Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp.

Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and Buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 μg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.


Introduction
Invasive candidiasis (IC) is the most prevalent fungal disease in intensive care unit (ICU) patients, accounting for 75% of all fungal infections. IC affects about 250,000 people each year, and its mortality rate is estimated to be 70% [1,2]. In the United States, candidemia is the third leading cause of bloodstream infections in ICU patients [3].
In these scenarios, the most frequently isolated yeast is Candida albicans, causing about 60% of all genital, oral, and cutaneous candidiasis infections [4]. C. albicans has been described as an innocuous commensal microorganism that is part of the microbiota of the gastrointestinal tract, genitourinary tract, oral cavities, and conjunctiva. When the host is immunosuppressed and/or has microbiota imbalance, this yeast can cause superficial infections (skin, mucous membranes of the mouth, and vagina). The hematogenous spread of the fungus can lead to invasive infections in almost all organs, which without effective treatment are life-threatening [5]. evaluated against the reference strain C. albicans ATCC SC5314, sensitive to FLC and the clinical isolate of C. albicans 256 HUSI-PUJ, which is resistant to FLC. Considering their primary structure and polyvalence, the chimeras evaluated in this study were classified into three groups (Table 1): (I) The C1 and C3 peptides were synthesized and evaluated in order to establish whether the antifungal activity is affected by the position of the RRWQWR or RLLR sequences in the chimera. Chimeras C2 and C4 contained Ahx (6-aminohexanoic acid residue) as a spacer for the two motifs. This is intended to establish whether the inclusion of the Ahx spacer between the two motifs affects the antifungal activity. The inclusion of the Ahx spacer facilitates the synthesis of the chimera and separates the two motifs so that each of them can interact independently with the cell surface of the pathogen. (II) Chimeras C5, C6, and C7 were synthesized and their antifungal activity was evaluated in order to determine whether partial or total replacement of Arg residues with Lys in RRWQWR and/or RLLR sequences affects their antifungal activity. Replacing Arg residues with Lys has been shown to facilitate and reduce the cost of chimeric synthesis [18]. (III) Chimeras C8 and C9 were synthesized in order to establish whether the polyvalence of the RRWQWR motif increased the antifungal activity. Previous reports have shown that the polyvalence of the RRWQWR sequence increases antibacterial and anticancer activity [20,31].

