Antibiofilm activity of peptides and synergistic effect with antifungal drugs
The peptides (50 µg. mL-1) presented different behaviors toward the biofilm of Candida ssp (Fig. 1). Either the peptides, antifungal drugs, or combinations had any activity against the biofilm of C. parapsilosis or C. tropicalis (data not shown). The first assay was done to evaluate the ability of peptides to inhibit the biofilm formation of C. krusei and C. albicans (Fig. 1A and B). To C. krusei both peptides barely reach 10% of inhibition. ITR and NYS reached, respectively, 20 and 0% of inhibition of biofilm formation by C. krusei (Fig. 1A). In contrast, the combinations of both PepGAT (50 µg mL-1) + ITR (1000 µg mL-1) and PepKAA (50 µg mL-1) + ITR (1000 µg mL-1) led to an inhibition, respectively, of 80 and 76% of C. krusei biofilm (Fig. 1A). The combination of peptides with NYS was not effective. Regarding C. albicans NYS, PepGAT, and PepKAA inhibited, respectively, 40, 10, and 20% of biofilm formation. An interesting result was the synergism found in the combinations made by PepGAT + ITR, PepGAT + NYS, and PepKAA + NYS that inhibited, respectively, 40%, 95%, and 98% the biofilm formation of C. albicans (Fig. 1B).
Regarding the degradation of formed biofilm, no results were found to C. albicans, C. parapsilosis, and C. tropicalis (data not shown), only toward C. krusei (Fig. 1C). ITR, PepGAT, and PepKAA alone did reduce biomass of preformed biofilm of C. krusei (Fig. 1C). In contrast, NYS was able to reduce in 20% the biomass of C. albicans preformed biofilm (Fig. 1C). Except for the PepGAT + NYS combination that did not show a significant difference in biomass reduction, PepGAT + ITR, PepKAA + ITR, and PepKAA + NYS reduced in, respectively, 50%, 30%, and 15% the preformed biofilm of C. albicans (Fig. 1C).
Biofilm integrity and ROS overproduction
To evaluate how peptides alone and the combination with drugs affect the membrane of cells composing the biofilm, fluorescence microscopy was employed (Figs. 2-5). Propidium iodide (PI) is a fluorophore that binds to DNA releasing red fluorescence. However, PI only crosses the damaged cell membrane, healthy membrane blocks the movement of PI leading to no fluorescence at all. As expected, all controls made by DMSO-NaCl solution (the vehicle of peptides) presented no fluorescence, suggesting the membranes have no type of more formed (Figs. 2-5). Another interesting result was that, alone, none of the drugs (NYS and ITR) produced any kind of fluorescence (Figs. 2-5).
Regarding the synthetic peptides, PepGAT did induce fluorescence in all treatments either in the inhibition of biofilm formation although at different intensities (Figs. 2-4) and degradation of preformed biofilm (Fig. 5). The PepKAA peptides presented a different behavior. In the case of the inhibition of biofilm formation by C. krusei, PepKAA alone was not able to induce damage in the membrane to allow PI to pass by the membrane and emits fluorescence (Fig. 2). In contrast, PepKAA induced the fluorescence in the inhibition of biofilm formation by C. albicans (Fig. 4). In the case of combinations made by peptides and antifungals drugs, all of them presented fluorescence to some extent (Figs. 2-5). This is an interesting result for three reasons: 1) drugs alone did not show any fluorescence (Figs. 2-5); 2) in the case of PepKAA, which did not show fluorescence, the combination with ITR lead to a release of fluorescence (Figs. 2 and 3) in some cases, such as PepGAT+ITR (Figs. 3 and 5) fluorescence produced by the combination was higher than that produced by drugs alone, suggesting a bigger number of cell damage in the combination.
The experiments to evaluate the ROS overproduction in the biofilms revealed a different pattern than PI experiments. All controls either positive or negative did not produce any type of fluorescence indicating no ROS production (Figs. 2-5). In the inhibition of biofilm formation of C. krusei, both peptides induced ROS production at different intensities, as revealed by fluorescence. In this case, the cells treated with PepGAT were brighter than the ones produced by PepKAA. Following this pattern, the combinations made to inhibit the biofilm formation by C. krusei produced only a slight fluorescence, indicating a low level of ROS was produced (Fig. 2). In the inhibition of biofilm formation by C. albicans to ROS production, only a slight production of ROS was indicated by a faint fluorescence in the treatment with PepKAA (Fig. 4). In contrast, brighter fluorescence indicates a higher production of ROS in both PepGAT alone and combination with ITR in the degradation of preformed biofilm of C. krusei (Fig. 5), suggesting that the ROS overproduction is, indeed, an important mechanism to degrade the biofilm.
Scanning electron microscopy (SEM) analyses of biofilm
SEM images revealed severe that all control made with DMSO-NaCl solution the biofilm was in a good spherical shape, with no cracks or damage, and cells were seen with no visible damage to membrane or cell wall (Figs. 6-8). The SEM analysis revealed that the cells involved in biofilm formation (Figs. 6 and 7) and preformed biofilm (Fig. 8) were not affected by the treatment with either ITR or NYS both at 1000 µg mL-1.
