Introduction

Dermatophytosis is a common disease that can affect a large proportion of the human population (Vander Straten et al. 2003). The main causative agent of dermatophytosis is Trichophyton rubrum (Elewski et al. 2002), a widespread filamentous fungus that can infect keratinized tissue such as skin, nails and, rarely, hair (Costa et al. 2002; Jennings et al. 2002; Monod et al. 2002). T. rubrum is known to account for up to 69.5% of all dermatophyte infections (Chen and Friedlander 2001; Coloe and Baird 1999), which are often intractable, and relapse occurs frequently after cessation of antifungal therapy (Mukherjee et al. 2003). The prevalence of T. rubrum infections and its anthrophilic nature make it a good model for the study of human pathogenic filamentous fungi.

Among the antifungal agents, allylamines such as terbinafine (TRB) are very widely used, both orally and topically, in the therapy of superficial mycoses, including dermatophyte onychomycosis, dermatomycoses, tinea, and piedra (Ryder 1992; Jessup et al. 2000; Aly 2001). Common therapeutic strategies based on the use of TRB are generally considered effective (Degreef and DeDoncker 1994; Crawford et al. 2002), but resistance against the antifungal agents has been reported (Mukherjee et al. 2003). The allylamines are potent inhibitors of the fungal squalene epoxidase enzyme (Ryder 1992), which is involved in the conversion of squalene to squalene epoxide, a precursor in the biosynthesis of ergosterol (Favre and Ryder 1996). As a consequence of this inhibition, fungal cells accumulate squalene and become depleted of ergosterol (Paltauf et al. 1982; Leber et al. 1995).

The cDNA microarray is a good tool for drug target identification in a survey of the global effects mediated by antifungal agents. Although the mechanisms of action of antifungal agents against some model fungi such as Saccharomyces cerevisiae and Candida albicans have been studied by cDNA microarray (Bammert and Fostel 2000; De Backer et al. 2001; Zhang et al. 2002), the inhibitory mechanism of TRB against T. rubrum is poorly understood.

In our experiment, a cDNA microarray was constructed from the expressed sequence tags (ESTs) of the cDNA library of T. rubrum. To identify class-specific changes in gene expression, we used a cDNA microarray to examine changes in transcriptional profiles of T. rubrum in response to TRB.

Materials and methods

Fungus and material

The T. rubrum clinical isolate BMU 01672 used in this study was provided by Professor Ruoyu Li (Research Center for Medical Mycology, Peking University). The isolate was confirmed as T. rubrum by morphologic identification of both microscopic and macroscopic characteristics (De Hoog et al. 2000; Larone 2002) as well as by PCR amplification and sequencing of the 18S ribosomal DNA and internal transcribed spacer (ITS) regions (Jackson et al. 1999). Potato dextrose agar (PDA), yeast extract, peptone, and d-glucose used for the strain cultures were from Difco. TRB was obtained from Sigma (St. Louis, MO, USA). Stock solutions of various concentrations were made in dimethyl sulfoxide (DMSO; Sigma).

Preparation of cell cultures, construction of cDNA libraries, sequencing, and annotation

Potato dextrose agar (39 g/l) was inoculated with a few hyphae of T. rubrum and incubated at 28°C for 2 to 3 weeks until good conidiation was produced. The mixture of conidia and hyphal fragments was collected in distilled water. Construction of cDNA libraries, cDNA sequencing, Phred quality assessment, and computational analysis were done as described (Wang et al. 2006; Yu et al. 2007).

cDNA microarray

T7 and SP6 universal primers were used for amplification of PCR fragments used for printing the microarray chip. Purified PCR products were resuspended in 12 Cl of spotting solution containing 50% dimethyl sulfoxide to produce the microarrays. A set of microarrays containing a total of 11,232 spots (10,250 clones in the form of PCR products and 982 controls, including blank, negative, and positive controls) were spotted in duplicate on b amino propylsilan-coated GAP II slides (Corning) with a Cartesian arrayer. Slides were subsequently placed in a blocking solution containing 0.2 M succinic anhydride and 0.05 M sodium borate prepared in 1-methyl-2-pyrrolidinone for 20 min, washed for 2 min in 95°C water, and rinsed five times in 95% ethanol. Slides were spin dried and stored for future hybridizations (Diehl et al. 2001; Peng et al. 2006).

