In Vitro and In Vivo Effect of Peptides Derived from 14-3-3 Paracoccidioides spp. Protein

Background: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. Methods: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. Results: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings.


Introduction
Globally, 1.2 million people are affected by fungal infections; however, it is difficult to estimate the real incidence of fungal infections since there is no obligatory reporting of these diseases in many countries [1,2]. Paracoccidioidomycosis (PCM) is a fungal disease restricted to Latin American countries, caused by the dimorphic fungi from the Paracoccidioides genus, composed of the species Paracoccidioides brasiliensis, Paracoccidioides lutzii, Paracoccidioides americana, Paracoccidioides restrepiensis and Paracoccidioides venezuelensis [3][4][5]. PCM is not a compulsory notification infection, which makes difficult the determination of real incidence of this disease. In Brazil it is estimated that the annual incidence ranges from 0.71 to 3.7/100,000 inhabitants [6,7].

Microorganisms and Culture Conditions
Paracoccidioides brasiliensis (São Paulo, Brazil) strain 18 and P. lutzii Pb01-like (American Type Culture Collection-ATCC MYA-826/Goiania, Brazil) strain 01 from Laboratory of Clinical Mycology, Faculty of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, Brazil were maintained in Fava-Netto agar at 37 °C for 4-5 days. For the experiments, the Fava-Netto agar culture was transferred to a brain heart infusion (BHI) broth supplemented with 1% glucose for 3-4 days at 37 °C at 150 rpm.

Cytotoxicity of 14-3-3 Peptides
Peptides cytotoxicity was performed using the resazurin method, according to Pavan et al. 2010 [43]. For this assay, HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, all acquired from the collection of the Banco de Células do Rio de Janeiro-BCRJ, Brazil, were used. For monolayer formation, 10 6 cells/mL were plated in a 96-well microplate using Dulbecco's Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and incubated for 24 h at 37 °C with 5% CO2. Next, the supernatant was removed, the peptides diluted in DMEM at concentrations from 19.5 to 2500 µg/mL were added. As controls were used DMEM medium, cells plus DMEM medium, and cells treated with 30% dimethyl sulfoxide (DMSO). The plate was incubated for 24 h at 37 °C with 5% CO2. After the incubation, 30 µL/well of 0.01% resazurin were added and incubated for six h at 37 °C with 5% CO2. The readings were taken at 570/600 nm, and the results expressed as a percentage of cell viability.

Antifungal Activity of 14-3-3 Peptides
The antifungal activity of the 14-3-3 peptides was evaluated by broth microdilution method, according to de Paula e Silva, et al. [44]. For this, Roswell Park Memorial Institute (RPMI) 1640 medium with L-glutamine and without sodium bicarbonate buffered at pH 7 with 4-Morpholinepropanesulfonic acid (MOPS) and supplemented with 2% glucose was used. The Paracoccidioides spp. inoculum concentration was 0.5-2.5 × 10 3 cells/mL, and the antifungal activity of the peptides P1, P2 and P3 from 4.9 to 2500 µg/mL were evaluated. Amphotericin B from 0.016 to 8 µg/mL was used as a control. The plates were incubated at 37 °C with shaking at 150 rpm for 72 h. After that, 20 µL/well of Alamar Blue TM (BioSource International, Invitrogen, Eugene, OR, USA) was added, followed by another 24 h incubation. The readings were taken at 570/600 nm, and the results expressed as a percentage of fungal cell viability.

