Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity

Marx, Florentine; Haas, Hubertus; Reindl, Markus; Stöffler, Georg; Lottspeich, Friedrich; Redl, Bernhard

doi:10.1016/0378-1119(95)00701-6

Cited by 91 publications

(89 citation statements)

References 18 publications

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“…Determination of antifungal activity. The antifungal protein PAF was purified from the supernatant of a 72-h culture of P. chrysogenum Q176 as described elsewhere (47). The activity assays were performed according to the protocols of Broekaert et al (10) and Ludwig and Boller (42).…”

Section: Methodsmentioning

confidence: 99%

Antifungal Protein PAF Severely Affects the Integrity of the Plasma Membrane ofAspergillus nidulansand Induces an Apoptosis-Like Phenotype

Leiter¹,

Szappanos

Oberparleiter

et al. 2005

Antimicrob Agents Chemother

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The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the ␤-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulans hyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulans protoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulans strain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.A large number of small, basic, cysteine-rich antimicrobial proteins are produced by organisms throughout all kingdoms. They display a great variety in their primary structure, in species specificity, and in the mechanism of action.Few ascomycetes secrete strongly related antifungal proteins, which do not show any sequence homology with other antimicrobial proteins, but most of these proteins exhibit structural similarities (46): a net positive charge and numerous cysteine residues that are involved in disulfide bond formation. These properties contribute to a compact tertiary structure and a high stability against environmental impact and finally support the model of a membrane-disturbing nature. The antifungal protein PAF from P. chrysogenum and AFP from A. giganteus are the two most intensively studied peptides in the group of antifungals from ascomycetes, but the information available on their exact mechanism of action is still rather limited (32,55,68,69). PAF inhibits the growth of various important plant pathogenic and zoopathogenic filamentous fungi, e.g., Aspergillus fumigatus, A. niger, A. nidulans, and Botrytis cinerea. Previous studies revealed the induction of multifactorial detrimental effects on target organisms that include growth inhibition, reduction of cellular metabolism, severe changes in hyphal morphology, increased K ϩ efflux, and the generation of intracellular reactive oxygen species (ROS) (32, 48). PAF was found to...

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Section: Methodsmentioning

confidence: 99%

Antifungal Protein PAF Severely Affects the Integrity of the Plasma Membrane ofAspergillus nidulansand Induces an Apoptosis-Like Phenotype

Leiter¹,

Szappanos

Oberparleiter

et al. 2005

Antimicrob Agents Chemother

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151

142

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“…The Penicillium antifungal protein PAF is abundantly secreted into the supernatant of the ␤-lactam-producing mold Penicillium chrysogenum (22). This small, basic, and cysteine-rich protein specifically inhibits the growth of numerous filamentous fungi (16).…”

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confidence: 99%

Active Internalization of the Penicillium chrysogenum Antifungal Protein PAF in Sensitive Aspergilli

Oberparleiter¹,

Kaiserer²,

Haas³

et al. 2003

Antimicrob Agents Chemother

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The Penicillium chrysogenum antifungal protein PAF inhibits the growth of various filamentous fungi. In this study, PAF was found to localize to the cytoplasm of sensitive aspergilli by indirect immunofluorescence staining. The internalization process required active metabolism and ATP and was prevented by latrunculin B, suggesting an endocytotic mechanism.Antimicrobial proteins are produced by many organisms, including humans, amphibians, arthropods, plants, and fungi (6,11,13,32,37). The Penicillium antifungal protein PAF is abundantly secreted into the supernatant of the ␤-lactam-producing mold Penicillium chrysogenum (22). This small, basic, and cysteine-rich protein specifically inhibits the growth of numerous filamentous fungi (16). Although its primary amino acid structure resembles that of antifungal proteins isolated from other molds, e.g., Aspergillus giganteus (AFP), A. niger (ANAFP), and P. nalgiovense (NAF), significant differences exist in their species specificity (12,20,38). Knowledge of the bases of this selectivity is essential for determining strategies by which to overcome the resistance of important pathogens and to design new antifungal agents. Most data on the possible mechanism of action derive from studies on plant antifungal proteins and AFP which support the hypothesis of an interaction with the plasma membrane that would result in its permeabilization (18,34,35). So far, experiments have addressed such an interaction only indirectly. Therefore, we were interested in elucidating the site of action of the antifungal protein PAF from P. chrysogenum in order to gain better insight into putative target structures that might play a role in the activity of the protein. Here we report the localization of PAF in the sensitive molds A. nidulans, A. fumigatus, and A. niger and give evidence for an active transport of the antifungal protein into affected hyphae.Purification of PAF and generation of polyclonal antiserum. PAF was purified as described previously (16), and polyclonal antibodies against PAF were raised in rabbits as reported elsewhere (26). To determine antibody specificity and the optimal antibody concentration, 25 to 200 ng of PAF was immobilized on Biotrace-NT 0.45-m nitrocellulose membrane strips (PALL Corp., Ann Arbor, Mich.) and detected with various dilutions of rabbit anti-PAF serum and alkaline phosphataseconjugated goat anti-rabbit immunoglobulin G (IgG; 1:10,000; Sigma, Vienna, Austria) in accordance with the protocol previously described (21). The polyclonal antiserum reacted specifically with PAF, and no signals were obtained with the control serum that was collected before the first injection (data not shown).Localization of PAF in aspergilli. Fungi were grown overnight on glass coverslips at 30°C in complete medium CM (16) inoculated with 10 6 conidia/ml. The samples were stained in accordance with the protocol of Fischer et al. (9). In brief, prior to fixation, hyphae were treated with 10 to 50 g of PAF/ml in CM for 90 min at room temperature. The samples were incubat...

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“…The amino acid and cDNA sequences of AFP have been reported, revealing a high content of disulfide bridges (fours bonds) and tyrosines and lysines (6 and 12 residues, respectively) (5-7). AFP shows a significant degree of sequence similarity only with both the abundantly secreted antifungal PAF protein (55 amino acid residues) from Penicillium chrysogenum (47% sequence identity) (8) and the antifungal peptide Anafp (56 amino acid residues) secreted by Aspergillus niger (31% sequence identity) (9). AFP has been tested, at concentrations as large as 0.2 mM, against a wide variety of microorganisms, including prokaryotes and eukaryotes (3).…”

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confidence: 99%

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

Pozo

Lacadena²,

Mancheño

et al. 2002

Journal of Biological Chemistry

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The antifungal protein AFP is a small polypeptide of 51 amino acid residues secreted by Aspergillus giganteus. Its potent activity against phytopathogenic fungi converts AFP in a promising tool in plant protection. However, no data have been reported regarding the mode of action of AFP. The three-dimensional structure of this protein, a small and compact ␤ barrel composed of five highly twisted antiparallel ␤ strands, displays the characteristic features of the oligonucleotide/oligosaccharide binding (OB fold) structural motif. A comparison of the structures of AFP and OB fold-containing proteins shows this structural similarity despite the absence of any significant sequence similarity. AFP, like most OB fold-containing proteins, binds nucleic acids. The protein promotes charge neutralization and condensation of DNA as demonstrated by electrophoretic mobility shift and ethidium bromide displacement assays. Nucleic acid produces quenching of the protein fluorescence emission. This nucleic acid interacting ability of AFP may be related to the antifungal activity of this small polypeptide.The mold Aspergillus giganteus produces two major extracellular proteins, ␣-sarcin, a potent cytotoxic ribonuclease, and the so-called antifungal protein (AFP) 1 (1-3). AFP is a small polypeptide of 51 amino acid residues that is secreted under the form of a rather inactive larger precursor containing 6 extra amino acid residues at the NH 2 -terminal end, which is processed in the extracellular medium (4). The amino acid and cDNA sequences of AFP have been reported, revealing a high content of disulfide bridges (fours bonds) and tyrosines and lysines (6 and 12 residues, respectively) (5-7). AFP shows a significant degree of sequence similarity only with both the abundantly secreted antifungal PAF protein (55 amino acid residues) from Penicillium chrysogenum (47% sequence identity) (8) and the antifungal peptide Anafp (56 amino acid residues) secreted by Aspergillus niger (31% sequence identity) (9). AFP has been tested, at concentrations as large as 0.2 mM, against a wide variety of microorganisms, including prokaryotes and eukaryotes (3). This protein inhibits the growth of some filamentous fungi, the minimal protein concentration for total inhibition being in the range of 6 -25 M (3), but it does not promote any effect on the producing mold or on bacteria or yeast. AFP is not active against P. chrysogenum or A. niger (3), the organisms producing PAF and Anapf proteins, respectively. Although these three proteins have been described as antifungal molecules, no data regarding their mode of action have been so far reported. We herein report the interaction of AFP with DNA. EXPERIMENTAL PROCEDURESUnless otherwise stated, all materials and reagents were molecular biology grade. Calf thymus double-stranded DNA (dsDNA) was purified as described previously (10, 11). The DNA fraction selected for the experiments described in this work (kept at Ϫ20°C until used) was that containing 200 bp dsDNA on average according to their electrophoret...

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Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity

Cited by 91 publications

References 18 publications

Antifungal Protein PAF Severely Affects the Integrity of the Plasma Membrane ofAspergillus nidulansand Induces an Apoptosis-Like Phenotype

Antifungal Protein PAF Severely Affects the Integrity of the Plasma Membrane ofAspergillus nidulansand Induces an Apoptosis-Like Phenotype

Active Internalization of the Penicillium chrysogenum Antifungal Protein PAF in Sensitive Aspergilli

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

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