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1 Department of Dermatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
2 Department of Medical Microbiology and National Reference Center for Systemic Mycoses, University Hospital of Göttingen, Germany
3 Department of Biology, Faculty of Medicine, Olomouc, Czech Republic
4 Clinical Chemistry Laboratory, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
Correspondence
Michel Monod
Michel.Monod{at}chuv.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the AfuSSU1, AfuSSUL1, AfuSSUL2, TruSSU1 and AbeSSU1 sequences reported in this paper are AY861352, AY861353, AY861354, DQ777768 and EF035480, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Weitzman & Summerbell, 1995). These highly specialized pathogenic fungi are almost exclusively found in the epidermal stratum corneum, hair or nails. The battery of proteases secreted by dermatophytes is similar to that of Aspergillus, but differs by multiple endoprotease members of the S8 (subtilisins) and M36 (fungalysins) family (see MEROPS proteolytic enzyme database; http://merops.sanger.ac.uk) (Jousson et al., 2004a, b). Although it is evident that the secreted proteolytic activity of these fungi is important for degradation of compact keratinous tissues, fungal secreted proteases are incapable by themselves of dissolving structures made of hard keratin which are rich in cystine. Efficient keratin degradation by hydrolytic enzymes has to be accompanied by the simultaneous reduction of cystine disulphide bridges, which are mainly responsible for the resistant nature of keratin (Kunert, 1992, 2000). Moreover, efficient degradation in vitro of the hair structure of keratin azure by hydrolytic enzymes is only possible in the presence of a reducing agent, e.g. 1 % 
-mercaptoethanol or DTT (Jousson et al., 2004b).
During keratin degradation, dermatophytes and filamentous fungi have been shown to excrete sulphite as a reducing agent (Kunert, 1972a, 1976). In the presence of sulphite, disulphide bonds of the keratin substrate are directly cleaved to cysteine and S-sulphocysteine. The presence of sulphocysteine has been confirmed by histochemical methods in human hair attacked by Microsporum gypseum (Kunert, 1972b). This compound was found in the free form and in oligopeptides of 
700–2500 Da which were products of keratinolysis (Kunert, 1976; Ruffin et al., 1976). As highly specialized fungi, the dermatophytes are able to metabolize free cystine added to a nutrient broth, and excrete excess sulphur as sulphate and sulphite. Because sulphite is immediately consumed by sulphitolysis of disulphide bridges, this compound is only detectable when its amount exceeds that of cystine (Kunert, 1975, 2000).
The necessity to reduce disulphide bridges in the digestion of cornified tissues instigated our investigation of the mechanism of sulphite secretion in dermatophytes. In dermatophytes, as well as in the opportunistic mould Aspergillus fumigatus, we have identified genes encoding sulphite efflux pumps by heterologous expression in Saccharomyces cerevisiae, which results in a sulphite-resistant phenotype. Sulphite transporters in keratinolytic fungi could be a new target for antifungal drugs in dermatology.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and A. fumigatus D141 (NRRL 6585, US Department of Agriculture, Peoria, IL) were used in this study. All plasmid subcloning experiments were performed in Escherichia coli XL-1 Blue using plasmids pMTL21 (Chambers et al., 1988), pUC18 and pCL1920. The diploid S. cerevisiae strain INVSc1 (MATa, his3D1, leu2, trp1-289, ura3-52; MAT
, his3D1, leu2, trp1-289, ura3-52) and the expression vector pYES2 (Invitrogen) were used to express heterologous transporters.
Growth media.
T. rubrum, Arth. benhamiae and A. fumigatus were grown on Sabouraud agar and liquid medium (Bio-Rad), or in soy protein liquid medium (SP) and keratin liquid medium (KSP) (Monod et al., 2005) to promote production of proteolytic activity, as previously described.
Cystine–arginine liquid medium (CAM) [0.8 % (w/v) glucose, 0.2 % (w/v) arginine, 0.04 % (w/v) KH2PO4, 3 mM (0.072 %, w/v) cystine] (Kunert, 1982) was used to demonstrate secretion of sulphite. Cystine was added to the sterile medium in the form of a 10x concentrated solution in HCl (1 M) and the medium was neutralized with KOH. A volume of 100 ml liquid medium was inoculated with a plug of freshly growing mycelium in 800 ml tissue-culture flasks. The cultures were incubated for 10 days at 30 °C without shaking.
