Mold Aspergillus Giganteus Research Articles

Antifungal Activity of α-Sarcin against Penicillium digitatum: Proposal of a New Role for Fungal Ribotoxins.

Among the putative defense proteins that occur in fungi, one of the best studied is α-sarcin, produced by the mold Aspergillus giganteus. This protein is the most significant member of the ribotoxin family, which consists of extracellular rRNA ribonucleases that display cytotoxic activity toward animal cells. Ribotoxins are rRNA endonucleases that catalyze the hydrolysis of the phosphodiester bond between G4325 and A4326 from the rat 28S rRNA. The results of several experimental approaches have led to propose ribotoxins as insecticidal agents. In this work, we report that α-sarcin displays a strong antifungal activity against Penicillium digitatum, being able to enter into the cytosol where it inactivates the ribosomes, thus killing the cells and arresting the growth of the fungus. This is the first time that a ribotoxin has been found to display antifungal activity. Therefore, this protein could play, besides the already proposed insecticidal function, a role in nature as an antifungal agent.

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (lf-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP *) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. Recombinant AFP * produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will permit investigation of the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches.

Rust and Downy Mildew Resistance in Pearl Millet (Pennisetum glaucum) Mediated by Heterologous Expression of the afp Gene from Aspergillus giganteus

The cDNA encoding the antifungal protein AFP from the mould Aspergillus giganteus was introduced into two pearl millet (Pennisetum glaucum) genotypes by particle bombardment. Stable integration and expression of the afp gene was confirmed in two independent transgenic T0 plants and their progeny using Southern blot and RT-PCR analysis. In vitro infection of detached leaves and in vivo inoculation of whole plants with the basidomycete Puccinia substriata, the causal agent of rust disease, and the oomycete Sclerospora graminicola, causal agent of downy mildew, resulted in a significant reduction of disease symptoms in comparison to wild type control plants. The disease resistance of pearl millet was increased by up to 90% when infected with two diverse, economically important pathogens. This is the first report of genetic enhancement of Pennisetum glaucum against fungal infections.

Biotechnologically relevant enzymes and proteins

The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.

Activity of the Antifungal Protein from Aspergillus giganteus Against Botrytis cinerea

ABSTRACT Botrytis blight (gray mold), caused by Botrytis cinerea, is one of the most widely distributed diseases of ornamental plants. In geranium plants, gray mold is responsible for important losses in production. The mold Aspergillus giganteus is known to produce and secrete a basic low-molecular-weight protein, the antifungal protein (AFP). Here, the antifungal properties of the Aspergillus AFP against various B. cinerea isolates obtained from naturally infected geranium plants were investigated. AFP strongly inhibited mycelial growth as well as conidial germination of B. cinerea. Microscopic observations of fungal cultures treated with AFP revealed reduced hyphal elongation and swollen hyphal tips. Washout experiments in which B. cinerea was incubated with AFP for different periods of time and then washed away revealed a fungicidal activity of AFP. Application of AFP on geranium plants protected leaves against Botrytis infection. Cecropin A also was active against this pathogen. An additive effect against the fungus was observed when AFP was combined with cecropin A. These results are discussed in relation to the potential of the afp gene to enhance crop protection against B. cinerea diseases.

New insights in the regulation of the afp gene encoding the antifungal protein of Aspergillus giganteus.

The antifungal protein (AFP) secreted by the mould Aspergillus giganteus is a small, highly basic polypeptide with antifungal activity. Previous work has shown that transcription of the corresponding afp gene is regulated by ambient pH, being suppressed under acidic and strongly induced under alkaline conditions. This observation suggested that the afp gene is regulated by the wide-domain transcriptional factor PacC. Here, we show that two putative PacC binding sites within the afp promoter, denoted afpP1 and afpP2, are efficiently recognised in vitro by a PacC fusion protein of A. nidulans. In addition, we found that phosphate, which was used as a buffering agent during cultivation, plays an important role in regulating afp expression. AFP production was nearly completely inhibited in the presence of external phosphate. The results of Northern analysis indicate that the inhibitory effect of phosphate is mediated at the transcriptional level.

Purification of α-Sarcin and an Antifungal Protein from Mold ( Aspergillus giganteus) by Chitin Affinity Chromatography

A simple method for preparation of α-sarcin and an antifungal protein (AFP) from mold ( Aspergillus giganteus MDH 18894) has been developed. α-Sarcin and AFP were purified simultaneously by chitin affinity column chromatography and gel filtration. By this method, 4.5 mg of pure α-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Compared with other purification methods such as ion-exchange column chromatography, this procedure was very simple and specific. The purified α-sarcin and AFP were homogeneous as characterized by SDS-polyacrylamide gel electrophoresis. Both α-sarcin and AFP exhibited the binding activity to generated chitin. Soluble glycochitin decreased the intensity of fluorescence of α-sarcin and made the λ emm shift from 340 to 347 nm. Titration of α-sarcin with N-bromosuccinimide under native conditions revealed that two tryptophans (Trps) were all located in the core part of α-sarcin molecule. This indicated that Trps were not involved in the binding of α-sarcin to chitin. Glycochitin in the culture medium increased the expression of α-sarcin, while it had no effect on the expression of AFP. Unlike other ligands such as Cibacron blue for the affinity purification of α-sarcin and AFP, glycochitin increased the nuclease activity of α-sarcin.

Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.

Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin

α-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis—histidine-50, glutamic acid-96, and histidine-137—were changed to glutamine. Three dif- ferent single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of α-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of α-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type α-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α-sarcin. Proteins 1999;37:474–484. ©1999 Wiley-Liss, Inc.

Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin.

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.

A peptide of nine amino acid residues from alpha-sarcin cytotoxin is a membrane-perturbing structure.

A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein alpha-sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of alpha-sarcin in its interaction with lipid membranes.

Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus

Two major proteins, α-sarcin and an antifungal polypeptide (AFP), are secreted by the mould Aspergillus giganteus MDH 18894 when it is cultured for 70–80 h. A third major protein is also found in the extracellular medium at 48–60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH 2-terminal primary structure, amino acid content, spectroscopical features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP (lf-AFP). Its amino acid composition is identical to that of AFP but containing six extra residues. NH 2-terminal sequence analysis of the first eight amino acid residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be Ile/Leu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue (Asp/Glu) and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.

Overproduction of fungal ribotoxin α-sarcin in Escherichia coli: generation of an active immunotoxin

α-Sarcin is a ribonucleolytic protein secreted by the mold Aspergillus giganteus. DNA encoding α-sarcin was isolated from the host and cloned into T7 promoter based E. coli expression vectors. Using bacterial outer membrane protein A (OmpA) signal sequence, properly processed recombinant (re-) protein was secreted into the culture medium while in the absence of a signal sequence protein remained insoluble in the bacterial inclusion bodies. The re-α-sarcin was purified to homogeneity by simple chromatographic techniques both from the insoluble and soluble sources with respective yields of 40–50 μg/ml and 2–3 μg/ml. The re-ribotoxin was functionally as active as the native toxin and preserved its specificity. The re-α-sarcin was used in the construction of an active immunotoxin targeted at the human cancer cells overexpressing transferrin receptor (TFR).

Characterization of the Antifungal Protein Secreted by the MouldAspergillus giganteus

An antifungal polypeptide (AFP) of 51 amino acid residues, secreted by the mouldAspergillus giganteus,has been purified to homogeneity and characterized. The inhibitory effect of this protein on the growth of different microorganisms has been studied. Whereas the growth of many of the filamentous fungi assayed is inhibited, no effect has been observed against yeasts or bacteria. The minimal concentration for total inhibition of the growth is in the range 6 to 25 μM. The antifungal polypeptide does not produce any effect on the growth of the producing mould. The polypeptide promotes aggregation of acidic phospholipid vesicles. A remarkable resistance to proteolysis and a low hydrogen × deuterium exchange have been observed for this protein. The protein does not show any thermal transition up to 80°C when studied by differential scanning calorimetry and infrared spectroscopy. The uv absorbance, fluorescence emission, and circular dichroism (CD) characteristics of this protein have been studied. The protein exhibits a strong positive band at 230 nm as a prominent feature of the CD spectrum in the far uv region. All the spectroscopical properties of the antifungal protein are highly influenced by the abundance of tyrosine residues. These can be grouped in two different populations, buried and exposed, based on the results of pH-titration experiments. Fourier-transform infrared spectroscopy reveals a high content of β-structure in AFP. Reduction and carboxyamidomethylation produces a rather unstructured polypeptide as deduced from its spectroscopical properties.

Overproduction and purification of biologically active native fungal α-sarcin in Escherichia coli

An efficient system was developed to produce, in Escherichia coli, large amounts of native α-sarcin (αSar), a cytotoxin from the mold Aspergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 μg/ml of original culture. The constructed expression vector (pINPGαS) is based on the synthesis of a fusion protein between αSar and a modified version of the OmpA signal peptide. This peptide seems to favour the postranslational processing of the fusion protein. The purified recombinant a-sarcin (re-αSar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, Chromatographie behaviour, immunoreactivity and spectroscopic features. Indeed, the recombinant protein recovered is completely functional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal αSar.

