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Int. J. Mol. Sci. 2012, 13(11), 15042-15053; doi:10.3390/ijms131115042
Published: 15 November 2012
Abstract: The presence of lysine (Lys) or arginine (Arg) and tryptophan (Trp) are important for the antimicrobial effects of cationic peptides. Therefore, we designed and synthesized a series of antimicrobial peptides with various numbers of Lys (or Arg) and Trp repeats [(KW and RW)n-NH2, where n equals 2, 3, 4, or 5]. Antifungal activities of these peptides increased with chain length. Light microscopy demonstrated that longer peptides (n = 4, 5) strongly inhibited in vitro growth of Fusarium solani, and Fusarium oxysporum, at 4–32 μM. Furthermore, longer peptides displayed potent fungicidal activities against a variety of agronomical important filamentous fungi, including F. solani and F. oxysporum, at their minimal inhibitory concentrations (MICs). However, RW series peptides showed slightly higher fungicidal activities than KW peptides against the two strains. Taken together, the results of this study indicate that these short peptides would be good candidates for use as synthetic or transgenic antifungal agents.
Cationic antifungal peptides have been developed as novel biocidal agents in the battle against pathogenic microorganisms . Generally, cationic peptides are 12 to 50 amino acids in length as well as amphiphilic, as they contain two to nine basic residues (arginine (Arg) or lysine (Lys)) and approximately 50% hydrophobic residues [2,3]. However, the large size of antimicrobial peptides (AMPs) hinders their use due to high manufacturing costs. Therefore, selective short AMPs have been developed based on their amino acid combinations, charge, and hydrophobicity [4–8]. In this context, AMPs containing cationic and hydrophobic amino acids constitute a promising tool to combat plant fungal pathogens. For example, both defensin and MtDef4 require cationic and hydrophobic amino acids for their antifungal activities . Furthermore, this requirement has been demonstrated by the loss of antimicrobial activity upon substitution of basic residues in AMPs . Other short tryptophan (Trp)-rich cationic peptides, PAF26, PAF38, PAF40, and BM0, also display antifungal activity [11,12]. The peptide indolicidin, which belongs to the cathelicidin family of AMPs, is an Arg and Trp-rich peptide with potent, broad-spectrum antimicrobial activity [13,14]. Further, a large number of cationic plant defensins exhibit inhibitory activities against filamentous fungi both in vitro and in transgenic plants [15–20]. It has been reported that short peptides show antifungal activity as they contain cationic and hydrophobic amino acids [21,22]. This is clearly evident based on previous reports that hexapeptides containing predominately cationic and hydrophobic amino acids show the highest antifungal activity [11,23]. Therefore, we synthesized a series of peptides containing a repeated pattern of Lys (K) or Arg (R) and Trp (W) residues, (KW)n and (RW)n (where n equals 2, 3, 4, or 5) (Figure 1), and determined their antifungal and fungicidal activities.
2. Results and Discussion
Antifungal peptides have been used in both transgenic plant and human pharmaceutical applications. Furthermore, it has been extensively reported that many short AMPs rich in Lys or Arg and Trp show antifungal activity [4,6,8,24–26]. Although it is not yet clear that these peptides kill plant fungal pathogens, it is important to optimize chain length of AMPs for the purpose of large-scale production. In the present study, we investigated the antimicrobial effects of small KW and RW series peptides against plant pathogens such as Fusarium solani and Fusarium oxysporum. Specifically, F. solani is a phytopathogenic fungus as well as an important causal agent of several crop diseases, including root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean, and dry rot of potato [27–30]. F. oxysporum is a phytopathogenic fungus that affects tomato crops, causing huge losses to farmers .