Complete medium for S. cerevisiae was YEPD [2 % (w/v) Difco Bacto peptone, 1 % (w/v) Difco Bacto yeast extract, 2 % (w/v) glucose]. S. cerevisiae synthetic minimal medium supplemented with histidine, leucine and tryptophan (20 µg ml–1) was prepared according to Sherman (1991). For expression of genes cloned in pYES2 under the control of the GAL promoter, galactose was added instead of glucose as the carbon source. YEPD+TA (YEPD containing 75 mM L-tartaric acid and buffered at pH 3.5) was prepared as described elsewhere (Park et al., 1999). YEPG+TA was prepared in the same way as YEPD+TA with 2 % galactose instead of glucose. YEPG+TA plates containing sulphite were prepared by spreading an appropriate amount of freshly made Na2SO3 stock solution (0.25 or 0.5 M) onto agar plates containing 25 ml medium, and allowing the sulphite to dry and diffuse overnight at room temperature.
Sulphite and S-sulphocysteine analysis.
Supernatants of cultures in SP and KSP media were first treated by Pronase (10 µg ml–1) (Roche Diagnostics) and porcine kidney aminopeptidase M (2 U ml–1) (Calbiochem) for 24 h at 37 °C. Free S-sulphocysteine was determined after deproteinization treatment of the sample with 32 % (w/v) sulphosalicylic acid. Amino acid analysis was performed by HPLC with a fluorescence detector, after precolumn derivatization with O-phthaldialdehyde-3-mercaptopropionate and 9-fluorenylmethyl-chloroformate (FMOC) (Henderson et al., 2000). The concentration of free sulphite was determined by the fuchsin–formaldehyde method (Scaringelli et al., 1967).
Expression of tellurite-resistance/dicarboxylate transporters (TDTs) in S. cerevisiae.
cDNAs encoding the transporters AfuSsu1p, AfuSsul1p, AfuSsul2p, TruSsu1p and AbeSsu1p were obtained by RT-PCR using a OneStep RT-PCR kit (Qiagen). Briefly, 20 ng total RNA, 10 µl of the supplied 5x OneStep RT-PCR buffer (12.5 mM MgCl2, pH 8.7), 2 µl deoxynucleotide mix containing 10 mM each dNTP, 5 µl sense and antisense primers (P1–P8, Table 1) at a concentration of 6 µM, and 2 µl OneStep RT-PCR enzyme mix were mixed on ice, and subsequently incubated at 50 °C for 30 min and 95 °C for 15 min. The reaction mixtures were then subjected to 35 cycles of 0.5 min at 94 °C, 0.5 min at 55 °C and 1 min at 72 °C, and finally incubated for 10 min at 72 °C. Alternatively, a cDNA library of A. fumigatus D141 (Denikus et al., 2005) was used to amplify cDNAs encoding A. fumigatus transporters. PCR was performed with the homologous primers P1–P6 (Table 1) and 200 ng of DNA prepared from 106 clones of the cDNA library, using a standard protocol (Jousson et al., 2004a, b).
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), and S. cerevisiae INVSc1 genomic DNA as a target. For amplification, a standard protocol was used (Jousson et al., 2004a, b). Expression plasmids were constructed by cloning PCR products in the S. cerevisiae expression vector pYES2. The PCR products were purified with a PCR purification kit (Roche Diagnostics) and then digested with restriction enzymes for which a site was previously designed at the 5' end of the primers. The cloned fragments were further sequenced and the absence of possible PCR-induced errors was confirmed. Plasmid DNA was prepared from one E. coli clone harbouring a correct construct. S. cerevisiae transformations were performed using a transformation kit and according to the recommendations of the supplier (Invitrogen). Selection of URA3 transformants was performed using minimal medium with glucose and required amino acids.
Transformants were tested for sulphite resistance using low-pH medium, as described by Park et al. (1999). Yeasts were grown to the mid-exponential phase (OD600 1.0) at 30 °C in minimal medium with galactose and required amino acids. Each culture was diluted to OD600 1.0. Subsequently, 5 µl of serial dilutions (100–104) were spotted onto YEPG+TA plates containing the desired concentration of sulphite. The plates were incubated at 30 °C for 2 days.
