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.     

Fluorescence method for determining the mechanism and speed of action of surface-active drugs on yeast cells

New antifungal agents are needed to treat life-threatening fungal infections, particularly with the development of resistance. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a time-dependent drug exposure assay that allows us to rapidly investigate the mechanism of surface-active antifungal drug action. The assay uses a multidrug pump-deficient strain of Saccharomyces cerevisiae and the potentiometric dye 3,3'-dipropylthiacarbocyanine iodide [diS-C?(3)] and can assess whether cells are depolarized, hyperpolarized, or permeabilized by drug exposure. In this work, we investigated the mechanisms of action of five surface-active compounds: SDS, nystatin, amphotericin B, octenidine dihydrochloride, and benzalkonium chloride. The diS-C?(3) time-dependent drug exposure assay can be used to identify the mechanisms of action of a wide range of drugs. It is a fast and cost-effective method for screening drugs to determine their lowest effective concentrations.     

Comparison of phospholipase activity with direct and indirect lytic effects of animal venoms upon human red cells.

1. The venoms of 22 species of arthropods, saurians, elapids and crotalids were studied concerning the phospholipase activity and the presence of a direct and an indirect lytic effect upon human red cells. 2. The venoms from the spiders Latrodectus and "tarantula", and the venoms from the scorpions of the genus Centruroides are not haemolytic and do not have phospholipase activity. 3. Only the venoms of Apis mellifera and Naja naja siamensis have shown direct lytic effect. 4. All other venoms studied have an indirect haemolytic effect associated to a phospholipase activity, but there is indication that other agents might be implicated in the haemolytic processes.     

Tyrosine 331 and phenylalanine 334 in Clostridium perfringens alpha-toxin are essential for cytotoxic activity

Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.     

Synthesis, antifungal and antimicrobial activity of alkylphospholipids

The antifungal, antibacterial and haemolytic activity of a series of alkylphosphocholines (e.g., miltefosine) and alkylglycerophosphocholines (e.g., edelfosine) has been investigated. These compound classes exhibit significant antifungal and moderate antibacterial activities. Several new alkylphosphocholine derivatives with amide or ester bonds in the alkyl chain have been synthesised. These compounds show much lower haemolytic activity than miltefosine. Alkylphosphocholines and alkylglycerophosphocholines show significant promise as novel orally available antifungal and antibacterial therapeutics.     

Facilitated diffusion of glucosamine-6-phosphate synthase inhibitors enhances their antifungal activity

N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) are strong inhibitors of the essential fungal enzyme, glucosamine-6-phosphate synthase, but their antifungal activity is poor, due to slow penetration of these agents through the cytoplasmic membrane. In the present studies we have exploited the possibility of enhancement of ADGP and FMDP antifungal activity by improving their transport properties. It has been found that membrane-permeabilising polyene macrolides amphotericin B (AMB) and its N-methyl-N-fructosyl methyl ester derivative (MF-AME), at subinhibitory concentrations, facilitate diffusion of ADGP through the fungal cell membrane, thus allowing a decrease of its minimal inhibitory concentration (MIC). Synergistic effects have been observed for combinations of ADGP with AMB or MF-AME. Fractional inhibitory concentration (FIC) indexes, determined against a number of Candida spp., have been in the 0.18-0.81 range. Weak antifungal synergistic effects have been found for combinations of FMDP with AMB or MF-AME. ADGP can be easily encapsulated into unilamellar lipid vesicles. Liposomal preparations of ADGP demonstrated stronger antifungal activity against some fungal strains than free ADGP.     

Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.     

Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene.

Lecithin-dependent haemolysin (LDH) of Vibrio parahaemolyticus was purified from Escherichia coli C600 transformed with a plasmid (pHL591) ligated with a 1.5 kb DNA fragment of V. parahaemolyticus. The final preparation comprised two LDH proteins with different molecular masses which were immunologically cross-reactive and had the same enzymic activity. The LDH was a phospholipase hydrolysing both fatty acid esters of phospholipid, i.e. it hydrolysed phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) and then LPC to glycerophosphorylcholine (GPC). From this point of view, LDH should be classified as a phospholipase B. Phospholipase B, however, does not usually show haemolytic activity, because the intermediate (LPC), which is the actual haemolytic agent, is immediately hydrolysed to the final product (GPC). On the other hand, LPC formed by LDH action was comparatively stable, because the rates of the two reactions catalysed by LDH, PC to LPC and LPC to GPC, are almost the same. This is the reason that LDH shows haemolytic activity. Therefore, LDH of V. parahaemolyticus is an atypical phospholipase to be designated as phospholipase A2/lysophospholipase.     

