1. Introduction
Candidiasis is one of the most medically important mycoses worldwide with different clinical manifestations; one of the most frequent is the invasive candidiasis, responsible for about 75% of opportunistic yeast infections in hospitalized patients with inherent risk factors [
1]. Although there are about 200 species of
Candida described, only some can cause infection and, in some cases, present reduced susceptibility to antifungals as well as a recognized intrahospital environment infectivity. The main etiological agent of invasive candidiasis is
C. albicans, representing about 50% of cases. However, the prevalence of
non-albicans Candida species such as
C. glabrata in the United States and northwest Europe and
C. parapsilosis in Latin America, southern Europe, India, and Pakistan is increasing [
2].
Candidiasis is a common cause of morbidity and mortality. In the United States, nosocomial infection by
Candida spp. is the fourth most common cause of hospital admission [
2]. In Colombia (2017), there were 753,000 cases of fungal infections, of which about 600,000 cases were candidiasis, 130,000 were aspergillosis, and 16,000 cases were of opportunistic infections associated to patients infected with HIV [
3].
Epidemiological data obtained in Latin America indicate that the incidence rates of candidiasis are higher than those described in North America and Europe. A study carried out in 11 Brazilian hospitals showed a candidemia global incidence of 2.49 cases per 1000 hospital admissions [
4]. Another study performed in 22 hospitals in different Latin American countries showed an incidence of 0.98 cases per 1000 hospital admissions with a wide variation among countries (from 0.32 in Chile to 1.75 in Argentina) [
5].
The global increase in fungal infections leads to a proportional increase in resistance to antifungal drugs such as those of the azole family, frequently used in clinics. Troublingly, resistance to these drugs has been detected in different clinically relevant
Candida species [
4], generating the requirement to investigate and implement new antifungal therapy alternatives to mitigate this problem. Recently, the use of antimicrobial peptides from different organisms such as insects, amphibians, mammals, plants, and even the same host (human) with fungicidal effect has been proposed, specifically against yeasts of the genus
Candida with clinical importance [
5]. Glands, structural cells, and the immune system have the ability to produce and secrete some molecules that have antimicrobial properties, such as ß-defensins, histatins, and cathelicidins [
6].
The LL-37 peptide is a cationic human cathelicidin with antimicrobial properties. Its chemical structure begins with two leucines and is composed of 37 amino acids (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) [
7]. Its α-helical cationic amphipathic structure gives it an antimicrobial effect that acts on the microorganism’s cellular structures [
8]. The LL-37 peptide has exhibited a broad spectrum of antimicrobial activity against
S. aureus,
M. tuberculosis, and
L. monocytogenes [
7] among other Gram-positive and Gram-negative bacteria [
9], viral agents such as herpes simplex virus type 1 (HSV-1) and adenovirus [
10], fungi such as
C. albicans [
11], and some parasites such as
Leishmania sp. [
12]. The antimicrobial profile of the LL-37 peptide is mediated in two ways: transmembrane pore formation and intracellular damage. Additionally, LL-37 has other activities related to host defense, including inflammatory response regulation, adaptive immune cells’ chemotactic migration to the infection site, endotoxin neutralization, angiogenesis, and phagocytic cell activation in the presence of different reactive oxygen species (ROS), thus producing the cell death [
10,
11,
13].
The LL-37 peptide originates from a precursor, an 18 kD molecule called hCAP-18, which exists constitutively in the neutrophil granules [
14]. LL-37 can also be secreted by epithelial cells present in the skin, gastrointestinal tract, and respiratory tract. Additionally, it can be produced by natural killer cells [
7] as well as by subpopulations of monocytes and lymphocytes [
15]. The LL-37 peptide is an important component in the epidermis and can act on keratinocytes to induce the pro-inflammatory cytokines’ and chemokines’ production [
16].