Paracoccidioides spp. Adhesion Inhibition Assay to the Extracellular Matrix Component or Pneumocytes (A549) Cells
Then adhesion inhibition assay was performed according to De Oliveira et al. [45]. For this, 96-well Corning ® plates were coated with 50 µg/mL laminin (Sigma Aldrich, Saint Louis, MO, USA) for 18 h at 4 °C. Wells plates were washed three times with phosphate-buffered saline (PBS). Then, the laminin-coated plate was treated with 100 µg/mL of peptides P1, P2, and P3 for 1 h at 37 °C. Paracoccidioides spp. at 10 6 cells/mL were stained with 100 µM of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Sigma Aldrich, Saint Louis, MO, USA) for 20 min at 37 °C, then 100 µL of this inoculum was added to laminin-coated wells and treated with the peptides and incubated for 3 h at 37 °C. After incubation, the wells were washed carefully three times with PBS to remove fungal cells not adhered to the laminin. One hundred microliters of PBS were added to each well, and with the aid of a tip, the bottom of each well was scraped to remove the yeast adhered to the laminin, this process was repeated twice. The samples were analyzed by flow cytometry using BD FACSCanto equipment (BD Biosciences, San Jose, CA, USA) by acquiring 30 s in medium mode; afterward, the data were calculated for milliliters. For the evaluation of the effects of peptides in Paracoccidioides spp. adhesion to pneumocytes (A549) cells, 10 6 cells/mL were plated in 96-well microplate using Modified Eagle Medium supplemented with 10% FBS and incubated for 24 h at 37 °C with 5% CO2. Treatments were realized as described above in the laminin assay. The samples were analyzed by flow cytometry and expressed in fungal-pneumocyte percentage of interaction.

G. mellonella Rearing and Experiment Conditions
G. mellonella larvae were cultivated in the laboratory, according to Jorjão, et al. [46]. Larvae weighting 150-200 mg, active and without dark spots, were selected and incubated at 37 °C, and protected from the light before the experiments. Before treatment/infection, aseptic treatment of the pro-legs was performed with 70% ethanol. The larvae were inoculated and/or treated using 10 µL Hamilton syringes (Hamilton, Reno, NV, USA). In all treatments, larvae were incubated at 37 °C. For each experimental condition, 10-15 larvae were used, and each experiment was repeated at least three times.

Hemocytes Concentration
G. mellonella were treated with 100 µg/larva of peptides P1, P2, and P3 and incubated for 3 h at 37 °C. After this period, the larvae hemolymph was removed through an incision with the aid of a scalpel, 10 µL of hemolymph was diluted in 90 µL of anticoagulant solution (2% NaCl; 0.1 M glucose; 30 mM sodium citrate; 26 mM citric acid and 10 mM Ethylenediamine tetraacetic acid (EDTA)) and then to 200 µL of FACs fluid. Hemocyte counts were performed by flow cytometry in BD FACSCanto equipment (BD Biosciences, San Jose, CA, USA). For that, the number of events was recorded in 60 s on medium acquisition mode, which is equivalent to 60 µL of the sample; afterward, the data were calculated for milliliters. The hemocytes from larvae injected with PBS were compared with the infected ones.

Induction of Phenoloxidase Activity by Peptides
The quantification of the phenoloxidase enzyme was performed according to the protocol described by Laughton and Siva-Jothy [47]; for that, the larvae of G. mellonella were treated with 100 µg/larva for 3 h at 37 °C. Then the hemolymph of 5 larvae was collected and pooled; 50 µL of each hemolymph pool was added to 150 µL anticoagulant solution and centrifuged for 5 min at 500 rpm to remove the cells. Aliquots of 50 µL of the supernatant were collected, and 50 µL of PBS was added to the samples; for positive control, 50 µL LPS (lipopolysaccharide) at 2.5 mg/mL, was added instead of PBS, and the plate was incubated for 5 min at 25 °C. After incubation, 25 µL of 6 mM L-dopa was added as a substrate in all samples and incubated for an additional 1 h at 25 °C. The reading was performed at 490 nm, and the optical density values obtained were analyzed.

