Pep5-Cpp, a Cyclin D2-Derived Antimicrobial

TL;DR

A new antimicrobial peptide derived from cyclin D2 shows promise against bacteria and fungi with minimal toxicity.

Contribution

Pep5-Cpp, a cyclin D2-derived peptide, demonstrates antimicrobial activity and selectivity against pathogens.

Findings

Pep5-Cpp showed antimicrobial activity against Staphylococcus aureus, Candida albicans, and Aspergillus niger with varying MIC values.

Only ΔC3-Pep5-Cpp inhibited biofilm formation in Staphylococcus aureus by over 50%.

The peptides did not cause hemolysis at their minimum inhibitory concentrations.

Abstract

Peptidomic studies in HeLa cells identified the antitumoral activity of Pep5, and our group observed that coupling Pep5 to a carrier peptide (Cpp) provides antimicrobial potential. This study evaluated the antimicrobial activity of Pep5-Cpp and its derivatives (ΔC3-Pep5-Cpp and ΔN-Pep5-Cpp) and investigated their potential mechanisms of action. For Staphylococcus aureus, MIC values were 6.25 µM for Pep5-Cpp and ΔC3-Pep5-Cpp and 12.5 µM for ΔN-Pep5-Cpp. Against Candida albicans, Pep5-Cpp showed a MIC of 3.125 µM, while both derivatives presented 6.25 µM. For Aspergillus niger, MIC values were 1.56 µM for Pep5-Cpp and ΔN-Pep5-Cpp, and 25 µM for ΔC3-Pep5-Cpp. Pep5-Cpp interacted with S. aureus DNA, increased fluorescence in S. aureus, and showed no change in C. albicans. Only ΔC3-Pep5-Cpp inhibited biofilm (>50%). Microbicidal assays showed that high concentrations were needed to kill S. aureus (>100 µM), while lower concentrations completely inhibited C. albicans (<6.25 µM). In the hemolytic assay, the peptides at their MIC values did not induce measurable hemolysis. These findings indicate that Pep5-Cpp and its derivatives have antimicrobial potential and selectivity, supporting future pharmacological development.

Figures

Full text

1. Introduction

The introduction of antibiotics revolutionized infection treatment and represented a major advancement in public health. Currently, however, this progress is threatened by limited access to essential antibiotics in many low- and middle-income countries, as well as by the global rise in resistance to antimicrobial therapies [1]. Moreover, the inappropriate use and subtherapeutic administration of these drugs contribute to the development and spread of antimicrobial resistance [1,2]. This resistance problem also affects clinically relevant fungi, against which available antifungal agents often exhibit limited efficacy [3]. Antimicrobial resistance is characterized by the ability of pathogens to survive when exposed to antibiotics currently used in clinical practice that can inhibit, restrict their proliferation, or eliminate them [4]. Consequently, there is a need for new therapeutic alternatives to combat these pathogens, among which antimicrobial peptides have stood out as promising candidates.

Antimicrobial peptides (AMPs) are a class of molecules naturally produced as the first line of defense by multicellular organisms [5], and are widely distributed across diverse biological groups, including bacteria, archaea, protists, fungi, animals, and plants [6]. These molecules exhibit broad activity, not only directly killing bacteria, yeasts, fungi, viruses, and even cancer cells, but also modulating immune responses relevant to normal homeostasis [5].

Goldberg and collaborators demonstrated in a recent study that there is a role for the proteasome in the constitutive and bacterial-induced generation of defense peptides that prevent bacterial growth both in vitro and in vivo [7]. The peptides studied were cleaved by the proteasome and characterized as antimicrobial peptides. An in silico prediction by this same group of researchers identified hundreds of other cationic peptides that are potential defense peptides generated by the proteasome [7].

Among these peptides, Pep5 (WELVVLGKL) is particularly noteworthy. Derived from protein degradation mediated by the ubiquitin–proteasome system, Pep5 was identified and characterized as a cell death inducer in tumor cells. Initially detected in HeLa cell extracts, this peptide fragment had its intracellular production intensified during the S phase of the cell cycle. Its protein of origin, cyclin D2, needs to be degraded by the proteasome during the G1/S transition to allow cell cycle progression. Furthermore, the amino acid sequence of Pep5 is exclusive to cyclin D2, which suggests that the peptide is likely generated endogenously by the proteasome [8].

Sensitivity to Pep5-Cpp was markedly higher in human MDA-MB-231 breast cancer cells synchronized at the G1/S boundary or in the S phase compared with asynchronous cultures. Under these synchronized conditions, Pep5-Cpp (WELVVLGKL-YGRKKRRQRRR) triggered a sustained activation of ERK1/2, evidenced by prolonged phosphorylation of the kinase. Affinity chromatography coupled with mass spectrometry revealed that only two proteins—CLIC1 and plectin—consistently associated with Pep5-Cpp in both asynchronous and synchronized cells. These interactions likely underlie the persistent ERK1/2 phosphorylation and the cytoskeletal alterations observed after treatment, including the disruption of stress-fiber architecture in MDA-MB-231 cells [9].

When covalently linked to a cell-penetrating peptide also called Cpp, in this case with the sequence YGRKKRRQRRR, Pep5 induced cell death in all tumor cell lines investigated, with variable efficacy. Initially, this activity was observed in HeLa cells, in which the peptide was reintroduced via Cpp conjugation. Based on these results, it was investigated whether the effect was restricted to HeLa cells or whether it also occurred in other tumor cell lines. It was then observed that Pep5–Cpp induced cell death in all cell lines tested, although with different levels of efficacy [8].

A later study also demonstrated that Pep5–Cpp was able to induce cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi, in addition to decreasing the percentage of infected cells without causing any detectable toxic effects in mammalian host cells [10]. Collectively, these findings indicate promising therapeutic potential and reinforce the idea that peptides derived from intracellular proteolytic processing, including those generated by proteasomes, can act as bioactive molecules with therapeutic relevance.

Peptides with antitumor activity, especially cationic ones, may exhibit cross-antimicrobial activity due to their affinity for negatively charged membranes, a characteristic common to both cancer cells and bacteria [11,12,13]. Bacterial membranes are negatively charged because they contain lipids such as cardiolipin, phosphatidylglycerol, or phosphatidylserine and, in the specific case of Gram-negative bacteria, anionic lipopolysaccharides. The electrostatic attraction between anionic lipids and cationic AMPs allows cationic peptides to bind to bacterial membranes [14].

In cancer cells, the presence of anionic molecules such as sialylated gangliosides, phospholipid phosphatidylserine (PS), O-glycosylated mucins, and heparin sulfate results in a net negative charge on the outside of the plasma membrane. This contrast is important, especially considering that healthy mammalian cells have a cell membrane with a zwitterionic nature. This shows that in addition to antitumor peptides having antimicrobial potential, they are also selectively toxic, not reaching healthy mammalian cells [11,15]. The cationic characteristic is therefore presented as the main requirement for the antimicrobial activity of the peptide, and its specific sequence would be linked to the spectrum of action [13].