MIC determinations

Test concentrations of TRB ranged from 0.001 to 0.5 µg/ml. MIC is defined as the lowest concentration at which the growth of the organism was inhibited 100% compared with growth in the control. All tests were done in duplicate and the results were read visually. MIC results were recorded in units of micrograms per milliliter. The antifungal susceptibility tests were done as described (Zhang et al. 2007).

Cell culture and drug exposure for microarray experiments

For a single microarray experiment, six 100-ml cultures were prepared (three independent 100-ml cultures were grown for TRB). TRB was added to three cultures at a concentration equivalent to 0.5 MIC (0.01 µg/ml) and incubated at 30°C. Three control cultures were treated with DMSO (0.01 µg/ml). Six hours after the addition of TRB, three control cultures and three drug-treated cultures were harvested and frozen in liquid nitrogen. The culture of T. rubrum clinical isolate BMU 01672 and the experimental protocol were as described (Yu et al. 2007).

RNA preparation

Total RNA was isolated using the QIAGEN RNeasy plant mini kit (QIAGEN, Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Three independent sets of RNA from control and three independent sets of RNA from drug-treated cells were used to prepare six independent cDNA sets. An aliquot of poly(A)+ mRNA was isolated with the Oligotex mRNA mini kit (QIAGEN) following the manufacturer’s instructions.

Microarray hybridization

First-strand cDNA was synthesized using Superscript II RT (Life Technologies/Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized as follows: 5× second-strand buffer; 0.2 mM deoxynucleoside triphosphate mix; Escherichia coli DNA ligase (10U); E. coli DNA polymerase I (40 U); and E. coli RNase H (2 U) were added to a first-strand reaction tube and incubated for 2 h at 16°C. Double-stranded cDNA (dscDNA) was purified using QIAquick columns (QIAGEN) by following the manufacturer’s instructions. dscDNA was then fluorescently labeled using BioPrime DNA labeling system (Life Technologies/Invitrogen). Those representing RNA from drug-treated cells were labeled using Cy5, and those representing RNA from control cells were labeled using Cy3. Labeled cDNA was purified using QIAquick columns and mixed with 2 Cg poly(A), 5× Denhardt’s, 3× SSC, 24 Cg yeast tRNA, 25 mM HEPES, pH 7.0, and 0.25% SDS. The mixture was heated at 100°C for 2 min, cooled to room temperature, and applied to the array slides under glass coverslips. Hybridization was performed at 65°C overnight in a Micro hybridization incubator (Robbins Scientific, Sunnyvale, CA). Slides were washed in 2× SSC (0.1% SDS), 1× SSC, and 0.2× SSC sequentially and then were scanned at 5 Cm on a GenePix 4000B scanner (Axon Instruments, Inc.). The Cy5- and Cy3-labeled DNA samples were scanned at 635 and 532 nm, respectively.

Data analysis

GenePix 6.0 software (Axon Instruments) was used for image analysis and data visualization. Before data analysis, signals were normalized using a locally weighted scatterplot smoothing regression algorithm in the MIDAS software package (Cleveland 1979; Saeed et al. 2003), with the smoothing parameter set to 0.33. Genes were considered to be expressed differentially if: (1) average expression changed by at least 1.5-fold in three independent experiments with triplicate RNA samples; and (2) the change in gene expression was in the same direction (increased or decreased) in three experiments.

Quantitative real-time RT-PCR

Quantitative real-time reverse transcription (RT)-PCR was used to verify the microarray results. Gene-specific primers were designed for the genes of interest and are shown in Table 1. All the RT-PCR reactions were performed as described (Yu et al. 2007).

Table 1 Gene-specific primers used for real-time RT-PCR assays
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Results

MIC determination

Assays were first done in microplates as described in ‘Materials and methods’. The inhibition assays were repeated in large-scale experiments because the microarray experiments use 100-ml cultures and the MIC values may be different in small-scale and large-scale experiments. The MIC value was determined as 0.02 µg/ml for TRB, and the microplate and large-scale assay results were in agreement. These results showed that the T. rubrum clinical isolate BMU 01672 used here was susceptible to TRB.

Global analysis of the transcriptional response to TRB

A large number of genes were affected, both positively and negatively, by exposure to TRB. In total, 337 genes were expressed differentially upon exposure to TRB; 193 showed increases in expression and 144 showed decreases in expression. (The data sets’ GEO number is GSE6951.) The distribution of TRB-responsive genes and their biological roles are shown in Table S1 of the Electronic supplementary material.

Validation of microarray data by real-time RT-PCR

In order to validate the differential gene expression obtained by microarray analysis, ten genes were tested by real-time RT-PCR using the same RNA as that used in the original microarray experiments. There was a strong positive correlation between the two techniques, and different levels of induction or repression were found, five genes showed up-regulation and five genes showed down-regulation in response to treatment with TRB, which confirmed the reliability of the microarray data (Fig. 1).

Fig. 1
figure 1

The relative fold change for ten genes listed in Table 1 determined by quantitative real-time RT-PCR

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Discussion

The number and characteristics of the responsive genes are shown according to biological function in Table S1 of the Electronic supplementary material. In our large data pool, most responsive genes are classed as being of unknown function (141 genes), indicating that the EST has not been identified and there is no amino acid sequence homology with other proteins of known function.

In our experiment, transcription of some genes involved in lipid metabolism was affected in response to TRB (Table 2). Firstly, some genes of the ergosterol biosynthesis pathway, including genes encoding C-8 sterol isomerase (ERG2), sterol C-24 reductase (ERG4), C-4 sterol methyl oxidase (ERG25), and C-14 sterol reductase (ERG24), were down-regulated. Additionally, transcription of some genes involved in lipid metabolism, including ERG13 (3-hydroxy-3-methylglutaryl CoA synthase), ERG10 (acetoacetyl-coenzyme A thiolase), INO1 (myo-inositol-1-phosphate synthase), EB801711 (chloride peroxidase), DW696584 (1-acyl-sn-glycerol-3-phosphate acyltransferase), and DW703474 (acyl-CoA oxidase), was increased.

Table 2 Some genes differentially expressed upon exposure to TRB
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Ergosterol is a vital component of the fungal cell, but very little is known about the genetics and biochemistry of the ergosterol biosynthesis pathway in T. rubrum. Some ESTs have been sequenced, and we know that there are about 20 genes involved in the biosynthesis of ergosterol in T. rubrum. We were able to identify six of these genes as having their transcription modulated during the response to TRB. The common ergosterol biosynthesis pathway of the fungus and the effect of TRB on the expression of T. rubrum genes involved in the pathway are shown in Fig. 2.

Fig. 2
figure 2

Effect of TRB on the expression of T. rubrum genes involved in the ergosterol biosynthetic pathway. Boldfaces indicate genes with increased transcript levels and shaded areas indicate genes with decreased transcript levels. The sterol biosynthesis inhibitor (TRB) examined in this study was underlined and shown to the right of the gene encoding the targeted enzyme. CoA coenzyme A, HMG-CoA synthase 3-hydroxy-3-methylglutaryl CoA synthase, FPP synthase farnesyl pyrophosphate synthetase

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It is known that squalene epoxidase is the specific target of the antifungal drug TRB, which is a FAD-containing monooxygenase encoded by ERG1 (Favre and Ryder 1997; Klobucnikova et al. 2003; Leber et al. 2003). Squalene epoxidase is an essential enzyme in the ergosterol biosynthesis pathway. It catalyzes the epoxidation of squalene to 2,3-oxidosqualene. Squalene epoxidase plays a key role in the synthesis of essential sterol compounds, and homozygous disruption of ERG1 was found to have deleterious effects in yeast cells (Leber et al. 1998; Tsai et al. 2004). It has been shown that TRB interferes with ergosterol biosynthesis by inhibiting a membrane-bound squalene epoxidase (Ryder 1992). Incubation of yeast cells with these compounds leads to accumulation of squalene and depletion of ergosterol (Paltauf et al. 1982), which may be responsible for the fungicidal activity observed in vitro (Petranyi et al. 1987; Gupta and Shear 1997).

