- Snake venom antimicrobial peptides present highly conserved motifs
A total of 170 antimicrobial peptide sequences from ant, bee, centipede, cone snails, scorpion, snake, spider or wasp venoms were analyzed. Sequence alignments within each taxonomic group were performed using ClustalX (Fig. 1 and Additional files 1-7). The alignment of snake sequences (Naja atra - Cathelicidin NA-CATH; Bothrops atrox - Batroxicidin; Crotalus durissus terrificus - Crotalicidin; Pseudonaja textilis - Cathelicidin Pt_CRAMP1; Ophiophagus hannah - Cathelicidin OH-CATH; Ophiophagus hannah - Cathelicidin OH-CATH30, and; Bungarus fasciatus - Cathelicicin-BF) revealed a greater number of conserved motifs among snake peptides than that found in other groups . Subsequently, 17 conserved amino acid sequences and their close variants (Fig. 1) were selected as derived peptides for antibiofilm and antimicrobial activity evaluations.
- Peptides 1, 2, and 3 have antibiofilm activity in epidermidis
The first aim of this work was to perform a screening for antibiofilm activity in 17 synthetic small peptides derived from snake venom sequences. For that, we chose two different species of bacteria, one Gram-negative (Pseudomonas aeruginosa PAO1), and one Gram-positive (S. epidermidis ATCCC 35984). The selection took into account their biofilm production capabilities, and their recognized value as good models for the study of biofilm formation and structure (12, 27). For screening, a crystal violet stain protocol was used in the absence or presence of peptides at different concentrations. No effects were detected on biofilm formation by P. aeruginosa (Additional file 8). On the other hand, peptides 1, 2, and 3 demonstrated strong activity against S. epidermidis biofilm. After 24 hours of exposure to different concentrations, there was a considerable reduction in biofilm mass (Fig. 2A). At a concentration of 100 µM, the biofilm mass was reduced by 77%, 95%, and 78% for peptides 1, 2, and 3, respectively (Fig. 2A).
Peptide 2 demonstrated greater antibiofilm activity than peptides 1 and 3. The considerable reduction of 63% in biofilm formation in the presence of 25 µM of peptide 2 led us to select that particular molecule at that specific concentration for the following experiments. We named the peptide “pseudonajide” after the name of the snake it was derived from, Pseudonaja textilis. In order to test its biofilm eradication activity, we pre-cultured S. epidermidis cells for 24 h, adding pseudonajide to pre-formed biofilm and incubating for another 24 h. The final quantification of biofilm mass showed a reduction of about 30% in the presence of the molecule (Fig. 2B).
- Pseudonajide has antimicrobial activity against epidermidis
We decided to test the antimicrobial activity over a shorter period of time, because no difference had been observed after 24 h. Growth and colony-forming unit (CFU) tests were performed. Cells were incubated in the same conditions as for the antibiofilm tests, with or without 25 µM pseudonajide. After 1, 2, 4, and 24 h incubation, we measured the optical density at 600 nm (OD600) and assessed the CFU counts. Fig. 3 shows clearly that the molecule caused a huge decrease in bacterial growth as compared to the control. Accordingly, the number of viable cells determined by CFU counts decreased after 1, 2, or 4 h of incubation with pseudonajide.
- Pseudonajide binds to the cell wall and membrane, causing permeabilization
To better understand pseudonajide’s binding site, we synthesized peptides tagged with fluorescein isothiocyanate (FITC) for use in confocal microscopy experiments. Cells were incubated with 25 µM FITC-tagged pseudonajide for 1, 4, or 24 h. After incubation, confocal microscopy showed that the molecule was located around or inside the bacterial cell, but not in the biofilm matrix (Fig. 4). Another important finding was the reduction in the number of fluorescent cells over time, with decreased peptide-tagged cell counts after 4 h and 24 h incubation.
To confirm that the interaction occurs between pseudonajide and S. epidermidis cell wall and membrane, we did LIVE/DEAD experiments. It is demonstrated in the literature that propidium ions can enter cells with high membrane potential (28). Cells were cultured for 4 h with or without 25 µM pseudonajide. Confocal microscopy image analysis demonstrated an increase in cell death when in the presence of pseudonajide. Moreover, statistical analysis shows that there was a significant decrease in the number of impermeable cells (green) when the peptide is present (Fig. 5). These data suggest that pseudonajide is interfering with cell wall and membrane integrity.
- Pseudonajide damages epidermidis cell wall and membrane
To check for morphological changes in S. epidermidis cells after exposure to the peptide, microscopy experiments were then performed after 1, 4, and 24 h incubation with or without 25 µM pseudonajide. We chose to approach this in two distinct ways, using both scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM experiments were performed by culturing the cells in the same conditions as before, with plastic slides added to the culture well for cell adherence. Our most notable result was that after 4 and 24 h incubation, cell adhesion was much weaker when cultured with the peptide, although no difference was observed after just 1 h (Fig. 6). Another important characteristic we noted was that several cells exposed to this molecule had a shrunken morphology and were smaller than non-exposed cells (Fig. 6, white arrows). Again, this morphology was only noted after 4 h or 24 h incubation. A final point that must be highlighted is that some extravasated material was present surrounding the shrunken cells, as was apparent in the SEM images (Fig 6, white arrows). None of these phenomena were observed in untreated cultures. After the SEM experiments and analysis, two questions remained unanswered: how does pseudonajide causes cells to shrink? Is there any damage to the cell wall or to the membrane? To address these questions, we performed TEM. This imaging method allows for the analysis of cell component ultrastructures and thus the analysis of cell wall and membrane integrity. Analysis of the resulting images demonstrated disrupted cells after pseudonajide exposure (Fig. 7, dark arrows). Specifically, after 4 and 24 h of peptide exposure, the cell wall is not intact, and the cell sizes are completely different than those of the control. Moreover, cytoplasmic material looks condensed in peptide-exposed cells. (Fig. 7).
- Pseudonajide increases the expression of genes coding for teichoic acid synthesis
The results obtained from microscopy analysis led us to hypothesize that pseudonajide acts on cell walls and membranes. Indeed, cationic peptides are known to be able to interact with the cell wall of Gram-positive bacteria (29) and to influence membrane fluidity when engaging with the phospholipid bilayer (30). One of the first molecules that is supposed to interact with cationic peptides is teichoic acid, a negatively charged molecule present in Gram-positive cell walls (31). To investigate this, real-time quantitative PCR tests were done, with S. epidermidis cultured in the same conditions as the previous experiments.
Upon testing 4 h incubation with serially diluted doses of pseudonajide (3 µM to 100 µM), the concentration of 6.25 µM, sufficient to inhibit 50% of bacterial growth (Fig 8), was selected for gene expression studies. Three genes involved in teichoic acid biosynthesis (UgtP, LtaA and LtaS) were tested and showed increased expression levels in the presence of sub-lethal concentration of pseudonajide (Fig. 8B). These results led us to hypothesize that pseudonajide interacts with teichoic acid in the S. epidermidis cell wall, causing a strong interaction with this structure, and leading to cell permeability. The same extracted RNA sample was used for expression analysis of nine biofilm-related genes: atlE, agrC, aap, embP, icaA, leuA, saeR, saeS, and sarA. No significant differences in expression were observed between control and peptide-treated conditions for these nine genes (Fig. 8C).
- Pseudonajide interacts with lipoteichoic acid (LTA) in vitro
To probe the interaction between pseudonajide and LTA, we performed binding experiments by fluorescence polarization (FP) using FITC-tagged pseudonajide. Peptide concentration was set constant to 5 μM while LTA monomer concentration varied from 2.5 μM to 5 mM. Titration series was set in triplicates. FITC-pseudonajide FP increased with LTA concentration, an indication of binding between the two molecules (Fig. 9). The LTA concentration range used allowed us to observe an almost complete titration curve. Mid titration occured at around 50 μM LTA. The sigmoid rise is sharper than expected for a 1:1 binding model, indicating cooperative binding of more than one molecule of LTA per peptide. Albeit observed in vitro, these results strongly support the conclusion derived from microscopy and RT-qPCR that pseudonajide interacts with the cell walls and membranes of Gram-positive bacteria by binding to lipoteichoic and teichoic acids. This would also explain why the peptide is not active against Gram-negative bacteria like Pseudomonas aeruginosa.
- Pseudonajide is not cytotoxic to human cells
One of the main challenges in the development of antimicrobial peptides as therapeutics is their potential toxicity to human cells (32, 33). We therefore performed toxicity tests using seven human cell lines: HuH7 (hepatocellular carcinoma); Caco-2 (colorectal adenocarcinoma); MDA-MB231 (breast adenocarcinoma); HCT116 (colorectal carcinoma); PC3 (prostatic adenocarcinoma); NCL-H727 (lung carcinoma); and MCF7 (breast cancer). After 24 h incubation with pseudonajide at 25 µM, there was no decrease in live cell counts compared to the control conditions (Fig. 10), indicating that pseudonajide is not cytotoxic to human cells.