Considering the cationic nature of Pep5-Cpp and its history of antitumor and antiparasitic efficacy, we hypothesized that this peptide also has antimicrobial activity. In this work, we investigated the antimicrobial potential Pep5-Cpp and its variants (ΔC3–Pep5-Cpp, ΔN–Pep5–Cpp) against four microorganisms from distinct pathogenic groups: Pseudomonas aeruginosa (Gram-negative bacteria), Staphylococcus aureus (Gram-positive bacteria), Candida albicans (yeast-like fungus), and Aspergillus niger (filamentous fungus). In addition, we aimed to elucidate the antimicrobial action mechanisms of the peptide in two of these targets: S. aureus and C. albicans.

2. Results

2.1. Predicted Physicochemical Parameters of the Peptides

In order to obtain predictions about the structure and physicochemical properties of the peptides of interest in this study, bioinformatics tools from Pepcalc (Innovagen, Lund, Sweden) and ProtParam (ExPASy) [16,17] platforms were used, and the results are presented in Table 1.

The Cpppeptide has a molecular weight of 1559.85 Da and is composed of 11 amino acid residues in the following order: YGRKKRRQRRR. Previously, this peptide was described as a short cationic peptide from HIV and its respective TAT protein, with cell penetration properties [18,19]. Pep5-Cpp, the main peptide of interest in this work, had its molecular weight calculated at 2598.15 Da and is formed from the fusion of the sequence of interest (WELVVLGKL) to Cpp, obtaining the following sequence of 20 amino acid residues: WELVVLGKLYGRKKRRQRRR.

Observing the results for the modified fractions of this peptide, ΔC3-Pep5-Cpp (WELVVLYGRKKRRQRRR) and ΔN-Pep5-Cpp (ELVVLGKLYGRKKRRQRRR) are composed of 17 and 19 amino acid residues, respectively, with molecular weights determined at 2299.76 Da and 2411.93 Da. In its conformation, the coupling with Cpp was maintained at the C-terminus, and strategic deletions were introduced: at the C-terminal ends (ΔC3), three amino acid residues were removed, or at the N-terminal (ΔN), one residue was removed from the Pep5-derived fragment [8].

When the theoretical isoelectric point (pI) property is analyzed, the value for the four peptides ranges from 12.12 to 12.31, suggesting an alkaline predominance. This characteristic is confirmed by the presence of arginine and lysine residues, which provide a net positive charge between 8 and 9.

2.2. Antimicrobial Activity—Liquid Medium Inhibition Assay

To characterize the four peptides for their antimicrobial properties, the minimum inhibitory concentration (MIC) assay was performed using the liquid microdilution method. The four peptides in this present study were serially diluted and then incubated with strains of Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (MDM8), and Aspergillus niger (A296). The resulting Table 2 shows the following:

The values indicated represent the lowest concentration of peptides used, which was able to inhibit the turbidity of the medium measured at an OD of 595 nm. As the table shows, the Cpp peptide exhibits antimicrobial activity against the microorganisms tested, but this activity occurs at the highest concentrations, except for P. aeruginosa, which may indicate that the greater inhibitory effect of the other peptides on this microorganism is linked to binding with Cpp. It is worth noting that this experiment was performed only in technical triplicate for P. aeruginosa.

As the table shows, the main peptide of interest and its minor fractions demonstrated inhibition of microbial growth at low concentrations. Pep5-Cpp and ΔN-Pep5-Cpp in particular showed a significant reduction in microbial proliferation at low inhibitory concentrations. The peptide ΔC3-Pep5-Cpp showed moderate efficacy against S. aureus and C. albicans, but showed lower activity against A. niger.

2.3. Kinetic Evaluation of Microbial Growth Under Peptide Treatment

Considering experimental criteria and the availability of strains, two of the four microorganisms evaluated in the MIC assay were selected for further analyses: one bacterial (Staphylococcus aureus) and one fungal (Candida albicans) representative.

After identifying the minimum concentrations required to inhibit microbial growth, curves were analyzed. Figure 1 shows that Pep5-Cpp and ΔC3-Pep5-Cpp, with a three-amino-acid deletion at the C-terminus, at concentrations equivalent to their MICs, were able to almost completely inhibit the growth of S. aureus throughout the 24 h incubation period, maintaining optical density (OD) values close to those observed in the antibiotic-treated control, demonstrating that their microbial growth inhibition time exceeded the antimicrobial activity assay time.

The ΔN-Pep5-Cpp peptide, on the other hand, which has a deletion at the N-terminus, presented an inhibition profile closely resembling that of the original peptide. However, after 18 h, it showed evidence of increased microbial growth through increased OD, indicating a possible impairment of antimicrobial activity over time. Nevertheless, the peptide allowed a delay in the growth curve when compared to the untreated control.

When analyzing the curve data for C. albicans, the pattern for the three peptides is quite similar. Figure 2 shows that all three peptides, at their minimum inhibitory concentration, maintain low OD values, even after 24 h of experimentation. The curve for all peptides remains below the values for the antifungal agent amphotericin B used. The growth control containing untreated yeast, in contrast, showed a consecutive increase in OD until the end of the experiment, reflecting continued growth. Unlike what was observed for S. aureus, the modified smaller fractions of the original peptide, ΔC3-Pep5-Cpp and ΔN-Pep5-Cpp, maintained the same inhibition pattern against C. albicans.

2.4. Determined Minimum Bactericidal and Fungicidal Concentration (MBC/MFC)

The kinetic graphs suggest that the peptides’ activity is microbicidal, as they cause inhibition similar to antibiotics and antifungals throughout the 24 h period, with the exception of ΔN-Pep5-Cpp for S. aureus. Therefore, an assay was performed to analyze bactericidal and fungicidal concentrations over time.

The bactericidal and fungicidal effects were impaired in this work starting from liquid culture assays immediately after determining microbial growth kinetics. After exposure to the peptides, the culture medium was replaced with fresh medium without peptides, and microbial growth was monitored by optical density to assess the ability of surviving cells to recover and resume growth. Consequently, the initial optical density values increased between conditions, reflecting residual growth from the growth kinetics assay rather than differences in the initial inoculum. A lower peptide concentration at which no visible growth was observed over time was considered indicative of loss of microbial targets. Table 3 indicates the concentrations of the peptides of interest that caused microbial death for S. aureus and C. albicans.

As the table shows, the peptides had microbicidal effects in different concentrations for the two types of microorganisms. For S. aureus, high molar concentrations of the peptide were required to inhibit subculture growth, resulting in the death of the bacteria present in the medium. For C. albicans, low concentrations, including those corresponding to the MIC, were able to inhibit yeast growth.