Minimum Inhibitory and Fungicidal Concentration
In this investigation, chimeric peptides containing the precursor sequences LfcinB (20-25): RRWQWR and BFII (32-35) Pal : RLLRRLLR were synthesized and purified, and their antifungal activity against reference strain C. albicans ATCC SC5314 sensitive to FLC (MIC= 1 µg/mL) and a clinical isolate of C. albicans 256 HUSI-PUJ resistant to FLC (MIC = 64 µg/mL) was evaluated. Antifungal activity of the peptides RLLR or RLLR-RRLLR has not been reported in the literature consulted to date.
In group I, the C1 (RRWQWRRLLR) and C3 (RLLRRWQWR) chimeras lacked the Ahx spacer and the RRWQWR and RLLR motifs were alternatively bound in the N-terminal or C-terminal region of the sequence (Table 1). Moreover, the C1 and C3 chimeras showed higher antifungal activity against C. albicans strain SC5314 and the FLC-resistant clinical isolate C. albicans 256 than the precursor peptides and the C2 and C4 chimeras, suggesting that the inclusion of Ahx affects the antifungal activity against this strain. It was previously reported that the C1 and C3 chimeras showed antibacterial activity against Gram-positive and Gram-negative ATCC bacterial strains [18], suggesting that these chimeric peptides exhibit a broad spectrum of antimicrobial action. The C3 chimera showed higher antifungal activity (MIC~100 µg/mL) against C. albicans SC5314 and the clinical isolate C. albicans 256 than the C1 peptide, suggesting that the position of the LfcinB (20)(21)(22)(23)(24)(25) and BFII (32)(33)(34)(35) motifs in the sequence might be relevant for antifungal activity against C. albicans. These results suggest that chemical linkage of the two precursor sequences in the C3 chimera enhances antifungal activity against the two C. albicans strains tested.
In group II, peptides C5 (RRWQWR-Ahx-KLLKKLLK) and C6 (KKWQWK-Ahx-RLLRRLLR) exhinited greater antifungal activity against the two strains evaluated than peptides C1, C2, C3, C4, and C7 (Table 1). These results indicate that the substitution of Arg with Lys in the RRWQWR sequence significantly increased the antifungal activity, and the substitution of the Arg residues with Lys residues in the RLLRRLLR motif also increased the antifungal activity against both strains. On the other hand, the substitution of all Arg residues with Lys in chimera C7 did not increase the antifungal activity (MIC 200 µg/mL, 101 µM). Moreover, the results reported for C5 and C6 are similar to those obtained by Pineda et al.: C5 (MIC 100 µg/Ml, 48 µM) and C6 (MIC 50 µg/mL,24 µM) showed greater antibacterial activity than chimera C2 (MIC 200 µg/mL,122 µM) [18,33]. According to the above, although for chimera C5 and C7 the MIC values can be considered similar to those of the precursors (controls), the C6 chimera exhibited greater activity, obtaining a MIC value up to 4 times lower than RRWQWR and RLLRRLLR peptides.
In group III, peptides C8 ((RRWQWR) 2 K-Ahx-RLLR) and C9 ((RRWQWR) 2 K-Ahx-RLLRRLLR) exhibited antifungal activity against the strains evaluated. Peptide C9 exhibited the greatest antifungal activity against both strains of C. albicans. These results suggest that the polyvalence of the motif RRWQWR plus the lineal dimer of the sequence RLLR increased the antifungal activity (Table 1).
Peptides C3, C5, C6, C8, and C9 showed moderate antifungal activity against the reference strain C. albicans SC5314 and the clinically isolated C. albicans 256, resistant to FLC. According to the categories proposed by Alves et al., synthetic antifungal molecules with MIC values between 26-100 µg/mL are considered to have moderate antifungal activity [34].
The results suggest that the partial replacement of Arg with Lys in one of the two motifs or the polyvalence of the RRWQWR motif enhances the antifungal activity. Peptides C6 and C9 showed the best results; both peptides exhibited the highest antifungal activity against the strains evaluated. Peptide C6 has more advantages for synthesis than peptide C9, due to the fact that peptide C6 has lesser residues than peptide C9; however, peptide C9 had the lowest MIC value against the clinically isolated C. albicans 256 resistant to FLC.
The results above described are in agreement with previous reports, in which peptides containing the minimal motif of LfcinB presented MICs between 0.8 and 400 µg/mL for C. albicans [35]. In addition, Chang et al. [36] reported that the viability of cells treated with LfcinB decreased as the concentration of LfcinB increased; similarly, the antifungal activity of the chimera was concentration dependent. Furthermore, Jang et al. [37] reported that peptides containing the minimum motif of BFII showed antifungal activity, with MIC values of 32 to >64 µg/mL in strains of C. albicans and Cryptococcus neoformans. This is the first report describing the antifungal activity of chimeras containing sequences of two AMPs (LfcinB and Buforin II). It is possible that chimeras composed of two AMP sequences exert their antifungal activity through the combination of their mechanisms of action. It has been suggested that LfcinB acts by permeating the cell membrane [21]. On the other hand, BFII is internalized and interacts with nucleic acids by inhibiting processes of replication of genetic material and protein translation, leading to cell death [38]. The branched chimeric peptide C9 (MIC = 50 µg/mL/15 µM) exhibited greater antifungal effect compared to linear chimeras C2 (MIC = 200 µg/mL/122 µM) and C5 (MIC = 100 µg/mL/48 µM), possessing the same linker as the branched chimeras.
Taking into account the definition of fungistatic activity (decrease in the growth of a fungus by <99%) and fungicidal activity (decrease in the growth of a fungus by ≥99%) [39], our results show that chimeras C5, C6, C8, and C9 exhibited fungistatic and fungicidal activity in C. albicans SC5314 and C. albicans 256 (Figure 1). When a molecule exhibits fungicidal and/or fungistatic activity, therapeutic and prophylactic options, which can be monitored clinically, increase. It is important to highlight that chimeras C5, C6, C8, and C9 inhibited the growth of the C. albicans strains evaluated by more than 90%, being that these strains belong to the species most frequently involved in invasive candidiasis forms. The effect generated by the chimeric peptides depends on the concentration used; thus, it can be seen that higher concentrations are required to achieve the fungicidal effect compared to the concentrations that cause fungistatic activity ( Table 2). 13 (4) 25 (8) 13 (4) 25 (8) * The values correspond to the fungistatic and fungicidal activity after 48 h of incubation.
Chimera C9 exhibited fungicidal activity at 25 µg/mL (8 µM) on both C. albicans strains during 48 h of incubation, while at 12.5 µg/mL (4 µM) it exerted a fungistatic effect on the strains evaluated; the strain growth was completely inhibited up to approximately 20 h of treatment (Figure 1g,h). These results suggest that the antifungal activity is enhanced by the joining of two polyvalent motifs, the linear dimer of the RLLR sequence and the branched dimer of the RRWQWR.   However, C. albicans 256 (resistant to FLC) and C. albicans SC5314 (sensitive to FLC) exhibited susceptibility to chimeras C3, C5, C6, C8, and C9, suggesting that the antifungal activity of both peptide chimeras is not affected by the resistance mechanisms that the isolated strains employ against FLC, a conventional antifungal. This particularity is of great relevance, since these chimeras could be considered promising candidates for developing potential treatments against even resistant yeasts.

Hemolytic Effect
Chimeras C3, C5, and C6 did not show a significant hemolytic effect (2-4%) at MIC concentrations, indicating that these peptides are not toxic to normal erythrocytes. The precursor peptide RLLR (200 μg/mL) exhibited a hemolytic effect of 63%, and the peptides RRWQWR (200 µ g/mL) and RLLRRLLR (200 µ g/mL) exerted a hemolytic effect of 1% and 2%, respectively, while chimeras containing the motif RLLR or RLLRRLLR did not show a significant hemolytic effect at MIC concentrations (hemolytic activity data of chimeras C1 to C7 were previously published by [18]). Similarly, the C8 chimera did not show a hemolytic effect on erythrocytes at any peptide concentration evaluated, even at concentrations where a fungicidal effect occurs (200 μg/mL), indicating that this peptide is not toxic to human red blood cells at the concentrations evaluated. Chimera C9, at 12.5 μg/mL (4 μM) and 25 μg/mL (8 μM), did not show hemolysis; at these chimeric concentrations, fungistatic and fungicidal effects, respectively, were observed (Table 3). Our results suggest that the inclusion of sequences with hemolytic activity in chimeras improves antifungal activity and decreases the hemolytic effect.

Hemolytic Effect
Chimeras C3, C5, and C6 did not show a significant hemolytic effect (2-4%) at MIC concentrations, indicating that these peptides are not toxic to normal erythrocytes. The precursor peptide RLLR (200 µg/mL) exhibited a hemolytic effect of 63%, and the peptides RRWQWR (200 µg/mL) and RLLRRLLR (200 µg/mL) exerted a hemolytic effect of 1% and 2%, respectively, while chimeras containing the motif RLLR or RLLRRLLR did not show a significant hemolytic effect at MIC concentrations (hemolytic activity data of chimeras C1 to C7 were previously published by [18]). Similarly, the C8 chimera did not show a hemolytic effect on erythrocytes at any peptide concentration evaluated, even at concentrations where a fungicidal effect occurs (200 µg/mL), indicating that this peptide is not toxic to human red blood cells at the concentrations evaluated. Chimera C9, at 12.5 µg/mL (4 µM) and 25 µg/mL (8 µM), did not show hemolysis; at these chimeric concentrations, fungistatic and fungicidal effects, respectively, were observed (Table 3). Our results suggest that the inclusion of sequences with hemolytic activity in chimeras improves antifungal activity and decreases the hemolytic effect.

Antifungal Activity of Chimeric Peptides Mixed with FLC
The mixture of chimera C6 (25 µg/mL) and FLC (0.5 µg/mL) showed an additive effect on antifungal activity (FICI = 1) in C. albicans SC5314, showing a twofold reduction in the MIC values of the peptide and the FLC. In the FLC-resistant clinical isolate C. albicans 256, the combination of peptide C6 and FLC induced an indifferent effect (FICI = 1.03). These results suggest that the combination of the C6 chimera with FLC, although it did not exhibit a synergistic effect against C. albicans SC5314, can increase the activity of FLC by a factor of two when mixed with a C6 chimera, while no significant antifungal activity was evidenced for the FLC-resistant strain C. albicans 256 (Table 4). In contrast, a synergistic relationship was observed when both branched chimeric peptides (C8 and C9) were evaluated in combination with FLC in C. albicans SC5314, decreasing the MIC by a factor of between four and eight for the peptides and four for FLC (Table 4); this feature makes it possible to potentiate the antifungal activity of both molecules. The FLC-sensitive and -resistant C. albicans SC5314 and 256 strains, respectively, show equal susceptibility to each of the branched chimeras, evidence that the antifungal activity of the evaluated peptides is not affected by conventional antifungal resistance mechanisms.
The C8 peptide combined with FLC showed synergy for the antifungal activity against C. albicans SC5314, decreasing the MIC of the peptide by a factor of eight and a factor of four for FLC; however, the combination of the C8 peptide with FLC induces an indifferent effect.
C. albicans 256 (Table 4). These results suggest that the synthesis of chimeras containing MAP sequences is a promising strategy for combating resistant fungal mycoses, in order to extend the shelf life and efficacy of currently used drugs through the use of combination therapy. The results suggest that FLC-combined chimeras have the advantage of producing antifungal activity against resistant strains and may also alter the fungistatic activity of many compounds [43].
The emergence of the multidrug-resistant yeast C. auris and the circulation of resistant clones C. glabrata make it imperative to search for new therapeutic alternatives [44][45][46]. The C6 chimera was evaluated in combination with FLC against C. glabrata ATCC 2001 and 1875 and C. auris 001 and 537, obtaining an additive effect in three of them. For the reference strain C. glabrata 2001, an additive effect (FICI: 0.6) was observed, decreasing the MIC of the peptide by half and the FLC up to 10 times, in a way similar to what occurred in clinical isolates C. auris 001 and C. auris 537. In both cases, the MIC of FLC decreased by a factor of 2. Finally, for C. glabrata, an indifferent effect was obtained (Table 5). The results obtained by combining AMPs with FLC are consistent with the effect found by Vargas et al. [47], who also observed an additive effect against C. albicans and C. auris by the combination of a palindromic peptide derived from LfcinB with FLC, achieving an 8-fold enhancement of the activity of the AMPs through this mixture.

Reagents and Materials
The strain C. albicans SC5314 was purchased from ATCC (Manassas, VA, USA), and the clinical isolate C. albicans 256 HUSI-PUJ was obtained from the oral mucosa of a patient in the San Ignacio Hospital and deposited in the strain bank of the MICOH-P group of the PUJ

Peptides
Chimeric peptides containing the LfcinB and BFII motifs were (i) synthesized using solid-phase peptide synthesis using Fmoc/tBu strategy, (ii) purified using RP-SPE chromatography, and (iii) characterized by RP-HPLC and MS (Supplementary material), following the protocol reported by [19].

In Vitro Antifungal Susceptibility Test
Antifungal susceptibility testing was carried out using the broth microdilution (BMD) method, following the CLSI M27-A3 guidelines with slight modifications [48]. Briefly, cells (0.5-2.5 × 10 3 CFU/mL) were incubated with peptides (200, 100, 50, 25, 12.5, and 6.25 µg/mL) at 37 • C for 48 h. MICs were visualized and densitometry (595 nm, microplate reader, Bio-Rad, i Mark TM ) was used to determine the lowest concentration of peptide that caused a significant decrease (MIC/2 or ≥50%) compared with the growth control (cells incubated in absence of peptide) (n = 3). In order to verify that the peptides were able to kill the yeast cells, the plates were also evaluated for minimum fungicidal concentration (MFC). Briefly, aliquots from each well from susceptibility testing assays were transferred to plates containing Sabouraud dextrose agar (SDA), which were then incubated at 37 • C for 24 h. The highest dilution with no growth on the agar plate was considered to be the MFC.

Time-Kill Curves
A time-kill kinetic assay was carried out according to the method previously described by Pfaller and coworkers [49] with minor modifications. The peptides (C5, C6, C8, and C9) diluted in RPMI were tested at a range of concentrations: 0 (control), 0.2, 0.5, 1, and 2 times the MIC value for each strain. The isolates were subcultured on SBD, and then an inoculum was adjusted to a 0.5-2.5 × 10 3 CFU/mL in an RPMI 1640 medium. Yeast inoculum (150 uL) was added to a 100-well plate containing serial dilutions of the peptides. The plates were incubated with agitation at 37 • C in a plate reader (Bioscreen C MBR automated turbidometric analyzer, Growth Curves Ltd., Helsinki, Finland), which takes hourly absorbance readings. Moreover, fluconazole MICs were used as a control. The readings were analyzed with Bioscreen software (Growth Curves USA, Piscataway, NJ, USA) and GraphPad Prism 8.0.1 (GraphPad Soft-ware, San Diego, CA, USA). Statistical comparisons were made using an analysis of variance (two-way ANOVA) followed by a Tukey-Kramer post hoc test. P values less than 0.005 were considered significant. To determine the fungistatic and fungicidal effects, a growth inhibition of >70% for 48 h was considered fungistatic, and a yeast kill of ≥99% for 72 h was considered fungicidal. Moreover, previously determined FLC MICs (data not shown) were used as a control.

Hemolysis Assays
The hemolysis assays were carried out following the methodology reported by Vargas et al. [20] with some modifications, as follows. First, 5 mL of heparinized peripheral blood was centrifuged at 1000 g for 7 min. The erythrocyte fraction was suspended in 10 mL of saline solution (SS) and washed twice by centrifugation at 1000 g for 7 min. The erythrocytes (4% hematocrit) were incubated with peptide (ranging from 6.2 to 200 µg/mL), for 2 h at 37 • C. SS was used as negative control, while distilled water was used as a positive control. The mixtures were centrifuged, the supernatants were collected, and the absorbance was determined to be 450 nm.

Checkerboard Test
The in vitro interactions between the peptides (C6, C8, and C9) and the FLC drug were evaluated in a checkerboard assay, as previously described by Cokol et al. [50]. Briefly, each isolate was prepared by picking colonies from an overnight culture in SBD at 37 • C and suspended in a sterile normal saline PBS buffer. The fungal inoculum was adjusted to 0.5 MacFarland and the inoculum was adjusted to 0.5-2.5 × 10 3 CFU/mL. The drugpeptide combinations were formed over a range of concentrations: 0 (control) and 0.06 to 2 times the MIC [51]. The fractional inhibitory concentration index (FICI) was calculated as follows: The calculated FICI was reported as synergy with values FICI ≤ 0.5, additive FICI > 0.5-1, indifference FICI > 1-4, and antagonism FICI ≥ 4 [52].
A synergistic effect was considered to be when the effect of the combination exceeded the effects of the individual components. An additive effect was when the effect of the combination was equal to the sum of the effects of the individual components. An indifferent effect was one in which the activity was equal to the effects of the most active component. Finally, an antagonism was a reduced effect of a drug combination observed compared to the effect of the single most effective substance [52].

Conclusions
In this study, we report for the first time the antifungal activity of nine chimeras containing the LfcinB and BFII minimal motifs. The chimeras C9: (RRWQWR) 2 K-Ahx-RLLRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the highest antifungal activity (MIC 50 µg/mL) than the precursors, no medically significant results were observed (MIC 200 µg/mL) for the other chimeras evaluated. The peptide C9 showed promising antifungal properties being both fungistatic and fungicidal (25-12.5 µg/mL) against C. albicans. We also observed that the inclusion of Ahx as a spacer affects the antifungal activity against this strain. Furthermore, changing Arg to Lys in the LfcinB or BFII motifs enhanced the antifungal activity of the chimeras against the reference strain C. albicans SC5314 and the clinically isolated FLC-resistant C. albicans 256. The chimera containing the dimeric peptide LfcinB showed the highest antifungal activity against the tested strains, suggesting that this chimera design could be a successful strategy to enhance antifungal activity against reference strains or clinical isolates of Candida spp. It was also observed that these modifications help to reduce the toxicity of the peptides against mammalian cells, such as erythrocytes.
Finally, it could be demonstrated that the chimeric peptides could be considered as a therapeutic alternative that acts individually, but also when combined with antifungals such as fluconazole.