Fungicidal effect of pleurocidin by membrane-active mechanism and design of enantiomeric analogue for proteolytic resistance

Pleurocidin (Ple) is a 25-residue peptide which is derived from the skin mucous secretion of the winter flounder (Pleuronectes americanus). In this study, we investigated antifungal effects and its mode of action of Ple on human pathogenic fungi. Ple showed potent antifungal activity with low hemolytic activity. To investigate the antifungal mechanisms of Ple, the cellular localization and membrane interaction of Ple were examined. Protoplast regeneration and membrane-disrupting activity by DPH-labeled membrane support the idea, that Ple exerts fungicidal activity against the human pathogenic fungus Candida albicans with the disruption of a plasma membrane. To aim for which was the application of a therapeutic agent, we designed a synthetic enantiomeric peptide composed of all-d-amino acids to enhance proteolytic resistance. The synthetic all-d-Ple also displayed two-fold more potent antifungal activity than that of all-l-Ple, and its antifungal activity showed proteolytic resistance against various proteases. Therefore, these results suggest a therapeutic potential of all-d-Ple with regard to its proteolytic resistance against human fungal infections.     

Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis

Histatin 5 (Hst‐5) is an antimicrobial peptide with strong antifungal activity against Candida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused by C. albicans. However, Hst‐5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced by C. albicans. Single‐residue substitutions can significantly affect the proteolytic resistance of Hst‐5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single‐residue modifications can be combined in a single peptide, K11R–K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single‐residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap‐dependent effects on degradation. Both single‐ and double‐substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.     

Pleurocidin-derived antifungal peptides with selective membrane-disruption effect

Pleurocidin (Ple) is a peptide derived from the winter flounder. In our previous study, we reported the antifungal effect of Ple and its mode of action. To develop novel antifungal peptides useful as therapeutic agents, two analogs, with amino acid substitutions, were designed to decrease the net hydrophobicity by Arg (R) or Ser (S)-substitution at the hydrophobic face of Ple without changing the amphipathic structure. By substituting Ser, the hydrophobicity of the peptide (anal-S) was decreased, and by substituting Arg, though the hydrophobicity of the peptide (anal-R) was decreased, the cationicity of this peptide was increased. CD measurements showed the substitution of Arg or Ser decrease the α-helical conformation of analog peptides. Studies with analog peptides have shown decreases in hydrophobicity and α-helicity do not affect antifungal activity but decrease hemolytic activity. These results suggest that highly hydrophobic and α-helical natures are not desirable in the design of antimicrobial peptides.     

Antifungal effect of CopA3 monomer peptide via membrane-active mechanism and stability to proteolysis of enantiomeric d-CopA3

In our previous study, coprisin, a 43-mer defensin-like peptide, was derived from the dung beetle, Copris tripartitus, and a 9-mer CopA3 (monomer), truncated coprisin analog peptide, was designed. However, the antifungal effects of CopA3 are not known yet. In this study, the antifungal activity and mechanism of CopA3 were investigated and to develop a more effective antimicrobial peptide under physiological conditions, the enantiomeric d-CopA3 was designed. l- and d-CopA3 had a similar antifungal activity without chiral selectivity, and their activity was more potent than that of melittin used as a positive control. Furthermore, l- and d-CopA3 did not even show any hemolysis against human erythrocytes. Membrane studies using propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], suggested that the antifungal effect of l- and d-CopA3 was due to the membrane-active mechanism, by contrast with coprisin possessing apoptotic mechanism without membrane permeabilization. Finally, the proteolytic resistance and antifungal activity of l- and d-CopA3 against trypsin was analyzed by HPLC and colony count assay. The results showed that only d-CopA3 maintained a potent antifungal activity despite the proteolytic condition. Therefore, this study suggests that d-CopA3 has potential as a novel antimicrobial agent.     

Effective antibacterial action of tat (47-58) by increased uptake into bacterial cells in the presence of trypsin

In a previous study, we found an antifungal effect on human pathogenic fungi by the cell-penetrating peptide Tat (47-58) derived from HIV-1. Tat (47-58) immediately entered into the fungal nucleus and affected some physiological changes on the intracellular condition. In this study, Tat (47-58) showed a broad spectrum of antibacterial activity against pathogenic bacteria including bacterial clinical isolates. To improve resistance against proteases for use in vivo, we synthesized an analog of Tat (47-58) by substituting the L-amino acid for the D-amino acid. The D-enantiomer of Tat (47-58) also exhibited a broad spectrum of antibacterial activity at almost the same level of L-Tat (47-58) concentration. Unlike L-Tat (47-58), D-Tat (47-58) showed a significant proteolytic resistance against all proteases tested and antimicrobial activities in the presence of trypsin. Moreover, D-Tat (47-58) inhibited MRSA infection in human HeLa cells whereas L-Tat (47-58) partially allowed MRSA infection, and the results were due to the proteolytic resistance of D-Tat (47-58).     