Figure 3 illustrates that Pep5-Cpp and ΔC3-Pep5-Cpp retain activity against S. aureus regardless of a twofold elevation in MIC values. A delay in the increase in the OD is observed at both doses; the OD initially takes a long time to rise, stopping growth after reaching a stable plateau around 18 h. The twofold MIC was also used in this analysis because the MIC does not necessarily eliminate microorganisms, but only prevents their proliferation, requiring a slightly higher concentration. In this case, it is possible to note that even the increased concentration still allowed microbial growth, albeit delayed compared to the control.

For ΔN-Pep5-Cpp, the curve illustrates a more pronounced growth restriction at twice the MIC. While at the MIC, bacterial growth increases from time 0; at the 2× MIC curve, growth only increases around 8 h. The results suggest that concentrations tested had only an inhibitory effect, slowing microbial growth over time but without completely eradicating all microorganisms capable of multiplication. According to Table 3, the concentrations required to eliminate microbial growth in the subculture were much higher, being around 200 µM.

Analyzing C. albicans, the pattern of microbial inhibition and killing changes. The curves indicate that Pep5-Cpp and its lower active fractions exhibit antifungal activity against C. albicans at both the MIC and 2× MICs. Although treatment with Pep5-Cpp at 2× MIC did not result in a noticeably greater inhibitory effect compared to MIC in the kinetic assay, the standard deviation of the MIC curve overlaps with the growth levels observed at 2× MIC. Therefore, no statistically significant difference can be inferred between these concentrations. The growth curves for all three peptides maintain stable optical density values very close to zero throughout the 24 h period. These data suggest that the MIC is sufficient to promote complete growth inhibition, corroborating the peptide’s fungicidal potential (Figure 4).

2.5. Evaluation of the Amount of Biofilm Inhibition

Quantitative analysis of biofilm formation inhibition was performed with concentrations of half and twice the MIC for the peptides Pep5-Cpp, ΔC3-Pep5-Cpp, and ΔN-Pep5-Cpp. Table 4 presents the percentage inhibition values of biofilm formation. The peptide Pep5-Cpp exhibited low activity at all tested concentrations, and the ΔN-Pep5-Cpp variant showed a similar profile. This variant also showed low overall activity and no clear dose–response relationship. On the other hand, the ΔC3-Pep5-Cpp variant showed a dose-dependent effect, characterized by a progressive increase in inhibition as the concentration rose, reaching approximately 43% at 2× MIC. This pattern could indicate that C-terminal deletion favored the interaction with the cell, resulting in greater antimicrobial efficacy. Taken together, the data suggest that modifications at the C-terminal end of Pep5-Cpp can optimize its action against biofilms.

2.6. Assessment of Membrane Damage in Microorganisms

In order to elucidate the possible mechanisms of action, considering the cationic characteristic of the peptides of interest and the possibility of interaction with the membrane, the membrane permeability test and fluorescence and scanning electron microscopy images were performed to correlate the results.

The graphs in Figure 5 show the membrane permeabilization of S. aureus over time after treatment of bacteria from cultures grown with the MIC and 2× MIC of the peptides. The positive control represents Sytox Green permeabilization, with increased fluorescence levels over 18 min following isopropanol treatment, while the negative control represents the absence of permeabilization, with low fluorescence values resulting from buffer treatment.

Analyzing the curves for the four peptides, initially for S. aureus, it can be observed that Cpp, even at high concentrations, did not promote an increase in fluorescence over the 18 min when compared to the negative control. This indicates a lack of permeabilization of the bacterial membrane. Fluorescence microscopy images corroborate this result by showing the absence of green fluorescent bacterial cells. It should be noted that, due to this result and the fact that Cpp was used as a comparative control, its use in scanning electron microscopy was discontinued, as the high concentration required would have exceeded the available supply.

When moving on to the main peptide of interest, Pep5-Cpp, a more pronounced increase is observed with the treatment that used 2× MIC, but there is still a large increase in the treatment that used the MIC value. Fluorescence microscopy images confirm this result by showing intensely fluorescent cells, as the Sytox Green used here binds to the microorganism’s genetic material only when there is sufficient membrane damage for the fluorophore to permeate.

In the case of ΔC3-Pep5-Cpp, both concentrations produced increasing and comparable levels of membrane permeabilization over the 18 min signal capture period. Again, dark-field images of fluorescent cells indicate the same result, displaying greenish fluorescence. Finally, for the ΔN-Pep5-Cpp peptide, a slight increase in membrane permeabilization occurs at 2× MIC, while MIC values remain low, albeit increasing. Here, the fluorescence images show permeabilized cells in both cases, which may indicate delayed permeabilization, since the experiments were performed sequentially.

Analyzing the SEM images, morphological changes were observed in the bacterial cells treated with the peptides compared to the controls. In this study, photos were taken after 30 min of incubation with and without the treatment and in zero time of incubation. Although referred to as time zero, the samples were exposed to the peptides before to SEM preparation. The time required to prepare, fix, wash, and dry the blade already allows for sufficient interaction to cause initial changes.

In the positive controls, both with and without 30 min incubation, isopropanol treatment revealed numerous bacterial aggregates and a large amount of extracellular excreted material, including amorphous debris and elongated structures. The negative control, treated with PBS buffer, revealed cocci-shaped bacteria in pairs, little debris, and a relatively clean laminar surface, indicating the morphological integrity typical of viable cells.

Evaluating the treatments, the Pep5-Cpp group displayed debris and cellular aggregates even before incubation, suggesting that the treatment had already affected the bacterial cells. The ΔC3-Pep5-Cpp variant showed a slide with less debris than the unincubated group; however, morphological changes were still observed after 30 min, including spherical structures and cellular fragments.

Finally, treatment with ΔN-Pep5-Cpp showed a lower degree of cell aggregation, but debris were still present with little visible difference between incubation times.

In the second analysis (Figure 6), this time for C. albicans, the graphs show a similar profile for the four peptides. All showed curves below the negative control level, which is reaffirmed in the fluorescence microscopy images by the absence of yeast cells positive for membrane permeabilization with Sytox Green, especially compared to the isopropanol-treated groups that show greenish fluorescence. This suggests that, unlike S. aureus, the effect of the peptides on C. albicans does not include plasma membrane permeabilization.

SEM images of C. albicans also showed less debris than those of S. aureus. The positive control showed cells with slight roughness and cellular aggregation, while the negative control showed cells with a regular surface and little debris, representing the expected morphology of viable, intact cells.

The Pep5-Cpp-treated group showed very little debris and cells arranged in pairs from time zero to 30 min. Analyzing the slides with the ΔC3-Pep5-Cpp variant, slightly more debris were released before and after incubation, albeit in small quantities. Treatment with ΔN-Pep5-Cpp resulted in fewer aggregates and cells with more preserved morphology, resulting in a relatively clean slide for both treatments with and without incubation.

