Máté Virágh

Isolation and characterization of Neosartorya fischeri antifungal protein (NFAP)

A novel 6.6 kDa antifungal peptide (NFAP) from the culture supernatant of the mold, Neosartorya fischeri (anamorf: Aspergillus fischerianus), and its encoding gene were isolated in this study. NFAP is a small, basic and cysteine-rich protein consisting of 57 amino acid residues. It shows 37.9-50% homology to similar proteins described in literature from Aspergillus clavatus, Aspergillus giganteus, Aspergillus niger, and Penicillium chrysogenum. The in silico presumed tertiary structure of NFAP, e.g. the presence of five antiparallel β-sheet connected with filaments, and stabilized by three disulfide bridges, is very similar to those of the defensin-like molecules. NFAP exhibited growth inhibitory action against filamentous fungi in a dose-dependent manner, and maintained high antifungal activity within broad pH and temperature ranges. Furthermore, it exhibited relevant resistance to proteolysis. All these characteristics make NFAP a promising candidate for further in vitro and in vivo investigations aiming at the development of new antifungal compounds.

Insight into the antifungal mechanism of Neosartorya fischeri antifungal protein

ABSTRACTSmall, 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.

Antifungal peptides homologous to the Penicillium chrysogenum antifungal protein (PAF) are widespread among Fusaria

Putative antifungal peptide encoding genes containing Penicillium chrysogenum antifungal protein (PAF) characteristic amino acid motifs were identified in 15 Fusarium isolates, representing 10 species. Based on the predicted sequences of mature peptides, discrepancy in one, two or three amino acids was observed between them. Phylogenetic investigations revealed that they show high amino acid sequence similarity to PAF and they belong to the group of fungal derived antifungal peptides with PAF-cluster. Ten from the 15 partially purified <10 kDa peptide fraction of Fusarium ferment broths showed antifungal activity. The presence of approximately 6.3 kDa molecular weight peptides was detected in all of the antifungally active ferment broths, and this peptide was isolated and purified from Fusarium polyphilaidicum. The minimal inhibitiory concentrations of F. polyphilaidicum antifungal protein (FPAP) were determined against different filamentous fungi, yeasts and bacteria. Filamentous fungal species were the most susceptible to FPAF, but some yeasts were also slightly sensitive.

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.

In vitro antifungal activity of phenothiazines and their combination with amphotericin B against different Candida species

Candidiosis is a mycosis that is currently increasingly affecting the population in consequence of its frequency and the severity of its complications, especially among immunocompromised hosts. In this work, the in vitro anticandidal activities of two phenothiazines (PTZs), chlorpromazine (CPZ) and trifluoperazine (TFP), and their combinations with amphotericin B (AMB) were tested against 12 different Candida strains representing 12 species (Candida albicans, Candida glabrata, Candida guillermondii, Candida inconspicua, Candida krusei, Candida lusitaniae, Candida lypolitica, Candida norvegica, Candida parapsilosis, Candida pulcherrima, Candida tropicalis and Candida zeylanoides). When used alone, both tested PTZs exerted antifungal effects against these strains. In their combinations, these PTZs and AMB mainly acted antagonistically at higher concentrations, but additively and synergistically at lower concentrations as concerns the clinically most important species (C. albicans and C. parapsilosis). For C. albicans, only synergistic interactions were revealed between CPZ and AMB. Synergistic, additive or no interactions were demonstrated between the investigated compounds for the most PTZ‐susceptible (C. glabrata to TFP and C. krusei to CPZ) and insusceptible strains (C. glabrata to CPZ and C. lypolitica to TFP).

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.

In vitro interactions of amantadine hydrochloride, R-(-)-deprenyl hydrochloride and valproic acid sodium salt with antifungal agents against filamentous fungal species causing central nervous system infection

The mortality rates of fungal infections that affect the central nervous system are high in consequence of the absence of effective antifungal drugs with good penetration across the blood-brain barrier and the blood-cerebrospinal fluid barrier. In the present work in vitro antifungal activities of three good penetrating non-antifungal drugs (amantadine hydrochloride, R-(-)-deprenyl hydrochloride, valproic acid sodium salt) and their combinations with three antifungal agents (amphotericin B, itraconazole, terbinafine) were tested with broth microdilution method against eight fungal isolates belonging to Zygomycetes (Lichtheimia corymbifera, Rhizomucor miehei, Rhizopus microsporus var. rhizopodiformis, Saksenaeavasiformis) and Aspergillus genus (A. flavus, A. fumigatus, A. nidulans, A. terreus). These are known to be possible agents of central nervous fungal infections (CNFI). When used alone, the investigated nonantifungal drugs exerted slight antifungal effects. In their combinations with antifungal agents they acted antagonistically, additively and synergistically against zygomyceteous isolates. Primarily antagonistic interactions were revealed between the investigated drugs in case of Aspergilli, but additive and synergistic interactions were also observed. The additive and synergistic combinations allowed the usage of reduced concentrations of antifungal agents to inhibit the fungal growth in our study. These combinations would be a basis of an effective, less toxic therapy for treatment of CNFI.

Transcriptomic atlas of mushroom development highlights an independent origin of complex multicellularity

We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall (FCW) remodeling, targeted protein degradation, signal transduction, adhesion and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, protein kinases and cadherin-like proteins, showed massive expansions in Agaricomycetes, with many convergently expanded in multicellular plants and/or animals too, reflecting broad genetic convergence among independently evolved complex multicellular lineages. This study provides a novel entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms.