Description of Chryseobacterium fluminis sp. nov., a keratinolytic bacterium isolated from a freshwater river.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated MMS21-Ot14T, was isolated from a freshwater river, and shown to represent a novel species of the genus Chryseobacterium on the basis of the results from a polyphasic approach. The 16S rRNA gene sequence analysis revealed that MMS21-Ot14T represented a member of the genus Chryseobacterium of the family Weeksellaceae and was closely related to Chryseobacterium hagamense RHA2-9T (97.52 % sequence similarity), Chryseobacterium gwangjuense THG A18T (97.46 %) and Chryseobacterium gregarium P 461/12T (97.27 %). The optimal growth of MMS21-Ot14T occurred at 25-30 °C, pH 6.0-7.0 and in the absence of NaCl. MMS21-Ot14T was capable of hydrolysing casein, starch, DNA, Tween 20 and tyrosine. The strain also showed keratinolytic activity with keratin azure and decolourising activity with remazol brilliant blue R (RBBR), which indicated potential ability to degrade keratin and lignin. The main polar lipids of MMS21-Ot14T were phosphatidylethanolamine, unidentified aminophospholipids, unidentified aminolipids, an unidentified phospholipid and several unidentified lipids. The predominant fatty acids of MMS21-Ot14T were iso-C15 : 0 and iso-C17 : 0 3-OH, and the major isoprenoid quinone was menaquinone 6 (MK-6). The whole genome of MMS21-Ot14T was 5 062 016 bp in length with a DNA G+C content of 37.7 %. The average nucleotide identity and digital DNA-DNA hybridisation values between MMS21-Ot14T and phylogenetically related members of the genus Chryseobacterium were well below the threshold values for species delineation. It is evident from the results of this study that MMS21-Ot14T should be classified as representing a novel species of the genus Chryseobacterium, for which the name Chryseobacterium fluminis sp. nov. (type strain, MMS21-Ot14T = KCTC 92255T = LMG 32529T) is proposed.

Comprehensive insights into the mechanism of keratin degradation and exploitation of keratinase to enhance the bioaccessibility of soybean protein.

Keratin is a recalcitrant protein and can be decomposed in nature. However, the mechanism of keratin degradation is still not well understood. In this study, Bacillus sp. 8A6 can completely degrade the feather in 20 h, which is an efficient keratin degrader reported so far. Comprehensive transcriptome analysis continuously tracks the metabolism of Bacillus sp. 8A6 throughout its growth in feather medium. It reveals for the first time how the strain can acquire nutrients and energy in an oligotrophic feather medium for proliferation in the early stage. Then, the degradation of the outer lipid layer of feather can expose the internal keratin structure for disulfide bonds reduction by sulfite from the newly identified sulfite metabolic pathway, disulfide reductases and iron uptake. The resulting weakened keratin has been further proposedly de-assembled by the S9 protease and hydrolyzed by synergistic effects of the endo, exo and oligo-proteases from S1, S8, M3, M14, M20, M24, M42, M84 and T3 families. Finally, bioaccessible peptides and amino acids are generated and transported for strain growth. The keratinase has been applied for soybean hydrolysis, which generates 2234 peptides and 559.93 mg/L17 amino acids. Therefore, the keratinases, inducing from the poultry waste, have great potential to be further applied for producing bioaccessible peptides and amino acids for feed industry.

Genome-wide analysis of Keratinibaculum paraultunense strain KD-1 T and its key genes and metabolic pathways involved in the anaerobic degradation of feather keratin.

Keratinibaculum paraultunense strain KD-1 T (= JCM 18769 T = DSM 26752 T) is a strictly anaerobic rod-shaped bacterium. Under optimal conditions, feather keratin can be completely degraded by strain KD-1 within 24 h. Genomic sequencing showed that the genome was a single circular chromosome consisting of 2,307,997 base pairs (bp), with an average G + C content of 29.8% and no plasmids. A total of 2308 genes were annotated, accounting for 88.87% of the genomic sequence, and 1495 genes were functionally annotated. Among these, genes Kpa0144, Kpa0540, and Kpa0541 encoding the thioredoxin family members were identified, and may encode the potential disulfide reductases, with redox activity for protein disulfide bonds. Two potential keratinase-encoding genes, Kpa1675 and Kpa2139, were also identified, and corresponded to the ability of strain KD-1 to hydrolyze keratin. Strain KD-1 encoded genes involved in the heterotrophic metabolic pathways of 14 amino acids and various carbohydrates. The metabolic pathways for amino acid and carbohydrate metabolism were mapped in strain KD-1 based on KEGG annotations. The complete genome of strain KD-1 provided fundamental data for the further investigation of its physiology and genetics.