In comparison to the results obtained for S. aureus, the morphological changes observed were less pronounced, with greater emphasis on the difference in cell organization than on the formation and presence of cellular debris.

2.7. Evaluation of Interaction with Genetic Material

To investigate possible interactions between the peptides and the genomic DNA of microorganisms, an electrophoretic mobility assay, or DNA gel retardation assay, was performed. Three concentrations of the peptides and their smallest fractions were tested to evaluate the electrophoretic mobility of the DNA bands, and the minimum inhibitory concentration (MIC), one above and one below, when possible. The images of Figure 7 show that when the peptide interacted with the genetic material, its migration was affected, suggesting possible interference with intracellular functions through interaction with DNA.

In the Staphylococcus aureus DNA assay, all concentrations of the Cpp peptide resulted in complete retention of genetic material in the electrophoretic well, indicating strong interaction with the DNA. The Pep5-Cpp peptide showed partial interaction at all concentrations tested, as evidenced by the presence of visible bands associated with drag patterns and partial retention of DNA in the well. For the ΔN-Pep5-Cpp and ΔC3-Pep5-Cpp variants, retention of genetic material in the well was observed at all concentrations evaluated.

For Aspergillus niger DNA, the Cpp peptide caused retention of genetic material in the well at all concentrations. For Pep5-Cpp, the two highest concentrations resulted in DNA retention, while the lowest concentration showed partial retention, with the simultaneous formation of a visible band. The ΔC3-Pep5-Cpp variant promoted retention of genetic material in the well at all concentrations, while the ΔN-Pep5-Cpp variant induced retention only at the two highest concentrations tested.

In the case of Candida albicans DNA, with a 10 min incubation, all Cpp concentrations resulted in retention of genetic material, as in the other assays. For Pep5-Cpp, the highest concentration promoted DNA retention in the well associated with a drag pattern, while the intermediate and lowest concentrations showed drag with a visible band. The ΔC3-Pep5-Cpp variant caused retention of genetic material only at the highest concentration, allowing band formation at the two lowest concentrations. For ΔN-Pep5-Cpp, the highest concentration resulted in DNA retention in the well, the intermediate concentration showed a drag pattern, and the lowest concentration allowed band formation, still with slight drag. To verify if the incubation time could increase the interaction of the peptide with the genetic material, the assay was repeated with a 30 min incubation.

When the incubation time for Candida albicans DNA was increased, the pattern remained similar to the assay with 10 min. The Cpp peptide promoted retention of genetic material in the well at all concentrations. For Pep5-Cpp, partial retention of DNA in the well was observed at all concentrations, which differed from the previous assay, but was simultaneous with the presence of visible bands. The ΔC3-Pep5-Cpp variant caused retention of genetic material at the highest concentration, while the two lowest concentrations allowed band formation. For ΔN-Pep5-Cpp, the highest concentration promoted DNA retention in the well, the intermediate concentration showed a drag pattern, and the lowest concentration allowed band formation (Figure 7).

2.8. Hemolytic Effects of the Peptides on Human Erythrocytes

Considering the possibility of a future pharmacological combination, a hemolytic activity assay was performed to determine whether the peptides of interest lyse human erythrocytes. Hemolytic activity is measured as an indicator of analyzing the cytotoxicity of Pep 5-Cpp and its variants on blood cells. Red blood cells were incubated with serial dilutions of the peptides Cpp, Pep5-Cpp, ΔC3-Pep5-Cpp, and ΔN-Pep5-Cpp, and the percentage of hemolysis was quantified as an indicator of cellular toxicity, always in comparison with the Triton and PBS treatments.

As can be seen in Figure 8, hemolysis values were less than 40% for all concentrations of the peptides tested. When observing the values obtained for the MIC, it can be seen that the percentage of hemolysis is even lower.

For the Pep5-Cpp peptide, the highest MIC value obtained was for the microorganism S. aureus, with a MIC of 6.25 μM, but still with a hemolysis percentage of less than 10%. Similar results were observed for the ΔN-Pep5-Cpp peptide.

The ΔC3-Pep5-Cpp peptide, on the other hand, presented its highest MIC for A. niger at a concentration of 25 μM. Despite having a higher value compared to the other peptides tested, its hemolysis was less than 20%.

Overall, the results shown here indicate that the peptides Pep5-Cpp, ΔC3-Pep5-Cpp, and ΔN-Pep5-Cpp exhibit low toxicity in human blood cells while maintaining their antimicrobial activity, especially at MICs. These data are promising for the development of therapies based on these peptides, as they combine antimicrobial efficacy with low cytotoxicity.

To analyze the relationship between the antimicrobial activity and the hemolytic percentage, the selectivity ratio (SR) was calculated as the ratio between the concentration inducing 10% hemolysis (HD_10_) and the highest minimum inhibitory concentration (MIC) observed for each peptide (Table 5). When 10% hemolysis was not reached within the tested concentration range, the highest concentration evaluated was used as a conservative estimate of HD_10_, and values were expressed as minimum limits (≥). In cases where this threshold was not reached, the highest concentration evaluated was used. The selectivity ratio (SR) was calculated as the quotient between HD_10_ and the highest MIC observed for each peptide. This approach allows the inclusion of all peptides in the analysis and indicates that the actual selectivity may be higher than the estimated values.

As Table 4 shows, between the peptides evaluated, ΔN-Pep5-Cpp exhibited the highest selectivity ratio (SR = 16), indicating a favorable therapeutic potential and suggesting that N-terminal deletion enhances antimicrobial selectivity while reducing hemolytic activity. ΔC3-Pep5-Cpp, however, displayed an SR of 1, indicating overlapping antimicrobial and hemolytic concentrations and limited therapeutic potential.

3. Discussion

Regarding their therapeutic potential, currently, the clinical application of antimicrobial peptides is mainly focused on the treatment of infections caused by pathogenic bacteria, as well as promoting wound healing and reducing inflammation. Some of these have already reached clinical phases, such as in cases of cancer treatment, and others have even reached commercial applications, such as P113 and NP213 as antifungal treatments. However, there are still inherent limitations to these peptides that represent obstacles to the development of compounds with real therapeutic efficacy [19,20,21]. Cell-penetrating peptides are generally small and cationic, capable of transporting various molecules, such as proteins, peptides, or oligonucleotides, into cells that would not normally absorb high-molecular-weight compounds [22]. In previous studies, the peptide Pep5, when coupled with Cpp, induced cell death in various tumor cells, with results, including in vivo assays, that reduced the volume of rat C6 glioblastoma by almost 50% [8]. Furthermore, Pep5-Cpp has also been characterized as an inducer of cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi [10]. Therefore, this study characterized the activity of Pep5 and two of its variants when coupled with Cpp.