A. fumigatus gene disruption.
Gene disruption vectors were constructed using pAN7.1 (Punt et al., 1987) and 1.0 kb-sized internal fragments of the respective A. fumigatus gene. In detail, gene fragments were obtained by PCR using appropriate primers (P11–P16, Table 1) and genomic A. fumigatus DNA as the template. The PCR products were first cloned into pCR-Script Amp SK(+) (Stratagene). In a second step, the A. fumigatus fragments were excised from the plasmid constructs with StuI and BstEII, for which a site was introduced into the primers, and ligated to the larger fragment of pAN7.1 digested with the same restriction enzymes. The generated plasmids were termed p
SSU1, pSSUL1 and pSSUL2. Undigested plasmids were used for subsequent gene-targeted disruption experiments.
Transformation of A. fumigatus D141 was performed according to a protocol that has been used for Aspergillus nidulans and A. fumigatus (Tilburn et al., 1983; Paris, 1994) with 107 protoplasts and 5 µg of plasmid. After overnight expression of the hygromycin B phosphotransferase gene (HPH), the transformants were incubated on agar based on minimal medium (Cove, 1966) containing 200 µg hygromycin ml–1 (Sigma), and selected after 5 days incubation at 20 °C followed by an overnight incubation at 42 °C. Transformants initially identified as hygromycin resistant were chosen and subcultured again on agar containing hygromycin. Typically, 100–200 hygromycin-resistant colonies were obtained for each transformation using 107 protoplasts and 5 µg plasmid DNA.
The A. fumigatus disruptants were identified by PCR of genomic DNA from various numbers of hygromycin-resistant colonies as a template and two pairs of specific primers (P17–P28, Table 1). Each primer pair was designed to yield a product of a predicted size when the respective plasmid had integrated at a homologous site. In each primer pair, one primer hybridized with the transformation plasmid and the other primer hybridized with genomic DNA near the desired homologous integration locus, as exemplarily shown for the AfuSSU1 disruption (Fig. 1). The primer pairs P17 to P20, P21 to P24 and P25 to P28 (Table 1) were used for screening ssu1, ssul1 and ssul2 mutants, respectively.
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Sulphite resistance of A. fumigatus and dermatophytes.
A. fumigatus and dermatophytes were grown for 4 and 10 days, respectively, at 30 °C on Sabouraud medium. YEPD+TA plates containing the desired concentration of sulphite were inoculated with A. fumigatus conidia or dermatophyte mycelium. Alternatively, 100 ml liquid YEPD+TA medium containing sulphite was inoculated in 800 ml tissue-culture flasks. Dermatophyte and A. fumigatus cultures were incubated for 10 and 3 days, respectively, at 30 °C without shaking.
T. rubrum and Arth. benhamiae gene cloning.
Recombinant plaques (2x104) of a previously constructed T. rubrum 
EMBL3 genomic DNA library were immobilized on GeneScreen nylon membranes (NEN Life Science Products). The filters were hybridized with a 32P-labelled AfuSSU1 probe under low-stringency conditions (Monod et al., 1994). All positive plaques were purified, and the associated bacteriophage DNAs were isolated as described elsewhere (Grossberger, 1987). Agarose gel electrophoresis of restricted recombinant bacteriophage EMBL3 DNA, Southern blotting and subcloning of hybridizing fragments from bacteriophages into pMTL21 or pUC19 were performed using standard protocols (Sambrook et al., 1989). DNA sequencing was performed by Microsynth. T. rubrum and Arth. benhamiae cDNAs were obtained by RT-PCR using homologous primers designed from genomic DNA sequences.
RNA preparations and Northern-blotting experiments.
The RNA of filamentous fungi was prepared from 10-day-old 100 ml liquid cultures in different media. The mycelium was ground in liquid nitrogen to a fine powder using a mortar and pestle, and the total RNA was isolated using an RNeasy Plant Mini Kit (Qiagen). Northern-blotting experiments were performed as described by Sambrook et al. (1989).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of genes encoding A. fumigatus and dermatophyte sulphite efflux pumps
The high concentrations of S-sulphocysteine and sulphite in dermatophyte culture supernatants in cystine media led us to identify sulphite efflux pumps in these fungi. A sulphite efflux pump, Ssu1 (called in this paper SceSsu1p), has been characterized elsewhere in S. cerevisiae (Avram & Bakalinsky, 1997). Overexpression of the gene SceSSU1 enhances the resistance of S. cerevisiae to sulphite, which is used as a preservative agent in fermentations (Park et al., 1999; Park & Bakalinsky, 2000; Donalies & Stahl, 2002). SceSsu1p belongs to the TDT family, which includes the E. coli tellurite transporter and the Schizosaccharomyces pombe malate transporter encoded by the genes TEHA and MAEI, respectively (Walter et al., 1991; Grobler et al., 1995; see the TransportDB database, http://www.membranetransport.org/). Our strategy to find sulphite efflux pumps in dermatophytes consisted of trying to isolate the SceSSU1 orthologue in A. fumigatus for which the genome sequence is available. If a hit was obtained, the orthologues in dermatophyte genomes could subsequently be sought. Indeed, it is known that the nucleotide sequences of dermatophyte genes exhibit 50–70 % identity to the orthologous genes of Aspergillus spp. (Jousson et al., 2004a, b; Monod et al., 2005).
