Frog Skin-Derived Peptides Against Corynebacterium jeikeium: Correlation between Antibacterial and Cytotoxic Activities

Corynebacterium jeikeium is a commensal bacterium that colonizes human skin, and it is part of the normal bacterial flora. In non-risk subjects, it can be the cause of bad body smell due to the generation of volatile odorous metabolites, especially in the wet parts of the body that this bacterium often colonizes (i.e., groin and axillary regions). Importantly, in the last few decades, there have been increasing cases of serious infections provoked by this bacterium, especially in immunocompromised or hospitalized patients who have undergone installation of prostheses or catheters. The ease in developing resistance to commonly-used antibiotics (i.e., glycopeptides) has made the search for new antimicrobial compounds of clinical importance. Here, for the first time, we characterize the antimicrobial activity of some selected frog skin-derived antimicrobial peptides (AMPs) against C. jeikeium by determining their minimum inhibitory and bactericidal concentrations (MIC and MBC) by a microdilution method. The results highlight esculentin-1b(1-18) [Esc(1-18)] and esculentin-1a(1-21) [Esc(1-21)] as the most active AMPs with MIC and MBC of 4–8 and 0.125–0.25 µM, respectively, along with a non-toxic profile after a short- and long-term (40 min and 24 h) treatment of mammalian cells. Overall, these findings indicate the high potentiality of Esc(1-18) and Esc(1-21) as (i) alternative antimicrobials against C. jeikeium infections and/or as (ii) additives in cosmetic products (creams, deodorants) to reduce the production of bad body odor.


Membrane Permeabilization
Esculentin peptides are known to display an antibacterial activity mainly through a membraneperturbing mechanism of action. To verify their ability to perturb the cytoplasmic membrane of Gram-positive bacteria, such as C. jeikeium, a Sytox Green assay was performed. Sytox Green is a fluorescent probe unable to cross intact membranes, and its fluorescence intensity significantly increases upon binding to nucleic acids. As shown in Figure 2, the rapid increase of the fluorescent signal immediately after peptide addition to the bacterial cells (arrow) indicated that the perturbation of the membrane was the result of the peptide-induced membrane damage, allowing the internalization of the probe with a consequent binding to the bacterial DNA. Both esculentin AMPs manifested a dose-dependent fast kinetic membrane destabilization with a total perturbation of the phospholipid bilayer already within the first minutes of treatment at the highest concentration of 32 µM. According to the MIC and MBC values, the most active Esc(1-21) exhibited a stronger effect compared to Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), even at a concentration as low as 0.5 µM with about 60% membrane damage within the first 2 min. Antibiotics 2020, 9, x 4 of 13 AMPs with a higher cationicity and lower hydrophobicity, as indicated by the corresponding grand average of hydropathicity index (GRAVY; Table 1), which is used to represent the hydrophobicity value of a peptide [38]. Interestingly, when the most active esculentin peptides were tested for their bactericidal activity, they were found to cause almost the complete killing of the bacterial population with an MBC (i.e., the lowest peptide concentration able to cause a ≥3 log reduction in the number of bacterial cells) 2fold higher than the corresponding MIC, i.e., 8 µM and 0.25 µM for Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and Esc(1-21), respectively.

Membrane Permeabilization
Esculentin peptides are known to display an antibacterial activity mainly through a membraneperturbing mechanism of action. To verify their ability to perturb the cytoplasmic membrane of Gram-positive bacteria, such as C. jeikeium, a Sytox Green assay was performed. Sytox Green is a fluorescent probe unable to cross intact membranes, and its fluorescence intensity significantly increases upon binding to nucleic acids. As shown in Figure 2, the rapid increase of the fluorescent signal immediately after peptide addition to the bacterial cells (arrow) indicated that the perturbation of the membrane was the result of the peptide-induced membrane damage, allowing the internalization of the probe with a consequent binding to the bacterial DNA. Both esculentin AMPs manifested a dose-dependent fast kinetic membrane destabilization with a total perturbation of the phospholipid bilayer already within the first minutes of treatment at the highest concentration of 32 µM. According to the MIC and MBC values, the most active Esc(1-21) exhibited a stronger effect compared to Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), even at a concentration as low as 0.5 µM with about 60% membrane damage within the first 2 min.

Hemolytic Activity
To evaluate the potential cytotoxicity of esculentin peptides in the short term, they were tested for the ability to lyse mammalian red blood cells after 40 min of treatment at 37 • C. Both AMPs caused a weak and similar hemolysis with an~10% release of hemoglobin at a concentration of 64 µM, which is significantly higher than the antimicrobial dosages, thus suggesting their safety profile for short-term treatment ( Figure 3). measurements from a single experiment representative of three independent experiments. Controls (Ctrl) are cells not treated with the peptides.