The two variants were also chosen because, as previously described by Araújo et al. in 2014 [8], the deletion of three amino acids at the C-terminal end (ΔC3) was characterized as the smallest fraction of the original peptide capable of inducing cell death. On the other hand, the deletion of only one amino acid residue at the N-terminal end (ΔN) was able to extinguish this antitumor induction [8].

Modifications were made to the Pep5 sequence to investigate the relationship between structure and activity. Sequential deletions were made, starting with one amino acid residue, increasing to two or three subsequently, at the N or C ends. The removal of a single amino acid from the N end (ΔN-pep5-Cpp) completely abolished the induction of cell death by pep5-Cpp, while the activity was slightly reduced by the removal of two or three amino acids from its C end. When four amino acid residues were depleted from the C-terminal, the activity was completely abolished [8]. Therefore, the ΔC3-Pep5-Cpp variant corresponds to the smallest fraction of the original peptide that retained antitumor activity and was selected to evaluate whether this minimal bioactive sequence would maintain activity when subjected to antimicrobial tests. In contrast, the ΔN-Pep5 variant was designed based on evidence that the deletion of a single amino acid residue at the N-terminal end completely abolished the antitumor activity of Pep5, indicating the critical role of this region. Thus, ΔN-Pep5-Cpp was included as a structurally related control peptide based on the hypothesis of similar activity between antimicrobial and antitumor peptides [11].

When analyzing the physicochemical properties of the four peptides, there is an indication that they are small in size, with fewer than 50 amino acids and a net positive charge ranging between +8 and +9 at physiological pH, with a predominance of basic amino acid residues. Therefore, the peptides of interest in this work are within the electrochemical profile compatible with cationic antimicrobial peptides. The high affinity for negatively charged membranes, a common characteristic in both microbial and cancer cells, reinforces the hypothesis of the possible antimicrobial action of antitumor peptides, such as Pep5-Cpp [19].

The antimicrobial action of Pep5-Cpp and its variations studied here was confirmed with the MIC assay, which indicated inhibition of microbial growth at low peptide concentrations, similar to studies previously reported for other peptides that are active below a concentration of 32 μg/mL [23].

Although the exclusion of a single amino acid at the N-terminal end of ΔN-Pep5-Cpp has previously been shown to abolish its antitumor activity, this modification did not impair its antimicrobial effects in the present study. Our initial hypothesis was that electrostatic similarities between bacterial and tumor membranes would yield similar results when interacting with cationic peptides. However, the result differs for this peptide. This apparent discrepancy may be due to different mechanisms of action.

Antitumor activity may depend on interaction with intracellular targets, while antimicrobial activity is often driven by specifically transmitted electrostatic interactions with microbial membranes, leading to membrane destabilization or changes in permeability [11,13].

When characterized as antitumor, Pep5-Cpp was identified as an inducer of cell death. The results suggested that Pep5-Cpp significantly induced apoptosis and necrosis in HeLa cells. Similar results were obtained using its minimal active sequence (ΔC3-Pep5-Cpp), with depletion of three amino acids from the C-terminal, in which the potency of the original Pep5-Cpp peptide was only diminished [8]. In this study, it was also characterized that cells treated with Pep5-Cpp showed a significant increase in the activities of caspases 3/7 and 9, while the activity of caspase 8 remained unchanged. When a single amino acid was removed from the N-terminus (ΔN-Pep5-Cpp), the induction of cell death was completely abolished [8].

Thus, N-terminal deletion may be critical for cell capture and cytotoxic signaling in tumor cells, but less relevant for membrane-mediated antimicrobial action. Consistently, ΔN-Pep5-Cpp was able to delay the onset of the exponential growth phase and inhibit microbial control for up to 18 h, providing a sublethal and time-dependent antimicrobial effect. These findings highlight that the mechanisms of action for antitumor and antimicrobial activities may differ, despite electrostatic similarity and even within the same peptide sequence.

The results for P. aeruginosa showed inhibition at low concentrations also for Cpp, which may indicate that its coupling to the original peptide influences the inhibition of this strain. The high inhibitory activity of Pep5-Cpp and its two smaller active fractions, therefore, reinforces its potential as an antimicrobial agent and highlights the relevance of further investigating its properties and possible therapeutic applications.

The results of microbial growth kinetics under peptide treatment, CBM, and CFM, combined with the results of membrane permeabilization and interaction with genetic material, show that the peptides acted with different mechanisms of action depending on the microorganism. Even so, they were able to inhibit the growth of four distinct classes: Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi.

The action on the plasma membrane and its disruption by peptides can lead to its destabilization, permeabilization, and eventually to the lysis of microbial cells, which points to a bactericidal mechanism. This effect occurs through electrostatic interactions between positive charges of the peptide and negative charges of the bacterial membrane, potentially involving different modes of action, such as toroidal, barrel-stave, and carpet pores [20,24,25]. In this study, however, it was observed that the microbial group that showed membrane permeability when treated with the peptides also showed microbial growth after removal of the treatment and subculturing, as was the case with S. aureus. Conversely, the group that did not show new growth in the subculture also did not show membrane permeability, as was the case with C. albicans. This reinforces the idea that different mechanisms of action must be affecting the microorganisms, with the possibility of more than one simultaneously affecting the same microorganism.

A study conducted by Lee et al. [26] compared different membrane-destabilizing properties caused by peptides. In this study, model membranes were used under the Dual Polarization Interferometry technique to monitor in real time the structural changes deposited on a biosensor. The results showed that some peptides caused partially reversible effects and a tendency to insert into the bilayer, promoting membrane expansion or pore formation. Thus, membrane disruption may be an intermediate mechanism of action for others or even a reversible mechanism. This is especially relevant, as there are considerable discussions regarding antimicrobial peptides that are able to translocate across the membrane, accumulate within the cell, and alter intracellular targets, affecting nucleic acid and protein synthesis as further mechanisms of action [26,27].

Through SEM analysis, the typical morphology of S. aureus was observed, consisting of spherical cells arranged in grape-like clusters [28], which can be altered according to the treatment provided. For C. albicans, the reference strain presented colonies of smooth cells [29].

In the study by Ajayakumar et al. [30], SEM images revealed that the control cells had a smooth, shiny, and intact surface, while the cells treated with the B1CTcu3 peptide showed blister formation, distortions, and, at concentrations corresponding to the MIC, time-dependent membrane alteration, which reinforced the idea that the peptide’s action occurs through a membrane disruption process [29].

Similar results were described by Lan et al. [31], who evaluated the effects of ε-PL on Shewanella putrefaciens [31]. Untreated bacterial cells showed regular morphology, with a smooth and intact surface, while treatment with ε-PL led to cell deformation, wrinkling, the presence of grooves, and signs of rupture and leakage of intracellular content, corroborating the hypothesis that the mechanism of action is related to membrane disorganization.