Recently, a group of short peptides derived from LL-37 composed of 12, 20, and 30 amino acids was designed and showed significant antimicrobial activity, such as that observed in an experimental model against
Entamoeba hystolitica, in which these short peptides were used in a range of concentration between 10 and 50 µM to control the parasite [
17]. Additionally, other studies by our group showed promising antimicrobial effects of the LL-37 analogue peptides that were tested in medically important bacterial strains such as
Staphylococcus spp. and
Pseudomonas aeruginosa.
An important characteristic of these short peptides derived from LL-37 is that they have a smaller hemolytic effect than its natural structure [
17]. The analogue peptides of LL-37 (LL37-1, AC-1, AC-2, and D) have a shorter peptide structure, which confers them protection against proteases secreted by some microorganisms as well as those secreted by the host. The LL37-1 peptide is 24 amino acids long and amidated at the carboxyl-terminal position. The ACL 37-1 (AC-1) peptide is 23 amino acids long and presents acetylation at the amino terminal portion and amidation at the carboxyl-terminal domain. The ACL 37-2 (AC-2) peptide, 24 amino acids long, begins with glycine and presents acetylation at the amino terminal domain and amidation at the carboxyl-terminal position. Finally, the DL 37-2 (D) peptide with 25 amino acids has a modification in the amino terminal position that turns this analogue peptide into a D enantiomer.
It is important to note that the positive charge, peptide structural variations (for example, D enantiomer), and the short peptide chains could decrease the active sites where the endoproteases (released by the microorganisms as a defense mechanism) give the analogue peptides an intrinsic resistance to microorganisms [
18]. Therefore, the LL-37 analogue peptides mentioned were selected in this study in order to evaluate their possible antifungal effect against different reference species of
Candida as well as 20 strains with clinical importance that cause vulvovaginal candidiasis.
2. Materials and Methods
2.1. Synthesis and Purification of Antimicrobial Peptides
The human cathelicidin LL-37-derived peptides (AC-1, AC-2, LL37-1, and D) with the amidated C-terminal portion were obtained in Peptide 2.0 (Chantilly, VA, USA). Analyses with a high-performance liquid chromatography system (HPLC) and mass spectrometry (MS) performed by the manufacturer showed that the analogue synthetic human cathelicidin was 98% pure. Peptides derived from the LL-37 bioinformatic design were investigated on an anti-BP server (
http://www.imtech.res.in/raghava/antibp/index.htmL) (accessed on 20 January 2019) using the APD database (
http://aps.unmc.edu/AP/main.php) (accessed on 20 January 2019). To carry out the in silico experiments, we analyzed 15 different fragments and then selected the four peptides with the best antimicrobial profile: LL37-1 (GRKSAKKIGKRAKRIVQRIKDFLR) and AC-2 (GRKSAKKIGKRAKRIVQRIKDFLR), both with 24 amino acids, with the difference being that the AC-2 peptide presented acetylation in the terminal amino group; AC-1 (RKSKEKIGKEFKRIVQRIKDFLR) with 23 amino acids; and D ((d-PHE) GRKSAKKIGKRAKRIVQRIKD (d-F) LR) with 25 amino acids.
2.2. Microorganisms
Standard strains of C. albicans (SC5314 and ATCC 10231), C. parapsilosis ATCC 22019, C. krusei ATCC 6558, and C. tropicalis ATCC 750 were challenged. An azole-resistant strain of C. albicans 256 was identified by MALDI-TOF and then incorporated into the study. Additionally, 20 clinical isolates of C. albicans from patients with vulvovaginal candidiasis from Bogotá, Colombia, were analyzed. All strains were preserved in 10% glycerol at −80 °C. Three days before the experiments started, each fungal strain was sub-cultured in Sabouraud dextrose agar (Becton, Dickinson and Company; Sparks, NV, USA) and maintained at 37 °C for 24–48 h. Subsequently, isolated colonies of each strain were sub-cultured in brain–heart infusion liquid medium (BHI, Becton Dickinson, New Jersey, NJ, USA) and shaken at 100 rpm for 24 h at 37 °C in order to recover exponentially growing yeasts.
2.3. Susceptibility Assay of Candida Planktonic Cells
The minimum inhibitory concentration (MIC) was determined by the liquid medium microdilution technique, described in the M27-S4 document (Clinical and Laboratory Standards Institute (CLSI), 2012). The MIC was defined as the lowest necessary concentration of the LL-37 analogue peptides (AC-1, AC-2, LL37-1, and D) capable of inhibiting the different Candida species’ growth (C. albicans SC5314 and ATCC 10231, C. parapsilosis ATCC 22019, C. krusei ATCC 6558, and C. tropicalis ATCC 750) and the clinical strains evaluated in this study. Yeasts were re-suspended in RPMI 1640 medium (BiowHITTAKER®, Lonza, Belgium) supplemented with (3-(n-morpholino) propanesulfonic acid (MOPs, Sigma-Aldrich, Missouri, MO, USA) at a 0.5 McFarland scale representing 1 × 108 colony-forming units per milliliter (CFU/mL). Antimicrobial-derived peptides of LL-37 were added at 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.12 µM, and 1.5 µM concentrations, diluted in RPMI medium, and placed into sterile 96-well microtiter polystyrene plates (Corning Incorporated, New York, NY, USA) until adjusting the volume to 100 µL. Fluconazole (FLZ) (Pfizer, New York, NY, USA) at an initial concentration of 64 µg/mL and amphotericin B (AMB) (Sigma-Aldrich, Missouri, MO, USA) at an initial concentration of 16 µg/mL were used as antifungal controls since they are frequently used in the treatment of different mycoses including candidiasis. As growth control, we utilized different Candida yeasts without any antifungal treatment or the LL-37 analogue antimicrobial peptides. After 48 h of samples’ incubation, the 492 nm optical density measurement was carried out using a FC multiscan spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
2.4. Determination of Growth Phases Using the LL-37-Derived Peptides
Growth curves were performed using different Candida species: C. albicans ATCC 10231, C. albicans SC5314, C. krusei ATCC 6558, and C. parapsilosis ATCC 22019. These yeasts were re-suspended in RPMI 1640 medium supplemented with MOPs in a scale of 0.5 McFarland or its equivalent in optical density from 0.08 to 0.1, which represents 1 × 108 CFU/mL, utilizing an FC multiscan spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) to measure the samples at a wavelength of 600 nm. Subsequently, in sterile 100-well microtiter polystyrene plates (Honeycomb, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 150 μL of MOPs-supplemented RPMI medium and 150 μL of each yeast inoculum were added separately to determine the yeasts’ growth in the presence of the LL-37-derived peptides (AC-1, LL37-1, AC-2, and D) at concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.62 μM. For this experiment, the amphotericin B (Sigma-Aldrich, USA) was used as antifungal control in an initial concentration of 40 μg/mL. Samples were incubated and analyzed in a BioScreen C piece of equipment (Thermo Labsystems Type FP-1100-C, Waltham, MA, USA) at a constant temperature of 37 °C with continuous shaking for 48 h. All measurements were carried out in an automated way every hour through the presence of turbidity at a wavelength of 600 nm. To increase the reproducibility, each assay parameter was performed in triplicate.
2.5. Preformed Biofilm Eradication Assay
The yeast concentration of
C. albicans 10231 was adjusted to 1 × 10
6 cells/mL in RPMI medium supplemented with MOPs. Subsequently, 100 μL/well of this solution was placed in 96-well plates and incubated for 24 h at 37 °C to induce the biofilm formation. Afterward, 100 µL of AC-1, LL37-1, AC-2, and D analogue peptides were added at different concentrations (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.62 μM, 0.31 μM, 0.15 μM), respectively, as well as the antifungal control (amphotericin B) in concentrations of 64 to 0.5 µg/mL, which is equivalent to 0.069 to 0.0005 ≈ µM. After this process, samples were incubated again for 24 h at 37 °C. Finally, the analysis of the peptides anti-biofilm effect was carried out by means of the reduction assay with 2,3-bis (2-methoxy-4-nitro-5sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT, Sigma-Aldrich, Missouri, MO, USA) in incubation for 3 h and subsequent OD measurement at 620 nm as described by Pierce and coworkers (2008) [
19].
2.6. Scanning Electron Microscopy (SEM)
C. albicans ATCC 10231 was treated with a concentration lower than the MIC of each antimicrobial peptide, analogous to LL-37 for 24 h at 37 °C. Afterward, the samples were fixed in 2.5% glutaraldehyde for 3 h at room temperature. The samples were then applied on a polylysine-coated coverslip, serially dehydrated in alcohol, and subsequently observed in a scanning electron microscope and focused ion beam FE-MEB LYRA3 of TESCAN (Brno, Czech Republic), which has an integrated X-ray energy dispersive spectroscopy (EDS) microanalysis system (energy dispersive X-ray spectroscopy).
2.7. Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 7.05 (GraphPad Software, San Diego, CA, USA). Statistical comparisons were carried out by the analysis of variance (one-way ANOVA) followed by a Tukey–Kramer post hoc test. The p-values of <0.05 indicated statistical significance.
4. Discussion
In this study, we showed the in vitro antifungal activity of four analogue peptides to human cathelicidin LL-37 against yeasts of the genus Candida that reflects a possible therapeutic alternative, favoring advances in the rational design of new peptides as possible therapies to treat superficial and deep mycoses.
The derived peptides herein analyzed (AC-1, AC-2, LL37-1, and D) showed inhibitory activity in vitro against different
Candida species and even against 20 clinical strains obtained from patients with vulvovaginal candidiasis. The differences observed in the inhibitory processes of each peptide are possibly related to some intrinsic characteristics of the antimicrobial peptides, such as their positive charge (generally +2 to +9) [
20] and their cationic nature, which allows these peptides to bind ideally to the anionic charges of cell membranes [
21]. On the other hand, the peptides’ hydrophobicity provides them with the ability to insert into microbial membranes causing structural damage. Finally, the amphipathicity or duality presented by these peptides, as they contain apolar and polar regions, results in an increased antimicrobial activity [
22,
23].
Regarding the synthetic variants of cathelicidin LL-37, the AC-1 peptide showed an outstanding antifungal effect at the five concentrations utilized against
Candida albicans 256 and at 10 μM for
Candida albicans SC5314. The AC-1 peptide has an acetylated N-terminal domain in its chemical structure that allows it to insert itself with more affinity in the fungal cell membrane, avoiding an adequate lipid packing and, therefore, increasing its lytic capacity [
24].
The AC-2 peptide showed an important antifungal activity against different Candida spp. strains, as observed in C. tropicalis ATCC 750, C. krusei ATCC 6558, C. parapsilosis ATCC 22019, and C. albicans ATCC 10231. Probably, the action mechanism of AC-2 is different from that of AC-1 since, within its chemical structure, it has one glycine more and it is acetylated and amidated in the carboxyl-terminal position, which confers protection against the microorganism’s proteolytic systems.
Tsai and coworkers (2014) [
25] announced the antifungal activity of LL-37 against
C. albicans, detecting the fungicidal effect from 20 μg/mL (~4.4 μM) to 40 μg/mL (~8.8 μM). Our results corroborate this information since the LL-37 analogue peptides such as LL-37-1 strongly inhibited yeast growth, with MICs observed from 0.07 μM for
C. tropicalis ATCC 750 to 5 μM for
C. albicans clinical isolates. It should be noted that when the
Candida albicans 256 strain was exposed to the LL37-1 peptide, the inhibitory effect was evidenced in the five concentrations analyzed. Possibly, the fact that LL37-1 is amidated in the C-terminal position provides it a protective effect (temporary) and increases its stability against microorganism exonucleases, enhancing its biological activity [
26].