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.     

Damage to the cytoplasmic membrane and cell death caused by lycopene in Candida albicans

Lycopene, an acyclic carotenoid found in tomatoes (Lycopersicon esculentum) and a number of fruits, has shown various biological properties, but its antifungal effects remain poorly understood. The current study investigated the antifungal activity of lycopene and its mode of action. Lycopene showed potent antifungal effects toward pathogenic fungi, tested in an energy-independent manner, with low hemolytic effects against human erythrocytes. To confirm the antifungal effects of lycopene, its effects on the dimorphism of Candida albicans induced by fetal bovine serum (FBS), which plays a key role in the pathogenesis of a host invasion, were investigated. The results showed that lycopene exerted potent antifungal activity on the serum-induced mycelia of C. albicans. To understand the antifungal mode of action of lycopene, the action of lycopene against fungal cell membranes was examined by FACScan analysis and glucose and trehalose-release test. The results indicated that lycopene caused significant membrane damage and inhibited the normal budding process, resulting from the destruction of membrane integrity. The present study indicates that lycopene has considerable antifungal activity, deserving further investigation for clinical applications.     

Antifungal effect and action mechanism of antimicrobial peptide polybia‐CP

The incidence of life‐threatening invasive fungal infections increased significantly in recent years. However, the antifungal therapeutic options are very limited. Antimicrobial peptides are a class of potential lead chemical for the development of novel antifungal agents. Antimicrobial peptide polybia‐CP was purified from the venom of the social wasp Polybia paulista. In this study, we synthesized polybia‐CP and determined its antifungal effects against a series of Candidian species. Our results showed that polybia‐CP has potent antifungal activity and fungicidal activity against the tested fungal cells with a proposed membrane‐active action mode. In addition, polybia‐CP could induce the increase of cellular reactive oxygen species production, which would attribute to its antifungal activity. In conclusion, the present study suggests that polybia‐CP has potential as an antifungal agent or may offer a new strategy for antifungal therapeutic option. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

PARP-1 regulates the expression of caspase-11

Lariciresinol is an enterolignan precursor isolated from the herb Sambucus williamsii, a folk medicinal plant used for its therapeutic properties. In this study, the antifungal properties and mode of action of lariciresinol were investigated. Lariciresinol displays potent antifungal properties against several human pathogenic fungal strains without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of action of lariciresinol, the membrane interactions of lariciresinol were examined. Fluorescence analysis using the membrane probe 3,3′-diethylthio-dicarbocyanine iodide (DiSC₃-5) and 1,6-diphenyl-1,3,5-hexatriene (DPH), as well as a flow cytometric analysis with propidium iodide (PI), a membrane-impermeable dye, indicated that lariciresinol was associated with lipid bilayers and induced membrane permeabilization. Therefore, the present study suggests that lariciresinol possesses fungicidal activities by disrupting the fungal plasma membrane and therapeutic potential as a novel antifungal agent for the treatment of fungal infectious diseases in humans.     

Antifungal cyclopeptolide from fungal saprophytic antagonist Ulocladium atrum

The saprophytic fungus Ulocladium atrum Preuss is a promising biological control agent for Botrytis cinerea in greenhouse- and field-grown crops. However, despite its known potent antifungal activity, no antifungal substance has yet been reported. In an effort to characterize the antifungal substance from U atrum, we isolated an antibiotic peptide. Based on extensive spectroscopic analyses, its structure was established as a cyclopeptolide with a high portion of N-methylated amino acids, and its 1H and 13C chemical shifts were completely assigned based on extensive 1D and 2D NMR experiments. Compound 1 exhibited potent antifungal activity against the plant pathogenic fungus Botrytis cinerea and moderate activity against Alternaria alternate and Magnaporthe grisea.     

The comparison of characteristics between membrane-active antifungal peptide and its pseudopeptides

By the introduction of various amide surrogates, novel pseudopeptides corresponding to a membrane active depsipeptide were synthesized and their native characteristics compared with that of the peptide. The pseudopeptides had more resistance to serum proteases than the peptide and similar antimicrobial activities to that of the peptide without hemolytic activity. The pseudopeptides like the peptide were active against current drug resistant fungi and pathogenic fungi isolated from patients, and also had a strong synergism with current antifungal drugs against Candida albicans. The leakage assay suggested that the pseudopeptides also acted on the lipid membrane of pathogenic cells. These results indicated that the novel pseudopeptides had advantages over the peptide as a candidate for a novel antifungal drug and backbone modifications can be a tool in the development of a novel antifungal agent from membrane-active peptides isolated from natural sources or chemically synthesized.     

