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.     

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.     

Functional assignment of YvgO, a novel set of purified and chemically characterized proteinaceous antifungal variants produced by Bacillus thuringiensis SF361

This study reports a novel class of antifungal protein derived from bacterial origin. Bacillus thuringiensis SF361, the strain also responsible for producing the novel bacteriocin thurincin H, exhibits broad antifungal activity against select members of several fungal genera, including Aspergillus, Byssochlamys, and Penicillium, as well as the pathogenic yeast Candida albicans. Optimal antifungal production and secretion were observed after-log phase growth when incubated at 37°C in a carbohydrate-free growth medium. High-performance liquid chromatography purification was performed after pH-selective ammonium sulfate precipitation and size-exclusion chromatography. Intact mass analysis and peptide mass fingerprinting identified the 13,484-Da protein to be a mass homolog to the YvgO protein construct sequenced from Bacillus cereus AH 1134. Further analysis via amino-terminal sequencing also revealed the existence of four distinct yet equally efficacious YvgO variants differing only within the first four N-terminal residues. YvgO was found to be remarkably stable, maintaining its antifungal activity under a wide pH and temperature range. When assayed against the toxigenic species Byssochlamys fulva H25, the selected primary filamentous fungal indicator, the MIC was estimated to be 1.5 ppm. Candida albicans 3153 was more resistant, exhibiting MICs between 25 and 800 ppm, depending on growth conditions. YvgO is unique among antifungals, showing no known sequential or functional homology to the typical classes of antifungal proteins, including common membrane-acting agents such as cellulases and glucanases. Due to its activity against an array of pathogenic and spoilage fungi, the potentials for clinical, agricultural, and food-processing applications are encouraging.     

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.     

A synthetic peptide as a novel anticryptococcal agent

An engineered, killer decapeptide (KP) has been synthesized based on the sequence of a recombinant, single-chain anti-idiotypic antibody (KT-scFv) acting as a functional internal image of a yeast killer toxin. Killer decapeptide exerted a strong fungicidal activity against Candida albicans, which was attributed to peptide interaction with beta-glucan. As this polysaccharide is also a critical component of the cryptococcal cell wall, we wondered whether KP was also active against Cryptococcus neoformans, a human pathogen of increasing medical importance. We found that KP was able to kill both capsular and acapsular C. neoformans cells in vitro. Furthermore, KP impaired the production of specific C. neoformans virulence factors including protease and urease activity and capsule formation, rendering the fungus more susceptible to natural effector cells. In vivo treatment with KP significantly reduced fungal burden in mice with cryptococcosis and, importantly, protected the majority of immunosuppressed animals from an otherwise lethal infection. Given the relevance of cryptococcosis in immunocompromised individuals and the inability of conventional drugs to completely resolve the infection, the results of the present study indicate KP as an ideal candidate for further studies on novel anticryptococcal agents.     

Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50μM of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized.     

Structure and fungicidal activity of a synthetic antimicrobial peptide, P18, and its truncated peptides

P18 (KWKLFKKIPKFLHLAKKF-NH2) is an antimicrobial peptide designed from a cecropin A-magainin 2 hybrid that has potent antibacterial activity without hemolytic activity against human erythrocytes. In this study, P18 displayed potent fungicidal activity (MIC: 12.5 approximately 25 microM) against pathogenic fungi, Candida albicans, Trichosporon beigelii, Aspergillus flavus and Fusarium oxyspovrum. The central Pro9 residue and the entire sequence of P18 are essential for its full fungicidal activity. Circular dichroism analysis suggested that the higher alpha-helical content of the peptides did not correlate with the stronger fungicidal activity.     

A melittin-related peptide from the skin of the Japanese frog,Rana tagoi,with antimicrobial and cytolytic properties

Two peptides with antimicrobial and cytolytic properties were purified from an extract of the skin of Tago's brown frog Rana tagoi. The primary structure of one peptide (FLPILGKLLS(10)GIL.NH(2)) identifies it as a member of the temporin family, whereas the second peptide (AIGSILGALA(10)KGLPTLISWI(20)KNR.NH(2)) displays 78% sequence identity to melittin from the venom of the honeybee Apis florea. Compared with melittin, the melittin-related peptide (MRP) was equipotent in inhibiting the growth of the Gram-positive bacterium Staphylococcus aureus, 5-fold less potent against the Gram-negative bacterium Escherichia coli and against the fungal pathogen, Candida albicans. MRP was 13-fold less hemolytic than melittin against human erythrocytes and 4- and 5-fold less cytolytic against mouse EL4 T-lymphoma-derived cells and L929 fibroblasts, respectively. However, at non-cytotoxic concentrations (相似文献   

Antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from Psacothea hilaris

Psacotheasin is a 34-mer knottin-type peptide that is derived from Psacothea hilaris larvae. In this study, the antifungal activity and mechanism(s) by which psacotheasin affects human fungal pathogens were investigated. Psacotheasin shows remarkable antifungal properties without hemolytic activity against human erythrocytes. To understand the antifungal mechanism(s) of psacotheasin in Candida albicans, flow cytometric analysis with DiBAC₄(3) and PI was conducted. The results showed that psacotheasin depolarized and perturbed the plasma membrane of the C. albicans. Three-dimensional (3D)-flow cytometric contour-plot analysis, accompanied by decreased forward scatter (FS), which indicates cell size, confirmed that psacotheasin exerted antifungal effects via membrane permeabilization. The membrane studies, using a single GUV and FITC-dextran (FD) loaded liposomes, indicate that psacotheasin acts as a pore-forming peptide in the model membrane of C. albicans and the radius of pores were presumed to be anywhere from 2.3 to 3.3 nm. Therefore, the current study suggests that the mechanism(s) of psacotheasin’s antifungal properties function within the membrane.     

The signal peptide NPFSD fused to ricin A chain enhances cell uptake and cytotoxicity in Candida albicans

Microorganisms possess stringent cell membranes which limit the cellular uptake of antimicrobials. One strategy to overcome these barriers is to attach drugs or research reagents to carrier peptides that enter cells by passive permeation or active uptake. Here the short endocytosis signal peptide NPFSD was found to efficiently deliver both FITC and GFP into Saccharomyces cerevisiae and Candida albicans with uptake into the majority of cells in a population. The NPFSD signal is itself non-toxic, but when fused to the ricin A chain toxin (RTA) the peptide enhanced both cell uptake and toxicity against C. albicans, which like other yeasts is resistant to naked RTA. Cell entry required at least 1 h incubation, temperatures above 4 degrees C, and an energy source, and uptake was out-competed with free peptide. Therefore, the NPFSD peptide can carry a range of compounds into yeasts and this delivery route holds promise to enhance the activity of antifungals.     

Multiplicity of peptide permeases in Candida albicans: evidence from novel chromophoric peptides.

Evidence is presented for the presence of multiple peptide permeases in the eucaryotic organism Candida albicans. Instrumental in these studies were the peptides L-alanyl-L-2-thiophenylglycine (Ala-alpha-TPG) and L-alanyl-L-2-thiophenylglycyl-L-alanine (Ala-alpha-TPG-Ala), which contain thiophenol attached to the alpha-carbon of glycine. Subsequent to transport into the fungal cell, enzymatic hydrolysis of these peptides resulted in the release of free thiophenol, which was quantified by using Ellman reagent. Thiophenol release was shown to be directly correlated to peptide transport and hydrolysis, with transport being the rate-limiting step in intact cells. These peptides, whose uptake showed Michaelis-Menten kinetics, have been used to determine peptide uptake in C. albicans. In addition, we found that the intracellular peptidases can readily be assayed in permeabilized cells and that bestatin, an aminopeptidase inhibitor, inhibits all detectable peptidase activity. C. albicans 124 was able to transport and hydrolyze both Ala-alpha-TPG and Ala-alpha-TPG-Ala, whereas the mutant (124NIK5) was able to transport only the tripeptide. The intracellular peptidases of this mutant were unaffected. In wild-type C. albicans 124, oligopeptides were able to compete with uptake of Ala-alpha-TPG-Ala to a far greater extent than with that of Ala-alpha-TPG; dipeptides inhibited uptake of both Ala-alpha-TPG and Ala-alpha-TPG-Ala. These results provide complementary evidence for the existence of distinct transport systems.     

Synthesis, antimicrobial evaluation and QSAR studies of novel piperidin-4-yl-5-spiro-thiadiazoline derivatives

In an attempt to find a new class of antimicrobial agents, a series of new 1,3,4-thiadiazolines were synthesized from 2,6-diarylpiperidin-4-ones, via the corresponding 4'-phenylthiosemicarbazones. All the synthesized compounds (23-39) were virtually screened against bacterial (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi) and fungal strains (Candida albicans, Rhizopus sp, Aspergillus niger and Aspergillus flavus) by serial dilution method. QSAR study indicated that the increase in weakly polar component of solvent accessible surface area will favour antibacterial activity while increase in polarizability and decrease in ionisation potential and hydrogen bond donor will favour antifungal activity.     

Characterisation and expression of phospholipases B from the opportunistic fungus Aspergillus fumigatus

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (afplb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.     

