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.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20-25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitroexpressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein.Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.Abbreviations: FDR, false discovery rate; MFS, major facilitator superfamily; PAS, periodic acid Schiff; qRT-PCR, quantitative reverse-transcription PCR.

Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ⌬acuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ⌬acuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes.Dermatophytes represent a group of specialized filamentous fungi which account for the majority of superficial fungal infections. Millions of so-called dermatophytoses, which in many cases are long lasting and difficult to eradicate, are recorded for humans and animals every year (29). As a peculiarity, dermatophytes specifically infect keratinized host structures, such as stratum corneum, hair, and nails. At the molecular level, however, little is known of the nature of the pathogenicity mechanisms in dermatophytes (17,30). This drawback might be related to the fact that these fungi, which grow comparatively slowly under laboratory conditions, have so far not intensively been studied genetically, in contrast to other medically important fungi, such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. Full genome sequence information for dermatophytes have been available since only very recently (http://www.broadinstitute.org /annotation/genome/dermatophyte_comparative/MultiHome .html), and genetic tools have hardly been established. As a consequence, in dermatophyte species only a few genes have to date been analyzed by targeted inactivation, i.e., pacC and MDR2 in Trichophyton rubrum (8, 9), Ku80, areA, and Trim4 in Trichophyton mentagrophytes (Arthroderma vanbreuseghemii) (32), and areA in Microsporum canis (31). Specifically constructed dermatophyte mutants have, to our knowledge, not yet been tested ...

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1⌬ and ssu1⌬ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1⌬ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

Millions of superficial fungal infections are annually observed in humans and animals. The majority of these mycoses are caused by dermatophytes, a specialized group of filamentous fungi that exclusively infect keratinized host structures. Despite the high prevalence of the disease, dermatophytosis, little is known about the pathogenicity mechanisms of these microorganisms. This drawback may be related to the fact that dermatophytes have been investigated poorly at the molecular level. In contrast to many other pathogenic fungi, they grow comparatively slowly under in vitro conditions, and in the last decades, only a limited number of molecular tools have been established for their manipulation. In recent years, however, major promising approaches were undertaken to improve genetic analyses in dermatophytes. These strategies include efficient systems for targeted gene inactivation and gene silencing, and broad transcriptional profiling techniques, which have even been applied in sophisticated infection models. As a fundamental prerequisite for future genetic analyses, full genome sequences of seven different dermatophyte species have become available recently. Therefore, it appeared timely to review the available molecular tools and methodologies in dermatophyte research, which may provide future insights into the virulence of these clinically important pathogens.

Our results support the hypothesis that ABCB1 and possibly other ABC-transporters are involved in the process of Aβ accumulation in the aging brain and may modulate the risk for AD in an allele-specific manner, and thus might represent a new target for prevention and treatment of AD.