Hemolytic Activity
To evaluate the potential cytotoxicity of esculentin peptides in the short term, they were tested for the ability to lyse mammalian red blood cells after 40 min of treatment at 37 °C. Both AMPs caused a weak and similar hemolysis with an ~10% release of hemoglobin at a concentration of 64 µM, which is significantly higher than the antimicrobial dosages, thus suggesting their safety profile for shortterm treatment ( Figure 3).

Discussion
Having been considered harmless and protective to humans, C. jeikeium has turned into a relevant etiological agent of dangerous infections, especially in hospitalized people or patients under chemotherapy. These include skin and wound infections, meningitis, enteritis, osteomyelitis, pyelonephritis, prosthetic joint infections, peritonitis, pneumonia, and liver abscess in patients with AIDS [42][43][44][45][46][47][48][49][50]. Unfortunately, current antibiotic therapies are often ineffective due to the onset of antibiotic resistance [51,52]. Interestingly, AMPs are promising hits to open the door for the generation of a new class of antimicrobials. In recent years, we thoroughly investigated the activity of Esc(1-18) and Esc(1-21) against a large number of microorganisms. However, no studies have been conducted so far for these peptides, as well as for other AMPs, against C. jeikeium. Here, for the first time, we analyzed the effect of some frog skin AMPs against this bacterium and selected the esculentin peptides as the most active molecules with an MIC of 0.125 and 4 µM for Esc  and Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), respectively. This outcome is in sharp contrast with the weaker activity of Esc  previously recorded against other Gram-positive bacteria (e.g., Staphylococcus aureus, S. epidermidis), where MICs ranging from 1 to 64 µM were found [37]. Note that both the higher cationicity and lower hydrophobicity of esculentin peptides compared to the other AMPs used for comparison (Table 1) would promote the peptide interaction with the anionic phospholipid headgroups of the bacterial membrane, thus explaining the higher antimicrobial activity of esculentin peptides. However, we cannot exclude the contribution of other chemical/physical features of the peptides (i.e., amphipathicity, length, oligomeric state) for the final outcome. Analogously, among esculentin peptides, the significantly higher antibacterial activity of Esc(1-21) compared to the shorter analog Esc(1-18) is likely due to the higher net positive charge and longer size of the former. Indeed, a minimum length for a peptide in alpha-helical conformation to span and perturb a phospholipid bilayer (∼30 Å thick) is 20 amino acids. Both esculentin peptides displayed the capability to kill C. jeikeium at a concentration two-fold higher than the MIC. Remarkably, despite the obtained MICs being comparable to those of traditional antibiotics [53,54], we have to take into account the additional advantageous features owned by these AMPs. As an example, we already demonstrated the ability of Esc  to promote re-epithelialization of a pseudo-wound by stimulating the migration of human keratinocytes, a relevant property for the therapeutic development of these peptides. Indeed, besides contributing to the elimination of C. jeikeium skin/wound-associated infections, the peptide would help to repair the damaged epithelial tissue. In addition, the membraneperturbing activity makes these AMPs even more interesting compounds, as bacteria are less prone to develop resistance to them. This is because the acquisition of resistance to AMPs would imply a complete reorganization of the bacterial membrane; an energetically unfavorable process not