The direct observation of morphological changes, such as wrinkling, bubble formation, deformations, changes in typical clustering, and cell leakage, is evidence pointed out in the literature as an indication of membranolytic action. For this specific work, some of these changes were visualized with the incubation of the peptides, especially the change in clustering and cell leakage, with the formation of debris. Some other less frequent changes were also seen, such as bubble formation and deformations, especially for S. aureus.

Here, as shown in Figure 5, the peptides may be inducing a bacteriostatic effect or reversible damage to the membrane, since both the quantification and the fluorescence microscopy images, as well as the SEM images, show cellular leakage, which indicates membrane permeability. However, this mechanism may only be sufficient to temporarily interrupt cell viability or metabolism, without causing immediate cell lysis or death, which is supported by the CBM results for S. aureus. Therefore, the absence of visible morphological changes in the SEM, despite the presence of debris and detected permeabilization, reinforces the hypothesis that the peptide acts through complex mechanisms on the membrane, which need to be further investigated in future studies for a better understanding.

On the other hand, the lack of membrane permeabilization in C. albicans and its generalized cell death in the CFM tests demonstrate different mechanisms of action or, at least, variable interactions depending on the microorganism.

The biofilm formation inhibition assay was performed on S. aureus (MRSA PSA 144) because the virulence factors of this bacterium are linked to the biofilm phenotype, which is influenced by the acquisition of the methicillin resistance gene [32]. PAMs have already been described as playing a role in the disruption of biofilms, potentially acting on the bacterial membrane and causing degradation of the extracellular matrix [21]. In this work, it was evaluated that only the peptide with a 3-amino acid deletion at its C-terminus was able to inhibit biofilm formation. This may suggest that the observed response is related to the structure and composition of the peptide, as the C-terminal deletion variant may favor interactions that enhance antimicrobial efficacy. The data indicates that modifications at the C-terminal end of Pep5-Cpp can optimize its activity against biofilms, whereas alterations in the N-terminal region do not show the same trend. Further structural and biophysical studies will be necessary to elucidate the precise mechanism of action.

DNA gel retardation assays also indicate the possibility of intracellular interactions of the peptides. Variations of Pep5-Cpp bound to the DNA of microorganisms in vitro in different proportions, thus suggesting the possibility of inhibiting cellular functions through interference with DNA [23].

Studies using the DNA gel retardation assay for sarconesin II, a peptide isolated from Sarconesiopsis magellanica from its secretions, also showed that the DNA retention pattern can occur through electrostatic interaction. In this study, a spontaneous binding to DNA was suggested with subsequent inhibition of cellular repair function, leading to bacterial death [25].

Other studies involving antimicrobial peptides and interaction with DNA through electrophoretic mobility shift analysis also emphasize the presence of alkaline amino acids as a possibility to mediate DNA binding. In the case of the NK-18 peptide, which contains lysine and arginine, its binding to the phosphate fragments of DNA was also suggested through electrostatic interaction with these amino acids. Then, after membrane disruption and transfer to the cytoplasm, the interaction between NK-18 and the microorganism’s DNA would occur, being an intracellular target [33]. The presence of these same amino acids in the peptides of interest in this work is also noteworthy.

For Candida albicans, the interaction between the peptides and the genomic DNA did not show substantial changes when the incubation time was increased from 10 to 30 min. This suggests that the prolonged exposure does not significantly enhance binding with the peptide to the DNA under the conditions tested. When analyzing data from other peptides, such as rondonin, derived from the plasma of Acanthoscurria rondoniae, the ability to alter the migration of C. albicans genomic DNA is observed only at higher concentrations, suggesting that the interaction is graded according to the amount of peptide available [34]. Thus, there is a possibility that the interaction of Pep5-Cpp and its fractions is predominantly dose-dependent rather than time-dependent in this case.

The minimum inhibitory concentration values of the peptides not exceeding 20% hemolysis are an interesting factor when considering antimicrobial peptides described in the literature in studies that standardized the 20% threshold as non-hemolytic. The study by Capecchi et al. (2021) [35], for example, used data from the Antimicrobial Peptide Activity and Structure Database to design short non-hemolytic AMPs. In this study, peptides that caused up to 20% hemolysis at concentrations of 50 μM were considered non-hemolytic, while peptides that showed more than 20% hemolysis at any concentration were classified as hemolytic [35]. Other studies, such as that of Wang et al. (2018), also considered the 20% limit as low hemolytic toxicity for antimicrobial peptides, as in the case of HJH-1, a peptide derived from bovine erythrocytes P3 from the α subunit of hemoglobin [36].

The use of Cpp coupled to the peptides of interest in this study may have played a role in their low toxicity. This is because cell-penetrating peptides, such as the HIV TAT-derived Cpp used in this study, are known for their ability to efficiently cross the cell membranes of eukaryotic cells without exhibiting apparent toxicity. This property makes them promising tools for the intracellular delivery of various molecules, such as drugs and peptides. By avoiding significant damage or alterations to host cells, they offer a safe and effective method for transporting therapeutic compounds directly into the cell interior, which has enormous potential in biomedical and pharmaceutical applications [36,37].

When antimicrobial activity was correlated with cytotoxicity using the HD10, ΔN-Pep5-Cpp showed the most favorable profile, combining low MIC values (6.25 µM) with a high concentration required to induce 10% hemolysis (HD_10_ = 100 µM), resulting in an RS of 16. This suggests that the N-terminal deletion improved antimicrobial selectivity while reducing toxicity to human erythrocytes. In contrast, ΔC3-Pep5-Cpp showed overlapping antimicrobial and hemolytic concentrations (RS = 1), indicating limited selectivity and a narrow therapeutic margin. Pep5-Cpp showed intermediate selectivity (RS = 4), reflecting increased antimicrobial potency compared to Cpp, although accompanied by greater hemolytic activity at higher concentrations. Taken together, these findings highlight the importance of structural modifications in modulating the balance between efficacy and cytotoxicity, and reinforce the relevance of selectivity-based parameters for the rational development of antimicrobial peptides. Previous studies calculated selectivity indices based on HD_50_ values to relate hemolytic activity to antimicrobial efficacy, using the 50% hemolysis parameter. In this work, however, HD_10_ values were used in order to avoid overestimating selectivity and to better reflect the concentrations associated with acceptable levels of hemolysis with the results obtained [38].