Susceptibility of Treponema pallidum to host-derived antimicrobial peptides

LL-37 displays potent broad-spectrum activity against a number of pathogenic bacteria and is the only cathelicidin thus far identified in humans. In this study, we examined the capacity of human LL-37 and the similar CAP-18-derived peptide from rabbits to exert antimicrobial activity against the causative agent of syphilis, Treponema pallidum. We found that both peptides, as well as a truncated version of human LL-37 that contains its bactericidal domain, could exert rapid, but salt-sensitive antimicrobial activity against T. pallidum. Infectivity of T. pallidum in a rabbit model could effectively be blocked with the synthetic truncated LL-37-derived peptide WS22-N-amide.     

Effect of a novel antifungal peptide P852 on cell morphology and membrane permeability of Fusarium oxysporum

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens.     

Characterization of antimicrobial peptides against a US strain of the rice pathogen Rhizoctonia solani

AIM: To identify antimicrobial peptides with high lytic activity against Rhizoctonia solani strain LR172, causal agent of rice sheath blight and aerial blight of soyabeans in the US. METHODS AND RESULTS: Among 12 natural and synthetic antimicrobial peptides tested in vitro, the wheat-seed peptide, purothionin, showed the strongest inhibitory activity that was similar to the antifungal antibiotics, nystatin and nikkomycin Z. Cecropin B, a natural peptide from cecropia moth, and synthetic peptide D4E1 produced the highest inhibitory activity against R. solani among linear peptides. Membrane permeabilization levels strongly correlated with antifungal activity of the peptides. Noticeable changes in membrane integrity were observed at concentrations of >/=0.5 micromol l(-1) for purothionin, 2 micromol l(-1) for cecropin B, D4E1, D2A21, melittin, and phor21, and 8 micromol l(-1) for magainin II and phor14. An increase of nuclear membrane permeabilization was observed in fungal cells treated with cecropin B, but not with purothionin. Diffusion of nuclear content was observed by fluorescent microscopy 10 min after adding a lethal concentration of cecropin B. Evaluation by electron microscopy confirmed severe cytoplasmic degradation and plasma membrane vesiculation. Purothionin and cecropin B were the most stable against proteolytic degradation when added to liquid cultures of R. solani. CONCLUSIONS: Purothionin, cecropin B, D4E1 and phor21 were shown to exhibit high in vitro lytic activity against R. solani strain LR172 for rice and soyabean. These peptides are greater than 16 amino acids long and rapidly increase fungal membrane permeabilization. Resistance to proteolysis is important for sufficient antifungal activity of antimicrobial peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected antimicrobial peptides offer an attractive alternative to traditional chemicals that could be utilized in molecular breeding to develop crops resistant to rice sheath blight and aerial blight of soyabean.     

Fungicidal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Candida albicans

The antifungal activity and mechanism of a 23-mer peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed strong antifungal activity against yeast and mold. To investigate the antifungal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. Candida albicans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide(PI) staining, which was similar to that of Melittin than untreated cells. Confocal microscopy showed that the peptide was located in the plasma membrane. The action of peptides against fungal cell membranes was examined by treating prepared protoplasts of C. albicans with the peptide and lipid vesicle titration test. The result showed that the peptide prevented the regeneration of fungal cell walls and induced release of the fluorescent dye trapped in the artificial membrane vesicles, indicating that the peptide exerts its antifungal activity by acting on the plasma lipid membrane.     

Anti-Candida property of a lignan glycoside derived from Styrax japonica S. et Z. via membrane-active mechanisms

Styraxjaponoside C was investigated with respect to its antifungal activity and mechanisms of action. Devoid of hemolytic activity, Styraxjaponoside C demonstrated an antifungal effect against the human pathogenic yeast Candida albicans in an energy-independent manner. To characterize the mechanisms of the antifungal activity of Styraxjaponoside C, fluorescence analysis with membrane probe 1,6-diphenyl-1,3,5-hexatriene, and flow cytometric analysis on C. albicans were conducted. The results showed that Styraxjaponosdie C induced cytoplasmic membrane perturbation. The current study suggested that Styraxjaponoside C was active against C. albicans with membrane-active mechanisms.     

Antifungal mechanism of SMAP-29 (1-18) isolated from sheep myeloid mRNA against Trichosporon beigelii

The antifungal activity and mechanism of SMAP-29 (1-18) (SMAP-29), a cathelicidin-derived antimicrobial peptide deduced from N-terminal sequence of sheep myeloid mRNA, were investigated. SMAP-29 displayed a strong antifungal activity against various fungi. To understand the antifungal mechanism(s) of SMAP-29, we examined the interaction of SMAP-29 with the pathogenic fungus Trichosporon beigelii. Confocal microscopy showed that SMAP-29 was localized in the plasma membrane. The antifungal effects of SMAP-29 were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plasma membrane probe. Flow cytometric analysis revealed that SMAP-29 acted in an energy-dependent manner. This interaction is also dependent on the ionic environment. Furthermore, SMAP-29 caused significant morphological changes when testing the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w), as shown by scanning electron microscopy. The results suggest that SMAP-29 may exert its antifungal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers and irregularly disrupted fungal membranes in an energy- and salt-dependent manner.     

Cecropin A-melittin hybrid peptide exerts its antifungal effects by damaging on the plasma membranes of Trichosporon beigelii

To elucidate the effect of the peptide derived from cecropin A(1-8)-melittin(1-12) having potent antifungal activity without cytotoxicity against eukaryotic cell on the fungal cell membranes, Trichosporon beigelii protoplasts were prepared. The protoplasts treated with the peptide not only failed to regenerate the fungal cell walls but also disrupted the membrane, indicating that the peptide exerts its antifungal activity by acting on the plasma membranes. © Rapid Science Ltd. 1998     

Cecropin A–melittin mutant with improved proteolytic stability and enhanced antimicrobial activity against bacteria and fungi associated with gastroenteritis in vitro

Cecropin A–melittin (CAM), a chimeric antimicrobial peptide with potent antimicrobial activity, is threatened by some special extracellular proteases when used to deal with certain drug-resistant pathogenic microbes in the gastrointestinal tract. Thus, a four-tryptophan-substitution mutant (CAM-W) from CAM was developed via the replacement of special amino acid residues to enhance the antimicrobial potency and to improve the proteolytic stability of this agent. The pharmaceutical index of CAM-W was investigated, with a focus on biological potency, cytotoxicity, and proteolytic stability, as well as pH and thermal resistance. CAM-W exhibited potent antimicrobial activity and was approximately 3–12 times higher than that of CAM. CAM-W also exhibited a strong antifungal activity against a series of common pathogenic fungi, in a lower IC50 range between 2.1 mg/L and 3.3 mg/L than that of its reference CAM ranging from 9.8 mg/L to 14.2 mg/L. Besides, CAM-W showed moderate cytotoxicity (IC₅₀ > 300 mg/L) in erythrocyte lysis test. In addition, CAM-W overcame challenges under various conditions, including specific temperatures (20, 30, 40, 50, 60, 70, 80, and 90 °C), pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0), and proteases (trypsin, pepsin, human neutrophil elastase, Pseudomonas aeruginosa elastase, and Staphylococcus aureus V8 protease) that are commonly present in human gastrointestinal tract. These results suggest that the four-tryptophan-substitution can confer CAM-W with a high pharmaceutical index, which is important for CAM-W to become a potential alternative to conventional antibiotics against bacteria and fungi associated with gastroenteritis.     

Design of novel peptide analogs with potent fungicidal activity,based on PMAP-23 antimicrobial peptide isolated from porcine myeloid

PMAP-23 is a 23-mer peptide derived from porcine myeloid. To develop novel antifungal peptides useful as therapeutic drugs, it would require a strong fungicidal activity against pathogenic fungal cells. To this goal, several analogs, with amino acid substitutions, were designed to increase the net hydrophobicity by Trp (W)-substitution at positions 10, 13, or 14 at the hydrophilic face of PMAP-23 without changing the hydrophobic helical face. The Trp (W)-substitution (P6) showed an enhanced fungicidal and antitumor activities, with the fungicidal activity inhibited by salts and the respiratory inhibitor, NaN(3). The results suggested that the increase of hydrophobicity of the peptides correlated with fungicidal activity. The fungicidal effects of analog peptides were further investigated using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. In Candida albicans, the analog peptide (P6) exerted its fungicidal effect on the blastoconidia in 20% fetal bovine serum by disrupting the mycelial forms. Furthermore, P6 caused significant morphological changes, and these facts suggested that the fungicidal function of the novel analog peptide (P6) was by damaging the fungal cell membranes. Thus, this peptide may provide a useful template for designing novel antifungal peptides useful for the treatment of infectious diseases.