Phosphatase-degradable nanoparticles: A game-changing approach for the delivery of antifungal proteins.

HYPOTHESIS: Polyphosphate nanoparticles as phosphatase-degradable carriers for Penicillium chrysogenum antifungal protein (PAF) can enhance the antifungal activity of the protein against Candida albicans biofilm. EXPERIMENTS: PAF-polyphosphate (PP) nanoparticles (PAF-PP NPs) were obtained through ionic gelation. The resulting NPs were characterized in terms of their particle size, size distribution and zeta potential. Cell viability and hemolysis studies were carried out in vitro on human foreskin fibroblasts (Hs 68 cells) and human erythrocytes, respectively. Enzymatic degradation of NPs was investigated by monitoring release of free monophosphates in the presence of isolated as well as C. albicans-derived phosphatases. In parallel, shift in zeta potential of PAF-PP NPs as a response to phosphatase stimuli was determined. Diffusion of PAF and PAF-PP NPs through C. albicans biofilm matrix was analysed by fluorescence correlation spectroscopy (FCS). Antifungal synergy was evaluated on C. albicans biofilm by determining the colony forming units (CFU). FINDINGS: PAF-PP NPs were obtained with a mean size of 300.9 ± 4.6 nm and a zeta potential of -11.2 ± 2.8 mV. In vitro toxicity assessments revealed that PAF-PP NPs were highly tolerable by Hs 68 cells and human erythrocytes similar to PAF. Within 24 h, 21.9 ± 0.4 µM of monophosphate was released upon incubation of PAF-PP NPs having final PAF concentration of 156 µg/ml with isolated phosphatase (2 U/ml) leading to a shift in zeta potential up to -0.7 ± 0.3 mV. This monophosphate release from PAF-PP NPs was also observed in the presence of C. albicans-derived extracellular phosphatases. The diffusivity of PAF-PP NPs within 48 h old C. albicans biofilm matrix was similar to that of PAF. PAF-PP NPs enhanced antifungal activity of PAF against C. albicans biofilm decreasing the survival of the pathogen up to 7-fold in comparison to naked PAF. In conclusion, phosphatase-degradable PAF-PP NPs hold promise as nanocarriers to augment the antifungal activity of PAF and enable its efficient delivery to C. albicans cells for the potential treatment of Candida infections.

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.

Iminated aminoglycosides in self-emulsifying drug delivery systems: Dual approach to break down the microbial defense.

HYPOTHESIS: Aminoglycosides are well known, cationic antimicrobial drugs. However, biofilm-based antibiotic resistance significantly limits their efficacy. Masking the polycationic character of these drugs, followed by incorporation into self-emulsifying drug delivery systems (SEDDS) can improve biofilm eradication. EXPERIMENTS: Imine derivatives were synthesized via coupling with trans-cinnamaldehyde and characterized regarding degree of substitution, logP, cytotoxicity and antimicrobial efficacy on the opportunistic human pathogens Escherichia coli, Staphylococcus aureus and Candida albicans. Imines were loaded into newly developed SEDDS formulations and the antimicrobial efficacy was assessed on these pathogens in planktonic state and after biofilm formation. FINDINGS: Successful synthesis of imine derivatives with almost entirely masked amine groups was confirmed by NMR, FT-IR, TLC and MS. Imines exhibited a marked elevation in logP value of 8 units for kanamycin and 7.7 units for tobramycin. They showed low toxicity profiles while fully preserving antimicrobial efficacy on all tested pathogens. Incorporation into SEDDS resulted in nanoemulsions, which exhibited equal antimicrobial efficacy on the model germs compared to the corresponding aminoglycosides. Moreover, the biofilm eradication assay revealed superior anti-biofilm properties of the nanoemulsions. Native aminoglycosides were largely prone to reduced microbial susceptibility due to biofilm formation, while the combination of SEDDS with iminated aminoglycosides provided overall enhanced biofilm eradication.

The Membrane Activity of the Amphibian Temporin B Peptide Analog TB_KKG6K Sheds Light on the Mechanism That Kills Candida albicans.

Temporin B (TB) is a 13-amino-acid-long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We recently showed that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low micromolar concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions, showing a preference for anionic lipids, and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using fluorescein isothiocyanate-labeled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy-independent manner. Peptide-treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures had disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and supports its great potential as a future anti-Candida therapeutic. IMPORTANCE Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Small, cationic, membrane-active peptides are promising compounds to fight against resistance development, as many of them effectuate rapid fungal cell death. This fast killing is believed to hamper the development of resistance, as the fungi do not have sufficient time to adapt to the antifungal compound. We previously reported that the synthetic variant of the amphibian TB peptide, TB_KKG6K, rapidly kills C. albicans. In the current study, the mechanism of action of the TB analog was investigated. We show that this TB analog is membrane-active and impairs cell membrane function, highlighting its potential to be developed as an attractive alternative anti-C. albicans therapeutic that may hinder the development of resistance.

Small, Cationic Antifungal Proteins from Filamentous Fungi Inhibit Candida albicans Growth in 3D Skin Infection Models.

The emerging resistance of human-pathogenic fungi to antifungal drugs urges the development of alternative therapeutic strategies. The small, cationic antifungal proteins (AFPs) from filamentous ascomycetes represent promising candidates for next-generation antifungals. These bio-molecules need to be tested for tolerance in the host and efficacy against fungal pathogens before they can be safely applied in humans. Testing of the efficacy and possible adverse effects of new drug candidates in three-dimensional (3D) human-cell based models represents an advantageous alternative to animal experiments. In, this study, as a proof-of-principle, we demonstrate the usefulness of 3D skin infection models for screening new antifungal drug candidates for topical application. We established a cutaneous infection with the opportunistic human-pathogenic yeast Candida albicans in a commercially available 3D full-thickness (FT) skin model to test the curative potential of distinct AFPs from Penicillium chrysogenum (PAFopt, PAFB, and PAFC) and Neosartorya (Aspergillus) fischeri (NFAP2) in vitro. All tested AFPs were comparably well tolerated by the skin models. The infected 3D models exhibited reduced epidermal permeability barriers, allowing C. albicans to colonize the epidermal and dermal layers, and showed increased secretion of the pro-inflammatory cytokine IL-6 and the chemokine IL-8. AFP treatment diminished the fungal burden and penetration depth of C. albicans in the infected models. The epidermal permeability barrier was restored and the secretion of IL-8 was decreased following AFP treatment. In summary, our study proves that the tested AFPs exhibit antifungal potential against cutaneous C. albicans infection in a 3D FT skin model. IMPORTANCE Candida albicans represents one of the most prevalent opportunistic fungal pathogens, causing superficial skin and mucosal infections in humans with certain predisposing health conditions and life-threatening systemic infections in immunosuppressed patients. The emerging drug resistance of this human-pathogenic yeast and the limited number of antifungal drugs for prevention and treatment of infections urgently demands the identification of new antifungal compounds with novel mechanisms of action. Small, cationic antifungal proteins (AFPs) from filamentous fungi represent promising candidates for next-generation antifungals for topical application. These bio-molecules need to be tested for tolerance by the host and efficacy in pathogen clearance prior to being involved in clinical trials. In a proof-of-principle study, we provide evidence for the suitability of 3D human-cell based models as advantageous alternatives to animal experiments. We document the tolerance of specific AFPs and their curative efficacy against cutaneous C. albicans infection in a 3D skin model.

The combination of Neosartorya (Aspergillus) fischeri antifungal proteins with rationally designed γ-core peptide derivatives is effective for plant and crop protection.

Plant pathogenic fungi are responsible for enormous crop losses worldwide. Overcoming this problem is challenging as these fungi can be highly resistant to approved chemical fungicides. There is thus a need to develop and introduce fundamentally new plant and crop protection strategies for sustainable agricultural production. Highly stable extracellular antifungal proteins (AFPs) and their rationally designed peptide derivatives (PDs) constitute feasible options to meet this challenge. In the present study, their potential for topical application to protect plants and crops as combinatorial biofungicides is supported by the investigation of two Neosartorya (Aspergillus) fischeri AFPs (NFAP and NFAP2) and their γ-core PDs. Previously, the biofungicidal potential of NFAP, its rationally designed γ-core PD (γNFAP-opt), and NFAP2 was reported. Susceptibility tests in the present study extended the in vitro antifungal spectrum of NFAP2 and its γ-core PD (γNFAP2-opt) to Botrytis, Cladosporium, and Fusarium spp. Besides, in vitro additive or indifferent interactions, and synergism were observed when NFAP or NFAP2 was applied in combination with γNFAP-opt. Except for γNFAP2-opt, the investigated proteins and peptides did not show any toxicity to tomato plant leaves. The application of NFAP in combination with γNFAP-opt effectively inhibited conidial germination, biofilm formation, and hyphal extension of the necrotrophic mold Botrytis cinerea on tomato plant leaves. However, the same combination only partially impeded the B. cinerea-mediated decay of tomato fruits, but mitigated the symptoms. Our results highlight the feasibility of using the combination of AFP and PD as biofungicide for the fungal infection control in plants and crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s10526-022-10132-y.

