is dependent on the complementary action of the two peptides. It is therefore necessary to determine the optimal molar ratio between PLNC8 α and β against Staphylococcus. While the total concentration of both peptides was kept constant, the concentrations of L-PLNC8 α and L-PLNC8 β, respectively, were varied to obtain different molar ratios. Furthermore, since L-PLNC8 β alone was shown to permeabilize both liposomes and bacteria, the concentration of L-PLNC8 α was decreased from 50 to 1.5 µM whereas L-PLNC8 β was increased from 50 to 98 µM while keeping the total concentration of peptides at 50 µM, followed by two-fold serial dilutions. Optimal inhibitory and bactericidal activity of L-PLNC8 αβ against S. epidermidis ATCC was achieved at a molar ratio of 1:1. The effects decreased when the ratio was altered towards higher concentration of β (Table 1). Subsequent experiments in the study were thus performed with a molar ratio of 1:1 (α:β).

Table 1 The molar ratio of PLNC8 α and β is critical for optimal antimicrobial activity. Full size table

In order to further verify the antimicrobial activity of L-PLNC8 αβ, the effects in a number of different Staphylococcus strains were examined. L-PLNC8 αβ was found to target the bacteria with similar effects, irrespective of the characteristics of the bacteria, including antibiotic resistance (MRSA vs MSSA) and ability to form biofilms (Table 2A). Although S. epidermidis was more susceptible than S. aureus, the recorded MIC and MBC concentrations were generally comparable between the different strains.

Table 2 Susceptibility of Staphylococcus to PLNC8 αβ. Full size table

Development of bacterial resistance against L-PLNC8 αβ was investigated after exposure of S. aureus to sub-MIC concentrations of the peptides for 20 passages. The susceptibility of S. aureus towards L- and D-PLNC8 αβ was not altered as the inhibitory and bactericidal concentrations remained unchanged at 12.5 µM and 25 µM, respectively (Table 2B).

Effect of PLNC8 αβ on attached bacteria

Several species of the genus Staphylococcus are common pathogens in nosocomial infections associated with their ability to form biofilms and persist on medical devices, such as catheters and medical implants. The concentrations of traditional antibiotics required to treat bacterial biofilms are in the range of 100–1000-fold higher compared to bacteria in suspension, which may cause severe complications during treatment23. We show rapid disruption of attached bacterial cells by L-PLNC8 αβ (Fig. 4). L-PLNC8 α alone caused minor effects, while L-PLNC8 β caused substantial disruption at the highest concentration. L-PLNC8 α and β together were most effective and caused rapid and dose-dependent disruption of surface-associated S. epidermidis.

Figure 4 PLNC8 αβ is effective against surface-associated S. epidermidis. S. epidermidis RP62A were allowed to adhere followed by removal of suspended bacteria and addition of the peptides for 1 h. (A) Absorbance measurement of detached material. The dotted line indicates the baseline (LB broth). (B) Crystal violet (CV) staining of the remaining attached bacterial material. The lower dotted line is the negative control (LB broth) and the upper dotted line is the positive control (untreated bacterial biofilm). Results from three independent experiments are presented as mean with SEM. Full size image

Sequence and length optimization of PLNC8 αβ

Truncated versions of L-PLNC8 α and β were investigated with respect to membrane activity and antimicrobial activity using both liposomes and S. epidermidis. Among the truncated forms of L-PLNC8 α, the amino acid sequence of 1–22 was found to retain a lytic activity in the liposome model systems that was comparable to the full length α-sequence (Fig. 5A). Addition of full-length L-PLNC8 β together with the different truncated α-peptides showed only minor differences compared to the effects of full-length L-PLNC8 α and β (Fig. 5B).

Figure 5 Permeabilizing activity of truncated forms of L-PLNC8 α and β. (A) CF release from liposomes was obtained with α1–29 (full length) and α1–22. (B) Addition of full length L-PLNC8 β potentiated the effects of different truncated L-PLNC8 α peptides. (C) CF release from liposomes was obtained with β1–34 (full length), β7–34, β1–20 and β7–20. (D) Addition of full length L-PLNC8 α potentiated the effects of different truncated L-PLNC8 β peptides. Quantification of 50% CF release by truncated L-PLNC8 α or β peptides, with and without L-PLNC8 α or β are indicated, n = 3. Full size image

In addition to full length L-PLNC8 β, several of the truncated β-peptides were membrane active and considerably enhanced release of liposome encapsulated CF, including truncated-β7-34, β7-20 and β1-20 (Fig. 5C). Combination of the different truncated L-PLNC8 β peptides with full length L-PLNC8 α enhanced their activity in the liposome model, with truncated-β7-34 and β7-20 showing the most pronounced effects (Fig. 5D). The amino acid sequences of full length and truncated forms of L-PLNC8 α and L-PLNC8 β are illustrated in Fig. 1, and the required concentrations for 50% CF release from liposomes are indicated (Fig. 5).

The antimicrobial effect of full length and truncated-α1–22 against S. epidermidis could only be observed in the presence of full length L-PLNC8 β (Table 3A). However, truncated β-peptides displayed inhibitory activity against S. epidermidis, which was similar to the results obtained with the liposome model, i.e. truncated-β7–34, β7–20 and β1–20 inhibited bacterial growth (Table 3B). Furthermore, combination of truncated β-peptides with full length L-PLNC8 α enhanced the inhibitory activity of truncated-β7–34. Our results prompted us to combine the active sequences of both truncated L-PLNC8 α and truncated PLNC8 β to determine their antimicrobial effects on S. epidermidis. Although the peptides were able to efficiently inhibit bacterial growth, especially the combination α1–22/β1–20, they did not show any bactericidal activity (Table 3C).

Table 3 Antimicrobial activity of truncated forms of PLNC8 αβ. Full size table

Neither the full-length peptides of both L- and D-enantiomers, nor the truncated α1–22, α1–15, β7–34, β7–20 and β1–20 were cytotoxic at relevant concentrations (<50 µM), as determined by haemolytic activity on isolated human erythrocytes (Supplementary Fig. S7A–D). A concentration of 200 µM of L-PLNC8 αβ caused 9.8% erythrocyte cell lysis after 1 hour of incubation.

Cytotoxicity of full length L-PLNC8 αβ was evaluated on human keratinocytes and quantified by measuring LDH activity after stimulation with the peptides for 24 h. The peptides showed no cytotoxic effects and the cells exhibited normal morphology compared to the untreated control (Supplementary Fig. S7E).

PLNC8 αβ permeabilized bacteria and caused morphological changes

The cause of the remarkably rapid antimicrobial effect of the two-peptide bacteriocin PLNC8 αβ on S. epidermidis shown in the Sytox Green experiments (Fig. 3A) was further investigated using both transmission and scanning electron microscopy. Planktonic bacteria were exposed to the peptides for 5 min followed by fixation and analysis. The bacteria in the untreated sample were intact as indicated by the absence of Sytox Green fluorescence (Fig. 6A). The corresponding electron micrographs also showed normal bacterial cell morphology, where the cell wall and cell membrane could clearly be distinguished in the TEM image. Although L-PLNC8 α did not appear to affect cell membrane integrity, as no Sytox Green fluorescence was detected, the electron micrographs revealed a substantial amount of bleb-formation and secretion of micro vesicles that formed complex networks and bacterial aggregation. The effects of L-PLNC8 β were, however, distinct and caused rapid and severe morphological changes. The cell wall appeared swollen and the cell membrane was irregular, and consequently, large quantities of intracellular material leaked out and the bacterial size was considerably reduced. Exposure of S. epidermidis to L-PLNC8 αβ at a 1:1 molar ratio caused severe damage. Formation of large aggregates of collapsed bacteria was observed, which was detected by the fluorescent stain Sytox Green, and the effect of L-PLNC8 αβ was concentration-dependent. Besides the apparent effect of a swollen cell wall, the inner cell membrane was completely detached and no longer associated with the inner cell wall zone. Overview images of electron microscopy show the large amount of PLNC8 αβ-induced secretion of bacterial material and formation of large aggregates (Supplementary Fig. S8).

Figure 6 PLNC8 αβ damages the integrity of bacterial cell wall and cell membrane. (A) The uptake of Sytox Green by S. epidermidis ATCC 12228 after exposure for 5 min to different peptide combinations (25 µM, except for PLNC8 αβ where two concentrations were used, 12.5 µM and 25 µM), indicates damaged cell membrane, scale bar is 300 µm. Visualization of bacterial damage with transmission electron microscopy. The cell membrane (CM) was completely detached (red arrow heads) and leakage of intracellular content is observed (black arrow heads). The thickness of the cell wall (CW) was increased (blue arrow heads). Scale bar is 200 nm. Scanning electron microscopy demonstrates further the substantial amounts of leaked material from the bacteria, particularly by PLNC8 α, while PLNC8 β and the combination of both peptides results in bacterial lysis. Scale bar is 100 nm. (B) Quantification of the fluorescence intensity of Sytox Green. (C) Quantification of the thickness of the cell wall. (D) Quantification of the area covered by bacteria and leaked material. Full size image

Furthermore, the effects of the truncated peptides β7–20 and β1–20, alone or in combination with full length L-PLNC8 α, were visualized by fluorescence microscopy and electron microscopy. The results showed morphological changes that were more prominent when the truncated peptides were combined with full length L-PLNC8 α (Supplementary Fig. S9). A large amount of secreted or leaked material from the bacteria was observed, which formed complex thread-like networks and promoted bacterial aggregation. Furthermore, to quantify the antimicrobial effect of PLNC8 αβ, fluorescence intensity of Sytox Green (Fig. 6B), thickness of the cell wall in the TEM images (Fig. 6C) and area covered by bacteria and their leaked material in the SEM images (Fig. 6D) were determined.

Antimicrobial activity of PLNC8 αβ in combination with antibiotics

The continuously increasing prevalence of antimicrobial resistance is a global threat to modern human medicine. A strategy that may restrict the selection and emergence of antimicrobial resistance is combination therapy. The antimicrobial effects of conventional antibiotics combined with sub-MIC concentrations of both L- and D-enantiomers of PLNC8 αβ were investigated. A final concentration of 3 µM of L/D-PLNC8 αβ was used and the MIC and MBC values were determined for S. epidermidis ATCC 12228 and the clinical isolate S. epidermidis 154. Both enantiomers of PLNC8 αβ reduced the MIC and MBC values of vancomycin, teicoplanin, rifampicin and gentamicin (Table 4). Susceptibility testing by the checkerboard microdilution method showed primarily additive effects between L/D-PLNC8 αβ and the different antibiotics. However, it was the D-enantiomer that showed synergistic effects with vancomycin, rifampicin and gentamicin against the clinical isolate S. epidermidis 154.

Table 4 Antimicrobial activity of PLNC8 αβ in combination with antibiotics against Staphylococcus. Full size table

The antimicrobial effect of PLNC8 αβ in combination with vancomycin, teicoplanin and rifampicin was obtained even when the final concentration of the peptides was reduced to 2 µM and 1.5 µM (data not shown). These results encouraged us to investigate the antimicrobial activity of PLNC8 αβ against S. epidermidis strains isolated from patients with prosthetic joint infections. The different strains were divided into two groups based on their resistance patterns against the glycopeptide antibiotics vancomycin and teicoplanin. Five strains were non-resistant and five strains were classified as heterogeneous glycopeptide intermediate S. epidermidis (hGISE), a group that is characterized by expressing a thick cell wall. The inhibitory activity of PLNC8 αβ was equally potent against both groups, with concentrations ranging between 6.25–12.5 µM (Table 5). However, it is obvious that the thick cell wall in hGISE renders the bacteria to be more resistant against PLNC8 αβ. The bactericidal concentrations were ≥50 µM, while the non-resistant bacteria were efficiently eliminated at concentrations ranging between 6.25–25 µM.

Table 5 Effects of PLNC8 αβ on heterogeneous glycopeptide intermediate strains of S. epidermidis (hGISE). Full size table

Furthermore, two concentrations of L-PLNC8 αβ (5 and 10 µM) were used in combination with either vancomycin or teicoplanin, and the MIC, MBC and ΣFIC values were determined for MRSA and the hGISE strains 126 and 157. Interestingly, the presence of L-PLNC8 αβ markedly reduced the inhibitory and bactericidal concentrations of vancomycin and teicoplanin (Table 6). Similar results were achieved for all strains, except for S. epidermidis 157 that was unaffected. The combined effects of L-PLNC8 αβ and antibiotics against MRSA and hGISE 157 were additive, while the effects against hGISE 126 were synergistic.

Table 6 Antimicrobial effect of PLNC8 αβ with antibiotics against resistant strains of Staphylococcus. Full size table

In addition, the truncated peptides α1–22 and β1–20 were combined together, or with either full length PLNC8 α or full length PLNC8 β, in the presence or absence of teicoplanin and rifampicin, against the clinical isolate S. epidermidis 154. The different peptide combinations did not inhibit bacterial growth (Supplementary Table S1). However, full length α/β1–20 and α1–22/β1–20 enhanced the effects of teicoplanin, while α1–22/full length β enhanced the effects of both teicoplanin and rifampicin.

In addition, we investigated whether L-PLNC8 αβ exerts inhibiting effect in a gel-like formula containing glycerol and gelatine, and after long-term storage at 4 °C. Importantly, the peptides rapidly permeabilized the bacteria in a dose-dependent manner (Fig. 7A). Furthermore, the peptides in the gel retained their antimicrobial activity after long-term storage at 4 °C (>6 months), which was tested by applying the gel on a lawn of S. epidermidis on agar plates and incubated overnight (Fig. 7B).