State-of-the-Art Dermatophyte Infections: Epidemiology Aspects, Pathophysiology, and Resistance Mechanisms.

The burden of fungal infections is not widely appreciated. Although these infections are responsible for over one million deaths annually, it is estimated that one billion people are affected by severe fungal diseases. Mycoses of nails and skin, primarily caused by fungi known as dermatophytes, are the most common fungal infections. Trichophyton rubrum appears to be the most common causative agent of dermatophytosis, followed by Trichophyton interdigitale. An estimated 25% of the world's population suffers from dermatomycosis. Although these infections are not lethal, they compromise the quality of life of infected patients. The outcome of antidermatophytic treatments is impaired by various conditions, such as resistance and tolerance of certain dermatophyte strains. The adage "know your enemy" must be the focus of fungal research. There is an urgent need to increase awareness about the significance of these infections with precise epidemiological data and to improve knowledge regarding fungal biology and pathogenesis, with an emphasis on adaptive mechanisms to tackle adverse conditions from host counteractions. This review outlines the current knowledge about dermatophyte infections, with a focus on signaling pathways required for fungal infection establishment and a broad perspective on cellular and molecular factors involved in antifungal resistance and tolerance.

Assessment of the subtilisin gene profile in Trichophyton verrucosum isolated from human and animal dermatophytoses in two-stage multiplex PCR.

AIMS: Keratin is a fibrous and recalcitrant structural protein and the third most abundant polymer in nature after cellulose and chitin. Subtilisin-like proteases (SUB) are a group of serine endoproteases, coded by seven genes (SUB1-7), which decompose keratin structures and have been isolated from dermatophytes. Herein, we identified the SUB genes in 30 clinical isolates of Trichophyton verrucosum obtained from human and animal dermatophytosis as well as asymptomatic animal carriers. METHODS AND RESULTS: We designed and proposed a two-stage multiplex PCR technique to detect all seven genes encoding serine proteases in dermatophytes. The analysis revealed the presence SUB1 and SUB2 amplicons in all strains regardless of the host. In the group of isolates obtained from humans, all seven subtilisin genes were shown in 40% of the strains. In T. verrucosum from asymptomatic animals, none of the isolates showed the presence of all seven subtilisin genes, and only 30% had six genes. In turn, 10% of the isolates from symptomatic animals demonstrated all seven subtilisins amplicons. CONCLUSIONS: In conclusion, the severity of infection and ability of T. verrucosum to cause dermatophytosis in humans may not be related to specific genes but their accumulation and synergistic effects of their products. SIGNIFICANCE AND IMPACT OF THE STUDY: Dermatophytes are pathogenic filamentous fungi with capacity to attack keratinized structures such as skin, hair and nails, causing cutaneous superficial infections. Indeed, a biological characteristic of dermatophytes is their ability to invade keratin-rich tissues by producing enzymes. Various degrees of inflammatory responses can be induced exactly by the enzymes. Subtilisin-like proteases are endoproteases, which decompose keratin structures. Our study identifies SUB genes in clinical isolates of T. verrucosum obtained from human and animal dermatophytosis as well as asymptomatic animal carriers.

Functional Characterization of Primordial Protein Repair Enzyme M38 Metallo-Peptidase From Fervidobacterium islandicum AW-1.

