Rational Design of Antifungal Peptides Based on the γ-Core Motif of a Neosartorya (Aspergillus) fischeri Antifungal Protein to Improve Structural Integrity, Efficacy, and Spectrum.

Antifungal peptides offer promising alternative compounds for the treatment of fungal infections, for which new antifungal compounds are urgently needed. Constant and broad antifungal spectra of these peptides play essential roles in their reliable therapeutic application. It has been observed that rationally designed peptides using the evolutionarily conserved γ-core region (GXC-X3-9-C) of an antifungal protein from Neosartorya (Aspergillus) fischeri highly inhibit the growth of fungi. The cysteines in these peptides have free sulfhydryl groups, which allow cyclization and dimerization under oxidative conditions, thereby impairing antifungal efficacy. To overcome this problem, one or two cysteine residues were substituted by serines or S-tert-butyl was applied as a cysteine-protecting group. Furthermore, structural integrity and antifungal efficacy investigations before and after oxidative exposure revealed that substituting both cysteines with serines and S-tert-butylation helped maintain the structural integrity. However, it slightly decreased the antifungal efficacy against a yeast, Candida albicans. Interestingly, S-tert-butylation maintained the efficacy and could extend the antifungal activity to a mold, Aspergillus fumigatus. Usually, cyclization and dimerization did not influence the antifungal efficacy of most peptides. Additionally, hemolysis tests and Galleria mellonella toxicity model experiments indicated that none of the applied modifications made the peptides harmful to animals.

Confirmation of the Disulfide Connectivity and Strategies for Chemical Synthesis of the Four-Disulfide-Bond-Stabilized Aspergillus giganteus Antifungal Protein, AFP.

Emerging fungal infections require new, more efficient antifungal agents and therapies. AFP, a protein from Aspergillus giganteus with four disulfide bonds, is a promising candidate because it selectively inhibits the growth of filamentous fungi. In this work, the reduced form of AFP was prepared using native chemical ligation. The native protein was synthesized via oxidative folding with uniform protection for cysteine thiols. AFP's biological activity depends heavily on the pattern of natural disulfide bonds. Enzymatic digestion and MS analysis provide proof for interlocking disulfide topology (abcdabcd) that was previously assumed. With this knowledge, a semi-orthogonal thiol protection method was designed. By following this strategy, out of a possible 105, only 6 disulfide isomers formed and 1 of them proved to be identical with the native protein. This approach allows the synthesis of analogs for examining structure-activity relationships and, thus, preparing AFP variants with higher antifungal activity.

Pest and disease management by red light.

Light is essential for plant life. It provides a source of energy through photosynthesis and regulates plant growth and development and other cellular processes, such as by controlling the endogenous circadian clock. Light intensity, quality, duration and timing are all important determinants of plant responses, especially to biotic stress. Red light can positively influence plant defence mechanisms against different pathogens, but the molecular mechanism behind this phenomenon is not fully understood. Therefore, we reviewed the impact of red light on plant biotic stress responses against viruses, bacteria, fungi and nematodes, with a focus on the physiological effects of red light treatment and hormonal crosstalk under biotic stress in plants. We found evidence suggesting that exposing plants to red light increases levels of salicylic acid (SA) and induces SA signalling mediating the production of reactive oxygen species, with substantial differences between species and plant organs. Such changes in SA levels could be vital for plants to survive infections. Therefore, the application of red light provides a multidimensional aspect to developing innovative and environmentally friendly approaches to plant and crop disease management.

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312-0.5, 0.155-0.5, 0.037-0.375, 0.064-0.375, and 0.064-0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 µM2%, 2.16 µM2%, 33.31 µM2%, 10.72 µM2%, and 111.19 µM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.

Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC.

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical ß-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

The Penicillium chrysogenum Q176 Antimicrobial Protein PAFC Effectively Inhibits the Growth of the Opportunistic Human Pathogen Candida albicans.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes promise treatment alternatives to licensed antifungal drugs. In this study, we characterized the Penicillium chrysogenum Q176 antifungal protein C (PAFC), which is phylogenetically distinct to the other two Penicillium antifungal proteins, PAF and PAFB, that are expressed by this biotechnologically important ascomycete. PAFC is secreted into the culture broth and is co-expressed with PAF and PAFB in the exudates of surface cultures. This observation is in line with the suggested role of AMPs in the adaptive response of the host to endogenous and/or environmental stimuli. The in silico structural model predicted five ß-strands stabilized by four intramolecular disulfide bonds in PAFC. The functional characterization of recombinant PAFC provided evidence for a promising new molecule in anti-Candida therapy. The thermotolerant PAFC killed planktonic cells and reduced the metabolic activity of sessile cells in pre-established biofilms of two Candidaalbicans strains, one of which was a fluconazole-resistant clinical isolate showing higher PAFC sensitivity than the fluconazole-sensitive strain. Candidacidal activity was linked to severe cell morphology changes, PAFC internalization, induction of intracellular reactive oxygen species and plasma membrane disintegration. The lack of hemolytic activity further corroborates the potential applicability of PAFC in clinical therapy.

The potential use of the Penicillium chrysogenum antifungal protein PAF, the designed variant PAFopt and its γ-core peptide Pγopt in plant protection.

The prevention of enormous crop losses caused by pesticide-resistant fungi is a serious challenge in agriculture. Application of alternative fungicides, such as antifungal proteins and peptides, provides a promising basis to overcome this problem; however, their direct use in fields suffers limitations, such as high cost of production, low stability, narrow antifungal spectrum and toxicity on plant or mammalian cells. Recently, we demonstrated that a Penicillium chrysogenum-based expression system provides a feasible tool for economic production of P. chrysogenum antifungal protein (PAF) and a rational designed variant (PAFopt ), in which the evolutionary conserved γ-core motif was modified to increase antifungal activity. In the present study, we report for the first time that γ-core modulation influences the antifungal spectrum and efficacy of PAF against important plant pathogenic ascomycetes, and the synthetic γ-core peptide Pγopt , a derivative of PAFopt , is antifungal active against these pathogens in vitro. Finally, we proved the protective potential of PAF against Botrytis cinerea infection in tomato plant leaves. The lack of any toxic effects on mammalian cells and plant seedlings, as well as the high tolerance to harsh environmental conditions and proteolytic degradation further strengthen our concept for applicability of these proteins and peptide in agriculture.

Cysteine-Rich Antifungal Proteins from Filamentous Fungi are Promising Bioactive Natural Compounds in Anti-Candida Therapy.

The emerging number of life-threatening invasive fungal infections caused by drug-resistant Candida strains urges the need for the development and application of fundamentally new and safe antifungal strategies in the clinical treatment. Recent studies demonstrated that the extracellular cysteine-rich and cationic antifungal proteins (crAFPs) originating from filamentous fungi, and de novo designed synthetic peptide derivatives of these crAFPs provide a feasible basis for this approach. This mini-review focuses on the global challenges of the anti-Canidia therapy and on the crAFPs as potential drug candidates to overcome existing problems. The advantages and limitations in the use of crAFPs and peptide derivatives compared to those of conventional antifungal drugs will also be critically discussed.

Solution structure and novel insights into phylogeny and mode of action of the Neosartorya (Aspergillus) fischeri antifungal protein (NFAP).

Small, cysteine-rich and cationic antifungal proteins from natural sources are promising candidates for the development of novel treatment strategies to prevent and combat infections caused by drug-resistant fungi. However, limited information about their structure and antifungal mechanism hampers their future applications. In the present study, we determined the solution structure, dynamics and associated solvent areas of the Neosartorya (Aspergillus) fischeri antifungal protein NFAP. Genome mining within the genus revealed the presence of orthologous genes in N. fischeri and Neosartorya spathulata, and genes encoding closely related proteins can be found in Penicillium brasiliensis and Penicillium oxalicum. We show that the tertiary structure of these putative proteins can be resolved using the structure of NFAP as reliable template for in silico prediction. Localization studies with fluorescence-labelled protein pointed at an energy-dependent uptake mechanism of NFAP in the sensitive model fungus Neurospora crassa and subsequent cytoplasmic localization coincided with cell-death induction. The presented results contribute to a better understanding of the structure/function relationship of NFAP and related proteins and pave the way towards future antifungal drug development.

In Vivo Applicability of Neosartorya fischeri Antifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis.