Peptide D presented an outstanding antifungal effect, with MICs between 0.15 and 5 µM against different strains of
C. albicans 256 resistant to azoles,
C. tropicalis ATCC 750,
C. parapsilosis ATCC 22019, and some of the clinical isolates. Peptide D has a structural change in the right region of carbon-α (chiral carbon), classifying itself as a positively charged D enantiomer, which allows it to easily bind to the anionic charges of microbial membranes, causing the microorganism’s structural destabilization and promoting the D peptide stability in proteolytic systems, thus enhancing its antimicrobial effect [
27].
The present work shows the inhibitory effect of the LL-37 analogue peptides (LL37-1, AC-1, AC-2, and D) in small concentrations (0.62 μM) against strains with a broad resistance profile to azoles such as
C. albicans 256 and clinical strains from patients with vulvovaginal candidiasis as well as different
Candida species’ control strains. It should be noted that the purpose of proposing new antimicrobial peptides as possible therapeutic candidates consists of the search for minimum concentrations, in which the risk of cytotoxicity for the host cells could consequently be reduced [
25].
Additionally, the peptides analyzed in this work presented an important inhibitory effect on the yeast growth in the planktonic state (range of action from 0.62 µM to 20 µM). Additionally, they strongly decreased the biofilm formation process, which is one of the main virulence factors of different
Candida species, due to the biofilm limiting the antifungals’ penetration through the extracellular matrix and preventing the host’s proper immune response functioning [
28]. Inhibiting biofilm formation is undoubtedly a very important step to control
Candida infection since the ability to form biofilm leads to a fungal successful persistence that is associated with high mortality rates [
29].
AC-1, LL37-1, AC-2, and D peptides showed significant antifungal activity against yeast of the Candida genus. It is important to highlight the antifungal effect of these peptides, even in strains resistant to fluconazole, as well as clinical isolates that cause recurrent vulvovaginal candidiasis that have reduced susceptibility to this azole. However, more detailed studies are needed to better understand the interaction of these analogue peptides with pathogenic yeasts. It is crucial to continue with the search for these types of peptides with antifungal properties, optimizing biophysical parameters, maximizing their antimicrobial effect, and minimizing the toxicity to host cells. On the other hand, the analogue peptides may be interesting therapeutic candidates to control infections caused by multiresistant strains to different conventional antifungals.
Finally, scanning electron microscopy (SEM) analysis showed the negative effect of LL37 analogue peptides (AC-1, AC-2, LL37-1, and D) on
C. albicans yeasts. Important morphological alterations on the cell wall, among others, have been described by some authors studying different antimicrobial peptides against this yeast with promising results [
30] Through this methodology it was possible to verify the antifungal effect of the analogous peptides of LL37. However, in the future, it will be necessary to carry out other complementary assays to better understand the site and mechanism of action of peptides on fungal yeasts.
Some research groups have analyzed the LL37 peptide cytotoxicity [
31]. However, no data on the specific cytotoxicity of the analogous peptides herein studied have been published so far. Coworkers are currently carrying out studies focused on the possible toxicity of these peptides in human red blood cells and in the fibroblast cell line L929 with promising results after 24, 48, and 72 h after peptide exposure (data in process).
Other formulations could be developed in the future using delivery systems, such as nanoparticles, in order to improve the antifungal effect of the antimicrobial peptides herein studied. Peptides immobilized in nanoparticles are a promising option in fungal infections’ control since this could potentiate the effect of some conventional antimicrobial or antifungal molecules and present a synergistic or additive effect in the infection control [
32,
33]. It would be interesting to address, in subsequent studies, the possible synergistic or additive effects that the mentioned peptides may present if together they deliver antifungal drugs currently used in clinics. Finally, we suggest that peptides derived from human cathelicidin LL-37 are potential therapeutic candidates due to their rapid mechanism of action and in vitro efficiency in yeast control and are projected as a therapeutic option against candidiasis, a frequent and important mycosis due to the high morbidity and mortality it causes worldwide.