Biochimie 105 (2014) 58e63

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Research paper

Antifungal activity and pore-forming mechanism of astacidin 1 against Candida albicans Hyemin Choi, Dong Gun Lee* School of Life Sciences, BK 21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 702-701, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 March 2014 Accepted 13 June 2014 Available online 21 June 2014

In a previous report, a novel antibacterial peptide astacidin 1 (FKVQNQHGQVVKIFHH) was isolated from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. In this study, the antifungal activity and mechanism of astacidin 1 were evaluated. Astacidin 1 exhibited antifungal activity against Candida albicans, Trichosporon beigelii, Malassezia furfur, and Trichophyton rubrum. Also, astacidin 1 had fungal cell selectivity in human erythrocytes without causing hemolysis. To understand the antifungal mechanism, membrane studies were done against C. albicans and T. beigelii. Flow cytometric analysis and Kþ measurement showed membrane damage, resulting in membrane permeabilization and Kþ release-induced membrane depolarization. Furthermore, the calcein leakage from liposomes mimicking C. albicans membrane demonstrated that the membrane-active action was driven by pore-forming mechanism. Live cell imaging using fluorescein isothiocyanate-labeled dextrans of various sizes suggested that the radii of pores formed in the C. albicans membrane were 1.4e2.3 nm. Therefore, the present study suggests that astacidin 1 exerts its antifungal effect by damaging the fungal membrane via pore formation. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Astacidin 1 Antifungal activity Pore-forming mechanism Candida albicans

1. Introduction Recently, the morbidity and mortality of candidiasis have increased dramatically [1]. Candida albicans is the primary cause of candidiasis and is the fourth most common cause of nosocomial infection [2]. Candida infections are often recalcitrant to therapy and, unfortunately, amphotericin B- and fluconazole-resistant Candida strains have been reported [3]. In particular, the clinical resistance of fungi is commonly observed in patients with persistent and profound immune deficiency or infected prosthetic materials such as central venous catheters [4]. To overcome fungal diseases, the development of more effective antifungal agents is necessary. Every living organism, including bacteria, fungi, plants, insects, birds, crustaceans, amphibians, and mammals, produces

Abbreviations: DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid; MIC, minimum inhibitory concentrations; PBS, phosphate-buffered saline; DiBAC4(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol; LUV, large unilamellar vesicle; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; FITC, fluorescein isothiocyanate; FD, FITC-labeled dextran. * Corresponding author. Tel.: þ82 53 950 5373; fax: þ82 53 955 5522. E-mail address: [email protected] (D.G. Lee). http://dx.doi.org/10.1016/j.biochi.2014.06.014 0300-9084/© 2014 Elsevier Masson SAS. All rights reserved.

antimicrobial peptides as an important component of innate immunity [5]. Antimicrobial peptides are typically relatively short (10e50 amino acids), positively charged (net charge ranging from þ2 to þ9), and amphiphilic [6]. Antimicrobial peptides have several advantages compared to conventional antibiotics because of their broad-spectrum antimicrobial activity and slow resistance development [7]. Hence, they are promising candidates for the development of antibiotics. Crayfish live in environments in which they are continually exposed to pathogenic bacteria, fungi, and viruses. Similar to other crustaceans, crayfish lack the typical antibody- and T/B cellbased adaptive immunity of vertebrates. They thus depend entirely on their innate immune systems to fight against pathogenic invasion. Antimicrobial peptides are vital factors in the innate immunity of crayfish [8]. Astacidin 1 (FKVQNQHGQVVKIFHH), a 16-mer antibacterial peptide, was identified from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. The peptide corresponds to the carboxyl-terminal segment of hemocyanin and is produced by the proteolytic cleavage of hemocyanin under acidic conditions [9]. In this study, the antifungal activity of astacidin 1 and its mechanism against C. albicans were investigated, and its hemolytic effect against human erythrocytes was evaluated.

