Effects of the antifungal peptide Skh-AMP1 derived from Satureja khuzistanica on cell membrane permeability, ROS production, and cell morphology of conidia and hyphae of Aspergillus fumigatus

Journal Pre-proof Effects of the antifungal peptide Skh-AMP1 derived from Satureja khuzistanica on cell membrane permeability, ROS production, and cell morphology of conidia and hyphae of Aspergillus fumigatus Soghra Khani, Sima Sadat Seyedjavadi, Hamideh Mahmoodzadeh Hosseini, Mehdi Goudarzi, Shirin Valadbeigi, Shohreh Khatami, Soheila Ajdary, Ali Eslamifar, Jafar Amani, Abbas Ali Imani Fooladi, Mehdi Razzaghi-Abyaneh

PII:

S0196-9781(19)30173-1

DOI:

https://doi.org/10.1016/j.peptides.2019.170195

Reference:

PEP 170195

To appear in:

Peptides

Received Date:

27 July 2019

Revised Date:

2 November 2019

Accepted Date:

4 November 2019

Please cite this article as: Khani S, Seyedjavadi SS, Hosseini HM, Goudarzi M, Valadbeigi S, Khatami S, Ajdary S, Eslamifar A, Amani J, Imani Fooladi AA, Razzaghi-Abyaneh M, Effects of the antifungal peptide Skh-AMP1 derived from Satureja khuzistanica on cell membrane permeability, ROS production, and cell morphology of conidia and hyphae of Aspergillus fumigatus, Peptides (2019), doi: https://doi.org/10.1016/j.peptides.2019.170195

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Effects of the antifungal peptide Skh-AMP1 derived from Satureja khuzistanica on cell membrane permeability, ROS production, and cell morphology of conidia and hyphae of Aspergillus fumigatus Soghra Khania, Sima Sadat Seyedjavadia, Hamideh Mahmoodzadeh Hosseinib, Mehdi Goudarzic, Shirin Valadbeigid, Shohreh Khatamid, Soheila Ajdarye,Ali Eslamifarf , Jafar Amanib,Abbas Ali Imani Fooladib**, Mehdi Razzaghi-Abyaneha*

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Department of Mycology, Pasteur Institute of Iran, Tehran, Iran Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran c Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran d Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran e Department of Immunology, Pasteur Institute of Iran, Tehran, Iran f Department of Clinical Research, Pasteur Institute of Iran, Tehran, Iran

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*Correspondence to:

**Corresponding author

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Mehdi Razzaghi-Abyaneh, PhD Department of Mycology Pasteur Institute of Iran, Tehran 1316943551, IRAN E-mails: [email protected] & [email protected] E-mail: [email protected]

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Highlights  Skh-AMP1 showed sporicidal activity against nongerminated conidia of A. fumigatus. Skh-AMP1 targeted fungal cell membrane integrity and permeability.

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Skh-AMP1 induced ROS production which may cause apoptosis in fungal cells.

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Skh-AMP1 destructed cell membrane and cellular compartments of conidia and hyphae.

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Abstract Skh-AMP1 (GRTSKQELCTWERGSVRQADKTIAG) is an antifungal peptide isolated from Satureja khuzistanica which has been shown to have strong antifungal activity against Aspergillus and Candida species, but no obvious hemolytic effects or cell cytotoxicity in vitro. In the present study, Skh-AMP1 was synthesized, and its mode of action on the plasma membrane, mitochondria, and morphological and ultrastructural changes against conidia and hyphae of Aspergillus fumigatus were evaluated. The results indicated that Skh-AMP1 had sporicidal activities against the non-germinated conidia of A. fumigatus at concentrations of 40 and 80 µM. Skh-AMP1 induced the release of K+ and the uptake of propidium iodide and enhanced reactive 1

oxygen species (ROS) production in the conidia and hyphae of the fungus. Scanning and transmission electron microscopy showed deformation and shrinkage of the hyphae and conidia, cell membrane disruption and detachment from the cell wall, microvesicle formation, vacuolation and depletion of cytoplasm and organelles of the hyphae of A. fumigatus exposed to 40-80 µM of the peptide. The results further demonstrated that the antifungal activity of SkhAMP1 may be related to its ability to disrupt fungal cell membrane permeabilization and induce enhanced ROS production. Therefore, Skh-AMP1 can be introduced as a novel antifungal candidate for developing new therapeutic agents.

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Abbreviations AMPs, Antimicrobial peptides; AFPs, Antifungal peptides; ROS, reactive oxygen species; SDA, Sabouraud Dextrose Agar; SDB, Sabouraud Dextrose Broth; DCFH-DA, 2,7-dichlorofluorescein diacetate; PI, propidium iodide; TFA, Trifluoroacetic acid; PBS, Phosphate-buffered saline; TEM, transmission electron microscopy; SEM, scanning electron microscopy.

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Key words: Aspergillus fumigatus; Antifungal peptide; Mode of action; Sporicidal activity; ROS generation; Electron microscopy

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1. Introduction During recent years, the incidence of invasive fungal infections has dramatically increased due to the growth in the number of immunocompromised patients of such diseases as AIDS and acute leukemia, of chemotherapy for cancer, and transplant recipients [1]. Many fungal diseases, especially aspergillosis, are routinely treated with antifungal drugs such as triazoles. However, the emergence of drug resistance found mainly in Aspergillus fumigatus has complicated this situation and raised concerns over the handling of aspergillosis for global healthcare [2-4]. Antifungal drug resistance has prompted researchers to seek new antifungal agents [5].As a general role, novel antifungal agents should have broad-spectrum activity, low toxicity, high specificity, and lack of microbial resistance [6, 7]. Antimicrobial peptides (AMPs) are attractive compounds that have received notable attention as a novel class of antimicrobial agents [8-10]. AMPs are short peptides (12–100 residues) which are part of the innate immune defense mechanism of many organisms [11].These peptides are amphipathic molecules most of which have a positive charge, though some have a negative charge or are neutral [12]. AMPs exhibit a broad spectrum of activities against various targets including gram-positive and gram-negative bacteria, protozoa, yeast, fungi, and viruses. Moreover, they have cytotoxic activity against sperm and tumor cells [13, 14]. Antifungal peptides (AFPs) are a specialized group of AMPs with strong activity against pathogenic fungal species. AFPs are classified into different groups based on origin and mode of action [15]. Peptides have different modes of action, including induction of the formation of reactive oxygen species (ROS); inhibition of DNA, RNA, and protein synthesis; inhibition of protein folding; and inhibition of enzyme activity and mitochondrial dysfunction [6, 12, 16-18]. AFPs based on their effect on cell membrane are classified into two groups: (1) peptides that cross cell membranes and cause pore formation or act on particular targets such as β-glucan or chitin synthesis; and (2) peptides that cooperate with cell membranes and cause cell lysis [15, 19, 20]. AFPs from natural sources including a marine snail named Cenchritis muricatus [21], a medicinal plant i.e. Matricaria chamomilla L. [22] and frog skin [23] that target pathogenic yeasts, especially Candida species, have been reported earlier. However, little has been 2

documented about antifungal peptides which affect filamentous fungi and more strictly their mode of action against Aspergillus species [24-26]. In our previous study, Skh-AMP1 (APD ID: AP03082; http://aps.unmc.edu/AP/database/query_output.php?ID=03082) was isolated from Satureja khuzistanica, a native plant from Iran from the Lamiaceae family, subfamily Nepetoideae, which exhibited antifungal activity against Aspergillus and Candida species, while showing no obvious cell cytotoxicity or hemolytic activity in vitro [27]. In the present study, we aimed to determine the mode of antifungal action of Skh-AMP1against Aspergillus fumigatus with special attention to permeabilization and integrity of cell membrane in hyphae and conidia.

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2. Material and Methods 2.1. Strain and materials Aspergillus fumigatus was purchased from the Collection of Pathogenic Fungi of the Pasteur Institute. It was cultured in Sabouraud Dextrose Agar (SDA; HiMedia) under sterile conditions at 35 ºC. The conidia suspension was prepared by gently scraping the culture surface using a sterile glass rod after adding adequate amounts of 0.1% aqueous solution of Tween 80.Sabouraud Dextrose Broth (SDB; HiMedia), 2,7-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich), propidium iodide (PI; Sigma-Aldrich), and Triton X-100 were also used. All other reagents were of the highest purity available from commercial sources.

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2.2. Synthesis of peptide Skh-AMP1 was chemically synthesized by Biomatik Co. (Ontario, Canada), according to the solid-phase methods, using Fmoc (9-fluorenyl-methxycarbonyl) as previously described [27]. The purity of the peptide was checked by analytical reverse-phase HPLC on a Kromasil 100-5 C18 analytical column (4.6×250 mm) at a flow rate of 1 ml/min with a gradient from 20% to 100% solvent A (0.1% TFA in acetonitrile) and solvent B (0.1% TFA in water) for 30 min. The elution was monitored with a UV detector based on the absorbance at 220 nm.

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2.3. Sporicidal activity The sporicidal activity of Skh-AMP1 against nongerminated A. fumigatus conidia was assessed according to Badosa et al. [28] with slight modification. Test tubes containing 210 μl SDB were inoculated with 190 μl of conidia suspension of A. fumigatus (106conidia/ml) and exposed to 100 μl of Skh-AMP1 at MIC concentrations of 20 (MIC), 40 (2MIC), and 80 (4MIC) µM (determined previously [27] by modified CLSI documents M27-A3) for 0, 4, 6, 8, 12, and 24 h at 25 ºC. After incubation, 100 μl of the tube contents was cultured on SDA plates. Viable colonies were counted after incubation for 3 days at 35 °C. The values of viable colonies were expressed as percentages of survival from the beginning of the experiment. Untreated fungal conidia and 1% Triton X-100 were used as controls. 2.4. Extracellular leakage of potassium 2.4.1. Conidia The effect of Skh-AMP1 on the extracellular K+ leakage from A. fumigatus conidia was evaluated according to Tao et al. [29] with some modifications. Briefly, a conidia suspension of A. fumigatus (106conidia/ml) was mixed with 20, 40, and 80 µM of Skh-AMP1 and incubated at 25 °C for 2, 4, 6, 8, 10, and 12 h. Then, the cell suspension was precipitated by centrifugation at 5000 × g for 20 min and the supernatant was used to determine the level of extracellular K+ 3

released into the culture medium. The K+ concentration was estimated by flame atomic absorption spectroscopy (ELICO-CL-378, India)using a potassium filter. Untreated fungal conidia and 1% Triton X-100 were used as controls.

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2.4.2. Hyphae To determine extracellular K+ leakage from A. fumigatus hyphae, the fungus was cultured on SDB in 6-well microplates at 35 °C for 24 h. Then, 20, 40, and 80 µM concentrations of SkhAMP1 were separately added to wells and incubated at 35°C for 2, 4, 6, and 8h. Thereafter, the culture content of each well was filtered through filter paper, and supernatant was used to determine the amount of extracellular K+ released into the culture medium. The K+ concentration was estimated by flame atomic absorption spectroscopy (ELICO-CL-378, India) using a potassium filter. Untreated fungal hyphae and 1% Triton X-100 were used as controls. 2.5. Determination of fungal cell membrane integrity

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2.5.1. Flow cytometric analysis To determine the loss of membrane integrity of A. fumigatus conidia after being exposed to SkhAMP1, the procedure of Tian et al. [30] was used with slight modifications. The spore suspension of A. fumigatus (106conidia/ml) was treated with 40 µM and 80 µM of Skh-AMP1 and incubated for 8 h at 25°C. The cells were stained with propidium iodide solution (PI) with a final concentration of 1 μg/ml in PBS for 20 min at room temperature in the dark. Then, the cells were harvested by centrifugation, washed, and resuspended in PBS. The percentage of PIpositive conidia was determined using FACS flow cytometer (BDBD-FACSCalibur, BD Biosciences, USA).Untreated fungal conidia and 1% Triton X-100 were used as controls.

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2.5.2. Fluorescence microscopy To determine the loss of membrane integrity of A. fumigatus hyphae, a conidia suspension of A. fumigatus (106 conidia /ml) in SDB was poured on a 6-well microplate and incubated at 35 °C for 24 h. Then, the hyphae were exposed to 20 µM and 40 µM of Skh-AMP1 at 35 °C for 6 h. After harvesting and washing with sterile PBS, the fungal hyphae was transferred to a new microplate, and PI solution with a final concentration of 50 μg/ml was added to each well for 15 min at room temperature in the dark. Next, the stained hyphae specimens were visualized by fluorescence microscopy (Eclipse 80i- Nikon-Japan) with appropriate filters (excitation/emission at 530/590 nm). The amount of hyphae staining was correlated with impairment of cell membrane integrity. Untreated fungal hyphae and 1% Triton X-100 were used as controls.

