Sabina Berne

Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.

Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains.

Ostreolysin, a 15 kDa pore‐forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono‐ or di‐unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysins activity. It appears that the protein recognizes specifically a cholesterol‐rich lipid phase, probably the liquid‐ordered phase.

Aegerolysins: Structure, function, and putative biological role

Aegerolysins, discovered in fungi, bacteria and plants, are highly similar proteins with interesting biological properties. Certain aegerolysins possess antitumoral, antiproliferative, and antibacterial activities. Further possible medicinal applications include their use in the prevention of atherosclerosis, or as vaccines. Additional biotechnological value of fungal aegerolysins lies in their involvement in development, which could improve cultivation of commercially important edible mushrooms. Besides, new insights on microheterogeneity of raft‐like membrane domains could be gained by using aegerolysins as specific markers in cell and molecular biology. Although the exact function of aegerolysins in their producing organisms remains to be explained, they are biochemically well characterized all‐β structured proteins sharing the following common features: low isoelectric points, similar molecular weights (15–17 kDa), and stability in a wide pH range.

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP‐mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4‐hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP‐HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4‐hydroxybenzoic acid, and 3‐methoxybenzoic acid to 3‐hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4‐dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Antifungal activity of cinnamic acid derivatives involves inhibition of benzoate 4‐hydroxylase (CYP53)

CYP53A15, from the sorghum pathogen Cochliobolus lunatus, is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes.

Isolation and characterisation of a cytolytic protein from mucus secretions of the Antarctic heteronemertine Parborlasia corrugatus

From freeze dried mucus of the Antarctic nemertine Parborlasia corrugatus we have isolated 10.3 kDa basic (pI>9.0) cytolytic protein, referred to as parborlysin. Although the purified protein sample was homogeneous by reversed phase HPLC chromatography profiles and several gel electrophoretic techniques, N-terminal sequence and mass spectrometric analyses revealed that it consisted of few very similar isotoxins. The N-terminal sequence of the parborlysin sample shows a high degree of homology with the sequence of cytolysin A-III from the heteronemertine Cerebratulus lacteus. Parborlysin in micromolar concentration range disrupts mammalian erythrocytes with an apparent detergent mode of action. Hemolytic activity was inhibited by preincubation of parborlysin with pure phosphatidic acid or with rather high concentrations of small unilamellar vesicles composed of phosphatidylcholine (PC)/phosphatidyglycerol, PC/phosphatidylinositol, and PC/phosphatidylserine. Osmotic protectants as large as 3000 Da failed to protect red cells from lysis induced by parborlysin. Further structural and pharmacological analysis of the heteronemertine cytolysins may provide new insights regarding the mechanisms by which some water soluble proteins are able to penetrate into lipid membranes and form pores or, acting as detergents, disrupt their normal structure and function.

Antifouling Activity of Synthetic Alkylpyridinium Polymers Using the Barnacle Model

Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.

Induction of fruiting in oyster mushroom (Pleurotus ostreatus) by polymeric 3-alkylpyridinium salts

Polymeric 3-alkylpyridinium salts (poly-APS), surface-active compounds from the marine sponge Reniera sarai, have been shown to stimulate the fruit body formation from Pleurotus ostreatus mycelium. In nutrient media supplemented with poly-APS (>or= 0.01 microg ml(-1)), the formation of primordia and development of fruit bodies were detected approximately 10d earlier than in the absence of poly-APS, and also led to a considerably larger quantity of young mushrooms. This effect appears to be specific, as other surface-active compounds, lysophospholipids and fatty acids, showed no induction of fruiting.

Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase.

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.

Benzoic acid derivatives with improved antifungal activity: Design, synthesis, structure–activity relationship (SAR) and CYP53 docking studies

Previously, we identified CYP53 as a fungal-specific target of natural phenolic antifungal compounds and discovered several inhibitors with antifungal properties. In this study, we performed similarity-based virtual screening and synthesis to obtain benzoic acid-derived compounds and assessed their antifungal activity against Cochliobolus lunatus, Aspergillus niger and Pleurotus ostreatus. In addition, we generated structural models of CYP53 enzyme and used them in docking trials with 40 selected compounds. Finally, we explored CYP53-ligand interactions and identified structural elements conferring increased antifungal activity to facilitate the development of potential new antifungal agents that specifically target CYP53 enzymes of animal and plant pathogenic fungi.