As a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-forming Candida species, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of several Candida spp. in vitro; however, no information is available about their in vivo antifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis, Neosartorya fischeri antifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistant Candida albicans strain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilm in vitro We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MIC in vitro. In vivo murine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistant C. albicans cells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes represent ideal bio-molecules for the development of next-generation antifungal therapeutics. They are promising candidates to counteract resistance development and may complement or even replace current small molecule-based antibiotics in the future. In this study, we show that a 14 amino acid (aa) long peptide (Pγ) spanning the highly conserved γ-core motif of the Penicillium chrysogenum antifungal protein (PAF) has antifungal activity against the opportunistic human pathogenic yeast Candida albicans. By substituting specific aa we elevated the positive net charge and the hydrophilicity of Pγ and created the peptide variants Pγvar and Pγopt with 10-fold higher antifungal activity than Pγ. Similarly, the antifungal efficacy of the PAF protein could be significantly improved by exchanging the respective aa in the γ-core of the protein by creating the protein variants PAFγvar and PAFγopt. The designed peptides and proteins were investigated in detail for their physicochemical features and mode of action, and were tested for cytotoxicity on mammalian cells. This study proves for the first time the important role of the γ-core motif in the biological function of an AMP from ascomycetes. Furthermore, we provide a detailed phylogenetic analysis that proves the presence and conservation of the γ-core motif in all AMP classes from Eurotiomycetes. We emphasize the potential of this common protein motif for the design of short antifungal peptides and as a protein motif in which targeted aa substitutions enhance antimicrobial activity.

Anti-Candidal Activity and Functional Mapping of Recombinant and Synthetic Neosartorya fischeri Antifungal Protein 2 (NFAP2).

The increasing number of life-threatening Candida infections caused by antifungal drug-resistant strains urges the development of new therapeutic strategies. The small, cysteine-rich, and cationic Neosartorya fischeri antifungal protein 2 (NFAP2) effectively inhibits the growth of Candida spp. Limiting factors of its future application, are the low-yield production by the native producer, unavailable information about potential clinical application, and the unsolved relationship between the structure and function. In the present study we adopted a Penicillium chrysogenum-based expression system for bulk production of recombinant NFAP2. Furthermore, solid-phase peptide synthesis and native chemical ligation were applied to produce synthetic NFAP2. The average yield of recombinant and synthetic NFAP2 was 40- and 16-times higher than in the native producer, respectively. Both proteins were correctly processed, folded, and proved to be heat-stable. They showed the same minimal inhibitory concentrations as the native NFAP2 against clinically relevant Candida spp. Minimal inhibitory concentrations were higher in RPMI 1640 mimicking the human inner fluid than in a low ionic strength medium. The recombinant NFAP2 interacted synergistically with fluconazole, the first-line Candida therapeutic agent and significantly decreased its effective in vitro concentrations in RPMI 1640. Functional mapping with synthetic peptide fragments of NFAP2 revealed that not the evolutionary conserved antimicrobial γ-core motif, but the mid-N-terminal part of the protein influences the antifungal activity that does not depend on the primary structure of this region. Preliminary nucleic magnetic resonance measurements signed that the produced recombinant NFAP2 is suitable for further structural investigations.

Exophiala dermatitidis Endophthalmitis: Case Report and Literature Review.

We report a case of a 59-year-old male patient with a postoperative fungal infection of the left eye. A dark-pigmented yeast, Exophiala dermatitidis (previously known as Wangiella dermatitidis), was identified from the culture of the biopsy taken from the posterior capsule. The infection was successfully eradicated by a combination of surgical and medical (i.e., voriconazole and fluconazole) treatment. This is the first report of successfully treated E. dermatitidis endophthalmitis, which demonstrates that a prompt and aggressive antifungal therapy combined with surgical intervention is necessary to prevent vision loss in cases of endophthalmitis due to Exophiala species. Beside the case description, we also aim to provide a literature review of previously reported eye infections caused by Exophiala species in order to help the future diagnosis and management of the disease.

New Antimicrobial Potential and Structural Properties of PAFB: A Cationic, Cysteine-Rich Protein from Penicillium chrysogenum Q176.

Small, cysteine-rich and cationic proteins with antimicrobial activity are produced by diverse organisms of all kingdoms and represent promising molecules for drug development. The ancestor of all industrial penicillin producing strains, the ascomycete Penicillium chryosgenum Q176, secretes the extensively studied antifungal protein PAF. However, the genome of this strain harbours at least two more genes that code for other small, cysteine-rich and cationic proteins with potential antifungal activity. In this study, we characterized the pafB gene product that shows high similarity to PgAFP from P. chrysogenum R42C. Although abundant and timely regulated pafB gene transcripts were detected, we could not identify PAFB in the culture broth of P. chrysogenum Q176. Therefore, we applied a P. chrysogenum-based expression system to produce sufficient amounts of recombinant PAFB to address unanswered questions concerning the structure and antimicrobial function. Nuclear magnetic resonance (NMR)-based analyses revealed a compact ß-folded structure, comprising five ß-strands connected by four solvent exposed and flexible loops and an "abcabc" disulphide bond pattern. We identified PAFB as an inhibitor of growth of human pathogenic moulds and yeasts. Furthermore, we document for the first time an anti-viral activity for two members of the small, cysteine-rich and cationic protein group from ascomycetes.

Structural determinants of Neosartorya fischeri antifungal protein (NFAP) for folding, stability and antifungal activity.

The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAPΔC), hydrophobic core deletion (NFAPΔh), and N-terminal amino acids exchanges (NFAPΔN) were produced in Pichia pastoris. The recombinant NFAP showed the same features in structure, folding, stability and activity as the native protein. The data acquired with mass spectrometry, structural analyses and antifungal activity assays of NFAP and its mutants proved the importance of the disulphide bonding, the hydrophobic core and the correct N-terminus for folding, stability and full antifungal function. Our findings provide further support to the comprehensive understanding of the structure-function relationship in members of this protein group.

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.

NFAP2, a novel cysteine-rich anti-yeast protein from Neosartorya fischeri NRRL 181: isolation and characterization.

The increasing incidence of fungal infections and damages due to drug-resistant fungi urges the development of new antifungal strategies. The cysteine-rich antifungal proteins from filamentous ascomycetes provide a feasible base for protection against molds due to their potent antifungal activity on them. In contrast to this, they show no or weak activity on yeasts, hence their applicability against this group of fungi is questionable. In the present study a 5.6 kDa anti-yeast protein (NFAP2) is isolated, identified and characterized from the ferment broth of Neosartorya fischeri NRRL 181. Based on a phylogenetic analysis, NFAP2 and its putative homologs represent a new group of ascomycetous cysteine-rich antifungal proteins. NFAP2 proved to be highly effective against tested yeasts involving clinically relevant Candida species. NFAP2 did not cause metabolic inactivity and apoptosis induction, but its plasma membrane disruption ability was observed on Saccharomyces cerevisiae. The antifungal activity was maintained after high temperature treatment presumably due to the in silico predicted stable tertiary structure. The disulfide bond-stabilized, heat-resistant folded structure of NFAP2 was experimentally proved. After further investigations of antifungal mechanism, structure and toxicity, NFAP2 could be applicable as a potent antifungal agent against yeasts.

In vitro susceptibility of Scedosporium isolates to N-acetyl-L-cysteine alone and in combination with conventional antifungal agents.

In recent years, Scedosporium species have been more commonly recognized from severe, difficult-to-treat human infections, such as upper respiratory tract and pulmonary infections. To select an appropriate therapeutic approach for these infections is challenging, because of the commonly observed resistance of the causative agents to several antifungal drugs. Therefore, to find a novel strategy for the treatment of pulmonary Scedosporium infections the in vitro antifungal effect of a mucolytic agent, N-acetyl-L-cysteine and its in vitro combinations with conventional antifungals were investigated. Synergistic and indifferent interactions were registered in 23 and 13 cases, respectively. Antagonism was not revealed between the compounds.

Insight into the antifungal mechanism of Neosartorya fischeri antifungal protein.

Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great potential for the development of novel antifungal strategies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the antifungal mechanism of Neosartorya fischeri antifungal protein (NFAP), a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting-effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP activates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any influence on the cell wall integrity pathway, but an unknown cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken together, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related proteins from Aspergillus giganteus and Penicillium chrysogenum.