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HapX Mediates Iron Homeostasis in the Pathogenic Dermatophyte Arthroderma benhamiae but Is Dispensable for Virulence.

ABSTRACT: For many pathogenic fungi, siderophore-mediated iron acquisition is essential for virulence. The process of siderophore production and further mechanisms to adapt to iron limitation are strictly controlled in fungi to maintain iron homeostasis. Here we demonstrate that the human pathogenic dermatophyte Arthroderma benhamiae produces the hydroxamate siderophores ferricrocin and ferrichrome C. Additionally, we show that the iron regulator HapX is crucial for the adaptation to iron starvation and iron excess, but is dispensable for virulence of A. benhamiae. Deletion of hapX caused downregulation of siderophore biosynthesis genes leading to a decreased production of siderophores during iron starvation. Furthermore, HapX was required for transcriptional repression of genes involved in iron-dependent pathways during iron-depleted conditions. Additionally, the ?hapX mutant of A. benhamiae was sensitive to high-iron concentrations indicating that HapX also contributes to iron detoxification. In contrast to other pathogenic fungi, HapX of A. benhamiae was redundant for virulence and a ?hapX mutant was still able to infect keratinized host tissues in vitro. Our findings underline the highly conserved role of the transcription factor HapX for maintaining iron homeostasis in ascomycetous fungi but, unlike in many other human and plant pathogenic fungi, HapX of A. benhamiae is not a virulence determinant.

SUBMITTER: Krober A

PROVIDER: S-EPMC4784894 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Project description: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 address this hypothesis a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae which causes highly inflammatory cutaneous infections in humans and rodents. Microarray analysis revealed a distinct in vivo protease gene expression profile in the fungal cells, which is surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro expressed proteases others were activated specifically during infection. These enzymes are therefore suggested to fulfill important functions that are not exclusively associated with the degradation of keratin. As the most upregulated in vivo specific A. benhamiae sequence we discovered 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. 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. This first broad transcriptional profiling approach during dermatophyte infection gives new molecular insights into pathogenicity associated mechanisms that make these microorganisms the most successful etiologic agents of superficial mycoses. Keywords: Two-condition experiment, strong proteolytic activity in the supernatant versus no proteolytic activity or infected tissue versus no proteolytic activity Three independently prepared A. benhamiae replicates grown in each of the three media, Sabouraud, soy and keratin-soy medium (designated SabA/B/C, soyA/B/C and keratin-soyA/B/C) were used. ARN from skin samples and fungus together of Guinea Pig infected with A. benhamieae were prepared. Pairwise transcriptional comparisons, i.e. soy versus Sabouraud, keratin-soy versus Sabouraud and Guinea Pig infected versus Sabouraud were done. The total number of slides in this study was 18.

Project description:Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates.

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HapX Mediates Iron Homeostasis in the Pathogenic Dermatophyte Arthroderma benhamiae but Is Dispensable for Virulence.

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