Detection of Trichophyton rubrum and Trichophyton interdigitale in onychomycosis using monoclonal antibodies against Sub6 (Tri r 2).

BACKGROUND: Onychomycosis is the most prevalent nail disease and is mainly caused by two dermatophyte species Trichophyton rubrum and Trichophyton interdigitale with a frequency in the range of 80% and 20%, respectively. The secreted protease Sub6 of the subtilisin family, which was never detected in vitro growth conditions, was found to be a robust marker of onychomycosis. OBJECTIVE: The aim of this work was to detect tinea unguium using anti-Sub6 monoclonal antibodies in proteins extracted from clinical nail samples. METHODS: We produced monoclonal antibodies in mice using recombinant Sub6 as an antigen. Selected monoclonal antibodies were tested by Western blot analysis and ELISA on protein extracts from onychomycosis samples. RESULTS: Several monoclonal antibodies used to quantify Sub6 in proteins extracted from clinical nail samples were produced and characterised. We showed that these antibodies were very specific and allowed the detection of T. rubrum and T. interdigitale in onychomycosis. Sub6 was detected in clinical samples infected by T. rubrum and not detected in nails with trauma and other diseases. CONCLUSION: Anti-Sub6 monoclonal antibodies could be useful for a rapid diagnosis of tinea unguium and/or therapeutic survey of dermatophyte in onychomycosis by ELISA or an immunochromatography device such as a strip test.

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

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.