Potential of Antifungal Proteins (AFPs) to Control Penicillium Postharvest Fruit Decay.

Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

New Perspectives in the Antimicrobial Activity of the Amphibian Temporin B: Peptide Analogs Are Effective Inhibitors of Candida albicans Growth.

Temporin B (TB) is a short, positively charged peptide secreted by the granular glands of the European frog Rana temporaria. While the antibacterial and antiviral efficacy of TB and some of its improved analogs are well documented, nothing is known about their antifungal potency so far. We dedicated this study to characterize the antifungal potential of the TB analog TB_KKG6K and the newly designed D-Lys_TB_KKG6K, the latter having the L-lysines replaced by the chiral counterpart D-lysines to improve its proteolytic stability. Both peptides inhibited the growth of opportunistic human pathogenic yeasts and killed planktonic and sessile cells of the most prevalent human pathogen, Candida albicans. The anti-yeast efficacy of the peptides coincided with the induction of intracellular reactive oxygen species. Their thermal, cation, pH and serum tolerance were similar, while the proteolytic stability of D-Lys_TB_KKG6K was superior to that of its template peptide. Importantly, both peptides lacked hemolytic activity and showed minimal in vitro cytotoxicity in primary human keratinocytes. The tolerance of both peptides in a reconstructed human epidermis model further supports their potential for topical application. Our results open up an exciting field of research for new anti-Candida therapeutic options based on amphibian TB analogs.

Porous assembly of an antifungal protein mediated by zinc and sulfonato-calix[8]arene.

Controlled protein assembly holds great potential in the fabrication of biohybrid materials. However, the structural diversity and complexity of proteins present an obstacle to controlled assembly. Supramolecular chemistry is a possible solution as it offers tools to mediate self-assembly with molecular precision. This paper deals with the calixarene- and zinc-mediated assembly and crystallization of the histidine-rich Penicillium chrysogenum antifungal protein B (PAFB). We report crystal structures of pure PAFB, PAFB in complex with Zn2+, and the ternary complex of PAFB, Zn2+ and sulfonato-calix[8]arene (sclx8). A comparison of the three crystal structures revealed the structural plasticity of PAFB. While the flexible and highly anionic sclx8 resulted in large molecular weight aggregates of PAFB in solution, diffraction-quality crystals of PAFB-sclx8 were not obtained. We report crystals of PAFB-Zn2+-sclx8 in which a trinuclear zinc cluster occurred adjacent to a calixarene binding site. Interestingly, the combination of sclx8 complexation and zinc coordination resulted in a porous framework with channels of circa 2 nm diameter. Detailed analysis of the crystal structure highlighted novel molecular recognition features. This research enriches the set of supramolecular interactions available to promote protein assembly.

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.

Biofungicidal Potential of Neosartorya (Aspergillus) Fischeri Antifungal Protein NFAP and Novel Synthetic γ-Core Peptides.

Because of enormous crop losses worldwide due to pesticide-resistant plant pathogenic fungi, there is an increasing demand for the development of novel antifungal strategies in agriculture. Antifungal proteins (APs) and peptides are considered potential biofungicides; however, several factors limit their direct agricultural application, such as the high cost of production, narrow antifungal spectrum, and detrimental effects to plant development and human/animal health. This study evaluated the safety of the application of APs and peptides from the ascomycete Neosartorya fischeri as crop preservatives. The full-length N. fischeri AP (NFAP) and novel rationally designed γ-core peptide derivatives (PDs) γNFAP-opt and γNFAP-optGZ exhibited efficacy by inhibiting the growth of the agriculturally relevant filamentous ascomycetes in vitro. A high positive net charge, however, neither the hydrophilicity nor the primary structure supported the antifungal efficacy of these PDs. Further testing demonstrated that the antifungal activity did not require a conformational change of the ß-pleated NFAP or the canonically ordered conformation of the synthetic PDs. Neither hemolysis nor cytotoxicity was observed when the NFAP and γNFAP-opt were applied at antifungally effective concentrations in human cell lines. Similarly, the Medicago truncatula plants that served as toxicity model and were grown from seedlings that were treated with NFAP, γNFAP-opt, or γNFAP-optGZ failed to exhibit morphological aberrations, reduction in primary root length, or the number of lateral roots. Crop protection experiments demonstrated that NFAP and associated antifungal active γ-core PDs were able to protect tomato fruits against the postharvest fungal pathogen Cladosporium herbarum.

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.

Nutrient Excess Triggers the Expression of the Penicillium chrysogenum Antifungal Protein PAFB.

Nutrient limitation and nonfavorable growth conditions have been suggested to be major triggers for the expression of small, cysteine-rich antimicrobial proteins (AMPs) of fungal origin, e.g., the Penicillium chrysogenum antifungal protein (PAF), the Aspergillus giganteus antifungal protein (AFP), the Aspergillus niger antifungal protein (AnAFP). Therefore, these AMPs have been considered to be fungal secondary metabolite products. In contrast, the present study revealed that the expression of the PAF-related AMP P. chrysogenum antifungal protein B (PAFB) is strongly induced under nutrient excess during the logarithmic growth phase, whereas PAFB remained under the detection level in the supernatant of cultures grown under nutrient limitation. The efficiency of the pafB-promoter to induce PAFB expression was compared with that of two P. chrysogenum promoters that are well established for recombinant protein production: the paf-promoter and the xylose-inducible promoter of the xylanase gene, xylP. The inducibility of the pafB-promoter was superior to that of the xylP-promoter yielding comparable PAFB amounts as under the regulation of the paf-promoter. We conclude that (i) differences in the expression regulation of AMPs suggest distinct functional roles in the producer beyond their antifungal activity; and (ii) the pafB-promoter is a promising tool for recombinant protein production in P. chrysogenum, as it guarantees strong gene expression with the advantage of inducibility.

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.

Membrane Sphingolipids Regulate the Fitness and Antifungal Protein Susceptibility of Neurospora crassa.

The membrane sphingolipid glucosylceramide (GlcCer) plays an important role in fungal fitness and adaptation to most diverse environments. Moreover, reported differences in the structure of GlcCer between fungi, plants and animals render this pathway a promising target for new generation therapeutics. Our knowledge about the GlcCer biosynthesis in fungi is mainly based on investigations of yeasts, whereas this pathway is less well characterized in molds. We therefore performed a detailed lipidomic profiling of GlcCer species present in Neurospora crassa and comprehensively show that the deletion of genes encoding enzymes involved in GlcCer biosynthesis affects growth, conidiation and stress response in this model fungus. Importantly, our study evidences that differences in the pathway intermediates and their functional role exist between N. crassa and other fungal species. We further investigated the role of GlcCer in the susceptibility of N. crassa toward two small cysteine-rich and cationic antimicrobial proteins (AMPs), PAF and PAFB, which originate from the filamentous ascomycete Penicillium chrysogenum. The interaction of these AMPs with the fungal plasma membrane is crucial for their antifungal toxicity. We found that GlcCer determines the susceptibility of N. crassa toward PAF, but not PAFB. A higher electrostatic affinity of PAFB than PAF to anionic membrane surfaces might explain the difference in their antifungal mode of action.

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.

Do Antimicrobial Proteins Contribute to Overcoming the Hidden Antifungal Crisis at the Dawn of a Post-Antibiotic Era?

The incidence of fungal infections has been grossly underestimated in the past decades as a consequence of poor identification techniques and a lack of regular epidemiologic surveys in low- and middle-income countries [...].

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.

Calcium binding of the antifungal protein PAF: Structure, dynamics and function aspects by NMR and MD simulations.

Calcium ions (Ca2+) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca2+ levels reduce the toxicity of PAF in the sensitive model fungus Neurospora crassa in a concentration dependent way. However, little is known about the mechanistic details of the Ca2+ ion impact and the Ca2+ binding capabilities of PAF outside the fungal cell, which might be the reason for the activity loss. Using nuclear magnetic resonance (NMR), isothermal titration calorimetry and molecular dynamics (MD) simulations we demonstrated that PAF weakly, but specifically binds Ca2+ ions. MD simulations of PAF predicted one major Ca2+ binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca2+ binding site. The exchange of Asp19 to serine had little impact on the Ca2+ binding, however caused the loss of antifungal activity, as was shown in our recent study. Now we replaced the C-terminal aspartates and expressed the serine variant PAFD53S/D55S. The specific Ca2+ binding affinity of PAFD53S/D55S decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca2+ interactions, we investigated the NMR and MD structure/dynamics of the free and Ca2+-bound PAF and PAFD53S/D55S. Though we found some differences between these protein variants and the Ca2+ complexes, these effects cannot explain the observed Ca2+ influence. In conclusion, PAF binds Ca2+ ions selectively at the C-terminus; however, this Ca2+ binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF.