Arthroderma tuberculatum and Arthroderma multifidum Isolated from Soils in Rook (Corvus frugilegus) Colonies as Producers of Keratinolytic Enzymes and Mineral Forms of N and S.

Keratinolytic fungi representing the genus Arthroderma that were isolated from the soils of a rook (Corvus frugilegus) colony were used as biological agents for the disposal of waste feathers. The aim of this study was to assess the abilities of Arthroderma tuberculatum and Arthroderma multifidum fungi with a varied inflow of keratin matter to biodegrade waste feathers. The evaluation was based on the determination of feather mass loss, the activity of keratinolytic enzymes, and the content of mineral N and S forms. It was found that the activity of protease released by the fungi contributed to an increase in the level of soluble proteins and peptides and the concentration of ammonium ions, as well as alkalization of the culture medium. Keratinase activity was significantly correlated with sulfate release, especially in A. tuberculatum cultures. The strains of A. tuberculatum fungi isolated from the soil with the highest supply of organic matter, i.e., strains III, IV, and V, had the lowest enzymatic activity, compared to the A. multifidum strains, but they released mineral nitrogen and sulfur forms that are highly important for fertilization, as well as nutritionally important peptides and amino acids. A. tuberculatum strains can be used for the management of waste feathers that can be applied in agricultural practice.

Alarming India-wide phenomenon of antifungal resistance in dermatophytes: A multicentre study.

BACKGROUND: An alarming increase in recalcitrant dermatophytosis has been witnessed in India over the past decade. Drug resistance may play a major role in this scenario. OBJECTIVES: The aim of the present study was to determine the prevalence of in vitro resistance to terbinafine, itraconazole and voriconazole in dermatophytes, and to identify underlying mutations in the fungal squalene epoxidase (SQLE) gene. PATIENTS/METHODS: We analysed skin samples from 402 patients originating from eight locations in India. Fungi were identified by microbiological and molecular methods, tested for antifungal susceptibility (terbinafine, itraconazole, voriconazole), and investigated for missense mutations in SQLE. RESULTS: Trichophyton (T.) mentagrophytes internal transcribed spacer (ITS) Type VIII was found in 314 (78%) samples. Eighteen (5%) samples harboured species identified up to the T interdigitale/mentagrophytes complex, and T rubrum was detected in 19 (5%) samples. 71% of isolates were resistant to terbinafine. The amino acid substitution Phe397Leu in the squalene epoxidase of resistant T mentagrophytes was highly prevalent (91%). Two novel substitutions in resistant Trichophyton strains, Ser395Pro and Ser443Pro, were discovered. The substitution Ala448Thr was found in terbinafine-sensitive and terbinafine-resistant isolates but was associated with increased MICs of itraconazole and voriconazole. CONCLUSIONS: The high frequencies of terbinafine resistance in dermatophytes are worrisome and demand monitoring and further research. Squalene epoxidase substitutions between Leu393 and Ser443 could serve as markers of resistance in the future.

DNA topoisomerase 2 gene polymorphism in dermatophytes.

BACKGROUND: Dermatophytes are a group of keratinophilic fungi of medical importance. Despite a relatively long history of molecular taxonomic studies, there is still a need for information on genetic polymorphism in wider variety of genomic loci. OBJECTIVES: Our goal was to study partial DNA topoisomerase 2 gene (TOP2) polymorphism in dermatophytes. METHODS: We performed DNA sequencing of TOP2 in 26 dermatophyte species along with ribosomal internal transcribed spacer (ITS) sequencing. RESULTS: The number of polymorphic sites in TOP2 data set was similar to that one in ITS data set. Nannizzia species formed paraphyletic group in TOP2 tree. Trichophyton simii was paraphyletic in concatenated TOP2-ITS tree, one of its two clades contained solely Iranian isolates. CONCLUSIONS: Our results revealed several unresolved problems in the taxonomy of dermatophytes, including probable polyphyly of the genus Nannizzia and the species T simii.

Keratin hydrolysis by dermatophytes.

Dermatophytes are the most common cause of superficial fungal infections (tinea infections) and are a specialized group of filamentous fungi capable of infecting and degrading keratinised tissues, including skin, hair, and nail. Essential to their pathogenicity and virulence is the production of a broad spectrum of proteolytic enzymes and other key proteins involved in keratin biodegradation and utilization of its breakdown products. The initial stage of biodegradation of native keratin is considered to be sulfitolysis, in which the extensive disulfide bridges present in keratin are hydrolyzed, although some secreted subtilisins can degrade dye-impregnated keratin azure without prior reduction (Sub3 and Sub4). Sulfitolysis facilitates the extracellular biodegradation of keratin by the dermatophytes' extensive array of endo- and exoproteases. The importance of dermatophyte proteases in infection is widely recognized, and these enzymes have also been identified as important virulence determinants and allergens. Finally, the short peptide and amino acid breakdown products are taken up by the dermatophytes, using as yet poorly characterised transporters, and utilized for metabolism. In this review, we describe the process of keratin biodegradation by dermatophytes, with an especial focus on recent developments in cutting edge molecular biology and '-omic' studies that are helping to dissect the complex process of keratin breakdown and utilization.

Phenotypic characterization of enzymatic activity of clinical dermatophyte isolates from animals with and without skin lesions and humans.

AIMS: The pathogenesis of dermatophytoses is associated with the secretion of enzymes degrading the infected tissue components. Although many studies on enzymatic activity of dermatophytes have been conducted over the years, there have been no concrete proposals on the construction of the profile of enzymes characteristic of individual species, genus or ecological types of dermatophytes. The aim of this study was to assess the capability of clinical dermatophyte isolates from both symptomatic and asymptomatic animals and humans to produce different enzymes. METHODS AND RESULTS: Clinical isolates of 234 dermatophyte strains collected during routine examination of animal health were used in this study. The enzymatic production of keratinase, elastase, phospholipase, lipase, protease, DNase and gelatinase as well as the haemolytic activity were evaluated using specific test media. The overall degree of enzymatic activity of the analysed clinical isolates of the dermatophytes was 67%. All tested clinical isolates of different species of dermatophytes showed keratinase activity and 96% additionally exhibited phospholipase activity. The weakest activity among the tested enzymes was demonstrated for elastase and gelatinase. 83% of the isolates of the dermatophytes showed haemolytic activity. CONCLUSION: Our data indicate that clinical isolates of dermatophytes from different species produce enzymes with different levels of activities. SIGNIFICANT AND IMPACT OF THE STUDY: Profile of enzymes characteristic of individual species, genus or ecological types of dermatophytes is possibly dependent upon factors related to the host. The relationship between each enzyme and the occurrence of skin lesions in animals and humans or asymptomatic animal carriers varies on whether the infection is caused by Trichophyton mentagrophytes, Trichophyton verrucosum or Microsporum canis. Interestingly, only keratinase seems to be correlated with the appearance of dermatophyte infections, irrespective of the pathogen species, and elastase is a characteristic enzyme for dermatophyte strains infecting humans. Haemolysis seems to be dependent on host factors and is more common in the case of human dermatophyte isolates.

New insights in dermatophyte research.

Dermatophyte research has renewed interest because of changing human floras with changing socioeconomic conditions, and because of severe chronic infections in patients with congenital immune disorders. Main taxonomic traits at the generic level have changed considerably, and now fine-tuning at the species level with state-of-the-art technology has become urgent. Research on virulence factors focuses on secreted proteases now has support in genome data. It is speculated that most protease families are used for degrading hard keratin during nitrogen recycling in the environment, while others, such as Sub6 may have emerged as a result of ancestral gene duplication, and are likely to have specific roles during infection. Virulence may differ between mating partners of the same species and concepts of zoo- and anthropophily may require revision in some recently redefined species. Many of these questions benefit from international cooperation and exchange of materials. The aim of the ISHAM Working Group Dermatophytes aims to stimulate and coordinate international networking on these fungi.

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene.

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

The anti-dermatophyte activity of Zataria multiflora essential oils.

OBJECTIVE: Dermtophytes are a group of pathogenic fungi and the major cause of dermatophytosis in humans and animals. Fighting dermatophytes by natural essential oils is one important issue in new researches. MATERIALS AND METHODS: In this investigation, we evaluated the anti-dermatophyte activities of three samples of Z. multiflora essential oils against dermatophytes along with analysis of chemical compositions of the essential oils and their anti-elastase activities on elastase production in dermatophytes. RESULTS: Carvacrol (1.5-34.4%), thymol (25.8-41.2%), carvacrol methyl ether (1.9-28.3%) and p-cymene (2.3-8.3%) were the main components of Z. multiflora essential oils. Z. multiflora essential oils (100ppm) inhibited the mycelium growth of dermatophytes (6±1.7-47.0±1.4%) and had the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values of 0.03-0.25µl/ml against dermatophytes. Essential oils inhibited elastase produced in dermatophytes and pure porcine elastase. CONCLUSION: Z. multiflora essential oils can be used as natural anti-dermatophyte agent for fighting dermatophytes in further preclinical and clinical studies.

In vitro evaluation of antifungal susceptibility and keratinase, elastase, lipase and DNase activities of different dermatophyte species isolated from clinical specimens in Iran.

The aims of this study were to evaluate the enzymatic activity of various dermatophyte species and their antifungal susceptibility profiles. A total of 60 dermatophyte isolates, including Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum, were examined. Fungal isolates were analysed for the production of keratinase, lipase, elastase and deoxyribonuclease (DNase). A broth microdilution method was performed on the basis of M38-A2 Clinical and Laboratory Standards Institute (CLSI) guidelines. T. mentagrophytes, M. canis and M. gypseum isolates were capable of producing keratinase, lipase, elastase and DNase, while T. rubrum isolates were elastase negative. The highest mean diameter of the clear zone around the colonies (PZ) was associated with keratinase (PZ: 4.56 ± 1.29 mm), followed by lipase (PZ: 1.53 ± 0.90 mm), DNase (PZ: 0.65 ± 0.54 mm) and elastase (PZ: 0.22 ± 0.27 mm) (P < 0.05). The mean minimum inhibitory concentration 90 (MIC90 ) of all strains were as follows: itraconazole (MIC90 : 0.28 ± 0.31 µg ml-1 ), ketoconazole (MIC90 : 0.48 ± 0.51 µg ml-1 ), griseofulvin (MIC90 : 0.86 ± 1.00 µg ml-1 ) and fluconazole (MIC90 : 18.57 ± 20.10 µg ml-1 ). Dermatophyte isolates had higher keratinolytic activity than other enzymes. Itraconazole was the most effective antifungal drug and fluconazole had the poorest activity.

Genetic and Phenotypic analyses of Calcineurin A subunit in Arthroderma vanbreuseghemii.

Calcineurin is a serine/threonine protein phosphatase that consists of catalytic (calcineurin A) and regulatory (calcineurin B) subunits. The conserved protein plays important roles in various biological processes. Drug combination of fluconazole and the calcineurin inhibitor (FK506) showed synergistic effects against dermatophytes. In the current study, we identified the calcineurin A homologous gene (TmcanA) in the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes). Knockdown mutants were produced from A. vanbreuseghemii, resulting in a defection in growth properties in accordance with dose of the suppressing reagent. The TmcanA gene restored the ability of calcineurin A-deficient Cryptococcus neoformans strain to grow at elevated temperatures. Repression of TmcanA at 37°C resulted in severely stunted growth, suggesting that this protein plays a role in tolerance to elevated temperatures. In addition, TMCANA showed an interaction with high osmolarity glycerol (HOG) signalling pathway by governing the secretion of a secondary metabolite. Moreover, expression of the hydrophobin A gene (TmHF) decreased significantly under the TmcanA-repressive condition, suggesting that TMCANA is involved in its regulation. In conclusion, calcineurin A is a multifunctional gene that is involved in the regulation of several biological processes and therefore is worth being considered as a drug target for treatment of dermatophytoses.

Exploring the potential of fungal arylacetonitrilases in mandelic acid synthesis.

The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.

Inhibitory effect of linalool-rich essential oil from Lippia alba on the peptidase and keratinase activities of dermatophytes.

Abstract Lippia alba (Miller) N.E. Brown is an aromatic plant known locally as "Erva-cidreira-do-campo" that has great importance in Brazilian folk medicine. The aim of our study was to evaluate the antidermatophytic potential of linalool-rich essential oil (EO) from L. alba and analyze the ability of this EO to inhibit peptidase and keratinase activities, which are important virulence factors in dermatophytes. The minimum inhibitory concentrations (MICs) of L. alba EO were 39, 156 and 312 µg/mL against Trichophyton rubrum, Epidermophyton floccosum and Microsporum gypseum, respectively. To evaluate the influence of L. alba EO on the proteolytic and keratinolytic activities of these dermatophytes, specific inhibitory assays were performed. The results indicated that linalool-rich EO from L. alba inhibited the activity of proteases and keratinases secreted from dermatophytes, and this inhibition could be a possible mechanism of action against dermatophytes. Due to the effective antidermatophytic activity of L. alba EO, further experiments should be performed to explore the potential of this linalool-rich EO as an alternative antifungal therapy.

A prodrug approach to the use of coumarins as potential therapeutics for superficial mycoses.

Superficial mycoses are fungal infections of the outer layers of the skin, hair and nails that affect 20-25% of the world's population, with increasing incidence. Treatment of superficial mycoses, predominantly caused by dermatophytes, is by topical and/or oral regimens. New therapeutic options with improved efficacy and/or safety profiles are desirable. There is renewed interest in natural product-based antimicrobials as alternatives to conventional treatments, including the treatment of superficial mycoses. We investigated the potential of coumarins as dermatophyte-specific antifungal agents and describe for the first time their potential utility as topical antifungals for superficial mycoses using a prodrug approach. Here we demonstrate that an inactive coumarin glycone, esculin, is hydrolysed to the antifungal coumarin aglycone, esculetin by dermatophytes. Esculin is hydrolysed to esculetin ß-glucosidases. We demonstrate that ß-glucosidases are produced by dermatophytes as well as members of the dermal microbiota, and that this activity is sufficient to hydrolyse esculin to esculetin with concomitant antifungal activity. A ß-glucosidase inhibitor (conduritol B epoxide), inhibited antifungal activity by preventing esculin hydrolysis. Esculin demonstrates good aqueous solubility (<6 g/l) and could be readily formulated and delivered topically as an inactive prodrug in a water-based gel or cream. This work demonstrates proof-of-principle for a therapeutic application of glycosylated coumarins as inactive prodrugs that could be converted to an active antifungal in situ. It is anticipated that this approach will be applicable to other coumarin glycones.

The possible role of dermatophyte cysteine dioxygenase in keratin degradation.

Cysteine dioxygenase (CDO, EC 1.13.11.20) is a key enzyme involved in the homeostatic regulation of cysteine level and in production of important oxidized metabolites of cysteine such as pyruvate, sulphite, sulphate, hypotaurine, and taurine in all eukaryotic cells. The intracellular CDO concentration is regulated at both transcriptional and posttranslational levels. In several fungi, CDO plays an important role as a virulence factor involved in morphological transition from yeast to mycelial forms. CDO is crucial for oxidation of cysteine to cysteine sulphinic acid and therefore for sulphite production and secretion. Because sulphite cleaves disulphide bridges as a first unavoidable step in keratinolysis, it is hypothesized that in dermatophytes, CDO is a virulence factor crucial for keratin degradation.

Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump.