Discussion
Having been considered harmless and protective to humans, C. jeikeium has turned into a relevant etiological agent of dangerous infections, especially in hospitalized people or patients under chemotherapy. These include skin and wound infections, meningitis, enteritis, osteomyelitis, pyelonephritis, prosthetic joint infections, peritonitis, pneumonia, and liver abscess in patients with AIDS [42][43][44][45][46][47][48][49][50]. Unfortunately, current antibiotic therapies are often ineffective due to the onset of antibiotic resistance [51,52]. Interestingly, AMPs are promising hits to open the door for the generation of a new class of antimicrobials. In recent years, we thoroughly investigated the activity of Esc(1-18) and Esc(1-21) against a large number of microorganisms. However, no studies have been conducted so far for these peptides, as well as for other AMPs, against C. jeikeium. Here, for the first time, we analyzed the effect of some frog skin AMPs against this bacterium and selected the esculentin peptides as the most active molecules with an MIC of 0.125 and 4 µM for Esc  and Esc(1-18), respectively. This outcome is in sharp contrast with the weaker activity of Esc(1-21) previously recorded against other Gram-positive bacteria (e.g., Staphylococcus aureus, S. epidermidis), where MICs ranging from 1 to 64 µM were found [37]. Note that both the higher cationicity and lower hydrophobicity of esculentin peptides compared to the other AMPs used for comparison (Table 1) would promote the peptide interaction with the anionic phospholipid headgroups of the bacterial membrane, thus explaining the higher antimicrobial activity of esculentin peptides. However, we cannot exclude the contribution of other chemical/physical features of the peptides (i.e., amphipathicity, length, oligomeric state) for the final outcome. Analogously, among esculentin peptides, the significantly higher antibacterial activity of Esc(1-21) compared to the shorter analog Esc(1-18) is likely due to the higher net positive charge and longer size of the former. Indeed, a minimum length for a peptide in alpha-helical conformation to span and perturb a phospholipid bilayer (∼30 Å thick) is 20 amino acids. Both esculentin peptides displayed the capability to kill C. jeikeium at a concentration two-fold higher than the MIC. Remarkably, despite the obtained MICs being comparable to those of traditional antibiotics [53,54], we have to take into account the additional advantageous features owned by these AMPs. As an example, we already demonstrated the ability of Esc  to promote re-epithelialization of a pseudo-wound by stimulating the migration of human keratinocytes, a relevant property for the therapeutic development of these peptides. Indeed, besides contributing to the elimination of C. jeikeium skin/wound-associated infections, the peptide would help to repair the damaged epithelial tissue. In addition, the membrane-perturbing activity makes these AMPs even more interesting compounds, as bacteria are less prone to develop resistance to them. This is because the acquisition of resistance to AMPs would imply a complete reorganization of the bacterial membrane; an energetically unfavorable process not compatible with bacterial survival [55][56][57][58][59][60]. Moreover, in contrast with mammalian AMPs, esculentin peptides can preserve antimicrobial activity in the presence of biological fluids [37]. Esc  was found to induce a complete permeabilization of the bacterial membrane at a concentration range from 2 to 32 µM. Differently, only 50% of the membrane perturbation had been previously achieved by this peptide against other Gram-positive bacteria (i.e., Streptococcus agalactiae ATCC 13813) at the higher concentration of 50 µM [36]. Finally, both esculentin peptides displayed a non-toxic profile on different types of mammalian cells, e.g., erythrocytes, keratinocytes, alveolar epithelial cells, and macrophages, either after a short-or long-term treatment. Importantly, HaCaT cells are a reliable and helpful in vitro model to determine the toxicity of various agents on the skin layer because keratinocytes represent 95% of the epidermal cells [61,62]. In comparison, the evaluation of the peptides' effect against lung epithelial cells, i.e., the A549 alveolar cell line, and immune cells, like macrophages, is of great interest for their crucial role in orchestrating both immune defense and inflammatory responses [63][64][65]. It is noteworthy that esculentin peptides may be developed not only as alternative antimicrobials against C. jeikeium infections, but also as additives in cosmetic products (e.g., creams, deodorants) aimed at countering the colonization of C. jeikeium and, as a result, axillary malodor formation. In fact, this bacterium is involved in the generation of volatile odorous metabolites, attributed to the bacterial degradation of skin lipids and specific odor precursors that are responsible for the unpleasant human body smell, in sweat secretions [6,7,66]. Frequent showers and different soaps cannot solve the problem of the so-called "wild axilla", thus provoking serious psychological concerns. Patients suffering from bad smell can adopt various atypical behaviors minimizing social interactions. This can lead to anxiety, decreased self-esteem, and low quality of life due to social difficulties, e.g., avoiding intimacy [67,68]. Overall, the potent antimicrobial action of esculentin peptides, especially of Esc  [MIC, 0.125 µM], and their safety profile make them attractive molecules for therapeutic and/or cosmetic application. Based on the amino acid sequence analysis conducted through bioinformatic platforms (https://webs.iiitd.edu.in/raghava/algpred/submission.html and https://web.expasy.org/protparam, [39,69]), it has been predicted that esculentin peptides are devoid of allergenic properties and have an estimated half-life time in mammalian reticulocytes (in vitro) of about 30 h, which is a compatible time for possible daily usage. This is also consistent with our recent in vivo efficacy studies showing: (i) an antimicrobial efficacy of Esc(1-21), 36 h after intra-tracheal administration in murine models of acute bacterial lung infection; and (ii) the absence of immunogenicity in mice [70,71]. In the work of Rahnamaeian and Vilcinskas, the authors already emphasized the feasibility of short-sized AMPs as cosmetic ingredients of topical formulations such as creams, lotions, shampoos, and wound dressings to deter dermatological pathogens [72]. As proof of this, Haisma and coworkers designed cream/gel formulations (e.g., a water-in-oil cream with lanolin, an oil-in-water cream with polyethylene glycol hexadecyl ether, and a hypromellose gel) containing the LL-37-derived AMP P60.4Ac to successfully eradicate methicillin-resistant S. aureus from colonized human epidermal models [73]. Taken all together, the data presented in this work demonstrated for the first time the high potentiality of esculentin peptides as a new choice to fight the undesirable infections caused by C. jeikeium in both healthy and susceptible individuals.