Regarding the toxicity of peptides to normal cells beyond hemolysis, previous studies with Pep5-based peptides have reported moderate cytotoxic effects in non-tumor human cell lines. In the study by Wang et al. (2022) [39], Pep5-derived peptides showed IC_50_ values for two cell lines, normal liver cells (LO2) and bronchial epithelial cells (BEAS-2B), lower than in HepG2 and A549 tumor cells, resulting in selectivity indices around 1.5. This selective behavior was attributed to differences in membrane composition and surface charge density [39]. However, the authors emphasized that, although Pep5-based peptides have demonstrated some degree of tumor selectivity, further optimizations would be necessary to improve their safety profile in relation to normal cells. In the present study, the absence of significant hemolytic activity suggests a favorable preliminary safety profile. Further cytotoxicity assays using non-tumor cell lines will be needed to better assess the compatibility of the peptides.

In summary, the data presented here demonstrate that Pep5-Cpp and its variants show antimicrobial activity against different microorganisms, mediated by multifactorial mechanisms of action, including membrane permeability and possible interaction with DNA. The results, therefore, highlight the potential of these peptides as models for new antimicrobial agents, especially in the face of the growing scenario of resistance.

4. Materials and Methods

4.1. Pep5-Cpp and Microorganisms

The peptides used in this work were synthesized by the biotechnology company Proteimax Biotecnologia LTDA (São Paulo city, São Paulo state, Brazil), at a purity of ≥90%. The peptides used were Pep5 and two of its smaller fractions, all coupled to Cpp, and Cpp itself isolated, containing the following sequences: the isolated Cpp sequence (YGRKKRRQRRR), Pep5-Cpp (WELVVLGKL-YGRKKRRQRRR), its smallest active antitumor fraction with the deletion of three amino acid residues at the C-terminus ΔC3-Pep5-Cpp (WELVVL-YGRKKRRQRRR), and the ΔN-Pep5-Cpp fragment (ELVVLGKL-YGRKKRRQRRR) depleted of the tryptophan residue at the N-terminus, characterized as having no antitumor activity [8].

To evaluate growth inhibition in liquid medium, the biological activities of the peptides described above were assessed against representative strains of different groups of microorganisms: Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), Candida albicans (MDM8), and Aspergillus niger (A296).

4.2. Bioinformatic Prediction of Physicochemical Parameters

The physicochemical parameters, as well as the identification of hydrophobic residues and the analysis of positive and negative charges, were obtained using ProtParam tool available at the ExPASy web server (Swiss Institute of Bioinformatics, SIB), accessed on 2 October 2023 (https://www.expasy.org) [17]. Innovagen platform was used to calculate the properties of the peptides through the PepCalc tool available on the Innovagen platform (Innovagen AB, Sweden), accessed on 2 October 2023 (https://pepcalc.com) [16].

4.3. Antimicrobial Activity Assays—General Conditions

Antimicrobial assays for minimum inhibitory concentration (MIC), Kinetic Growth Evaluation, and Minimum Bactericidal and Fungicidal Concentration were conducted in 96-well microplates using microbial suspensions in the logarithmic growth phase, adjusted to 10^3^ CFU/mL for bacteria and 10^4^ CFU/mL for fungi. Mueller-Hinton broth or Potato Dextrose broth was used according to adapted CLSI guidelines, and Streptomycin and Amphotericin B were included as standard antibiotics for bacteria and fungi, respectively. Optical density (OD) at 595 nm was measured using a VICTOR^3^-1420 (PerkinElmer^®^, Shelton, CT, USA) or Multiskan (Thermo Fisher, Waltham, MA, USA) microplate reader, as specified for each methodology below.

4.4. Minimum Inhibitory Concentration (MIC)

In order to determine the minimum inhibitory concentration (MIC), serial dilutions of the four peptides were performed in ultrapure water in a 96-well microplate in a ratio of 20 μL of peptide with final concentrations from 200 to 1.56 μM and 80 μL of microbial suspension. Wells with 20 μL of ultrapure water and 80 μL of microbial suspension were used as a growth control, and 100 μL of culture medium was used as a background control. A volume of 20 μL of streptomycin at 10 mg/mL or amphotericin B at 100 mg/mL, and 80 μL of microbial suspension were used as a control for microorganism death. The plates were incubated at 30 °C for 18 to 48 h, the time depending on the type of microorganism, under constant shaking for bacteria and static growth for fungi. The representative strains used in this assay were: Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (MDM8), and Aspergillus niger (A296). Microbial growth was evaluated by measuring optical density (OD) using the VICTOR^3^-1420 microplate reader (PerkinElmer^®^, Shelton, CT, USA) [34].

4.5. Kinetic Assessment of Microbial Growth Under Peptide Treatment

To perform the time-kill assay, the peptides were used at MICs. The controls used in the MIC determination were maintained, and the OD at 595 nm was recorded every hour for a total period of 24 h, using a Multiskan microplate reader (Thermo Fisher). Plates with S. aureus were incubated under pulsed agitation, while plates with C. albicans were agitated only before each reading to minimize disruption of fungal growth. This assay used the following strains: Staphylococcus aureus (ATCC 29213) and Candida albicans (MDM8).

4.6. Minimum Bactericidal and Fungicidal Concentration (MBC/MFC)

To determine the Minimum Bactericidal Concentration (MBC) or Minimum Fungicidal Concentration (MFC), after 24 h of incubation with the peptides in serial dilution with concentrations from 200 μM to 1.56 μM and maintaining the initial MIC controls, the microplates were centrifuged at 800× g for 5 min. The supernatant was discarded and the precipitated biomass was resuspended in Mueller-Hinton broth, free of peptides. Again, the microplates were incubated for 24 h at 30 °C with pulsed 5 s agitation maintained for S. aureus and agitation applied only before the 595 nm OD readings for C. albicans. The MBC or MFC was defined as the lowest peptide concentration at which no visible microbial growth was observed after re-incubation [40].

4.7. Assessment of Biofilm Inhibition Amount

Quantitative analysis of biofilm formation in microtiter plates by the biofilm-producing sample of Methicillin-Resistant S. aureus (MRSA PSA 144) [41]. Pre-inocula were cultured for 18 h at 37 °C in trypticase soy broth (TSB) under static conditions. Bacterial cultures were inoculated into TSB supplemented with 1% glucose at a 2:100 dilution in 96-well flat-bottom polystyrene microplates (final volume of 200 µL) and incubated at 37 °C for 24 h, with or without peptide treatment at concentrations corresponding to ½×, 1×, and 2× the MIC. After incubation, the OD was read at 595 nm in a microplate reader.