SEM analysis strength the damages revealed by fluorescence microscopy in Candida cells (Figs. 2-5), which were confirmed by SEM analysis (Fig. 6-8). To inhibit the formation of C. krusei PepGAT induced several damages to cells. It was possible to see depression-like cavity on cells (Fig. 6, PepGAT and PepKAA Panels, white dashed circle) indicating damage to the cell wall, small blebs, new buds, scars on new buds and cells, and rings of truncated bud scars in the treatment with both peptides (Fig. 6, white arrowheads). In both treatments with peptides, cells present high levels of wrinkles and scars all over the structure. Additionally, in both treatment with peptides alone is possible to see a lack of constriction in the solid point of the septum (Fig. 6, white open arrows). In contrast to what was seen in the treatment with drugs alone, the combination of peptides and drugs lead to several damages to cells and thus inhibit biofilm formation. In both cases, many cells presented depression-like cavities (Fig. 6, PepGAT+ITR and PepKAA+ITR panels, white dashed circle) indicating damage to the cell wall, in addition to alterations in cell shape, wrinkles and scars all over the structure, small blebs, new buds, scars on new buds and cells, and rings of truncated bud scars in the treatment with both peptides (Fig. 6, white arrowheads), and no presence of a solid point in septum junction (Fig. 6, white open arrows).
SEM analysis revealed a different pattern in the inhibition of biofilm formation of C. albicans (Fig. 7). The controls DMSO-NaCl solution, ITR, and NYS at 1000 µg mL-1 showed no significant alteration on C. albicans cells (Fig. 7). Treatment with PepGAT induced small blebs, new buds, scars on new buds and cells (Fig. 7, white arrowheads). In contrast, the treatment with PepKAA is possible to damage all cells. Cells stick together but all the present bad conformation and with pieces of other cells on the top of them (Fig. 7, panel PepKAA). SEM analysis revealed that the combination of PepGAT + ITR killed almost all cells (Fig. 7), and the leftovers are completely damaged unable to form biofilm. The combination made of PepKAA + ITR is far less effective than PepGAT + ITR causing scars and wrinkles on cells, abnormal shape, and it is also possible to see the presence of small blebs, new buds, scars on new buds and cells, and rings of truncated bud scars in the treatment with both peptides (Fig. 7, white arrowheads). The combinations of both peptides + NYS were most efficient than with combination with ITR. In both cases all cells were dead, and it was only found isolated cell completely damaged, with the signal of loss of internal content and with no ability to form biofilm at all (Fig. 7, PepGAT and PepKAA panels).
In the degradation of C. krusei preformed biofilm, SEM analysis of ITR-treated biofilm revealed no damage at all on the biofilm (Fig. 8). In the treatment with peptides alone or combination with ITR the biofilm constituting cells presented damage to the cell wall, loss of internal content, small blebs, new buds, many scars, and wrinkles on new buds and cells (Fig. 8 white arrowheads), and rings of truncated bud scars in the treatment with both peptides (Fig. 8, white open arrows).
As shown in a previous study (Souza et al., 2020) the synthetic peptides had no hemolytic activity against any human blood type tested (Table 1), even at 50 μg. mL-1. In contrast, NYS at 1000 μg. mL-1 caused 100% hemolysis in all human blood types and ITR at 1000 μg. mL-1 caused 80, 75, and 69% of hemolysis, respectively, to Type-A, B, and O of red blood cells (Table 1).
In general, the combination of synthetic peptides with antifungal drugs decreased their hemolytic effect (Table 1). The combination of PepGAT with NYS resulted in a hemolytic effect of 54, 43, and 12%, respectively, to Type-A, B, and O of red blood cells, and combination of PepGAT with ITR caused in 17, 45, and 43% of hemolysis, respectively, to Type-A, B, and O of red blood cells (Table 1). The combination of PepKAA with NYS hemolyzed 15, 10, and 21%, respectively, of Type-A, B, and O of red blood cells, whereas the combination of PepKAA with ITR 21, 34, 12%, respectively, of Type-A, B, and O of red blood cells (Table 1).
Molecular docking between peptides and drugs
PepGAT interacts with ITR and NYS with a score of -4.7 kcal.mol-1 (Fig. 9A and B). The amino acid residues Gly1, Ala2, and Asn8 of PepGAT establish Van der Walls interactions with ITR, while the Arg5 residue establishes 4 Pi-Alkyl interactions with the dichlorophenyl (4.3 Å), triazole (4.7 Å), methoxyphenyl (4.9), and piperazine groups (4.2 Å). Ile4 and Ala6 residues of PepGAT interact through Pi-Alkyl interactions with the chlorophenyl (3.6 Å) and phenyl (3.5 Å) groups of ITR, respectively (Fig. 9A and C). The interaction of PepGAT with NYS is through hydrogen bonds with residues Asn8 (1.9 Å), Ser9 (2.7 Å), and Arg10 (2.0 Å), as well as through van der Walls interactions with residues Gly1, Ile4, Arg5, and Val7 (Fig. 9B and D).
PepKAA showed a docking score of -5.8 and -5.4 kcal.mol-1 with the drugs ITR and NYS, respectively (Fig. 9E and F). PepKAA-ITR complex is supported by Pi-Alkyl interaction of Arg5 and Lys1 interactions with the dichlorophenyl and triazole groups of ITR. Lys7 from PepKAA performs three Pi-Cation interactions with piperazine (5.0 Å), phenyl (3.8 Å), and triazolone (3.7 Å) groups from ITR. PepKAA residues Asn4, Tyr8, and Phe9 interact with ITR through Pi-Sigma (2.5 Å), Pi-Pi stacked (4.1 Å) and van der Walls interactions, respectively (Fig. 9E and G). In turn, the interaction of PepKAA with NYS is supported by hydrogen bonds of residues Arg5 (2.0 Å) and Tyr8 (2.9 Å) of PepKAA, as well as van der Walls interactions with residues Asn4 and Phe9. It was also noted 2 Pi-Alkyl interactions between the residues Lys1 (4.8 Å) and Ala2 (4.3 Å) of the peptide with NYS (Fig. 9F and H).