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2. Materials and methods 2.1. Solid-phase peptide synthesis Anygen Co. (Gwangju, Korea) performed the peptide synthesis. The assembly of the peptides was achieved with a 60-min cycle for each residue at ambient temperature using the following method: (1) 2-chlorotrityl (or 4-methylbenzhydrylamine amide) resin was charged to a reactor and then washed with dichloromethane and N,N-dimethylformamide (DMF), respectively, and (2) a coupling step with vigorous shaking using a 0.14 mM solution of Fmoc-Lamino acids and Fmoc-L-amino acids that were preactivated with a 0.1 mM solution of 0.5 M 1-hydroxybenzotriazole/diisopropylcarbodiimide in DMF for approximately 60 min. Finally, the peptide was cleaved from the resin using a trifluoroacetic acid (TFA) cocktail solution at ambient temperature [10,11]. 2.2. Peptide characterization Analytical and preparative reverse-phase high-performance liquid chromatography runs were performed with Shimadzu 20 A or 6 A gradient systems. Data were collected with an SPD-20 A detector at 230 nm. Chromatographic separations were achieved with a 1%/min linear gradient of buffer B in solution A (A ¼ 0.1% TFA in H2O; B ¼ 0.1% TFA in acetonitrile (CH3CN)) over 40 min at flow rates of 1 ml/min and 8 ml/min using Shimadzu C18 analytical (5 mm, 0.46  25 cm) and preparative C18 (10 mm, 2.5  25 cm) columns. Mass spectrometry was conducted with an AXIMA CFR MALDI-TOF Mass Spectrometer (Kratos/Shimadzu). 2.3. Fungal strains and antifungal activity assay C. albicans (ATCC 90028) was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Trichosporon beigelii (KCTC 7707), Malassezia furfur (KCTC 7744), and Trichophyton rubrum (KCTC 6345) were obtained from the Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) (Daejeon, Korea). The fungal strains were cultured in YPD broth (Difco) with aeration at 28  C, and M. furfur was cultured at 32  C in a modified YM broth (Difco) containing 1% olive oil. The cell suspensions were adjusted to obtain standardized population sizes by measuring the turbidity with a spectrophotometer (DU530; Beckman; Fullerton, CA, USA). Fungal cells (1  106 cells/ml) were inoculated into the broth, and 0.1 ml/well of the mixture was dispensed to microtiter plates. Minimum inhibitory concentrations (MICs) were determined with a serial two-fold dilution of the peptides, based on the Clinical and Laboratory Standards Institute (CLSI) method. The MIC values were determined in three independent assays. 2.4. Hemolytic activity assay A fresh human blood sample was diluted in phosphate-buffered saline (PBS: 35 mM phosphate buffer, 150 mM NaCl, pH 7.4) and centrifuged at 2000 rpm for 10 min to remove the plasma and buffy coat, and the supernatant was removed. This washing procedure was repeated three times, and the final concentration of the erythrocytes was 4%. The erythrocyte suspension was transferred into sterilized 96-well plates and incubated with peptides at 37  C for 1 h. The plate was centrifuged at 1500 rpm for 10 min. An aliquot of the supernatant was taken, and then the hemolytic activity of the peptides was evaluated by determining the release of hemoglobin from a 4% suspension of human erythrocytes at 414 nm with an enzyme-linked immunosorbent assay reader. Hemolytic levels of zero and 100% were determined in PBS alone and with 0.1%

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Triton X-100, respectively. The hemolysis percentage was calculated with the following equation: hemolysis (%) ¼ [(Abs414nm in the peptide solution  Abs414nm in PBS)/(Abs414nm in 0.1% Triton X100  Abs414nm in PBS)]  100 [12]. 2.5. Propidium iodide influx assay C. albicans and T. beigelii (1  106/ml YPD), suspended in PBS, were treated with 6.3 mM of the peptides and incubated for 4 h at 28  C, respectively. The cells were harvested by centrifugation and resuspended in PBS. Subsequently, the cells were treated with 9 mM propidium iodide and incubated for 5 min at room temperature. The cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson; San Jose, CA, USA) [13]. 2.6. Membrane depolarization assay C. albicans and T. beigelii (1  106/ml YPD) were harvested and suspended in PBS, respectively. After incubation with 6.3 mM of the peptides for 4 h at 28  C, the cells were harvested by centrifugation and resuspended in PBS. Subsequently, the cells were treated with 5 mg of bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] (Molecular Probes; Eugene, OR, USA). Flow cytometric analysis was conducted with a FACSCalibur flow cytometer [14]. 2.7. Potassium ion (Kþ) release assay C. albicans (1  106/ml YPD) was incubated with 6.3 mM of the peptides for 4 h at 28  C. After incubation, the cell suspensions were centrifuged at 10,000  g for 5 min, and the supernatant was collected to determine the extracellular Kþ content. The released Kþ was measured with a Kþ-selective electrode (Thermo Scientific) [15]. 2.8. Calcein leakage assay Large unilamellar vesicles (LUVs) encapsulating calcein, composed of phosphatidylcholine (PC):phosphatidylethanolamine (PE):phosphatidylinositol (PI):ergosterol (5:4:1:2, w/w/w/w), were prepared by vortexing the dried lipids in a dye buffer solution (70 mM calcein, 10 mM Tris, 150 mM NaCl, and 0.1 mM ethylenediamine tetraacetic acid [pH 7.4]). The suspension was freezethawed in liquid nitrogen for 11 cycles and extruded through polycarbonate filters (two stacked 200-nm pore-size filters) with a LiposoFast extruder (Avestin Inc.; Ottawa, Canada). Calceinentrapped LUVs were separated from free calcein by gel filtration chromatography on a Sephadex G-50 column. The leakage of calcein from the LUVs was monitored at 25  C by measuring the fluorescence intensity at an excitation wavelength of 490 nm and an emission wavelength of 520 nm with a spectrofluorophotometer. To determine 100% dye release, 30 ml of 10% Triton X-100 was added to the vesicles. The percentage of dye leakage caused by the peptides was calculated as follows: dye leakage (%) ¼ 100  (F  F0)/(Ft  F0), where F represents the fluorescence intensity obtained after addition of the peptides, and F0 and Ft represent the fluorescence intensities without any compound and with Triton X-100, respectively. The data represent the mean ± standard deviation of three independent experiments [16]. 2.9. Estimation of pore size in live C. albicans cells C. albicans (1  106/ml YPD) was harvested and suspended in PBS. After incubation with 6.3 mM of the peptides for 4 h at 28  C, the cells were harvested by centrifugation and resuspended in PBS. Fluorescein isothiocyanate (FITC)-labeled dextrans (FDs) including FD4, FD10, and FD20 were added to the cells to final concentrations

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Table 1 The antifungal activity of astacidin 1 and melittin. MIC (mM)

Fungal strains

C. albicans ATCC 90028 T. beigelii KCTC 7707 M. furfur KCTC 7744 T. rubrum KCTC 6345

Astacidin 1

Melittin

6.3 6.3 12.5 25.0

1.6 1.6 3.1 6.3

Table 2 Hemolytic activity of astacidin 1 and melittin against human erythrocytes. Peptides

% Hemolysis

T. beigelii, M. furfur, and T. rubrum. Melittin, a component of the venom of the honeybee Apis mellifera, was used as a positive control peptide for comparing the activity of astacidin 1. Melittin is one of the most potent antimicrobial peptides with membrane-disruptive action and has hemolytic activity [18e20]. As shown in Table 1, astacidin 1 showed strong antifungal activity with MIC values in the range of 6.3e25.0 mM; melittin had MIC values in the range of 1.6e6.3 mM. The cytotoxicity of astacidin 1 to human erythrocytes was evaluated. Astacidin 1 did not show hemolytic activity at any concentration, whereas melittin caused 12.7% hemolysis at its MIC value (1.6 mM) against C. albicans and T. beigelii; 100% hemolysis was observed with 12.5 mM melittin (Table 2).

100.0 mM 50.0 mM 25.0 mM 12.5 mM 6.3 mM 3.1 mM 1.6 mM Astacidin 1 0 Melittin 100.0

0 100.0

0 100.0

0 100.0

0 74.8

0 42.0

0 12.7

of 0.1 mg/ml, respectively. After incubation for 10 min, the influx of fluorescent molecules was observed with a fluorescence microscope (Nikon eclipse Ti-S; Japan) [17]. 3. Results 3.1. Physicochemical features and biological activities of astacidin 1 Astacidin 1 was chemically synthesized and characterized. The purity and observed mass of astacidin 1 were 97.5% and 1945.6 Da (Calculated mass ¼ 1946.2 Da), respectively. The net charge in physiological pH of astacidin 1 was þ2. The antifungal activity of astacidin 1 was investigated by measuring the MICs for the human pathogenic fungi C. albicans,

3.2. Damage to the fungal cytoplasmic membrane The effect of astacidin 1 on the integrity of fungal membranes was examined by monitoring the influx of propidium iodide. Propidium iodide, a membrane impermeant dye, only enters membrane-compromised cells, which results in an increase of the fluorescence of the probe by 20e30-fold [21]. In flow cytometric analysis, C. albicans cells treated with astacidin 1 showed increased fluorescence of propidium iodide (18.6%) compared to control cells, indicating that the membrane integrity was destroyed (Fig. 1A). T. beigelii cells treated with astacidin 1 also showed influx of propidium iodide (30.0%) compared to control cells (Fig. 1B). 3.3. Membrane depolarization involving Kþ release To investigate the ability of astacidin 1 to change the membrane potential of intact C. albicans and T. beigelii, a membrane depolarization experiment was conducted using DiBAC4(3). DiBAC4(3) is a

Fig. 1. C. albicans (A) and T. beigelii (B) (1  106/ml) were incubated with 6.3 mM of peptides for 4 h at 28  C. In flow cytometric analysis, membrane damage is revealed by propidium iodide influx. (a) Control cells, (b) astacidin 1-treated cells, and (c) melittin-treated cells.

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Fig. 2. (A) C. albicans cells (1  106/ml) were incubated with 6.3 mM of peptides for 4 h at 28  C. Membrane depolarization was detected by DiBAC4(3) staining. (a) Control cells, (b) astacidin 1-treated cells, and (c) melittin-treated cells. (B) The amount of Kþ released by peptides from C. albicans cells. The data represent the mean þ standard deviation of three independent experiments. (C) T. beigelii cells (1  106/ml) were incubated with 6.3 mM of peptides for 4 h at 28  C. Membrane depolarization was detected by DiBAC4(3) staining.

potential-sensitive dye, and the increase in fluorescence intensity is interpreted as membrane depolarization. In C. albicans cells exposed to astacidin 1, the fluorescence intensity increased by 27.2% compared to control cells, indicating dissipation of the membrane potential. Melittin, the positive control for membrane depolarization, showed higher depolarization activity (85.5%) on C. albicans cells (Fig. 2A). In T. beigelii cells treated with astacidin 1 or melittin, 21.0% or 60.9% increase in DiBAC4(3) fluorescence was observed compared to the control cells, respectively (Fig. 2C). To further characterize the ion movement, the amount of Kþ released from peptide-treated C. albicans cells was measured. As shown in Fig. 2B, peptide-induced depolarized cells showed Kþ release. When the cells were treated with astacidin 1 and melittin, a 2.2 ppm and 4.2 ppm release of intracellular Kþ was observed compared to control cells, respectively. 3.4. Pore-forming action To elucidate the membrane-active mechanism, calceinentrapping LUVs were formed. The composition of the liposomes mimics the outer leaflets of the plasma membrane of C. albicans composed of PC:PE:PI:ergosterol (5:4:1:2, w/w/w/w) [22]. The LUVs were treated with astacidin 1 and melittin at various concentrations (1, 2, and 4 MIC). At 6.3 mM, astacidin 1 and melittin induced 27.0% and 86.3% calcein leakage from LUVs, respectively, indicating pore-forming action. The entrapped calcein leaked from LUVs in a concentration-dependent manner when exposed to the peptides (Fig. 3).

