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.
Dermatophytes and other filamentous fungi excrete sulphite as a reducing agent during keratin degradation. In the presence of sulphite, cystine in keratin is directly cleaved to cysteine and Ssulphocysteine, and thereby, reduced proteins become accessible to hydrolysis by a variety of secreted endo-and exoproteases. A gene encoding a sulphite transporter in Aspergillus fumigatus (AfuSSU1), and orthologues in the dermatophytes Trichophyton rubrum and Arthroderma benhamiae (TruSSU1 and AbeSSU1, respectively), were identified by functional expression in Saccharomyces cerevisiae. Like the S. cerevisiae sulphite efflux pump Ssu1p, AfuSsu1p, TruSsu1p and AbeSsu1p belong to the tellurite-resistance/dicarboxylate transporter (TDT) family which includes the Escherichia coli tellurite transporter TehAp and the Schizosaccharomyces pombe malate transporter Mae1p. Seven genes in the A. fumigatus genome encode transporters of the TDT family. However, gene disruption of AfuSSU1 and of the two more closely related paralogues revealed that only AfuSSU1 encodes a sulphite efflux pump. TruSsulp and AbeSsulp are believed to be the first members of the TDT family identified in dermatophytes. The relatively high expression of TruSSU1 and AbeSSU1 in dermatophytes compared to that of AfuSSU1 in A. fumigatus likely reflects a property of dermatophytes which renders these fungi pathogenic. Sulphite transporters could be a new target for antifungal drugs in dermatology, since proteolytic digestion of hard keratin would not be possible without prior reduction of disulphide bridges.
Biochemical mechanism of keratin degradation by the actinomycete Streptomyces fradiae and the fungus Microsporum gypseum : A comparison J I R~ KUNERT Two keratinolytic organisms, the procaryote Streptomyces fradiae and the fungus Microsporum gypseum, were cultured on sterile sheep's wool in a mineral solution. The loss in substrate was recorded and the degradation products in the cultivation fluid were analyzed. In M. gypseum the key reaction was the cleaving of the substrate disulfide bridges by means of sulfite excreted into the medium. Keratin denatured by ,,sulfitolysis" was further attacked by extracellular proteases. A typical finding was the accumulation of peptides containing S-sulfocysteine, the product of sulfitolysis of cystine. The overall excess of sulfur was removed by oxidation to sulfite and to sulfate, which was the main and final product. In S . fradiae the degradation was faster. The results did not prove that sulfite formed and the concentration of sulfate in the medium remained negligible. Neither could cysteine desulfhydration and hydrogen sulfide excretion be demonstrated. The medium was found to contain relatively high concentrations of sulfhydryl compounds, evidently cysteine-containing peptides. Therefore, in this microorganism, keratin was most likely denatured by the direct reduction of cystine bridges. The main product of the elimination of excess sulfur was inorganic thiosulfate, which accumulated in the medium.
Zusammenfassung. Es wurde die Wirkung von Natriumsulfit, Cystein, Glutathion, Merkaptoethanol und Dithioerythritol (0.1‐10 mmol 1‐1) auf die Aktivität der Proteinasen von Microsporum gypseum untersucht. Azokasein, vernetztes Rinderserumalbumin und Keratin dienten als Substrate. Bei dem Substrat ohne Disulfidbindungen (Kasein) wurde keine Stimulierung der Proteolyse durch Reduktionsmittel gefunden; diese wurde meistens gehemmt. Beim Serumalbumin und Keratin wurde die Aktivität der Proteasen durch alle Reduktionsmittel erhöht, wahrscheinlich infolge der Spaltung von Disulfidbindungen des Substrats. Sulfit war wirksamer als die vier benutzten Thiole und erhöhte die Aktivität füur Serumalbumin bis 3.2 mal und füur Keratin bis 2.9 mal. Mit sulfitolysierter Schafwolle als Substrat wurde die keratinolytische Aktivität nach einer Spaltung von mehr als 20% aller Disulfidbindungen gesteigert. Bei der völlig sulfitolysierten Wolle wurde die Aktivität 43 mal erhöht. Die Ergebnisse stimmen mit der Hypothese des Autors über den Keratinabbau durch Sulfitausscheidung vor dem Angriff der Pilzproteinasen überein. Die Stimulierung der Proteolyse und Keratinolyse durch Spaltung von Disulfidbindungen ist nicht für die Dermatophyten‐Proteinasen spezifisch, weil Trypsin und Pronase ein ähnliches Verhalten aufwiesen. Summary. The effect of sodium sulphite, cysteine, glutathione, mercaptoethanol and dithioery‐thritol (0.1–10 mmol 1‐1) on the activity of proteases of Microsporum gypseum was studied using azocasein, cross‐linked bovine serum albumin and keratin as substrates. With the substrate without disulphide bonds (casein) no stimulation was found, and reducing agents inhibited proteolysis in most cases. With the remaining two substrates, all substances enhanced the activity of proteases probably through the cleavage of the substrate disulphide bonds. Sulphite was more effective than the four used thiols and enhanced the activity against serum albumin up to 3.2 times and against keratin up to 2.9 times. Using sulph‐itolysed sheep wool, keratinolytic activity increased after sulphitolysis of more than 20% of disulphide bonds. With the fully sulphitolysed wool the activity increased 43 times. The obtained results support the author's hypothesis on keratin degradation by sulphite excretion prior to attack by fungal proteases. Stimulation of proteolysis and keratinolysis by cleavage of disulphide bonds is not specific for dermatophytic proteases because trypsin and pronase behaved similarly in the experiments.
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