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.

Keywords: Arthroderma benhamiae; RNA-seq; Trichophyton; annotation; dermatophytes; infection; proteases; secreted proteins.

FIG 1

Experimental infection of the natural host of Arthroderma benhamiae. Cutaneously infected guinea pigs developed skin symptoms that were the most severe at 14 days postinfection (dpi) due to inflammation, while 8 dpi was the time point for the peak of infection.

FIG 2

Prediction and manual correction of the gene coding for the autophagy protein Atg27 (ARB_01857; a transmembrane protein). (A) Original gene prediction; (B) automatic prediction from Augustus (signal peptide is missing); (C) final (new) gene prediction after manual correction. The reannotation of this particular gene is remarkable, as it produced a new intron, an alternative stop codon, and a manually corrected start codon.

FIG 3

Characterization of the secretome. (A) Pie chart showing the main functional groups identified within the 457 proteins of the secretome. See detailed description in Data Set S1 in the supplemental material. (B) Pie charts showing the same functional groups as in panel A but within the 100 most expressed genes in Gp8 (in vivo 8 days postinfection), K (in vitro in keratin medium), and S (in vitro in soy medium). (C) Venn diagram of proteases (top) and carbohydrate/cell wall metabolism proteins (bottom) present in the 100 most expressed secreted proteins under the 3 conditions described for panel B. Proteases represent about 20% of the 100 most expressed proteins under the 3 conditions; however, the batch of proteins in Gp8 is clearly different from those in K and S. This trend is not as significant when comparing carbohydrate/cell wall metabolism proteins.

FIG 4

Hierarchical clustering (A and C) and principal component (PC) analysis (B and D) of RNA sequencing samples considering the genes from the complete genome (A and B) or only the secretome subset (C and D). The sample names reflect the growth conditions: Cb, in vivo in guinea pig; S, in vitro in soy medium; Sa, in vitro in Sabouraud medium; K, in vitro in keratin medium. The in vivo samples cluster together.

FIG 5

Number of differentially expressed genes versus the enumeration of all possible contrasting conditions in the genome and the secretome, using a cutoff of 1e−3 for FDR and 2 for the fold change.

FIG 6

(A) The twenty-five most highly expressed genes encoding secreted proteins during infection compared to in vitro expression. (B) The twenty-five most highly expressed genes encoding secreted proteins in vitro (keratin medium) compared to in vivo expression. Abbreviations are as defined in the Fig. 4 legend.