Furthermore, a cytotoxicity test on human erythrocytes was performed to assess the potential application of astacidin 1 in internal medicine because this is one of the key factors required in new drug candidate evaluations before using it in a clinical setting [24]. These bioactivity assays suggested that astacidin 1 has selective toxicity on fungal cells without causing hemolysis against human erythrocytes. C. albicans, one of the tested fungal strains, is an important nosocomial pathogen and causes diseases ranging from superficial mucosal infections to life-threatening systemic disease [25,26]. T. beigelii is considered to be the cause of a superficial hair infection called white piedra and also the cause of a more serious severe opportunistic infection (trichosporonosis) in immunocompromised individuals [27]. Because of its importance for human health, C. albicans and T. beigelii was selected as model organisms for the experiments of this study. Antifungal peptides have different mechanisms. (i) Most antifungal peptides bind to the membrane surface and trigger membrane permeabilization. (ii) Some antifungal peptides interfere with cell wall synthesis or the biosynthesis of essential cellular components such as glucan or chitin. (iii) Histatins from the human saliva and some other higher primates bind to a receptor on the fungal cell membrane, enter the cytoplasm and cause mitochondrial depletion. (iv) Pn-AMP1, a small cysteine-rich plant peptide, causes depolymerization of the actin cytoskeleton in fungi. (v) Some antifungal peptides induce apoptosis in yeast via ROS

3.5. Determination of the pore size in live C. albicans cells To estimate the size of pores formed in the live cell membrane, the influx of FD4 (average molecular weight ¼ 4 kDa, StokeseEinstein radius ¼ 1.4 nm), FD10 (average molecular weight ¼ 10 kDa, StokeseEinstein radius ¼ 2.3 nm), and FD20 (average molecular weight ¼ 20 kDa, StokeseEinstein radius ¼ 3.3 nm) [23] into C. albicans cells was visualized under a fluorescence microscope. When treated with astacidin 1, FD4 permeated into the cytoplasm (27.0%), but FD10 and FD20 did not enter the cytoplasm. Melittin induced the influx of all FDs (FD4, FD10, and FD20) (Fig. 4). These results indicate that astacidin 1 induced the formation of pores with radii of 1.4e2.3 nm in the C. albicans membranes. The radii of the pores made by melittin were larger than 3.3 nm. 4. Discussion In this study, the bioactive property of astacidin 1 was examined. Astacidin 1 exerted an inhibitory effect against various fungal strains in a manner similar to melittin, the positive control.

Fig. 3. Percentage of calcein leakage from LUVs [PC:PE:PI:ergosterol ¼ 5:4:1:2 (w/w/ w/w)] was measured after the peptides were applied to astacidin 1 for 10 min at various concentrations (6.3, 12.5, 25.0 mM). The data represent the mean þ standard deviation for three independent experiments.

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Fig. 4. C. albicans cells (1  106/ml) were incubated with 6.3 mM of peptides for 4 h at 28  C. Fluorescein isothiocyanate (FITC)-labeled dextrans (FDs) of various sizes were added to the cells. (A) The influx of fluorescent molecules was observed with a fluorescence microscope. (B) Percentage of FD influx into C. albicans cells induced by treatment with peptides. The data represent the mean þ standard deviation of three independent experiments.

production [28,29]. Astacidin 1 has two putative b-sheet structures in each terminus [9]. According to some reports, many antifungal peptides with b-sheet structures show a membrane-active mechanism [14,30]. Hence, a membrane study using propidium iodide was conducted to investigate the potential change in membrane permeability induced by astacidin 1. Propidium iodide only enters membrane-compromised cells and then intercalates between the bases within guanine and cytosine pairs or with a stoichiometry of one dye per 4e5 base pairs. After binding to nucleic acids, the fluorescence of propidium iodide increases [31]. The propidium iodide influx assay against fungal strains such as C. albicans and T. beigelii confirmed that the fungal membrane is indeed injured by astacidin 1, which increases membrane permeability. Damage to the cytoplasmic membrane of a cell by an antimicrobial peptide can result in loss of the functional integrity of the membrane [32]. To examine whether astacidin 1 affects the functions of the fungal membrane, changes in membrane potential were detected with the dye DiBAC4(3). DiBAC4(3), an anionic lipophilic dye, binds reversibly to lipid-rich intracellular components of only depolarized cells and then fluoresces [33]. After treating the fungal cells with astacidin 1, membrane depolarization was observed, as indicated by the increase in DiBAC4(3) fluorescence. Dissipation of membrane potential is caused by a change in ion movement across the cytoplasmic membrane [34]. Measurement of the amount of extracellular Kþ confirmed that astacidin 1 caused an imbalance of Kþ movement, leading to significant Kþ release. The sustained release of Kþ triggers membrane depolarization. Therefore, these results confirm that astacidin 1 exerts antifungal activity via membrane-active mechanisms. LUVs are used to characterize lipidepeptide interactions at the molecular level by monitoring the leakage of internal contents, such as fluorescent probes, induced by interaction with various kinds of substances such as peptides and drugs [35]. If watersoluble fluorescent probes are released by a substance from LUVs, this indicates that the substance induces pore formation in the lipid membrane [36]. The model membrane study using LUVs mimicking C. albicans membrane showed that astacidin 1 caused dosedependent leakage of calcein, a water-soluble probe. This result confirms that astacidin 1 exerts antifungal activity by forming pores in the fungal membranes. Live cell imaging is widely carried out with fluorescence microscopy, and provides information on cellular integrity, endocytosis, exocytosis, protein trafficking, signal transduction, and enzyme activity. FDs of various sizes were added to astacidin 1treated C. albicans cells to determine the average size of pores

formed in the membrane. The cells used in the influx assay were unfixed and living because the fixation process may significantly alter membrane permeability [37]. Astacidin 1 caused the influx of FD4, but did not induce the influx of FD10 and FD20. This indicated that the radii of the pores induced by astacidin 1 were 1.4e2.3 nm. In summary, astacidin 1 showed pore-forming action on the fungal membrane, resulting in increased permeability and excessive Kþ release, thereby inducing a cellular ionic imbalance. The loss of the cellular ion gradient causes membrane depolarization and inactivation of Kþ-dependent enzymes or pathways [38]. In this study, astacidin 1 exerted antifungal activity on various fungal strains without causing hemolysis. Membrane studies using C. albicans confirmed that the antifungal activity is caused through damage to the fungal membrane via pore formation. Conflict of interest No conflict of interest declared. Acknowledgments This work also supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008158), Rural Development Administration, Republic of Korea. References [1] G. Maschmeyer, The changing epidemiology of invasive fungal infections: new threats, Int. J. Antimicrob. Agents 27 (2006) 3e6. [2] M.A. Pfaller, D.J. Diekema, Epidemiology of invasive candidiasis: a persistent public health problem, Clin. Microbiol. Rev. 20 (2007) 133e163. [3] F.C. Odds, A.J. Brown, N.A. Gow, Antifungal agents: mechanisms of action, Trends Microbiol. 11 (2003) 272e279. [4] A.H. Groll, S.C. Piscitelli, T.J. Walsh, Clinical pharmacology of systemic antifungal agents: a comprehensive review of agents in clinical use, current investigational compounds, and putative targets for antifungal drug development, Adv. Pharmacol. 44 (1998) 343e500. [5] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature 415 (2002) 389e395. [6] R.E. Hancock, H.G. Sahl, Antimicrobial and host-defense peptides as new antiinfective therapeutic strategies, Nat. Biotechnol. 24 (2006) 1551e1557. [7] K. Matsuzaki, Control of cell selectivity of antimicrobial peptides, Biochim. Biophys. Acta 1788 (2009) 1687e1692. [8] X.Z. Shi, X.F. Zhao, J.X. Wang, A new type antimicrobial peptide astacidin functions in antibacterial immune response in red swamp crayfish Procambarus clarkii, Dev. Comp. Immunol. 43 (2014) 121e128. €derha €ll, Processing of an antibacterial peptide from [9] S.Y. Lee, B.L. Lee, K. So hemocyanin of the freshwater crayfish Pacifastacus leniusculus, J. Biol. Chem. 278 (2003) 7927e7933. [10] B. Merrifield, Solid phase synthesis, Science 232 (1986) 341e347.

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Antifungal activity and pore-forming mechanism of astacidin 1 against Candida albicans.

In a previous report, a novel antibacterial peptide astacidin 1 (FKVQNQHGQVVKIFHH) was isolated from hemocyanin of the freshwater crayfish Pacifastacu...
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