Synthesis and investigation of novel benzimidazole derivatives as antifungal agents

The rise and emergence of resistance to antifungal drugs by diverse pathogenic fungal strains have resulted in an increase in demand for new antifungal agents. Various heterocyclic scaffolds with different mechanisms of action against fungi have been investigated in the past. Herein, we report the synthesis and antifungal activities of 18 alkylated mono-, bis-, and trisbenzimidazole derivatives, their toxicities against mammalian cells, as well as their ability to induce reactive oxygen species (ROS) in yeast cells. Many of our bisbenzimidazole compounds exhibited moderate to excellent antifungal activities against all tested fungal strains, with MIC values ranging from 15.6 to 0.975 μg/mL. The fungal activity profiles of our bisbenzimidazoles were found to be dependent on alkyl chain length. Our most potent compounds were found to display equal or superior antifungal activity when compared to the currently used agents amphotericin B, fluconazole, itraconazole, posaconazole, and voriconazole against many of the strains tested.     

Diversity of antifungal and plant-associated Serratia plymuthica strains

A total of 21 plant-associated Serratia plymuthica strains were characterized phenotypically by their nutritional patterns, susceptibility to antibiotics, antifungal and haemolytic properties, and genotypically by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA, PCR fingerprints using BOX primers (BOX-PCR) and pulsed-field gel electrophoresis (PFGE) after digestion with SpeI. All of the investigated strains demonstrated antifungal activity in vitro against fungal pathogens while only six strains produced the antifungal antibiotic prodigiosin. Haemolytic activity and antibiotic resistance patterns were investigated to assess the risk associated with the use of isolates in plant protection. The strains were haemolytic at human-relevant temperatures. The level of resistance to antibiotics was low. This work shows that BOX-PCR and PFGE are useful fingerprinting methods to characterize Ser. plymuthica strains, although the discriminatory effect between the two methods differed. Computer-assisted analysis of phenotypic and genotypic features demonstrated relationships between the origin of isolation, the production of prodigiosin and the molecular fingerprint.     

Antifungal proteins and peptides of leguminous and non-leguminous origins

Antifungal proteins and peptides, as their names imply, serve a protective function against fungal invasion. They are produced by a multitude of organisms including leguminous flowering plants, non-leguminous flowering plants, gymnosperms, fungi, bacteria, insects and mammals. The intent of the present review is to focus on the structural and functional characteristics of leguminous, as well as non-leguminous, antifungal proteins and peptides. A spectacular diversity of amino acid sequences has been reported. Some of the antifungal proteins and peptides are classified, based on their structures and/or functions, into groups including chitinases, glucanases, thaumatin-like proteins, thionins, and cyclophilin-like proteins. Some of the well-known proteins such as lectins, ribosome inactivating proteins, ribonucleases, deoxyribonucleases, peroxidases, and protease inhibitors exhibit antifungal activity. Different antifungal proteins may demonstrate different fungal specificities. The mechanisms of antifungal action of only some antifungal proteins including thaumatin-like proteins and chitinases have been elucidated.     

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.     

Differential antifungal and calcium channel-blocking activity among structurally related plant defensins

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca(2+). By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca(2+) channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca(2+) channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.     

Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis.

Two lysophospholipases were isolated from the venom of an Australian elapid snake (subfamily Acanthophiinae), Pseudechis australis, by sequential chromatography on CM-52 cellulose, Sephadex G-75 and DE-52 cellulose columns. They were very similar to each other. One of them, lysophospholipase I, was obtained as a homodimer, the monomer of which consisted of 123 amino acid residues with seven disulphide bridges. The amino acid composition and the N-terminal amino acid sequence of the enzyme were similar to those of phospholipase A2, Ca2+ was required for its activity and the maximum activity was attained at 2 mM-CaCl2 in the presence of 1 mM-EDTA. The optimum pH was 7.5. Lysophospholipase I hydrolysed lysophosphatidylcholine more rapidly than lysophosphatidylethanolamine. It did not hydrolyse, however, phosphatidylcholine, 1-palmitoylglycerol, tripalmitoylglycerol or p-nitrophenyl acetate. Modification of the enzyme with p-bromophenacyl bromide or 2-nitrophenylsulphenyl chloride suppressed the activity. A strong direct haemolytic activity was exhibited when the lysophospholipase was present together with phospholipase A2.     