A BLASTP analysis with SceSsu1p on the A. fumigatus Af293 genome (www.tigr.org/tdb/e2k1/afu1) revealed seven genes encoding putative transporters of the TDT family. Three transporters with the highest similarity to SceSsu1p, corresponding to Afu7g01790, Afu3g14640 and Afu1g13360 in the TransportDB database, were retained for gene expression experiments in S. cerevisiae (Fig. 2). Three corresponding cDNAs could specifically be amplified using 5'-sense and 3'-antisense primers (P1–P6, Table 1) by RT-PCR of A. fumigatus D141 RNA, or by PCR based on DNA extraction from a pool of 106 clones of a previously constructed cDNA library as the template. The nucleotide sequences of the obtained cDNA fragments were 100 % identical to sequences in the A. fumigatus Af293 genome from which introns were removed. However, their deduced amino acid sequences revealed some differences from the deposited amino acid sequences of Afu7g01790, Afu3g14640 and Afu1g13360 because of differences in intron positions. The three A. fumigatus cDNAs were cloned into the S. cerevisiae expression vector pYES2 under control of the GAL promoter, and the resulting plasmids were transformed into S. cerevisiae. Only transformants encoding one of the three transporter genes, corresponding to Afu7g01790, showed increased sulphite resistance on galactose media (Fig. 3). This gene was called AfuSSU1 (GenBank accession no. AY861352). The two other genes were called AfuSSUL1 and AfuSSUL2, encoding Ssu-like protein 1 and Ssu-like protein 2 in A. fumigatus (GenBank accession nos AY861353 and AY861354, respectively). AfuSSU1, AfuSSUL1 and AfuSSUL2 revealed similar collinear structures with four introns and five exons.
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), while the resistance to sulphite of ssul1 and ssul2 mutants was comparable to that of the wild-type strain of the fungus. These results confirmed that only AfuSSU1 encodes a sulphite efflux pump among the three selected genes of A. fumigatus encoding transporters of the TDT family. However, the A. fumigatus ssu1 mutants grew in a similar manner to the wild-type strain in SP and KSP media, and S-sulphocysteine was detected in the culture supernatant in KSP medium.
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), the deduced amino acid sequences revealed proteins with hydrophilic N and C termini and 10 putative membrane-spanning helices which are typical of membrane transport proteins (Fig. 5). The length of the hydrophilic linkers ranged from 3 to 20 amino acids.
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EMBL3 phage T. rubrum genomic DNA library. All isolated hybridizing clones contained a nucleotide sequence encoding a gene 50 % identical to AfuSSU1. The corresponding cDNA encoding a putative sulphite transporter could be specifically amplified by RT-PCR using 5'-sense and 3'-antisense primers (Table 1) and total RNA extracted from T. rubrum mycelium in cystine–arginine medium. An Arth. benhamiae cDNA encoding a putative transporter differing from that of T. rubrum by two amino acids (Val63 and Ala206 in Arth. benhamiae instead of Ile63 and Val206 in T. rubrum) was also amplified using the same primers. S. cerevisiae became resistant to 1.6 mM sulphite when these amplified cDNAs were expressed under the control of the GAL promoter (Fig. 3). The genes encoding these sulphite transporters in T. rubrum and Arth. benhamiae were called TruSSU1 and AbeSSU1 (GenBank accession nos DQ777768 and EF035480, respectively). TruSSU1 and AbeSSU1 revealed an intron–exon structure similar to that of AfuSSU1. Both TruSSU1 and AbeSSU1 were predicted to encode proteins of 375 amino acid residues with molecular masses of approximately 42 kDa, hydrophilic N and C termini, and 10 putative membrane-spanning helices (Fig. 5).