Analysis of the Expression of Antimicrobial Peptides
Five G. mellonella larvae per group were treated with 100 µg/larva of peptides P1, P2, and P3 and incubated for 3 h at 37 °C. The hemolymph of all larvae was pooled and diluted in 100 µL of anticoagulant solution. RNA extraction was performed using the Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The synthesis of the cDNA strand was performed using reverse transcriptase (RevertAid H Minus Reverse Transcriptase, (Fermentas, Waltham, MA, USA). Real-time polymerase chain reaction (PCR) was performed with Maxima ® SYBR Green/ROX qPCR Master Mix (2X) (Fermentas, Waltham, MA, USA) using the Applied Biosystems 7500 equipment. The relative expression of the galiomicin and gallerimycin genes was calculated using values 2 CT according to Livak and Schmittgen [48], using s7 gene (ribosomal protein) as a housekeeping gene. The primers sequences are described in Table 1.  [49] Galiomicin TCG TAT CGT CAC CGC AAA ATG GCC GCA ATG ACC ACC TTT ATA [50] Gallerimycin TATCAT TGG CCT TCT TGG CTG GCA CTCGTA AAA TAC ACA TCC GG [50] 2.6.5. Efficacy of 14-3-3 Peptides in G. mellonella Model The larvae were treated with 100 µg/larva of peptides P1, P2, and P3 and incubated for 3 h at 37 °C. After that, the infection with 5 × 10 6 cells/larvae of P. brasiliensis or P. lutzii was done, and as a control, larvae were treated with PBS. After the infection, larvae were incubated at 37 °C, and death was monitored daily for seven days, checking the movement after touching them with forceps.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). The effects of peptides in hemocytes concentration and phenoloxidase liberation were analyzed by using Kruskal-Wallis and Dunn's tests, and C. elegans antimicrobial peptides real-time PCR. The survival test was analyzed using the Log-rank test (Mantel-Cox). The p > 0.05 is considered significant in all tests.

In Silico Prediction of Binding Peptides from Paracoccidioides spp. 14-3-3 Protein to Mouse MHC Class II Alleles
The NetMHCIIpan-4.0 algorithm revealed three peptides with a good prediction for binding to mouse MHC class II alleles. The sequences of these peptides are in red ( Figure  1), and these were called peptide 1 (P1), peptide 2 (P2), peptide 3 (P3).

Effect of 14-3-3 Peptides in Mammalian Cell
PCM affects the lungs and skin during the infection; because of this, the peptides from 14-3-3 protein were evaluated regarding its cytotoxicity in lung epithelial cells, lung fibroblasts, and skin keratinocytes at concentrations ranging from 19.5 to 2500 µg/mL (Figure 2A-C). The peptides treatment showed viability rates above 80% for all evaluated cell lines, regardless of concentration.

Effect of 14-3-3 Peptides on Paracoccidioides spp. Cells
The broth microdilution assay demonstrated that peptide P1 was the most active peptide and reduced P. brasiliensis and P. lutzii viability in a concentration-dependent manner. The treatment with 4.9 µg/mL of P1 reduced the P. brasiliensis viability by 30.5% and 2500 µg/mL was able to reduce P. brasiliensis viability by 66%. In a similar way, P1 could reduce the P. lutzii viability by 22% (39 µg/mL) to 70% (2500 µg/mL) ( Figure 3A,B). The peptide P2 did not affect Paracoccidioides spp. viability. On the other hand, P3 at high concentration (2500 µg/mL) reduced P. brasiliensis and P. lutzii viability by 20% and 30%, respectively.

Evaluation of the Immunomodulatory and Antifungal Effect in G. mellonella
Before the evaluation of immunomodulatory and antifungal effect in G. mellonella, the toxicity of the 14-3-3 peptides was assessed at concentrations of 10, 20, 40, 100 µg/larva. No larval death was observed in survival curves at the evaluated concentrations, demonstrating that these peptides were not toxic (data not shown). The peptides concentration of 100 µg/larva were selected for the next G. mellonella assays.

Effect of 14-3-3 Peptides in Hemocytes Concentration
After 3 h of treatment with 100 µg/larvae of 14-3-3 protein peptides, a 1.8-fold increase in G. mellonella hemocytes concentration (p < 0.05) was observed for peptides P1 and P2 (Figure 4) when compared to PBS. No statistical difference was observed for the treatment with P3.

Effect of 14-3-3 Protein Peptides in Humoral Response of G. mellonella
As described, phenoloxidase enzyme and antimicrobial peptides are essential factors in the humoral response of G. mellonella against microorganisms. The effect of 14-3-3 peptides in the humoral response of G. mellonella was evaluated in phenoloxidase enzyme and in the expression of genes that encode the antimicrobial peptides galiomicin and gallerimycin. P2 and P3 induce the production of phenoloxidase with a 7.0 and 6.3-fold increase (p < 0.05), respectively when compared to larvae treated with PBS ( Figure 5A). Also, P1, P2, and P3 increased galiomicin and gallerimycin genes expression ( Figure  5B,C). The most significant increase in peptide expression occurred with the treatment of P1 and P3, respectively, a 15-fold increase for galerimicin and a 6.8-fold increase in galiomycin expression compared to the untreated control. Figure 5. Effect of 14-3-3 protein peptides in humoral response of G. mellonella. Induction of G. mellonella phenoloxidase enzyme after treatment of 14-3-3 peptides using L-dopa as substrate (A). Relative expression G. mellonella of peptides galiomicin (B) and gallerimycin (C) genes after treatment of 14-3-3 peptides. Data expressed as mean ± standard deviation of three biological replicates and three independent experiments (* p < 0.05), using Kruskal-Wallis and Dunn's for phenoloxidase enzyme activity and gene expression using Student's t-test. Lipopolysaccharide (LPS).