Millions of people suffer from superficial infections caused by dermatophytes. Intriguingly, these filamentous fungi exclusively infect keratin-rich host structures such as hair, nails, and skin. Keratin is a hard, compact protein, and its utilization by dermatophytes for growth has long been discussed as a major virulence attribute. Here, we provide strong support for the hypothesis that keratin degradation is facilitated by the secretion of the reducing agent sulfite, which can cleave keratin-stabilizing cystine bonds. We discovered that sulfite is produced by dermatophytes from environmental cysteine, which at elevated concentrations is toxic for microbes and humans. We found that sulfite formation from cysteine relies on the key enzyme cysteine dioxygenase Cdo1. Sulfite secretion is supported by the sulfite efflux pump Ssu1. Targeted mutagenesis proved that dermatophyte mutants in either Cdo1 or Ssu1 were highly growth-sensitive to cysteine, and mutants in Ssu1 were specifically sensitive to sulfite. Most notably, dermatophyte mutants in Cdo1 and Ssu1 were specifically growth-defective on hair and nails. As keratin is rich in cysteine, our identified mechanism of cysteine conversion and sulfite efflux supports both cysteine and sulfite tolerance per se and progression of keratin degradation. These in vitro findings have implications for dermatophyte infection pathogenesis.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Genome mining reveals the presence of a conserved gene cluster for the biosynthesis of ergot alkaloid precursors in the fungal family Arthrodermataceae.

Genome sequence analysis of different fungi of the family Arthrodermataceae revealed the presence of a gene cluster consisting of five genes with high sequence similarity to those involved in the early common steps of ergot alkaloid biosynthesis in Aspergillus fumigatus and Claviceps purpurea. To provide evidence that this cluster is involved in ergot alkaloid biosynthesis, the gene ARB_04646 of the fungus Arthroderma benhamiae was cloned into pQE60 and expressed in Escherichia coli. Enzyme assays with the soluble tetrameric His(6)-tagged protein proved unequivocally that the deduced gene product, here termed ChaDH, catalysed the oxidation of chanoclavine-I in the presence of NAD(+), resulting in the formation of chanoclavine-I aldehyde. The enzyme product was unequivocally proven by NMR and MS analyses. Therefore, ChaDH functions as a chanoclavine-I dehydrogenase. K(m) values for chanoclavine-I and NAD(+) were 0.09 and 0.36 mM, respectively. Turnover number was 0.76 s(-1).

Role of pH in the pathogenesis of dermatophytoses.

The secretion of proteolytic enzymes by dermatophytes is a key factor in their invasion and subsequent dissemination through the stratum corneum of the host. During the first stages of infection, dermatophytes respond to the skin by de-repressing a number of genes coding for proteins and enzymes such as adhesins, lipases, phosphatases, DNAses, non-specific proteases, and keratinases. These proteins have their optimal activity at acidic pH values, which matches the acidic pH of human skin, allowing the pathogen to adhere and penetrate the host tissue, scavenge nutrients and overcome host defence mechanisms. The conserved PacC/Rim101p signal transduction pathway mediates diverse metabolic events involved in ambient pH sensing and in the virulence of pathogenic microorganisms. The seven dermatophyte genomes analysed here revealed the presence of the PacC/Rim101p pH-responsive signal transduction pathway, which consists of the six pal genes (palA, B, C, F, H and I) and the transcription factor PacC. The PacC binding site was present in the promoter regions of pacC, palB, palI and palH genes of all dermatophytes, suggesting functional equivalency with the signalling cascade of other fungi. Moreover, the promoter region of pacC gene of the seven dermatophytes had multiple PacC DNA-binding sites, suggesting that these genes, like their homologues in model fungi, are auto-regulated.

Mechanisms of skin adherence and invasion by dermatophytes.

Dermatophytes are keratinophilic fungi that can be pathogenic for humans and animals by infecting the stratum corneum, nails, claws or hair. The first infection step consists of adherence of arthroconidia to the stratum corneum. The mechanisms and the kinetics of adherence have been investigated using different in vitro and ex vivo experimental models, most notably showing the role of a secreted serine protease from Microsporum canis in fungal adherence to feline corneocytes. After germination of the arthroconidia, dermatophytes invade keratinised structures that have to be digested into short peptides and amino acids to be assimilated. Although many proteases, including keratinolytic ones, have been characterised, the understanding of dermatophyte invasion mechanisms remains speculative. To date, research on mechanisms of dermatophyte infection focused mainly on both secreted endoproteases and exoproteases, but their precise role in both fungal adherence and skin invasion should be further explored.