Design of novel analogue peptides with potent antibiotic activity based on the antimicrobial peptide,HP (2-20), derived from N-terminus of Helicobacter pylori ribosomal protein L1

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RPL1). In order to develop novel antibiotic peptides useful as therapeutic agents, potent antibiotic activities against bacteria, fungi and cancer cells without a cytotoxic effect are essential. To this end, several analogues with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, the substitution of Trp for the hydrophobic amino acids, Gln and Asp at positions 17 and 19 of HP (2-20) (Anal 3), caused a dramatic increase in antibiotic activity without a hemolytic effect.In contrast, the decrease of hydrophobicity brought about by substituting Ser for Leu and Phe at positions 12 and 19 of HP (2-20), respectively (Anal 4, Anal 5), did not have a significant effect on the antibiotic activity. The antibiotic effects of these synthetic peptides were further investigated by treating prepared protoplasts of Candida albicans and conducting an artificial liposomal vesicle (PC/PS; 3:1, w/w) disrupting activity test. The results demonstrated that the Anal 3 prevented the regeneration of fungal cell walls and induced an enhanced release of fluorescent dye (carboxyfluorescein) trapped in the artificial membrane vesicles to a greater degree than HP (2-20).The potassium-release test conducted on C. albicans indicated that Anal 3 induced greater amounts of potassium ion to be released than the parent peptide, HP (2-20) did. These results indicated that the hydrophobic region of peptides is prerequisite for its effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.     

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.     

Anidulafungin: experimental therapy of fungal infections in animal models

We have reviewed the existing data on the efficacy of anidulafungin, which is the most recent echinocandin in the experimental treatment of fungal infections. The scarce published data practically only refers to disseminated and pulmonary aspergillosis and to disseminated candidiasis. Anidulafungin shows fungistatic activity against Aspergillus fumigatus, and fungicidal activity against Candida albicans and Candida glabrata.     

Oxovanadium(IV) complexes of hydrazides: potential antifungal agents

Oxovanadium(IV) -derived antifungals have been prepared by condensing equimolar amounts of vanadyl sulfate with hydrazides. All the synthesized ligands and their metal complexes were characterized by IR, UV-Visible and micro analytical data. These synthesized compounds were screened for their antifungal activity against Aspergillus flavus (A. flavus), Trichophyton longifusus (T. longifusus), Candida albicans (C. albicans), Microsporum canis (M. canis), Fusarium solani (F. solani) and Candida glaberata (C. glaberata) fungal strains. All complexes showed promising antifungal activity against different fungal strains with the exception of F. Solani and C. glaberata. Minimum Inhibitory Concentration (MIC) of different complexes and ligands are in the range of 250 to 400 microg/mL. Complex 7a and ligand 13 exhibit lowest MIC of 250 microg/mL whereas, complex 5a and ligands 2, 7 and 14 showed highest MIC of 400 microg/mL.     

Structural and biological characterization of chromofungin, the antifungal chromogranin A-(47-66)-derived peptide.

Vasostatin-I, the natural fragment of chromogranin A-(1-76), is a neuropeptide able to kill a large variety of fungi and yeast cells in the micromolar range. We have examined the antifungal properties of synthetic vasostatin-I-related peptides. The most active shortest peptide, named chromofungin, corresponds to the sequence Arg(47)-Leu(66). Extensive (1)H NMR analysis revealed that it adopts a helical structure. The biophysical mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied, showing the penetration of this peptide with different lipid monolayers. In order to examine thoroughly the antifungal activity of chromofungin, confocal laser microscopy was used to demonstrate the ability of the rhodamine-labeled peptide to interact with the fungal cell wall, to cross the plasma membrane, and to accumulate in Aspergillus fumigatus, Alternaria brassicola, and Candida albicans. Our present data reveal that chromofungin inhibits calcineurin activity, extending a previous observation that the N-terminal region of chromogranin A interacts with calmodulin in the presence of calcium. Therefore, the destabilization of fungal wall and plasma membrane, together with the possible intracellular inhibition of calmodulin-dependent enzymes, is likely to represent the mechanism by which vasostatin-I and chromofungin exert antifungal activity.     

Antifungal activity of coumarins

The antifungal activity of 40 coumarins was tested against the fungal strains: Candida albicans (ATCC 14053), Aspergillus fumigatus (ATCC 16913) and Fusarium solani (ATCC 36031), using the broth microdilution method. Osthenol showed the most effective antifungal activity among all the compounds tested, with a MIC value of 125 microg/ml for Fusarium solani and 250 micro/ml for Candida albicans and Aspergillus fumigatus. The antifungal potential of this prenylated coumarin can be related to the presence of an alkyl group at C-8 position.