Gene Expression Profiling in the Human Pathogenic Dermatophyte Trichophyton rubrum during Growth on Proteins

Abstract

Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo-and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicityrelated host adaptation mechanisms of these human pathogenic fungi.Dermatophytes are highly specialized fungi which are the most common agents of superficial mycoses in humans and animals (13, 34). Among the human pathogenic dermatophytes, the anthropophilic species Trichophyton rubrum is clinically the most commonly observed. In contrast to most other medically important fungi, dermatophytes are not opportunists but are obligate pathogens, infecting exclusively the skin stratum corneum, nails, or hair. The ability of dermatophytes to degrade and utilize compact hard keratin within such host structures is presumably related to their common secreted keratinolytic activity, which is therefore a major putative virulence attribute of these fungi. Individual endo-and exoproteases secreted by dermatophytes are similar to those of species of the genus Aspergillus. However, in contrast to those of Aspergillus spp., dermatophyte-secreted endoproteases are multiple and members of two large protein families, the subtilisins (Subs) S8 (serine proteases) and the fungalysins M36 (metalloproteases) (see the MEROPS peptidase database, http://merops.sanger.ac.uk) (10, 11). More than 20 genes that encode secreted proteases have been identified in dermatophytes, some of which have been characterized in detail on the molecular level (reviewed in reference 23).In order to better understand the basic mechanisms of protein degradation by dermatophytes, the secretome of different dermatophyte species was previously analyzed during in vitro growt...

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection

“…The expression levels of six A. benhamiae genes were analysed by quantitative reverse-transcription PCR (qRT-PCR). Using PrimerExpress 2.0 software (Applied Biosystems), specific primers and 59-FAM-39BHQ probes were designed for genes encoding A. benhamiae subtilisins Sub3 and Sub6, which have been published previously (Jousson et al, 2004b); the sequence of primer Sub3AbenR is identical to that of primer Sub3TrubR (Zaugg et al, 2009). Sequences of primers and probes for detection of genes encoding McpA and isocitrate lyase as well as the controls encoding ADP-ribosylation factor ADPrf (TrMZC10ACH) and chitin-synthase ChitinS (TrMZG10ACO) were based on T. rubrum sequences (Zaugg et al, 2009).…”

“…Samples (3 mg) of total RNA isolated from liquid cultures and from infected tissue were amplified using the MessageAmp aRNA II kit (Ambion). Samples (2 mg) of amplified RNA were subsequently reverse-transcribed into cyanin 3 (Cy3)-or cyanin 5 (Cy5)-labelled cDNA with Superscript II reverse transcriptase (Invitrogen) by use of random primers (Invitrogen), purified with QIAquick columns (Qiagen) and hybridized on the cDNA microarrays which we had constructed previously (Zaugg et al, 2009). This microarray contains 2626 PCR products, which are spotted in triplicate on glass slides.…”

“…A. benhamiae was routinely grown on Sabouraud dextrose (hereafter referred to as Sabouraud) agar and liquid medium (Bio-Rad). For in vitro gene expression analyses, A. benhamiae was grown in liquid Sabouraud, soy [2 g soy protein l 21 (Supro 1711, Protein Technologies International)] and keratin-soy medium [0.2 g keratin 100 ml 21 (Merck; keratin is derived from animal hooves and horns), 0.01 g soy protein 100 ml 21 ], as previously described (Jousson et al, 2004a;Zaugg et al, 2009). A plug of fresh A. benhamiae mycelium grown on Sabouraud agar was inoculated into 100 ml liquid Sabouraud, soy and keratin-soy medium and incubated for 5, 10 and 24 days, respectively, at 30 uC without shaking.…”

“…The evolution of dermatophyte-specific protease gene families suggests particular functions of the individual isoenzymes for keratin degradation and pathogenicity. Distinct protease genes have been shown to be differentially expressed in dermatophytes during in vitro growth on proteins (Giddey et al, 2007;Zaugg et al, 2009), and the expression of some selected members of the subtilisins and fungalysins has been monitored in the zoophilic dermatophyte Microsporum canis during experimental guinea pig infection (Brouta et al, 2002;Descamps et al, 2002). However, the global in vivo protease gene expression pattern and the role of individual proteases in the pathogenicity of dermatophytes remain unknown.…”

Dermatófitos: interação patógeno-hospedeiro e resistência a antifúngicos

“…These studies also reveal interesting genes that should be evaluated as new targets for the development of antifungal drugs. 36 Chart 1 shows determining virulence factors identified in dermatophytes.…”