Antifungal peptides homologous to the Penicillium chrysogenum antifungal protein (PAF) are widespread among Fusaria

Putative antifungal peptide encoding genes containing Penicillium chrysogenum antifungal protein (PAF) characteristic amino acid motifs were identified in 15 Fusarium isolates, representing 10 species. Based on the predicted sequences of mature peptides, discrepancy in one, two or three amino acids was observed between them. Phylogenetic investigations revealed that they show high amino acid sequence similarity to PAF and they belong to the group of fungal derived antifungal peptides with PAF-cluster. Ten from the 15 partially purified <10 kDa peptide fraction of Fusarium ferment broths showed antifungal activity. The presence of approximately 6.3 kDa molecular weight peptides was detected in all of the antifungally active ferment broths, and this peptide was isolated and purified from Fusarium polyphilaidicum. The minimal inhibitiory concentrations of F. polyphilaidicum antifungal protein (FPAP) were determined against different filamentous fungi, yeasts and bacteria. Filamentous fungal species were the most susceptible to FPAF, but some yeasts were also slightly sensitive.     

Functional expression of a Drosophila antifungal peptide in Escherichia coli

Drosomycin is a key effector molecule involved in Drosophila innate immunity against fungal infection. This peptide is composed of 44 residues stabilized by four disulfide bridges. As the first step towards the understanding of the molecular basis for its specific antifungal activity, rapid and efficient production of the wild-type peptide and its mutants is needed. Here, we report a pGEX system for high-level expression of recombinant Drosomycin. The fusion Drosomycin protein with a carrier of Glutathione S-transferase (GST) was initially purified by affinity chromatography followed by Enterokinase cleavage. The digested product was separated by gel filtration and reverse phase HPLC. Mass spectrometry and circular dichroism spectroscopy analysis revealed that the recombinant peptide has identical molecular weight and correct structural conformation to native Drosomycin. Classical inhibition assay showed clear antifungal activity against Neurospora crassa with the IC(50) of 1.0muM. Successful expression of the CSalphabeta-type antifungal peptide in E. coli offers a basis for further studying its functional surface by alanine scanning mutagenesis strategy. Also, our work should be helpful in developing this peptide to an antifungal drug.     

Influence on the plasma membrane of Candida albicans by HP (2-9)-magainin 2 (1-12) hybrid peptide

A 20-residue hybrid peptide (HP (2-9)-MA (1-12): HP-MA), incorporating 2-9 residues of Helicobacter pyroli ribosomal protein L1 (HP) and 1-12 residues of magainin 2 (MA), has more potent antibacterial activity than parent peptide HP (2-20) and magainin 2. In this study, the antifungal activity and its mechanism of HP-MA were investigated. HP-MA displayed a strong antifungal activity in an energy-dependent manner. To elucidate the antifungal mechanism(s) of HP-MA, FACScan analysis and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe of Candida albicans were examined. The results indicated that the HP-MA exerts its antifungal effect by acting on the plasma membrane. Furthermore, the peptide induced remarkable morphological change when tested for membrane disrupting activity using liposomes (PC/Cholesterol; 10:1, w/w). In C. albicans, dimorphism plays a crucial role in pathogenesis but HP-MA could disrupt the mycelial forms and exert its antifungal effect on the blastoconidia in 20% fetal bovine serum.     

First report of a glutamine-rich antifungal peptide with immunomodulatory and antiproliferative activities from family Amaryllidaceae

This represents the first report of purification of a glutamine-rich antifungal peptide from family Amarylliaceace. The peptide, designated as nartazin, was purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis by means of ion-exchange chromatography and affinity chromatography. Its molecular mass was 7.1kDa, as determined by SDS-PAGE and gel filtration. Nartazin stimulated proliferation of mouse splenocytes and bone marrow cells but inhibited proliferation of leukemia L1210 cells. It also inhibited translation in a cell-free rabbit reticulocyte lysate system. The sequence of its first 20 N-terminal residues was characterized by an abundance of glutamine. The peptide possessed antifungal activity on four phytopathogenic fungi. Its activity was retained after incubation with bovine trypsin and chymotrypsin (enzyme: substrate ratio 1:10 w/w) at 37 degrees C for 1h but was attenuated after treatment with proteinase K. The data revealed its pronounced resistance to proteolytic digestion.     

Antifungal properties of a peptide derived from the signal peptide of the HIV-1 regulatory protein, Rev

Antifungal effects of nuclear entry inhibitory signal peptide of HIV-1 Rev protein (Rev-NIS) were investigated. Rev-NIS contained potent antifungal activities without hemolytic effects. To understand the antifungal mechanism(s), in vivo and in vitro fluorescent studies were conducted. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and calcein-leakage measurement from large unilamellar vesicles (LUVs) indicated that Rev-NIS depolarized and disrupted the fungal membranes. These results were further confirmed by using giant unilamellar vesicles (GUVs). The current study suggests that Rev-NIS exerts its antifungal activity with membrane-active mechanism(s).     

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC₅₀ values from 2.33 × 10⁻⁷ M to 36.0 × 10⁻⁷ M. SP6 peptide activity was neutralized by laminarin, a β-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall β-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.     

First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus

A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 degrees C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named "AcAFP" for A. clavatus antifungal peptide, has molecular mass of 5773Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 degrees C for 1h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.     

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.     

A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value

As fungal infections are becoming more prevalent in the medical or agricultural fields, novel and more efficient antifungal agents are badly needed. Within the scope of developing new strategies for the management of fungal infections, antifungal compounds that target essential fungal cell wall components are highly preferable. Ideally, newly developed antimycotics should also combine major aspects such as sustainability, high efficacy, limited toxicity and low costs of production. A naturally derived molecule that possesses all the desired characteristics is the antifungal protein (AFP) secreted by the filamentous ascomycete Aspergillus giganteus. AFP is a small, basic and cysteine-rich peptide that exerts extremely potent antifungal activity against human- and plant-pathogenic fungi without affecting the viability of bacteria, yeast, plant and mammalian cells. This review summarises the current knowledge of the structure, mode of action and expression of AFP, and highlights similarities and differences concerning these issues between AFP and its related proteins from other Ascomycetes. Furthermore, the potential use of AFP in the combat against fungal contaminations and infections will be discussed.     

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: Design,synthesis, activity against multidrug-resistant bacteria and Candida

Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials.     

Increased potency of a novel d-β-naphthylalanine-substituted antimicrobial peptide against fluconazole-resistant fungal pathogens

The antifungal activities of the known antimicrobial peptide, P-113, as well as a new type of Trp-rich peptide, Ac-KWRRWVRWI-NH₂, Pac-525, and its modified peptide, d -Nal-Pac-525, were determined using the broth microdilution method in three different media. All peptides had similar activities against yeast pathogens in low-salt LYM media. However, only d -Nal-Pac-525 retained its antifungal activity in the media containing high concentrations of salt. Hence, d -Nal-Pac-525 has the potential of becoming a promising antifungal agent, especially for fungal pathogens with intrinsic resistance to fluconazole.     

A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

Pn-AMP1, a plant defense protein, induces actin depolarization in yeasts

Pn-AMP1, Pharbitis nil antimicrobial peptide 1, is a small cysteine-rich peptide implicated in host-plant defense. We show here that Pn-AMP1 causes depolarization of the actin cytoskeleton in Saccharomyces cerevisiae and Candida albicans. Pn-AMP1 induces rapid depolarization of actin cables and patches within 15 min. Increased osmolarity or temperature induces transient actin depolarization and results in increased sensitivity to Pn-AMP1, while cells conditioned to these stresses show less sensitivity. Mutations in components of a cell wall integrity pathway (Wsc1p, Rom2p, Bck1p and Mpk1p), which regulate actin repolarization, result in increased sensitivity to Pn-AMP1. A genetic screen reveals that mutations in components of the alpha-1,6-mannosyltransferase complex (Mnn10p, Mnn11p and Och1p), which regulate mannosylation of cell wall proteins, confer resistance to Pn-AMP1. FITC-conjugated Pn-AMP1 localizes to the outer surface of the cell with no significant staining observed in spheroplasts. Taken together, these results indicate that cell wall proteins are determinants of resistance to Pn-AMP1, and the ability of a plant defense protein to induce actin depolarization is important for its antifungal activity.     

A radish seed antifungal peptide with a high amyloid fibril-forming propensity

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.     

Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba

An antifungal peptide from leaves of Ginkgo biloba, designated GAFP, has been isolated. Its molecular mass of 4244.0 Da was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation. GAFP exhibited antifungal activity towards Pellicularia sasakii Ito, Alternaria alternata (Fries) Keissler, Fusarium graminearum Schw. and Fusarium moniliforme. Its activities differed among various fungi. GAFP could also cause increased hyphal membrane permeabilization and a rapid alkalization of the medium when applied at 100 microgram/ml to Pellicularia sasakii Ito hyphae. The amino acid sequence of GAFP shows characteristics of the cysteine/glycine-rich chitin binding domain of many chitin binding proteins. The cysteine residues are well conserved.