Antifungal Protective Effect of 14-3-3 Protein Peptides against Paracoccidioides spp. Infection
We also evaluated whether the changes in the immune response of G. mellonella caused by pretreatment with the peptides would affect the survival of larvae infected with P. brasiliensis and P. lutzii, which are the most studied etiologic agents of PCM. Although there may have been no statistical difference between the curves, all three peptides led to a delay in the larvae death ( Figure 6). Figure 6. Survival curve of G. mellonella treated with immunogenic peptides at a concentration of 100 µg/larva at 3 h before the infection with P. brasiliensis (A) or P. lutzii (B). Log-rank test (Mantel-Cox) of eight to 10 biological replicates and three independent experiments (p < 0.05).

Evaluation of C. elegans Antimicrobial Peptide Expression after Treatment with 14-3-3 Peptides
The effect of peptides 14-3-3 was evaluated in the C. elegans model, and peptide P3 induced a significant increase of npl-31 (2.9 times), compared to other antimicrobial peptides (p < 0.05) (Figure 7).  Table 3 summarizes the results obtained in vitro and in G. mellonella and C. elegans invertebrate models.

Discussion and Conclusions
Mycoses are a threat to immunosuppressed and immunocompetent patients [2,54]. PCM is the most prominent systemic mycosis in Latin America due to its high incidence, long period of treatment, and the sequelae that can affect patients in the most productive life period [6][7][8]. Considering these factors, the search for alternatives to prevention and treatment is urgent. Different approaches have been investigated in the search for a safe and effective immunization or vaccination therapy for invasive mycoses such as the use of monoclonal antibodies [55], peptide vaccine [56,57], vaccines based on nanotechnology [58], and DNA vaccine [59]. The 14-3-3 protein is an important virulence factor of Paracoccidioides spp. acting as an essential adhesin [17][18][19]. Therefore, we aimed to determine candidates of immunogenic peptides from this protein and evaluate their biological activity and immunogenic potential in vitro and in vivo. Three peptides of 14-3-3 protein were obtained based on MHC class II alleles binding properties using in silico methods. The cytotoxicity was analyzed using skin keratinocytes, lung fibroblasts, and pneumocytes. The peptides were not toxic for these cells, leading to a small reduction (20%) viability. Therefore, a reduction in viability below 30% is acceptable in cytotoxicity testing, according to ISO 10993-5 (2009).
The antifungal activity against Paracoccidioides spp. showed that the peptide P1 was the most effective in reducing Paracoccidioides spp. viability around 30.5-70% at concentrations between 4.9 and 2500 µg/mL. On the other hand, the pneumocytes treatment with 14-3-3 peptides followed by Paracoccidioides spp. infection was not effective at inhibiting the host-cell interaction of P. brasiliensis or P. lutzii. Similar results were observed when the extracellular matrix component (laminin) was treated with 14-3-3 peptides and further infected ( Figure A1). This peptide originates from an adhesin of the fungus itself, and possibly is not associated with the adhesion role. Thus, inhibition of adhesion to the components of the extracellular matrix or interaction with cells is not a property of this peptide.
Next, 14-3-3 peptides characterization was expanded for in vivo assay using the G. mellonella invertebrate model. The 14-3-3 peptides were not toxic for the larvae and could increase the larvae hemocyte density compared to the control. The increase in the hemocyte density is an essential aspect of the cellular immune response in G. mellonella associated with eliminating infections [60]. Previous data showed that infection by Paracoccidioides spp. reduces the density of hemocytes [22]; therefore, the induction of these cells is an essential strategy for treating larvae infected with this fungus. The challenge of G. mellonella with a sublethal dose of Aspergillus fumigatus increased the hemocyte concentration and the resistance of larvae to this infection [61]. Hemocyte concentration increases with the peptides' treatment derived from a phage library with anti-adhesive activity for Paracoccidioides spp. [62]. The treatment of G. mellonella infected with Candida haemulonii with copper (II), manganese (II), and silver (I) 1,10phenanthroline chelates significantly increased the hemocyte concentration, larvae survival, and reduced fungal burden [63]. Treatment and pretreatment of larvae infected with Candida auris with crude extract and derivatives of the culture supernatant fraction of Lactobacillus paracasei 28.4, a postbiotic, increased hemocytes level of infected larvae [64].
