A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

BACKGROUND: Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. RESULTS: The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. CONCLUSION: This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.

Penicillium antifungal protein (PAF) is involved in the apoptotic and autophagic processes of the producer Penicillium chrysogenum.

PAF, which is produced by the filamentous fungus Pencicillium chrysogenum, is a small antifungal protein, triggering ROS-mediated apoptotic cell death in Aspergillus nidulans. In this work, we provide information on the function of PAF in the host P. chrysogenum considering that carbon-starving cultures of the Δpaf mutant strain showed significantly reduced apoptosis rates in comparison to the wild-type (wt) strain. Moreover, the addition of PAF to the Δpaf strain resulted in a twofold increase in the apoptosis rate. PAF was also involved in the regulation of the autophagy machinery of this fungus, since several Saccharomyces cerevisiae autophagy-related ortholog genes, e.g. those of atg7, atg22 and tipA, were repressed in the deletion strain. This phenomenon was accompanied by the absence of autophagosomes in the Δpaf strain, even in old hyphae.

Antifungal proteins: More than antimicrobials?

Antimicrobial proteins (AMPs) are widely distributed in nature. In higher eukaryotes, AMPs provide the host with an important defence mechanism against invading pathogens. AMPs of lower eukaryotes and prokaryotes may support successful competition for nutrients with other microorganisms of the same ecological niche. AMPs show a vast variety in structure, function, antimicrobial spectrum and mechanism of action. Most interestingly, there is growing evidence that AMPs also fulfil important biological functions other than antimicrobial activity. The present review focuses on the mechanistic function of small, cationic, cysteine-rich AMPs of mammals, insects, plants and fungi with antifungal activity and specifically aims at summarizing current knowledge concerning additional biological properties which opens novel aspects for their future use in medicine, agriculture and biotechnology.

The small molecular mass antifungal protein of Penicillium chrysogenum--a mechanism of action oriented review.

The ß-lactam producing filamentous fungus Penicillium chrysogenum secretes a 6.25 kDa small molecular mass antifungal protein, PAF, which has a highly stable, compact 3D structure and is effective against a wide spectrum of plant and zoo pathogenic fungi. Its precise physiological functions and mode of action need to be elucidated before considering possible biomedical, agricultural or food technological applications. According to some more recent experimental data, PAF plays an important role in the fine-tuning of conidiogenesis in Penicillium chrysogenum. PAF triggers apoptotic cell death in sensitive fungi, and cell death signaling may be transmitted through two-component systems, heterotrimeric G protein coupled signal transduction and regulatory networks as well as via alteration of the Ca(2+) -homeostasis of the cells. Possible biotechnological applications of PAF are also outlined in the review.

The paf gene product modulates asexual development in Penicillium chrysogenum.

Penicillium chrysogenum secretes a low molecular weight, cationic and cysteine-rich protein (PAF). It has growth inhibitory activity against the model organism Aspergillus nidulans and numerous zoo- and phytopathogenic fungi but shows only minimal conditional antifungal activity against the producing organism itself. In this study we provide evidence for an additional function of PAF which is distinct from the antifungal activity against putative ecologically concurrent microorganisms. Our data indicate that PAF enhances conidiation in P. chrysogenum by modulating the expression of brlA, the central regulatory gene for mitospore development. A paf deletion strain showed a significant impairment of mitospore formation which sustains our hypothesis that PAF plays an important role in balancing asexual differentiation in P. chrysogenum.

AtfA bZIP-type transcription factor regulates oxidative and osmotic stress responses in Aspergillus nidulans.

The aim of the study was to demonstrate that the bZIP-type transcription factor AtfA regulates different types of stress responses in Aspergillus nidulans similarly to Atf1, the orthologous 'all-purpose' transcription factor of Schizosaccharomyces pombe. Heterologous expression of atfA in a S. pombe Deltaatf1 mutant restored the osmotic stress tolerance of fission yeast in surface cultures to the same level as recorded in complementation studies with the atf1 gene, and a partial complementation of the osmotic and oxidative-stress-sensitive phenotypes was also achieved in submerged cultures. AtfA is therefore a true functional ortholog of fission yeast's Atf1. As demonstrated by RT-PCR experiments, elements of both oxidative (e.g. catalase B) and osmotic (e.g. glycerol-3-phosphate dehydrogenase B) stress defense systems were transcriptionally regulated by AtfA in a stress-type-specific manner. Deletion of atfA resulted in oxidative-stress-sensitive phenotypes while the high-osmolarity stress sensitivity of the fungus was not affected significantly. In A. nidulans, the glutathione/glutathione disulfide redox status of the cells as well as apoptotic cell death and autolysis seemed to be controlled by regulatory elements other than AtfA. In conclusion, the orchestrations of stress responses in the aspergilli and in fission yeast share several common features, but further studies are needed to answer the important question of whether a fission yeast-like core environmental stress response also operates in the euascomycete genus Aspergillus.

Functional aspects of the solution structure and dynamics of PAF--a highly-stable antifungal protein from Penicillium chrysogenum.

Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G-protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five beta-strands forming two orthogonally packed beta-sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S-S bond exhibit essentially the same fold. (15)N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site-directed mutagenesis, we demonstrate that the highly-conserved and positively-charged lysine-rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent-exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.

Effect of the Penicillium chrysogenum antifungal protein (PAF) on barley powdery mildew and wheat leaf rust pathogens.

The small molecular mass antifungal protein of Penicillium chrysogenum (PAF) inhibited the growths of two obligate biotrophic fungal pathogens, Blumeria graminis f. sp. hordei and Puccinia recondita f.sp. tritici and, hence, mitigated the symptoms of barley powdery mildew and wheat leaf rust infections, respectively. PAF also affected adversely the germination of B. graminis conidia and P. recondita uredospores causing degenerative branching of germ tubes. Since powdery mildews and rusts cause serious economic losses the potential applicability of PAF to control these plant diseases is promising.

In vitro activity of Penicillium chrysogenum antifungal protein (PAF) and its combination with fluconazole against different dermatophytes.

Strains of five dermatophyte species (Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton tonsurans) were selected for testing against Penicillium chrysogenum antifungal protein (PAF) and its combination with fluconazole (FCZ). Inhibition of microconidia germination and growth was detected with MICs of PAF ranging from 1.56 to 200 microg ml(-1) when it was used alone, or at constant concentration (100 microg ml(-1)) in combination with FCZ at from 0.25 to 32 microg ml(-1). The MICs for FCZ were found to be between 0.25 and 128 microg ml(-1). PAF caused a fungicidal effect at 200 microg ml(-1) and reduced growth at between 50 and 200 microg ml(-1). Total growth inhibition with fungistatic activity was detected at 64 microg ml(-1) of FCZ for M. gypseum, T. mentagrophytes, and T. tonsurans, and at 32 microg ml(-1) FCZ for M. canis and T. rubrum. PAF and FCZ acted synergistically and/or additively on all of the tested fungi except M. gypseum, where no interactions were detected.