Next, washes were performed with phosphate-buffered saline (PBS) to remove planktonic microorganisms. The biofilm was fixed with 200 µL of 75% ethanol for 10 min at room temperature. The fixative was removed, and the microorganisms were stained with 200 µL of 0.5% crystal violet solution for 5 min. After removal of the stain and washes with PBS, the plates were dried, and the dye retained in the formed biofilm was solubilized by adding 200 µL of 95% ethanol for 2 min. Absorbance was read at 595 nm in the microplate reader. Biofilm quantification was performed using the following formula:

$$[eqn]$$

4.8. Membrane Damage Assessment in Microorganisms

Staphylococcus aureus (ATCC 29213) and Candida albicans (MDM8) strains were inoculated onto Luria–Bertani (LB) agar plates and incubated overnight at 30 °C. The resulting bacterial colonies were suspended in Hanks’ Balanced Salt Solution (HBSS) containing 5 mM glucose at an optical density of 0.5 at 620 nm. A volume of 100 μL of each suspension was inoculated into a black 96-well microplate with 0.1 μM SYTOX Green (Molecular Probes) in HBSS and incubated at 35 °C for 15 min in the dark. Fluorescence was measured every minute for 18 min using a spectrofluorometric microplate reader (Fluorstar Omeg, BMG LABTECH, Ortenberg, Germany) with excitation and emission wavelengths of 485 and 520 nm, respectively. The maximum permeabilization control was defined using isopropanol, and treatment with HBSS was used as a negative control. Subsequently, microscopy slides were prepared to observe the fluorescence pattern under a microscope (Olympus BX51, Olympus Corporation, Tokyo, Japan) [42].

The membrane permeability to peptides was also analyzed by Scanning Electron Microscopy (SEM). The treatment used was double the MIC, isopropanol as a positive control, or PBS as a negative control. Microbial cells were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer, washed with 0.1 M sodium cacodylate buffer, post-fixed with 1% (v/v) osmium tetroxide (OsO_4_) in the same buffer solution, and dehydrated through a graded series of ethanol (50%, 75%, 85%, 95%, and 100%). The preparations were subsequently dried by the critical point drying method, mounted on SEM metal stubs, coated with gold by cathodic sputtering, and examined under scanning electron microscope (Quanta 250, FEI Company, Eindhoven, The Netherlands) operating at 12.5 kV with a working distance of 7 mm [43].

4.9. Assessment of Interaction with Genetic Material

To investigate whether the mechanism of action of Pep5-Cpp and its fractions involved intracellular targets of genetic material, the genomic DNA of the microorganisms was extracted, incubated with the peptide at different concentrations, and subjected to a nucleic acid retardation assay [34].

4.9.1. Agarose Gel DNA Retardation Assay

Genomic DNA (approximately 250 ng) was mixed with increasing amounts of peptide. The mixtures were incubated at room temperature for 10 or 30 min and then subjected to electrophoresis on 0.8% agarose gels in TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA) [34].

4.9.2. DNA Extraction

Genomic DNA was isolated using a centrifugation column-based fungal/yeast DNA isolation kit (Norgen Biotek Corp., Thorold, ON, Canada). The microorganism of interest was cultured on LB agar plates, in the case of Aspergillus niger, or in LB liquid medium, in the case of Staphylococcus aureus and Candida albicans. Strains preculture were grown in LB, and incubated overnight at 37 °C, with agitation. The resulting cultures were used to inoculate fresh medium, which was further incubated at 37 °C until it reached an optical density of 0.500 at 595 nm. After the centrifugation of 1 mL of the resulting culture at 14,000× g for 1 min, the supernatants were discarded, and the pellets resuspended in lysis buffer. Filamentous fungi, spores and mycelium were collected from plates using sterile collection solution. The material was subjected to mechanical lysis with beads and vortexing for 10 to 15 min, followed by incubation at 65 °C. After centrifugation, the supernatant containing the DNA was collected and mixed with ethanol for further purification.

The solution was transferred to purification columns, with centrifugation at 10,000× g, repeating the procedure until the entire volume was processed. The columns were washed twice with washing solution, centrifuging at 10,000× g, followed by drying through centrifugation at 14,000× g. The DNA was eluted in 100 µL of elution buffer, centrifuging at 10,000× g.

4.10. Hemolytic Activity Assay

The hemolytic activity assay was performed under the approval of the Research Ethics Committee, through the Brazil Platform (Opinion No. 6.830.203; CAAE: 76701023.6.0000.0086). For this assay, 20 μL of a peptide solution initially at 1 mM were serially diluted in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na_2_HPO_4_, 1.76 mM KH_2_PO_4_, pH 7.4), obtaining concentrations ranging from 200 μM to 1.56 μM. Peripheral blood samples from healthy human donors of type A and Rh-positive were collected in citrate buffer (150 mM, pH 7.4). After collection, the samples were centrifuged at 800× g for 15 min and washed three times with PBS. Then, a 3% (v/v) erythrocyte suspension in PBS was prepared and inoculated into a U-bottom microplate with the peptide solution diluted in PBS and incubated at 37 °C for one hour. Subsequently, the plate was centrifuged at 800× g and the supernatant transferred to a flat-bottom microplate to measure the absorbance at 405 nm using a VICTOR^3^-1420 plate reader (PerkinElmer^®^). Absorbance values corresponding to 0% and 100% hemolysis were obtained and compared with the negative control, which consisted of treatment with PBS, and the positive control, with treatment in 0.1% Triton X-100 solution. The absorbance value was calculated as the average of the triplicates. The percentage of hemolysis was calculated based on the average of technical triplicates and expressed as mean ± standard deviation of three independent experiments using blood samples from different donors (n = 3) with the same blood type A+.

The selectivity ratio (SR) was calculated for each peptide in order to relate cytotoxicity to antimicrobial efficacy [38]. The SR was defined as the quotient between the concentration inducing 10% hemolysis (HD_10_) and the minimum inhibitory concentration (MIC), according to the following formula:

$$[eqn]$$

When 10% hemolysis was not reached within the concentration range evaluated, the highest concentration tested was used as an estimate, and HD_10_ values were expressed as minimum limits (≥). In cases where average hemolysis values fell between two consecutive concentrations without reaching exactly 10%, the higher concentration of the interval was conservatively selected as the HD_10_ value. Likewise, to avoid overestimation of selectivity, the highest MIC observed for each peptide across all tested microorganisms was used for SR calculation.

5. Conclusions

In this study, the peptides Pep5-Cpp, ΔC3-Pep5-Cpp, and ΔN-Pep5-Cpp were shown to exhibit antimicrobial activity against a range of tested microorganisms, including Gram-positive and Gram-negative bacteria, yeasts, and filamentous fungi. The action appears to be different in each microorganism, involving both membrane and intracellular interactions, such as DNA binding. The finding that the ΔN-Pep5-Cpp variant, inactive against tumor cells, remains effective against bacteria and fungi indicates that, despite the initial hypothesis of similarities between the biophysical characteristics of bacterial and tumor cell membranes, the mechanism of action based on the peptide structure is likely different in each case. Furthermore, the peptide displayed minimal toxicity and considerable therapeutic potential in its interaction with human erythrocytes, important factors to be considered when developing new antimicrobial therapy. ΔC3-Pep5-Cpp also showed significant antibiofilm activity, differing from the others and opening up possibilities for further studies.