Microorganism and Cell Lines
The microorganism used in the study was the reference strain C. jeikeium ATCC BAA-949. The culture media used for bacterial growth and the various assays were tryptone soy broth and agar (TSB and TSA, respectively) supplemented with 0.1% Tween 80 (TSB + and TSA + ). fetal bovine serum (FBS), and 0.1 mg/mL of penicillin and streptomycin at 37 • C and 5% CO 2 , in 25 cm 2 or 75 cm 2 flasks. In the case of macrophages, sodium pyruvate and non-essential amino acids were also added to the culture medium.

Antimicrobial Assays
The MIC of the different AMPs against C. jeikeium ATCC BAA-949 were determined following the previously described procedure with some modifications [57]. The bacterium was grown in TSB + up to an optical density (O.D.) of 0.8 (λ = 590 nm) and diluted to reach a concentration of 2× 10 6 colony forming units (CFU) per mL. Aliquots (50 µL) of this dilution were added to 50 µL of TSB + supplemented with 2-fold serial dilution of peptides previously dispensed in the wells of a 96 well plate. Controls were bacteria not treated with the peptides. The plate was then incubated for 20 h at 37 • C, and the MIC was defined as the lowest peptide concentration that visually inhibits microbial growth (absence of turbidity) after 20 h incubation. For determining the MBC of the most potent peptides, i.e., Esc(1-18) and Esc , aliquots from MIC, 2× MIC and 4× MIC wells were spread onto TSA + plates for CFU counting after an overnight incubation. MBC was defined as the lowest peptide concentration able to cause a ≥3 log reduction in the number of cells of the initial inoculum after 20 h of incubation.

Cytotoxicity Test on Mammalian Cell Lines
The long-term cytotoxicity of Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) was investigated by the MTT assay according to [41], as previously carried out for Esc . MTT (Sigma-Aldrich, St. Luis, MO, USA) is a yellow dye that, upon intracellular entry, is converted into insoluble and dark purple formazan crystals by mitochondrial dehydrogenases. This reduction occurs only in metabolically-active cells; therefore, the colorimetric absorbance is directly proportional to the cell viability. About 4 × 10 4 cells, suspended in DMEM supplemented with glutamine (at the concentration indicated for each cell line) and 2% FBS without antibiotic, were plated in triplicate wells of a microtiter plate. After overnight incubation at 37 • C and 5% CO 2 , the medium was replaced by fresh serum-free medium containing the peptide at different concentrations. Cells not treated with the peptide were used as controls. After 24 h of incubation at 37 • C in a 5% CO 2 atmosphere, the medium was removed, and MTT solution in Hank's buffer (final concentration 0.5 mg/mL) was added to each well. The plate was incubated for 4 h at 37 • C and 5% CO 2 . Afterwards, the formazan crystals were dissolved by adding acidified isopropanol, and the absorbance of each well was measured at 570 nm using the microplate reader (Infinite M200; Tecan, Salzburg, Austria) (cell viability was calculated by assuming a percentage of 100% for control cells without any peptide treatment).

Statistical Analysis
Unless otherwise specified, all experiments were performed three times, and the obtained values were reported as the mean ± SEM.

Conclusions
AMPs are an effective alternative to conventional antibiotics in the battle to defeat microbial pathogens, due to their membrane-active microbicidal activity and to further biological properties. In the last few decades, C. jeikeium raised concerns in the clinical field because of the increased occurrence of its infections, especially in immunocompromised or hospitalized subjects. In non-risk patients, this bacterium normally colonizes skin (e.g., axilla), causing a bad smell. Here, for the first time, we characterized the efficacy of two derivatives of esculentin-1 peptides, i.e., Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and Esc(1-21), against C. jeikeium and highlighted their high potentiality as new antimicrobials with negligible cytotoxicity. Beyond the clinical relevance that these peptides can have in the scenario of antibiotic resistance, they represent excellent candidates to be used at low concentrations in the production of cosmetics designed to reduce bad body odors.