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2.6. Measurement of reactive oxygen species (ROS) production 2.6.1. Conidia The intracellular ROS content of A. fumigatus was determined using fluorescent dye 2′,7′dichlorofluorescin diacetate as previously described with some modifications [17]. Briefly, a conidia suspension of A. fumigatus (106conidia/ml) was exposed to 40 µM and 80 µM concentrations of Skh-AMP1 and incubated at 25°C for 6 h. After incubation, DCFH-DA prepared in PBS was added into the mixture with a final concentration of 10 µM. Next, the fungal cells were collected by centrifugation, and the pellet was washed twice with PBS and then resuspended in PBS. The fluorescence intensity of the fungal cells was measured using FACS 4

flow cytometer (BD-FACSCalibur, BD Biosciences, USA) at an excitation wavelength of 485 and emission wavelength of 530 nm. The unstained cells were used as the negative control, with H2O2 (4 mM) added to fungal cells instead of the peptide serving as the positive control.

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2.6.2. Hyphae To detect the formation of ROS in A. fumigatus hyphae, the method of Shekhova et al. [31] was employed with some modifications. Conidia suspension of A. fumigatus (106conidia /ml) was added to a 96-well F-bottom microplate containing SDB and incubated at 35 °C for 24 h. Then, the hyphae were exposed to 20 µM and 40 µM of Skh-AMP1 at 35 °C for 2 h. After a washing step with sterile PBS, DCFH-DA in PBS at a final concentration of 3 µM was added into each well and incubated at 25 °C for 30 min in the dark. After washing with sterile PBS, the fluorescence intensity was determined by a fluorescence plate reader (Gemini XPS/0200-5040Molecular Devices LLC, USA) at excitation and emission wavelengths of 485 nm and 530 nm, respectively. The unstained cells were used as the negative control, with H2O2 (4 mM) added to fungal cells instead of the peptide serving as the positive control. 2.7. Electron microscopy

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2.7.1. Preparation of fungal spores and hyphae To determine ultrastructural changes in A. fumigatus in presence of Skh-AMP1, the method of Leiter et al. [24] was used with slight modifications. To evaluate the effect of peptide on conidia morphology, the fungal conidia suspension prepared from cultures on SDA tubes (106conidia /ml) was exposed to 80 µM of the peptide in tubes containing 1 mL SDB and incubated at 25°C for 12 h. For hyphae, the conidia suspension of A. fumigatus (106conidia/ml) was cultured in 1 ml of SDB in 24-well microplates for 24 h at 35 °C and then exposed to 40 µM of Skh-AMP1 and incubated at 35 °C for 8 h. The non-treated conidia and hyphae were considered as controls.

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2.7.2. Transmission electron microscopy (TEM) The specimens (conidia and hyphae) were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer and pH 7.2 for 3 h at 4°C.After washing three times in the phosphate buffer and infiltrating with 2% molten agar, the specimens were post-fixed in 1% aqueous solution of osmium tetroxide in phosphate buffer, pH 7.2, for 1 h at room temperature. The specimens were dehydrated using a graded water acetone series (10% steps for 30-90% each lasting 60 min, 100% for 180 min, and finally 100% overnight).Next, they were embedded in Epon 812 and polymerized in Spurr’s resin (Epon 812 contained 1.50% hardening agent, DMP-30) at 45ºC for 24 h followed by 65ºC for 72 h. Ultrathin sections (80-nm thickness) were prepared using a Leica Ultracut UCP on 100-mesh and stained with uranyl acetate for 20 min followed by lead citrate for 5 min. Finally, the thin sections were examined under a ZEISS EM-900 transmission electron microscope. 2.7.3. Scanning electron microscopy (SEM) Fungal conidia and hyphae were fixed in 3% glutaraldehyde and then post-fixed in osmium tetroxide as mentioned for TEM. For dehydration, the specimens were submerged in gradually increasing ethanol concentrations (25%, 50%, 75%, 95%, and 100%) for 10 min at each step. The samples were critical–point dried and mounted on stubs using a carbon adhesive. Finally, the dehydrated specimens were coated with a thin layer (20–30 nm) of gold-palladium as a 5

conductive medium by sputter coating and were observed under a scanning electron microscope (ZEISS DSM-960A). 2.8. Statistical analysis All statistical analyses were performed using Graph Pad Prism version 8.01 for Windows (Graph Pad Software).The results were expressed as means ± standard deviation in all experiments. All tests were performed in triplicate.