To our surprise, the expression of squalene epoxidase (ERG1) was not affected by TRB in our study. The same result was reported by Bammert and Fostel (2000), who showed that ERG1 expression did not change significantly under the conditions that they used for inhibition of ergosterol synthesis, including inhibition by TRB. But Leber et al. showed that inhibition of ergosterol biosynthesis with TRB increased the expression of ERG1 in a concentration-dependent manner (Leber et al. 2001). Thus, the expression of ERG1 in this work was not affected by TRB maybe because we used a concentration equivalent to 0.5 MIC. Bammert et al. found that, after incubation with the inhibitor for 90 min, ERG1 expression was only a little higher than that in the untreated control. Such a small increase may not be detected by measuring transcript levels (Bammert and Fostel 2000). Late exponential phase yeast cells accumulate steryl esters, while the concentration of free sterols is unaltered (Leber et al. 1995; Parks and Casey 1995). Under conditions of ergosterol synthesis inhibition, steryl esters are hydrolyzed and ergosterol levels remain sufficiently high to maintain membrane formation and growth for some time (Leber et al. 1995), and, thus, do not lead to induced ERG1 expression (Leber et al. 2001). Ergosterol has been reported to regulate its own synthesis by negative feedback mechanisms (Pinto et al. 1985; Servouse and Karst 1986; Casey et al. 1992; Smith et al. 1996).

In this experiment, we observed that genes ERG10 and ERG13 were up-regulated by TRB, which indicates that low levels of ergosterol increase the enzymatic activities of acetoacetyl-CoA thiolase and HMG-CoA synthase, two early enzymes in the ergosterol biosynthesis pathway (Servouse and Karst 1986), and this result is consistent with a previous report (Pasrija et al. 2005). The expression of ERG24, ERG4, and ERG25 was down-regulated, perhaps because they are functionally downstream of ERG1, and their reduction is in response to ergosterol depletion.

In this study, TRB induced the transcription of multidrug-resistance (MDR) genes, including DW686642, TruMDR2, and MDR1. The reduced intracellular accumulation has been correlated with over-expression of MDR efflux transporter genes of the ATP-binding cassette (ABC), and the major facilitator superfamily (MFS) classes (Lupetti et al. 2002, Fachin et al. 2006). Multidrug- and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in the treatment of pathogenic bacteria, fungi, parasites, and cancer cells (Paulsen et al. 1996). It has long been speculated that fungi have efflux pumps that allow them to avoid the effects of metabolic poisons present in the environment. Urban et al. proposed that the fungal transporters initiate associations between host and fungal cells (Urban et al. 1999).

We found that transcription of some genes encoding ribosomal proteins, including DW688173 (mitochondrial ribosomal protein L17), MRP7 (mitochondrial ribosomal protein MRP7), and DW700063 (40S ribosomal protein S8), was down-regulated significantly (see Table S1 of the Electronic supplementary material). Small reductions in the expression of ribosomal protein genes allow energy to be redistributed, which allows increased expression of genes involved in protective responses, while maintaining a basal level of protein synthesis (Vanden Bossche et al. 1987).

It was found that TRB induced some genes that encode stress response-related proteins in T. rubrum, such as DW708782 (HSP70) and DW708095 (HSP104), and oxidative-stress proteins such as DW703584 (stress response RCI peptide).

In addition, expression of the gene DW701480 encoding the CFEM domain protein was inhibited. CFEM might serve as a characteristic signature for a subset of proteins that function in the extracellular environment. Some CFEM-containing proteins are proposed to have important roles in fungal pathogenesis (Kulkarni et al. 2003), which may be of benefit in studying the interaction between pathogen and host.

In conclusion, the T. rubrum microarray studies described here revealed the changes of the transcriptional level of genes exposed to the allylamine antifungal agent TRB and laid the groundwork for an antifungal drug development program employing cDNA microarrays to identify gene expression profiles.