Translocation of alpha-sarcin across the lipid bilayer of asolectin vesicles.

alpha-Sarcin is a cytotoxic protein produced by the mould Aspergillus giganteus. Insertion of alpha-sarcin into asolectin membranes has been demonstrated by protein labelling with photoreactive phospholipids. alpha-Sarcin added externally to tRNA-containing asolectin liposomes degrades the entrapped tRNA. Trypsin-containing asolectin liposomes were also prepared. Encapsulated trypsin degrades alpha-sarcin, even in the presence of a large excess of external hen egg-white trypsin inhibitor to prevent any alpha-sarin degradation outside the vesicles. These processes occur only with acidic phospholipids and were not observed when phosphatidylcholine vesicles were used. These results indicate that alpha-sarcin penetrates the lipid bilayer and becomes exposed to the lumen of negatively charged liposomes.

Kinetic study of the cytotoxic effect of alpha-sarcin, a ribosome inactivating protein from Aspergillus giganteus, on tumour cell lines: protein biosynthesis inhibition and cell binding.

alpha-Sarcin is a ribosome inactivating protein produced by the mould Aspergillus giganteus. The effect of this protein on eight different tumour cell lines has been studied in the absence of any agent affecting membrane permeability. The protein is cytotoxic for all the tumour cell lines considered. alpha-Sarcin modifies the cell proliferation pattern by inhibiting the protein biosynthesis of the cultured cells. No membrane damage produced by alpha-sarcin has been observed by measuring lactic dehydrogenase leakage. Alteration on the cell mitochondrial activity has not been detected upon treatment with alpha-sarcin. Differences on the extent of the protein binding to the cells have been observed by flow cytometric measurements. The kinetic analysis of the protein biosynthesis inhibition produced by alpha-sarcin reveals an alpha-sarcin concentration-dependent lag phase followed by a first order decrease of the protein synthesis rate. This parameter is dependent on the external alpha-sarcin concentration. A saturable component for the action of alpha-sarcin is also deduced from these experiments. Results are discussed in terms of the protein passage across the cell membrane as the potential rate-limiting step for the action of alpha-sarcin.

Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome‐inactivating protein α‐sarcin from Aspergillus giganteus

An immunotoxin was synthesized by the attachment of alpha-sarcin, the ribosome-inactivating protein derived from the mould Aspergillus giganteus, to a monoclonal mouse IgG2 antibody Fib75. The alpha-sarcin immunotoxin exerted toxic effects in tissue culture against the EJ human bladder carcinoma cell line, expressing the antigen recognised by the Fib75 antibody, inhibiting the incorporation of [3H]leucine by 50% at a concentration of 0.46 nM. The cytotoxic effects of the alpha-sarcin immunotoxin were indistinguishable from those of a Fib75 immunotoxin made with ricin A chain. Fib75-alpha-sarcin was cleared from the circulation of the rat with biphasic kinetics following intravenous administration. The alpha- and beta-phase half-lives were 0.8 h and 6 h, respectively, similar to the serum half-lives of analogous Fib75 immunotoxins made with ribosome-inactivating proteins derived from plants. alpha-Sarcin was completely stable in physiological saline buffer at 37 degrees C, whereas the ribosome-inactivating activity of ricin A chain was gradually lost under identical conditions. alpha-Sarcin may be a valuable alternative to ricin A chain for the construction of therapeutic immunotoxins because of its smaller size and greater thermostability.

Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus

A very basic secreted protein which displays antifungal activity was isolated from the medium of the mold Aspergillus giganteus. The protein consists of 51 amino acid residues whose sequence was determined as Ala-Thr-Tyr-Asn-Gly-Lys-Cys-Tyr-Lys-Lys-Asp-Asn- Ile-Cys-Lys-Tyr-Lys-Ala-Gln-Ser-Gly-Lys-Thr-Ala-Ile-Cys-Lys-Cys-Tyr-Val- Lys- Lys-Cys-Pro-Arg-Asp-Gly-Ala-Lys-Cys-Glu-Phe-Asp-Ser-Tyr-Lys-Gly-Lys-Cys- Tyr-Cys . Disulfide bonds were formed between Cys7-Cys33, Cys14-Cys40, Cys26-Cys28 and Cys49-Cys51. These results suggest that the antifungal protein forms a loop structure and is similar to phospholipase A2.

Conformational study of the antitumor protein α-sarcin

The antitmnor protein α-sarcin is a single polypeptide chain produced by the mold Aspergillus giganteus. It inhibits protein synthesis in some tumor cells by inactivating the larger ribosomal subunit. The secondary structure of the molecule has been studied by circular dichroism and predictive methods. The protein contains about 40% of periodic structures, mainly located at both extremes of the polypeptide chain, β-Turns and aperiodic conformation appear at the central part of the molecule. Two different tyrosine populations have been observed in α-sarcin. Attempts to correlate solvent accessibility and particular protein regions have been carried out by using CD in the near-ultraviolet region, fluorescence and absorbance spectroscopies as well as acrylamide quenching and hydropathy profiles. Five different pH-induced conformational transitions are detected. Two of them, at pH 2.5 and 10.2, are denaturing transitions. These results are explained in terms of the structural features of this molecule, and related to its ribonucleolytic activity and ability to cross cell membranes.