Systemic antifungals. Pharmacodynamics and pharmacokinetics

Invasive fungal infections are important causes of morbidity and mortality in critically ill non neutropenic patients. For many years, amphotericin B and flucytosine have been the only available antifungal agents for invasive fungal infections. Fortunately, the antifungal armamentarium has increased during the past two decades with the addition of several new agents. In addition to itraconazole and fluconazole, lipid formulations of amphotericin B, voriconazole, and caspofungin have been recently licensed. These various antifungal agents differ in their pharmacokinetic and pharmacodynamic profile.     

Total synthesis and antifungal evaluation of cyclic aminohexapeptides

The need for new therapies to treat systemic fungal infections continues to rise. Naturally occurring hexapeptide echinocandin B (1) has shown potent antifungal activity via its inhibition of the synthesis of beta-1,3 glucan, a key fungal cell wall component. Although this series of agents has been limited thus far based on their physicochemical characteristics, we have found that the synthesis of analogues bearing an aminoproline residue in the 'northwest' position imparts greatly improved water solubility (> 5 mg/mL). The synthesis and structure-activity relationships (SAR) based on whole cell and upon in vivo activity of the series of compounds are reported.     

A basic phospholipase A from the venom of the Australian king brown snake (Pseudechis australis) showing diverse activities against membranes

1. A basic phospholipase A (MSPA) was isolated from the venom of the Australian king brown snake, Pseudechis australis. 2. MSPA had an approximate Mr of 13,000 and consisted of a single polypeptide chain of 119 amino acid residues cross-linked by seven disulphide bridges. 3. MSPA exhibited direct haemolytic, anticoagulant and myotoxic activities. 4. Treatment of MSPA with p-bromophenacyl bromide modified a single histidine residue, resulting in complete loss of enzyme activity.     

Metal-based sulfonamides: their preparation, characterization and in-vitro antibacterial, antifungal & cytotoxic properties. X-ray structure of 4-[(2-hydroxybenzylidene) amino] benzenesulfonamide

Synthesis, characterization and biological studies of Schiff base-derived sulfonamides and their Co (II), Cu (II), Ni (II) and Zn (II) complexes have been reported and screened for in-vitro antibacterial activity against six Gram-negative; E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis, S. typhi and S. dysenteriae and four Gram-positive; B. cereus, C. diphtheriae, S. aureus and S. pyogenes bacterial strains and for in-vitro antifungal activity against T. longifusus, C. albicans, A. flavus, M. canis, F. solani, and C. glaberata. All compounds showed moderate to significant antibacterial activity, however, the zinc (II) complexes were found to be more active. Some of the compounds also showed significant antifungal activity against various fungal strains. Only compounds (6) and (10) displayed potent cytotoxic activity with LD(50) = 4.644 x 10(- 4) and 4.106 x 10(- 4) moles/mL respectively, against Artemia salina. The X-ray structure of 4-[(2-hydroxybenzylidene)amino]benzenesulfonamide is also reported.     

Antifungal effect of silver nanoparticles on dermatophytes

Spherical silver nanoparticles (nano-Ag) were synthesized and their antifungal effects on fungal pathogens of the skin were investigated. Nano-Ag showed potent activity against clinical isolates and ATCC strains of Trichophyton mentagrophytes and Candida species (IC80, 1-7 mug/ml). The activity of nano-Ag was comparable to that of amphotericin B, but superior to that of fluconazole (amphotericin B IC80, 1-5 mug/ml; fluconazole IC80, 10- 30 mug/ml). Additionally, we investigated their effects on the dimorphism of Candida albicans. The results showed nano-Ag exerted activity on the mycelia. Thus, the present study indicates nano-Ag may have considerable antifungal activity, deserving further investigation for clinical applications.