In our study, we demonstrated that P2 and P3 were able to induce phenoloxidase production. Melanization is a vital process in the immune response of invertebrates. It occurs by activating the pro-phenoloxidase cascade generating toxic intermediates such as quinones, and the final product is melanin. Various components of the membrane and cell wall of microorganisms such as PAMPS (pathogen associated molecular patterns), peptidoglycans, lipopolysaccharide, and β-1-3 glucan (cell wall lipopolysaccharides) participate in the activation of the pro-phenoloxidase. Also, enzymes released by the invertebrate itself due to tissue damage by microorganisms can stimulate the cascade [65]. These substances are very toxic to microorganisms and help in phagocytosis and invertebrate survival [66]. As already described, G. mellonella larvae infected with a sublethal dose of C. albicans could induce phenoloxidase activity [67].
Another essential attribute of the G. mellonella immune system is the production of antimicrobial peptides. In this study, 14-3-3 peptides increased the expression of gallerimycin and galiomicin peptides. A significant increase of galiomicin occurred after treatment with P1. The P3 peptide was able to increase the expression of both antimicrobial peptides. The relation between these antimicrobial peptides with infection control has already been demonstrated in different microorganisms. The pretreatment of G. mellonella larvae with Photorhabdus luminescens killed by heat with subsequent infection by this bacterium increased gallerimycin and galiomycin expression and, consequently, the survival of the larvae [68]. Another study demonstrated that sublethal doses of C. albicans and A. fumigatus induced the expression of the same antimicrobial peptides [50,61]. Furthermore, the infection of G. mellonella with Bacillus subtilis and Bacillus atrophaeus vegetative and sporulating forms also increased gallerimycin and galliomycin expression [69]. The pre-infection with B. subtilis and B. atrophaeus increased gallerimycin and galiomicin expression and survival of larvae infected with C. albicans. On the other hand, the treatment with peptides delayed larvae death when they were infected with P. brasiliensis or P. lutzii.
The effect of 14-3-3 peptides was also evaluated on the expression of C. elegans antimicrobial peptides. The treatment promoted an increase of peptides from Ascaris suum antibacterial factor (ASABF), caenacins (cnc), neuropeptide-Like protein (npl) class. Different signaling cascades are involved in the C. elegans response against pathogens, such as pathways involving protein kinases (MAPKs), homologous to TGF-β, insulin-like peptides, and Toll-like receptors. These cascades are responsible for the expression of antimicrobial peptides [52]. An increasing number of reports have demonstrated the importance of these peptides' expression in protecting C. elegans against different fungal and bacterial pathogens [51,53,70,71]; thus, 14-3-3 peptides proved to be beneficial in C. elegans.
Due to the fungus' complexity, attenuated, or inactivated, whole cells for immunization can generate unwanted responses. Predictions of epitope biology for selecting peptides capable of inducing the immune response are alternatives in searching for vaccines [57]. A significant advance in the research of a possible PCM vaccine was made using one peptide of the gp43 P. brasiliensis protein (P10), which is an adhesin and significant diagnostic antigen of this mycosis. By using different vaccine approaches, the P10 peptide was able to reduce the fungal burden and to increase cytokine production [59,[72][73][74][75].
This study characterized three peptides derived from the protein 14-3-3 of P. brasiliensis. The peptides induced an immune response in G. mellonella and C. elegans, invertebrate animal models demonstrating their potential. Although a positive correlation has been reported between invertebrates and mammals' models [30], further studies are needed to evaluate these peptides in this model.

Data Availability Statement:
The data presented in this study are available in the article.