2.1. Antifungal Activities against Hyphal Growth
In the present study, spectrophotometric and microscopic experiments were used to assess the antifungal activities of the peptides on hyphal growth of F. solani and F. oxysporum. As shown in Figure 2, chain length of (KW)n and (RW)n peptides strongly correlated with increasing antifungal activity. Decamer (KW and RW)5 peptides inhibited conidial germination completely (100% growth inhibition) at 4 μM for F. solani and 8 μM for F. oxysporum. Further, decamer peptides reduced growth rates compared to other peptides at all tested concentrations (1, 2, 4, 8, 16, 32, and 64 μM), although a concentration of 1 μM did not prevent sporulation. Similarly, (KW)4 and (RW)4 peptides exhibited antifungal effects against F. solani at concentrations as low as 4 μM, which is two-fold less than their minimal inhibitory concentration (MIC) (8 μM). (KW)4 and (RW)4 peptides also inhibited growth of F. oxysporum at concentrations below their MIC (16 μM). Specifically, at a concentration of 8 μM, (KW)4 and (RW)4 inhibited growth of F. oxysporum by 45% and 55%, respectively (Figure 2C,D). (KW)3 and (RW)3 peptides at a concentration of 16 μM completely inhibited germination of F. solani conidia. In contrast, F. oxysporum conidia were able to germinate and grow even in the presence of peptides at 16 μM, although 100% growth inhibition was observed at 32 μM (Figure 2C,D). Thus, (KW)5 and (RW)5 peptides exhibited significantly higher antifungal activities than other peptides and were almost as potent as melittin. Lastly, (KW)2 and (RW)2 at 64 μM did not inhibit growth of the fungal strains. The antifungal activities of peptides were in the following order: tetrameric peptides < hexameric peptides < octameric peptides < decameric peptides (Figure 2). These data suggest that a critical chain length may be required for significant antifungal activity. Melittin possesses strong antifungal activity and served as a positive control in this experiment. Photomicrographs of the mycelia of F. solani and F. oxysporum fungi were taken after a 24 h growth period. The effects of peptides on hyphal morphology were monitored and compared with the morphology of untreated hyphae (Figure 3A). Decameric and octameric peptides significantly inhibited spore germination and hyphal growth of phytopathogenic fungi in comparison with tetrameric peptides, which is consistent with the results on percentage inhibition of fungal growth. However, above 16 μM (for F. solani) or 32 μM (for F. oxysporum), (KW)3 and (RW)3 peptides noticeably inhibited conidial germination and hyphal development (Figure 3B). Significantly, hyberbranching of fungal hyphae, which is a typical morphological response of F. solani and F. oxysporum in response to (KW)2 and (RW)2, was not observed in the presence of other peptides at concentrations above or near their MICs. Therefore, we believe that antifungal activity increased with chain length. A previous study showed that (RW)n series peptides with increased chain length possess enhanced antimicrobial activity . Furthermore, decameric peptides and melittin showed similar antifungal activities against F. solani and F. oxysporum. On the other hand, hexametic and octameric peptides showed decreased antifungal activities against the same two strains in comparison with melittin. This result is consistent with previous data that also indicated that small cationic hexapeptides had lower antimicrobial activities compared to longer, naturally occurring AMPs such as magainin, cecropin, and melittin [11,32]. In addition, these hexameric peptides showed higher antifungal activities than the other hexameric peptides , suggesting that, in this (KW)3 or (RW)3, the composition of amino acid within the repeating motif seems to be more selective than the PAF26 or PAF32 for fungal membrane.
2.2. Effects of Longer Peptides on Cell Viability
To determine whether the antifungal peptides are fungicidal or fungistatic, F. solani and F. oxysporum cells were treated with or without peptides for 3 h. (KW)3 and (RW)3 peptides at their MICs showed moderate fungicidal activities against both F. solani and F. oxysporum. After treatment with (KW)4 and (RW)4 at their MICs, 68% and 69% of F. solani (or 74% and 76% of F. oxysporum) cells were killed, respectively (Figure 4). For (KW5) and (RW5) at their MICs, 72% and 75% of F. solani cells along with 76% and 79% of F. oxysporum cells were killed, respectively. The percentages for F. oxysporum further increased to 89% and 92% in the presence of (KW)5 and (RW)5 at 32 μM, respectively. Consistent with the results on percentage of growth inhibition, killing activity increased with peptide concentration as well as peptide length. Moreover, the results show that both (KW)n and (RW)n (n = 3, 4, and 5) had similar inhibitory activities against the two strains at corresponding MICs, but they differed in their fungicidal activities. In fact, RW series peptides showed slightly higher fungicidal activity than KW peptides. Some studies also reported that Arg-containing peptides were more active against plant fungal strains than Lys-containing peptides . Specifically, the guanidinium group of Arg strongly interacts with fungal strains due to its higher net positive charge compared to the protonated amine of lysine. In addition, Arg-containing peptides showed a two-fold greater activity against F. solani than F. oxysporum. Taken together, our results clearly indicate that the level of antimicrobial activity depends on the peptide sequence and fungal membrane composition.