Expression of sulphite transporter genes in A. fumigatus and dermatophytes
Northern-blot analysis revealed that TruSSU1 and AbeSSU1 encode single transcripts of approximately 2000 bp each. Expression of TruSSU1 and AbeSSU1 in cystine–arginine and KSP media was higher than that in Sabouraud and SP media (Fig. 6). Although AfuSSU1 DNA could be amplified by RT-PCR with RNA extracted from A. fumigatus grown in YEPD+TA containing 1.2 mM sulphite (300 mM below the MIC), SP and KSP media, no AfuSSU1 signal was detected in comparably sensitive Northern blotting experiments and utilizing the whole AfuSSU1 cDNA as a probe.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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AfuSsu1p, TruSsu1p and AbeSsu1p are only 20 % identical to SceSsu1p. In contrast, A. fumigatus transporters of the TDT family which are not sulphite efflux pumps are more similar to AfuSsu1p (Fig. 2). Alignments of TDT transporters included in Fig. 2 show that the identified sulphite efflux pumps have only two short conserved amino acid sequences (Fig. 7). Two potential signature sequences for sulphite transporters in the TDT family could be derived from the portions of the alignment shown in Fig. 7 as follows:
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(2) LP(I/L)GP(L/M)GQG(G/S)(F/Y)G
Sulphite is a product of the metabolism of cysteine compounds assimilated by fungi. Although a regular fungal metabolite, sulphite is potentially toxic in the cytoplasm (Thomas & Surdin-Kerjan, 1997). Excess sulphite is generally excreted by fungi after oxidation in the form of inorganic sulphate (Obata & Ishikawa, 1959; Kunert, 1989). Excretion of sulphite mediated by an efflux pump represents an alternative detoxification pathway for dermatophytes during infection of the epidermal stratum corneum, hair and nails, which are rich in cysteine. At the same time, the ability of dermatophytes to grow within hard keratin depends on the secretion of sulphite in order to reduce proteins. Once disulphide bridges are directly cleaved by sulphitolysis, reduced proteins become accessible to further digestion by various secreted endo- and exoproteases. Therefore, efflux-pump-mediated sulphite detoxification and sulphitolysis may be considered as complementary functions in the digestion of compact keratinous tissue.
The relatively high expression of TruSSU1 and AbeSSU1 compared to that of AfuSSU1 (Fig. 6) likely reflects a property of dermatophytes which renders these fungi pathogenic in the epidermal stratum corneum, hair and nails. Dermatophytes were able to grow more rapidly than A. fumigatus in KSP medium. Hard keratin grains were totally digested after 20 days under our culture conditions, while 40 days were necessary to observe clarification of the medium with A. fumigatus. Although sometimes isolated as a contaminant from nails, the latter fungus is not an aetiologic agent of infection in hard keratinous tissues. Apparently, A. fumigatus growth in keratin-containing medium does not depend on AfuSsu1 activity, since the A. fumigatus ssu1 mutants were able to grow well in KSP medium, and S-sulphocysteine was detected in the culture supernatant. Thus, the existence of another sulphite efflux pump cannot be excluded in A. fumigatus, but it is also possible that small amounts of sulphite can leave the mycelium by a route other than that of sulphite transporters.
No fewer than 16 genes encoding secreted endo- and exopeptidases have been found in dermatophytes (Jousson et al., 2004a, b; Monod et al., 2005). The hypothesis that one keratinase, alone or with further proteases, decomposes hard keratin, has now been abandoned (Kunert, 1992). Dermatophyte-secreted subtilisins have been shown to be at most 50 % more active towards keratin azure than proteinase K or subtilisin Carlsberg, with the last two enzymes both in the presence of -mercaptoethanol (Jousson et al., 2004b). Sulphitolysis is so far the sole known dermatophyte mechanism that allows the reduction of disulphide bridges, a bottleneck in the process of degradation of compact keratinized tissues. In contrast, the activities of the dermatophyte-secreted proteases are redundant for the digestion of reduced protein (Jousson et al., 2004b; Monod et al., 2005). Therefore, sulphite transporters, rather than secreted proteases, could be a target of choice to treat dermatophyte infection. Given the absence of TDT transporters in humans, mechanism-based toxicity would most likely be minimal.
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ACKNOWLEDGEMENTS |
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Edited by: N. L. Glass
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 10 October 2006;
revised 23 November 2006;
accepted 7 December 2006.
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