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

ABSTRACT: 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.

SUBMITTER: Tran VD

PROVIDER: S-EPMC5069957 | biostudies-literature | 2016 Jul-Aug

REPOSITORIES: biostudies-literature

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

Tran Van Du T VD De Coi Niccolò N Feuermann Marc M Schmid-Siegert Emanuel E Băguţ Elena-Tatiana ET Mignon Bernard B Waridel Patrice P Peter Corinne C Pradervand Sylvain S Pagni Marco M Monod Michel M

mSystems 20160701 4

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 <i>Arthroderma benhamiae</i> was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fu ...[more]

PMID: 27822542

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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:Background:Dermatophytosis represents one of the common infectious diseases worldwide and it is a major public health problem around the globe. The disease causes considerable morbidity and still continues to increase especially in developing countries. Objective:This study was undertaken to determine the prevalence of dermatophytes and the spectrum of fungal agents in patients attending Rank Higher Specialized Dermatology Clinic. Methods:A cross-sectional study has been conducted, in which 318 samples from 318 suspected patients were collected. Samples include hair, nail, and skin. A portion of each sample was examined microscopically and the remaining portion of each sample was cultured onto plates of Sabouraud's dextrose agar containing chloramphenicol with and without cycloheximide. Isolates were identified by studying the macroscopic and microscopic characteristics of the colonies. Results:Tinea capitis was the predominant clinical manifestation accounting for 53.4% of the cases. Patients with age group 1-14 years were more affected. Of 318 samples, fungi were detected in 133 (54.4%) by direct wet mount while 148/315 (46.5%) of them were culture positive. From these 72/148 (46.8%) were dermatophytes. T. tonsurans was the most common pathogen in tinea capitis, whereas T. mentagrophytes was the most common pathogen in tinea corporis. Among dermatophyte isolates, T. tonsurans 29/72 (40.2%) was the most common cause of infection. Among non-dermatophyte molds, Cladosporiumspp. 21/63 (33.3%) was predominant isolate followed by Neoscytalidim dimidatum 11/63 (17.4%) and Alternariaspp. 9/63 (14.2%), respectively. Yeasts also account for 13 (8.7%) of the total suspects of dermatophytosis. Conclusion:In this study, the prevalence of dermatophytes was higher in tinea capitis 46/72 (63.8%) and T. tonsurans 29/72 (40%) was the dominant-isolated dermatophyte. Recovery of a large number of dermatophytes and non-dermatophyte fungi in our study showed that non-dermatophyte fungi are emerging as important causes of dermatophytosis warranting further intensive epidemiological studies that have public health significance are needed.

Dataset Information

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

Publications

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

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