Our results also show that the peptides act as fungicidal compounds. Application of fungicidal agents is usual practice when fighting plant diseases . Specifically, antifungal peptides with fungicidal activity are often used in plant transgenic applications. For example, antifungal plant defensin is induced in radish leaves upon challenge with fungal pathogens, suggesting a role in plant defense . Further, the natural AMP magainin has been expressed in Nicotiana tabacum to develop resistance to phytopathogens . Other studies also reported that transgenic plants expressing AMPs exhibit broad-spectrum resistance to phytopathogen infection [36–38]. At present, the high cost of synthetic peptides constitutes an obvious limitation to agricultural and food applications. As a result, there have been many efforts to express short AMPs in transgenic plants [38,39], for example indolicidin [40,41]. Short hexapeptides are used to control postharvest diseases in fruits and vegetables caused by fungal phytopathogens [21,23,42]. In fact, we previously identified (KW)4 as a potentially non-toxic AMP  that can be produced on a large scale economically, although this short peptide has been produced in transgenic plants. Future research will determine the feasibility of these options. Meanwhile, the modes of action of these peptides are yet to be resolved, although studies have proposed various killing mechanisms for Lys-Arg/Trp rich AMPs, including cytoplasmic membrane disruption and inhibition of nucleic acid synthesis . Other studies have reported that Lys-Arg/Trp-rich AMPs kill fungal strains through nucleic acid binding or cell penetration [45,46]. Studies on the modes of action of these peptides are currently underway.
3. Experimental Section
Rink amide 4-methylbenzhydrylamine resin, fluoren-9-ylmethoxycarbonyl (Fmoc) amino acids, and other reagents for peptide synthesis were purchased from Calibiochem-Novabiochem (La Jolla, CA, USA). For the quantitative antifungal assay and fungicidal activity assay, the following fungal strains were obtained from the Korea Collection for Type Cultures (KCTC): F. solani (KCTC 6326) and F. oxysporum (KCTC 6076). Fungal cells were grown on PDA (potato dextrose agar) plate and subcultured for 2–3 weeks.
3.2. Peptide Synthesis and Purifications
The peptides KWKW-NH2 (KW)2, KWKWKW-NH2 (KW)3, KWKWKWKW-NH2 (KW)4, KWKWKWKWKW-NH2 (KW)5, RWRW-NH2 (RW)2, RWRWRW-NH2 (RW)3, RWRWRWRW-NH2 (RW)4, RWRWRWRWRW-NH2 (RW)5 and GIGAVLKVLTTGLPALISWIKRKRQQ (melittin) were synthesized by the solid-phase method using Fmoc chemistry on a solid support of rink amide 4-methylbenzhydrydrylamine resin. Then, 0.1 M N-hydroxy benzotriazole (HOBt) and 0.45 M 2-(1H-benzotriazole-1-yil)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) in dimethylformamide (DMF) along with 2 M N,N-diisopropyl ethylamine (DIEA) in N-methylpyrrolidone (NMP) were used as coupling reagents, and 10-fold excess Fmoc-amino acid was added during every coupling cycle. Following a final deprotection with a solution of 20% piperidine in DMF and cleavage with a mixture of TFA/water/triisopropylsilane (90:5:5) for 2 h at room temperature , the crude peptides were repeatedly extracted with diethyl ether and purified using reverse phase preparative high performance liquid chromatography (HPLC) on a Vydac C18 column (4.6 × 250 mm, 300 Å, 5 nm). The molecular masses of the peptides were confirmed by using a matrix-assisted laser desorption ionization mass spectrometer (data not shown) (MALDI II, Kratos Analytical Ins.). The purity of all peptides were found to be > 95%.
3.3. Computational Modeling
Chemical structures of the peptides were built using ChemOffice Desktop 2004 for Windows (CambridgeSoft (CS) Corporation, Cambridge, MA, USA).
3.4. Antifungal Assay
Fungal fragments, precultured in mycelial growth medium, were placed in the center of PDA plates, after which the cultures were incubated for 96 h at 25 °C in the dark. After incubation, spores were isolated from cultures growing in half-strength PDA. Spore concentrations were then adjusted to 5 × 104 spores/mL in half-strength PDA, after which 80 μL was added to the wells of sterile 96-well flat-bottomed microtiter plates along with 20 μL of peptide or media to give final concentrations of 1–64 μM. Several wells were kept untreated as a control to monitor fungal growth. Plates were incubated in the dark at 25 °C for 24 h before hyphal growth was determined by measuring optical density at 595 nm using a microtiter plate Elisa reader (Molecular Devices, Sunnyvale, CA, USA) . Each test was performed in triplicate. Percentages of inhibition were then calculated (Figure 1) (0% inhibition indicates growth equal to control sample (only media)) . The lowest concentration of peptide inhibiting fungal growth was monitored microscopically with an inverted light microscope (IX71, Olympus, Tokyo, Japan) .
3.5. Cell Viability Assay
The spore suspension at a concentration of 5 × 104 spores/mL (80 μL) was transferred to a 96-well microtiter plate along with 20 μL of (KW)n or (RW)n peptides (n = 3, 4, and 5), melittin, or media to give final peptide concentrations of 4–32 μM. Plates were incubated for 3 h before the addition of 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/mL; Sigma). Plates were then incubated again for 16 h at room temperature, followed by the addition of 100 μL of MTT solvent (0.1 N HCl in anhydrous isopropyl alcohol). Presence of MTT/formazan was monitored spectrophotometrically by measuring the absorbance at 570 nm and then subtracting the background absorbance at 690 nm using a Versa-Max microplate Elisa reader (Molecular Devices, Sunnyvale, CA, USA) . Each measurement was conducted in triplicate.
In summary, increased chain length along with a higher ratio between hydrophobicity and net charge resulted in increased antifungal and fungicidal activities. Our results confirm that KW and RW peptide elements could be incorporated into the development of an AMP with low cost due to their short lengths, making these peptides a promising alternative tool in the field of green biocides.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0017532) and the Human Resource Training Project for Regional Innovation.
- Laverty, G.; Gorman, S.P.; Gilmore, B.F. The potential of antimicrobial peptides as biocides. Int. J. Mol. Sci 2011, 12, 6566–6596.
- Brogden, K.A. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol 2005, 3, 238–250.
- Hancock, R.E.W.; Sahl, H.G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol 2006, 24, 1551–1557.
- Strom, M.B.; Rekdal, O.; Svendsen, J.S. Antimicrobial activity of short arginine- and tryptophan-rich peptides. J. Pept. Sci 2002, 8, 431–437.
- Storm, M.B.; Haug, B.E.; Skar, M.L.; Stensen, W.; Stiberg, T.; Svendsen, J.S. The pharmacophore of short cationic antibacterial peptides. J. Med. Chem 2003, 46, 1567–1570.
- Liu, Z.; Brady, A.; Young, A.; Rasimick, B.; Chen, K.; Zhou, C.; Kallenbach, N.R. Length effects in antimicrobial peptides of the (RW)n series. Antimicrob. Agents Chemother 2007, 51, 597–603.
- Jing, W.; Hunter, H.N.; Hagel, J.; Vogel, H.J. The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipids bilayers. J. Pept. Res 2003, 61, 219–229.
- Dathe, M.; Nikolenko, H.; Klose, J.; Bienert, M. Cyclization increases the antimicrobial activity and selectivity of arginine- and tryptophan-containing hexapeptides. Biochemistry 2004, 43, 9140–9150.
- Sagaram, U.S.; Pandurangi, R.; Kaur, J.; Smith, T.J.; Shah, D.M. Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against fusarium graminearum. PLoS One 2011, 6, doi:10.1371/journal.pone.0018550.
- Powell, W.A.; Catranis, C.M.; Maynard, C.A. Synthetic antimicrobial peptide design. Mol. Plant Microb. Interact 1995, 8, 792–794.
- Muñoz, A.; López-Garcia, B.; Marcos, J.F. Comparative study of antimicrobial peptides to control citrus postharvest decay caused by penicillium digitatum. J. Agric. Food Chem 2007, 55, 8170–8176.
- Muñoz, A.; López-Garcia, B.; Pérez-Payá, E.; Marcos, J.F. Antimicrobial properties of derivatives of the cationic tryptophan-rich hexapeptide PAF26. Biochem. Biophys. Res. Commun 2007, 354, 172–177.
- Chan, D.I.; Prenner, E.J.; Vogel, H.J. Tryptophan and arginine rich antimicrobial peptides: Structures and mechanism of action. Biochim. Biophys. Acta 2006, 1758, 1184–1202.
