2.1 Sequence alignment and peptide selection
The sequences of seven snake venom peptides having related antimicrobial activities (2) deposited in the Antimicrobial Peptide Database (APD) website (http://aps.unmc.edu/AP) were aligned using Clustal X software (24). After alignment analysis of the common sequences of these seven peptides, 17 small peptides were synthesized (Fig. 1).
2.2 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 small peptides derived from snake venom. For that, we chose two different species of bacteria, one Gram-negative (Pseudomonas aeruginosa PAO1), and one Gram-positive (S. epidermidis ATCCC 35984). This selection was based on their biofilm production capabilities, and both strains are known to be good models for the study of biofilm formation and structures (9, 25). For screening, a crystal violet stain protocol was used with or without different concentrations of peptides. No effects were detected on biofilm formation in P. aeruginosa (Fig. S2). On the other hand, peptides 1, 2, and 3 demonstrated strong activity on the S. epidermidis biofilm. After 24 hours of exposition 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 to 3, respectively (Fig. 2A).
Peptide 2 demonstrated greater antibiofilm activity than peptides 1 and 3. The considerable reduction of 63% of the biofilm mass 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 precultured 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).
2.3 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’s presence causes a huge decrease in bacterial growth as compared to the control. The same result was seen in the CFU experiments. After 1, 2, or 4 h incubation with pseudonajide, the number of viable cells vastly decreases as compared to the control conditions.
2.4 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), and then performed confocal microscopy. Cells were incubated with 25 µM FITC-tagged pseudonajide for 1, 4, or 24 h. After incubation, confocal microscopy showed that the molecule is located around or inside the bacterial cell, but not in the biofilm matrix (Fig. 4). Another important finding was the reduction of fluorescent cells over time, with decreased peptide-tagged cell counts after 4 and 24 h incubation.
To confirm that the interaction occurs between pseudonajide and S. epidermidis cell walls and membranes, we did LIVE/DEAD experiments. Because it was demonstrated that propidium ions can enter on cell with high membrane potential (26). Cells were cultured for 4 h with or without 25 µM pseudonajide. Confocal microscopy image analysis demonstrates an increase in cell death when in the presence of pseudonajide. Moreover, statistical analysis shows that there is a significant decrease in the number of impermeable cells when the peptide is present (Fig. 5). These data suggest that pseudonajide are interfering on cell walls and membranes integrity.
2.5 Pseudonajide damages epidermidis cell walls and membranes
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 and 24 h incubation. A final point that must be highlighted is that some extravasated material is present surrounding the shrunken cells, and this can be seen in the same figure in the cells that have arrows. None of these characteristics were seen in the control.
After the SEM experiments and analysis, two questions remained unanswered: how does pseudonajide cause cells shrink? Is there any damage to the cell walls 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 exposition (Fig. 7, dark arrows). Specifically, after 4 and 24 h of peptide exposition, the cell wall is not intact, and the cell sizes are completely different than those of the control. Moreover, in the peptide-exposed cells, the material inside the cytoplasm is condensed (Fig. 7).
2.6 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 walls of Gram-positive bacteria (27) and to influence membrane fluidity when engaging with the phospholipid bilayer (28). 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 (29). To investigate this, real-time quantitative PCR tests were done, with S. epidermidis cultured in the same conditions as the previous experiments. However, due to pseudonajide’s high antimicrobial activity, we decided to use a lower concentration. We therefore tested a series of dilutions ranging from 3 to 100 µM of the molecule at 4 h incubation. We found that a concentration of 6.25 µM is enough to inhibit about 50% of growth as compared to the control (Fig. 8A). To investigate the relative expression levels of genes when bacterial cells are cultured in subtoxic concentrations of this peptide, we selected three genes that code for teichoic acid molecules. By testing these, we were able to clearly see that cells cultured in the presence of 6.25 µM peptide had higher expression levels of UgtP, LtaA, and LtaS genes (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, leading to cell permeability. The same extracted RNA was used for biofilm-related gene expression analysis. We chose nine genes related to biofilm formation for expression analysis: AtleE, agrC, aap, EmbP, icaA, leuA, saeR, saeS, and sarA. No significant differences in expression were observed under control and peptide conditions for these nine genes (Fig. 8C).
One of main challenges in the development of antimicrobial peptides is their potential toxicity to human cells (30, 31). 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 in a concentration of 25 µM pseudonajide, there was no decrease in the living cell counts as compared to the control conditions (Fig. 9), demonstrating that pseudonajide is not cytotoxic to human cells.