The NA23_RS08100 gene of Fervidobacterium islandicum AW-1 encodes a keratin-degrading ß-aspartyl peptidase (FiBAP) that is highly expressed under starvation conditions. Herein, we expressed the gene in Escherichia coli, purified the recombinant enzyme to homogeneity, and investigated its function. The 318 kDa recombinant FiBAP enzyme exhibited maximal activity at 80°C and pH 7.0 in the presence of Zn2+. Size-exclusion chromatography revealed that the native enzyme is an octamer comprising a tetramer of dimers; this was further supported by determination of its crystal structure at 2.6 Å resolution. Consistently, the structure of FiBAP revealed three additional salt bridges in each dimer, involving 12 ionic interactions that might contribute to its high thermostability. In addition, the co-crystal structure containing the substrate analog N-carbobenzoxy-ß-Asp-Leu at 2.7 Å resolution revealed binuclear Zn2+-mediated substrate binding, suggesting that FiBAP is a hyperthermophilic type-I IadA, in accordance with sequence-based phylogenetic analysis. Indeed, complementation of a Leu auxotrophic E. coli mutant strain (ΔiadA and ΔleuB) with FiBAP enabled the mutant strain to grow on isoAsp-Leu peptides. Remarkably, LC-MS/MS analysis of soluble keratin hydrolysates revealed that FiBAP not only cleaves the C-terminus of isoAsp residues but also has a relatively broad substrate specificity toward α-peptide bonds. Moreover, heat shock-induced protein aggregates retarded bacterial growth, but expression of BAP alleviated the growth defect by degrading damaged proteins. Taken together, these results suggest that the viability of hyperthermophiles under stressful conditions may rely on the activity of BAP within cellular protein repair systems.

Degradation of keratin substrates by keratinolytic fungi

Background: The hydrolysis of keratin wastes by microorganisms is considered a biotechnological alternative for recycling and valorization through keratinolytic microorganisms. Despite their resistant structure, keratin wastes can be efficiently degraded by various microorganisms through the secretion of keratinases, which are promising enzymes for several applications, including detergents, fertilizers, and leather and textile industry. In an attempt to isolate keratinolytic microorganisms that can reach commercial exploitation as keratinase producers, the current work assesses the dynamics of keratin biodegradation by several keratinolytic fungal strains isolated from soil. The activity of fungal strains to degrade keratin substrates was evaluated by SEM, FTRIR-ATR spectra and TGA analysis. Results: SEM observations offered relevant information on interactions between microorganism and structural elements of hair strands. FTIR spectra of the bands at 1035­1075 cm-1 assigned to sulfoxide bond appeared because of S­S bond breaking, which demonstrated the initiation of keratin biodegradation. According to TGA, in the second zone of thermal denaturation, where keratin degradation occurs, the highest weight loss of 71.10% was obtained for sample incubated with Fusarium sp. 1A. Conclusions: Among the tested strains, Fusarium sp. 1A was the most active organism in the degradation process with the strongest denaturation of polypeptide chains. Because keratinolytic microorganisms and their enzymes keratinases represent a subject of scientific and economic interest because of their capability to hydrolyze keratin, Fusarium sp. 1A was selected for further studies.

Onychomycosis Associated with Exophiala oligosperma in Taiwan.

A fungus was isolated from a nail of a 54-year-old female patient with onychomycosis in Taiwan. Based on ITS rDNA as well as beta tubulin gene sequences and microscopic analyses, this fungus was identified as Exophiala oligosperma. This is the first record of E. oligosperma in Taiwan. Negative keratin azure test indicates that keratin degradation is not involved in cases of E. oligosperma associated with skin and nail diseases.

Biochemical and structural characterization of a keratin-degrading M32 carboxypeptidase from Fervidobacterium islandicum AW-1.

Comparative genomics of the keratin-degrading extremophilic eubacterium Fervidobacterium islandicum AW-1 and the closely related Fervidobacterium nodosum with no keratinolytic activity suggested that the FIAW1_1600 gene encoding a carboxypeptidase (CP) plays an important role in keratin degradation. The presumptive 489 amino acid sequence of the gene showed a conserved HEXXH motif with low levels of sequence identity (<38%) to reported thermostable M32 CPs. To identify its functional role, the FIAW1_1600 gene was overexpressed in Escherichia coli, and the recombinant enzyme was purified and characterized in detail. F. islandicum AW-1 CP (FisCP) formed a homodimer with a molecular mass of 107 kDa, and its apoenzyme exhibited maximal activity at 80 °C and pH 7.0 in the presence of Co(2+). This metalloenzyme mainly cleaved the C-termini of peptides with a basic amino acid sequence. The crystal structure of FisCP at 2.2 Å resolution showed high levels of structural similarities (root-mean-square deviations of <1.7 Å) to those of other M32 CP homologs. Remarkably, the enzyme significantly enhanced the degradation of native chicken feathers. This study suggests that FisCP, a keratinolytic member of the thermostable M32 CP family, plays an important role in keratin degradation for cellular metabolism in F. islandicum AW-1.