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3. Results 3.1. Peptide synthesis

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The purity of synthetic Skh-AMP1 was confirmed by recording a single sharp peak with the retention time of 15.66 min in HPLC analysis. The molecular weight of both the native and the synthesized peptides was identical (2778.10 Da) as previously determined by mass spectrometry [27].

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3.2. Sporicidal activity of Skh-AMP1 The fungicidal activity of Skh-AMP1against nongerminated A. fumigatus conidia was evaluated by comparing the survival percentage of the conidia after each time point of exposure to different peptide concentrations (Fig. 1). At the MIC concentration of 20 µM, Skh-AMP1 exhibited little sporicidal activity at 24 h, while at 40 µM (2MIC); the survival percentage of the conidia was reduced to about 40% at 12 h and 5% at 24 h. Finally, at the concentration of 80 µM (4MIC), the peptide killed 100% of conidia after 12 and 24 hours of incubation.

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3.3. Extracellular potassium leakage Extracellular K+ was measured to determine the permeability of the cell membrane. The results indicated that the concentration of K+ increased in the supernatant culture media of A. fumigatus conidia exposed to 40 and 80 µM of Skh-AMP1 as the incubation times were extended (Fig. 2a). At the concentration of 20 µM, little leakage of K+ was reported. At concentrations of 40and 80 µM after 12 h incubation, the K+ leakage for fungal conidia was about 68% and 79%, respectively. Hyphae of A. fumigatus exposed to Skh-AMP1 are shown in Fig. 2b. At all concentrations (20, 40, and 80 µM) of the peptide, K+ release was increased by the incubation time and reached a maximum of 88% in the concentration of 80 µM after 8 h, which revealed an almost similar behavior with Triton X-100 as the positive control (99%). The results of K+ leakage indicated thatSkh-AMP1 could induce membrane permeabilization of the fungal cell. 3.4. Fungal cell integrity (PI uptake) PI uptake by conidia and hyphae of A. fumigatus was monitored by FACS and fluorescence microscopy. As shown in Fig. 3b, the percentage of PI positive conidia was 66% and 49% at concentrations of 80 and 40 µM of Skh-AMP1, respectively. The results shown in Fig.4 suggest that fluorescent intensity increased in the hyphae of A. fumigatus incubated with 20 µM of SkhAMP1 compared with the negative control. Also, there were more PI-stained hyphae of A. fumigatus at 40 µM than with 20 µM of Skh-AMP1. Accordingly, the results of PI uptake 6

indicated thatSkh-AMP1 could disrupt the integrity of the cell membrane and induce PI uptake in the fungal cell. 3.5. Measurement of reactive oxygen species (ROS) production As shown in Fig. 5, the fluorescent signals at Skh-AMP1 concentrations of 40 and 80 µM were increased in a dose-dependent manner and reached 644 units at the concentration of 80 µM after incubation for 6 h. Furthermore, the percentages of fluorescence intensity in the hyphae of A. fumigatus incubated with 20and 40 µM of Skh-AMP1 were increased by raises in concentration and reached a maximum 74% at the concentration of 40 µM, which was a significant growth compared to the negative control (Fig. 5).

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3.6. Electron microscopy observations

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3.6.1. Conidia morphology TEM results, shown in Fig. 6a, indicated that all conidia compartments including the cell wall, cell membrane, cytoplasm, and organelles are normal in the untreated fungal conidia, while the vacuolation of conidia in treated sample exposed to 80 µM of the peptide was obvious through the formation of a large vacuole inside the cell and detachment of the fibrillar layer from the cell wall (Fig. 6b). In SEM micrographs, the topography of conidia was normal in the untreated sample (Fig. 6c), while shrinkage and depletion of conidia exposed to 80 µM of the peptide was evident, showing possible damage to cell membrane integrity (Fig. 6d).

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3.6.2. Hyphae ultrastructure The ultrastructural analysis of untreated hypha of A. fumigatus by TEM revealed that the cell wall, cell membrane, and cytoplasmic organelles including nuclei, mitochondria, and vacuoles were normal (Fig. 7a).In treated hyphae exposed to 40 µM of Skh-AMP1, morphological changes including complete disruption of hyphae and detachment of plasma membrane from the cell wall, microvesicle formation, and massive depletion of cytoplasmic organelles were evident (Figs. 7b and 7c). SEM micrographs of untreated hyphae revealed normal tubular structures with no obvious morphological changes (Fig. 7d), while in the treated samples exposed to 40 µM of Skh-AMP1, severe shrinkage and folding of the hyphae and depletion of the cytoplasm were evident (Fig. 7e).

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4. Discussion In the present study, the mechanism of action of Skh-AMP1 isolated from Satureja khuzistanica against conidia and hyphae of A. fumigatus was evaluated. Based on the findings, it can be concluded that Skh-AMP1has complete sporicidal activity against conidia of A. fumigatus at concentrations of 40 and 80 µM within 12 and 24 h of incubation, respectively. In relation to mycelia fungi (molds), it is crucial to check the antifungal activity of a peptide against both conidia and hyphae. Conidia are resting structures of fungi that are resistant to environmental conditions which should be addressed in order to control fungal diseases [28]. So far, several antifungal peptides have been found that have sporicidal activity, but at different concentrations and times and in several fungal species [28, 32, 33]. This discrepancy may be explained by differences in the structure and mechanism of action of various peptides. Badosa et al. [28] evaluated the sporicidal activities of several peptides against conidia of F. oxysporum, comparing the survival of conidia after 35 min of exposure at different peptide concentrations; all peptides 7

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exhibited sporicidal activities at concentrations around 20 µM in 35 min. Jiao et al. [32] indicated that a thaumatin-like protein isolated from banana at 60 µM has complete fungicidal activity against P. expansum conidia through plasma membrane disturbance and cell wall disorganization. Moreover, EcAMP1, an antifungal peptide isolated from the kernels of the barnyard grass Echinochloa crus-galli, showed antifungal activity against F. solani conidia at 4 µM by binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbing membrane integrity [33]. The sporicidal activity of Skh-AMP1 was also supported by measuring the uptake of extracellular K+ and PI in the conidia of A. fumigatus. When the cell membranes of conidia were exposed to 40 and 80 µM of Skh-AMP1, K+ efflux grew significantly compared with the negative control. The release of K+ is one of the most important reasons for membrane permeability and the apoptotic process [34]. In addition, the results of PI uptake revealed that Skh-AMP1 can disrupt the integrity of the cell membrane and enter conidia cells. PI is a DNAstaining dye which binds tightly to nucleic acids and gives a red fluorescence. PI can penetrate the cells only if the integrity of the plasma membrane of the cell is disrupted [35]. The results of Jiao et al. [32] indicated that BanTLP can induce the release of K+ and PI uptake by P. expansum conidia. Notably, the percentage of P. expansum conidia treated with 60 µM of BanTLP stained with PI after 6 h incubation was 100%. However, in the current study, the percentage of PI positive conidia at 80 µM of Skh-AMP1was 66% after 8 h incubation; this contradiction can be explained by the different structures of peptides and the different fungal species. Also, measuring the extracellular K+ and PI uptake in the hyphae of A. fumigatus indicated that Skh-AMP1 yields an effect similar to that of conidia but at lower concentrations and within a shorter time. In the past, researchers such as Kaiserer et al. [36] reported a significantly elevated K+ efflux in PAF-treated A. nidulans hyphae. The results of measuring ROS production suggested that increased membrane permeability could not be the sole cause of fungal cell death in the current study. The reason may be the fact that Skh-AMP1 could induce a significant elevation of ROS production in the conidia and hyphae of A. fumigatus in a dose-dependent manner. When fungal cells are exposed to specific stress conditions such as treatment with antifungal agents, ROS is generated as a natural byproduct from the intracellular metabolism of oxygen. The production of excess ROS, such as superoxide anion, hydrogen peroxide, and hydroxyl radicals, plays an important role as an early signal of programmed cell death or apoptosis [37]. Similarly, several studies have suggested that AMPs can induce increased ROS production in fungal cells [24, 34, 38, 39]. For example, the results of Leiter et al. and Kaiserer et al. [24, 36] indicated that PAF can induce enhanced generation of ROS in A. nidulans hyphae and A. niger hyphae. Moreover, Jiao et al. [32] showed that BanTLP can induce increased intracellular ROS production in the spores of P. expansum in a timedependent manner. TEM and SEM observations in the present study indicated that these changes in conidia may be related to digestion of the cell wall, enhanced membrane permeabilization, followed by release of K+ and other small molecules, and eventually increased ROS production [40]. The ultrastructural and morphological changes in hyphae such as the appearance of microvesicles and shrinkage may be related to programmed cell death (PCD) [31]. 5. Conclusion Skh-AMP1 exhibited potent antifungal activity through a multi-action mode-involved mechanism. Skh-AMP1 can cause the induction of leakage of K+ and an imbalance in 8

intracellular osmotic pressure by altering the membrane permeabilization. This, in turn, leads to the destruction of intracellular organelles and cell shrinkage. In addition, it can also induce PI uptake and enhance ROS production, which may contribute to PCD or apoptosis. Therefore, Skh-AMP1 can be introduced as a novel antifungal candidate for developing new therapeutic agents. However, further research on the exact PCD mechanism and stress oxidative of the antifungal effect of Skh-AMP1 is recommended.

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Funding This study was supported financially by a PhD grant to Soghra Khani by the Pasteur Institute of Iran. Research reported in this publication was supported by Elite Researcher Grant Committee under award numbers [958634 and 963646] from the National Institute for Medical Research Development (NIMAD), Tehran, Iran. The funders had no role in study design, data collection and decision to publish, or preparation of the manuscript.

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Acknowledgments Authors would like to thank Research Deputy of the Pasteur Institute of Iran for financial support of the study.

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Fig 1. Sporicidal activities of Skh-AMP1 on non-germinated A. fumigatus conidia. Spore suspension of A. fumigatus (106 spore/ml) was treated with several concentrations of SkhAMP1(MIC; 20 µM, 2MIC; 40 µM and 4MIC; 80 µM) and incubated at 25°C for 4, 6, 8, 12, and 24 h. Viable spores were counted after dilution and plating in SDA. Values are expressed as percentages of survival from the start of the experiment. The results are presented as mean± standard deviation (n = 3).

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Fig 2. Effects of different concentrations of Skh-AMP1 (MIC; 20 µM, 2MIC; 40 µM and 4MIC; 80 µM) on the leakage of intracellular potassium ions on a) conidia b) hyphae of A. fumigatus. Untreated cells were used as the negative control, and 1% Triton X-100 was employed as the positive control. The results are presented as mean ± standard deviation (n = 3).

Fig 3. Effects of different concentrations of Skh-AMP1 on membrane integrity of A. fumigatus conidia using PI uptake. PI uptake by conidia was monitored by FACS flow cytometry and expressed in both chromatographs and column graphs as PI positive cells. Untreated cells were used as the negative control, and 1% Triton X-100 was employed as the positive control. The results are presented as mean± standard deviation (n = 3). 13

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Fig 4. Effects of different concentrations of Skh-AMP1 on membrane integrity of hyphae of A. fumigatus using PI uptake monitored by fluorescence microscopy. Untreated cells were used as the negative control, and 1% Triton X-100 was employed as the positive control.

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Fig 5. Effects of Skh-AMP1 on intracellular ROS production of conidia and hyphae of A. fumigatus. As indicated in chromatograms, fluorescence intensity of the conidia and hyphae of A. fumigatus were increased significantly in treated samples compared with untreated controls as the negative control. Quantitative data of ROS production is shown in column graphs where H2O2 was employed as the positive control. The results are presented as mean ± standard deviation (n = 3).

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Fig 6. TEM and SEM images of the cellular ultrastructure and morphological characteristics of conidiaA. fumigatus. a and c) Untreated control shows normal conidium with intact cell wall and plasma membrane in (a) and normal round shape conidium in (c); b and d) Conidia treated with 80 µM of Skh-AMP1 for 12 h shows pathological changes including detachment of fibrillar layer from the cell wall and vacuolation of conidium in (b) and massive shrunken and folded conidia in (d).

Fig 7. TEM and SEM images of the cellular ultrastructure and morphological characteristics of hyphae A. fumigatus. a and d) Untreated control shows normal hypha with intact cell wall, plasma membrane, and organelles in (a) and tubular non-folded hyphae in (d); b, c, and e) Hyphae treated with 40 µM of Skh-AMP1 for 6 h shows pathological changes including detachment of plasma membrane from the cell wall, massive destruction and depletion of cellular compartments and organelles including mitochondria in (b and c), and shrunken and folded hyphae indicative of cell depletion in (e). 15

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