- Falla, T.J.; Karunaratne, D.N.; Hancock, R.E.W. Mode of action of the antimicrobial peptide indolicidin. J. Biol. Chem 1996, 271, 19298–19303.
- Lay, F.T.; Anderson, M.A. Defensins–components of the innate immune system in plants. Curr. Protein Pept. Sci 2005, 6, 85–101.
- Thomma, B.P.H.J.; Cammue, B.P.A.; Thevissen, K. Plant defensins. Planta 2002, 216, 193–202.
- Carvalho, A.O.; Gomes, V.M. Plant defensins–prospects for the biological functions and biotechnological properties. Peptides 2009, 30, 1007–1020.
- Murad, A.M.; Pelegrini, P.B.; Neto, S.M.; Franco, O.L. Novel findings of defensins and their utilization in construction of transgenic plants. Transgenic Plant J 2007, 1, 39–48.
- Stotz, H.U.; Thomson, J.G.; Wang, Y. Plant defensins: Defense, development and application. Plant Signal. Behav 2009, 4, 1010–1012.
- Tavares, L.S.; Santos Mde, O.; Viccini, L.F.; Moreira, J.S.; Miller, R.N.; Franco, O.L. Biotechnological potential of antimicrobial peptides from flowers. Peptides 2008, 29, 1842–1851.
- López-García, B.; Pérez-Payá, E.; Marcos, J.F. Identification of novel hexapeptide bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library. Appl. Environ. Microbiol 2002, 68, 2453–2460.
- Choi, J.; Moon, E. Identification of novel bioactive hexapeptides against phytopathogenic bacteria through rapid screening of a synthetic combinatorial library. J. Microbiol. Biotechnol 2009, 19, 792–802.
- Lóopez-Garcia, B.; González-Candelas, L.; Pérez-payá, E.; Marcos, J.F. Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits. Mol. Plant Microbe Interact 2000, 13, 837–846.
- Chen, P.W.; Shyu, C.L.; Mao, F.C. Antimicrobial activity of short hydrophobic and basic-rich peptides. Am. J. Vet. Res 2003, 64, 1088–1092.
- Wessolowski, A.; Bienert, M.; Dathe, M. Antimicrobial activity of arginine and tryptophan rich hexapeptides: The effects of aromatic clusters, d-amino acid substitution and cyclization. J. Pept. Res 2004, 64, 159–169.
- Gopal, R.; Kim, Y.J.; Seo, C.H.; Hahm, K.S.; Park, Y. Reversed sequence enhances antimicrobial activity of a synthetic peptide. J. Pept. Sci 2011, 17, 329–334.
- Cochrane, V.W.; Cochrane, J.C. Chlamydospore induction in pure culture in Fusarium solani. Mycologia 1971, 63, 462–477.
- Cho, J.H.; Rupe, J.C.; Cummings, M.S.; Gbur, E.E., Jr. Isolation and identification of Fusarium solani f. sp. glycines from soil on modified Nash and Snyder’s medium. Plant Dis. 2001, 85, 256–260.
- Aoki, T.; O’Donnell, K.; Homma, Y.; Lattanzi, A. Sudden-death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex—F. virguliforme in North America and F. tucumaniae in South America. Mycologia 2003, 95, 660–684.
- Zaccardelli, M.; Vitale, S.; Luongo, L.; Merighi, M.; Corazza, L. Morphological and Molecular Characterization of Fusarium solani Isolates. J. Phytopathol 2008, 156, 534–541.
- Gómez, Y.; Gil, K.; González, E.; Farías, L.M. Anti-fungi activity of organic extracts from the tree Fagara monophylla (Rutaceae) in Venezuela. Rev. Biol. Trop 2007, 55, 767–775.
- Blondelle, S.E.; Takahashi, E.; Dinh, K.T.; Houghten, R.A. The antimicrobial activity of hexapeptides derived from synthetic combinatorial libraries. J. Appl. Bacteriol 1995, 78, 39–46.
- Knight, S.C.; Anthony, V.M.; Brady, A.M.; Greenland, A.J.; Heaney, S.P.; Murray, D.C.; Powell, K.A.; Schulz, M.A.; Spinks, C.A.; Worthington, P.A.; et al. Rationale and perspectives in the development of fungicides. Annu. Rev. Phytopathol 1997, 35, 349–372.
- Terras, F.R.G.; Eggermont, K.; Kovaleva, V.; Raikhel, N.V.; Osborn, R.W.; Kester, A.; Rees, S.B.; Torrekens, S.; van Leuven, F.; Vanderleyden, J.; et al. Small cysteine rich antifungal proteins from radish: Their role in host defence. Plant Cell 1995, 7, 573–588.
- Ponti, D.; Mangoni, M.L.; Mignogna, G.; Simmaco, M.; Barra, D. An amphibian antimicrobial peptide variant expressed in Nicotiana tabacum confers resistance to phytopathogens. Biochem. J 2003, 370, 121–127.
- Huang, Y.; Nordeen, R.O.; Di, M.; Owens, L.D.; Mcbeath, J.H. Expression of an engineered cecropin gene cassette in transgenic tobacco plants confers resistance to Pseudomonas syringae pv. tabaci. Phytopathology 1997, 87, 494–499.
- Cary, J.W.; Rajasekaran, K.; Jaynes, J.M.; Cleveland, T.E. Transgenic expression of a gene encoding a synthetic antimicrobial peptide result in inhibition of fungal growth in vitro and in planta. Plant Sci 2000, 154, 171–181.
- Osusky, M.; Zhou, G.; Osuska, L.; Hancock, R.E.; Kay, W.W.; Misra, S. Transgenic plants expressing cationic peptide chimeras exhibit broad spectrum resistance to phytopathogens. Nat. Biotechnol 2000, 18, 1162–1166.
- Montesinos, E. Antimicrobial peptides and plant disease control. FEMS Microbiol. Lett 2007, 270, 1–11.
- Xing, H.Y.; Lawrence, C.B.; Chambers, O.; Davies, H.M.; Everett, N.P.; Li, Q.Q. Increased pathogen resistance and yield in transgenic plants expressing combinations of the modified antimicrobial peptides based on indolicidin and magainin. Planta 2006, 223, 1024–1032.
- Bhargava, A.; Osusky, M.; Hancock, R.E.; Forward, B.S.; Kay, W.W.; Misra, S. Antiviral indolicidin variant peptides: Evaluation for broad-spectrum disease resistance in transgenic Nicotiana tabacum. Plant Sci 2007, 172, 515–523.
- López-Garcia, B.; Veyrat, A.; Pérez-Payá, E.; González-candelas, L.; Marcos, J.F. Comparison of the activity of antifungal hexapeptides and the fungicides thiabendazole and imazalil against postharvest fungal pathogens. Int. J. Food Microbiol 2003, 89, 163–170.
- Gopal, R.; Park, J.S.; Seo, C.H.; Park, Y. Applications of circular dichroism for structural analysis of gelatin and antimicrobial peptides. Int. J. Mol. Sci 2012, 13, 3229–3244.
- Nan, Y.H.; Lee, S.H.; Kim, H.J.; Shin, S.Y. Mammalian cell toxicity and candidacidal mechanism of Arg- or Lys-containing Trp-rich model antimicrobial peptides and their d-enantiomeric peptides. Peptides 2010, 31, 1826–1831.
- Muñoz, A.; López-Garcia, B.; Marcos, J.F. Studies on the mode of action of the antifungal hexapeptide PAF26. Antimicrob. Agents Chemother 2006, 50, 3847–3855.
- Munoz, A.; Marcos, J.F.; Read, N.D. Concentration-dependent mechanisms of cell penetration and killing by the de novo designed antifungal hexapeptide PAF26. Mol. Microbiol 2012, doi:10.1111/j.1365–2958.201208091.x.
- Atherton, E.; Sheppard, R.C. Solid Phase Peptide Synthesis: A Practical Approach; IRL Press: Oxford, UK; p. 1989.
- Van der Weerden, N.L.; Hancock, R.E.; Anderson, M.A. Permeabilization of fungal hyphae by the plant defensin NAD1 occurs through a cell wall dependent process. J. Biol. Chem 2010, 285, 37513–37520.
- Park, S.C.; Lee, J.R.; Kim, J.Y.; Hwang, I.; Nah, J.W.; Cheong, H.; Park, Y.; Hahm, K.S. Pr-1, a novel antifungal protein from pumpkin rinds. Biotechnol. Lett 2010, 32, 125–130.
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