Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum

Olivier Jousson1, Barbara Léchenne1, Olympia Bontems1, Sabrina Capoccia1, Bernard Mignon2, Jachen Barblan3, Manfredo Quadroni3 and Michel Monod1

1 Dermatology Service (DHURDV), Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
2 Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, Belgium
3 Protein Analysis Facility, Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland

Correspondence
Michel Monod
Michel.Monod{at}chuv.hospvd.ch


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55–75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72–97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19–36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.


GenBank accession numbers are given in Table 1.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections (Weitzman & Summerbell, 1995). Among approximately 10 human pathogenic species isolated in Europe, Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis are most commonly observed, accounting for 72–95 % of the species isolated in hospital and private practices (Monod et al., 2002a). A characteristic of the dermatophytes is their ability to grow exclusively in the stratum corneum, nails or hair and to digest components of the cornified cell envelope. All investigated dermatophytes produce proteolytic activity in vitro (Monod et al., 2002b). There are many reports of the isolation and characterization of one or two proteases from an individual species of dermatophyte (e.g. Asahi et al., 1985; Tsuboi et al., 1989; Brouta et al., 2001), often described as keratinases without paying attention to the presence of other components in the cornified cell envelope. Different studies have shown that these enzymes play an important role in the provision of nutrients (Apodaca & McKerrow, 1989a), in host tissue invasion (Apodaca & McKerrow, 1989b) and in the control of host defence mechanisms (Grappel & Blank, 1972; Collins et al., 1973). With the exception of a 43·5 kDa M. canis zinc metalloprotease (Brouta et al., 2001), most of the proteases secreted by dermatophytes are characterized as serine proteases or, at least, the effect of various inhibitors supports the view that they belong to this class of proteases. The M. canis zinc metalloprotease was found to be homologous to the secreted 45 kDa A. fumigatus metalloprotease, for which the family of fungalysins was created [MEROPS database (http://merops.sanger.ac.uk/) family M36].

A small number of proteases from dermatophytes have been characterized at the gene level. In M. canis, a three-member metalloprotease (fungalysin) gene family (Brouta et al., 2002) including the previously characterized 43·5 kDa protease (Brouta et al., 2001), as well as a subtilisin gene family (Descamps et al., 2002) including a previously characterized 31·5 kDa protease (Mignon et al., 1998), were identified. In T. rubrum, a single serine protease has been characterized at gene level (Woodfolk et al., 1998).

To date, there has to our knowledge been no study comparing proteases isolated from different dermatophyte species. Here, we show that T. rubrum, T. mentagrophytes and M. canis possess a five-member gene family encoding secreted metalloproteases. A phylogenetic analysis revealed that the metalloproteases secreted in vitro by the three species are encoded by orthologous genes, strongly suggesting that the multiplication of an ancestral metalloprotease gene occurred prior to species divergence in dermatophytes.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and plasmids.
Clinical isolates of T. rubrum, T. mentagrophytes and M. canis from patients at the University Hospital Lausanne (Switzerland) were used. All isolates were grown on Sabouraud agar and were identified on the basis of macroscopic appearance, microscopic examination of the cultures and partial 28S rDNA sequencing (Ninet et al., 2003). One isolate of each species, T. rubrum (LAU862-01), T. mentagrophytes (LAU2434-02) and M. canis (LAU709-03) were chosen for preparing gene libraries and protein extract analysis.

Escherichia coli LE392 was used for the propagation of the bacteriophage {lambda}EMBL3 (Promega). All plasmid subcloning experiments were performed in E. coli XL-1 Blue with plasmids pMTL21 (Chambers et al., 1988) and pUC19 (Sambrook et al., 1989).

Growth media.
T. rubrum, T. mentagrophytes and M. canis isolates were grown on Sabouraud agar and liquid medium (Bio-Rad) or, to promote production of proteolytic activity, in liquid medium containing protein as a sole nitrogen and carbon source. Protein medium was prepared by dissolving 0·2 % soy protein (Supro 1711, Protein Technologies International) in distilled water. No salt was added. The medium was sterilized by autoclaving at 120 °C for 15 min. 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.

Proteolytic assays.
The proteolytic activity was measured using resorufin-labelled casein (Roche Diagnostics). The reaction mixture contained 0·02 % substrate, Tris/HCl buffer (20 mM; pH 7·4), and 20 µl of culture supernatant in a total volume of 500 µl. After incubation at 37 °C for 30 min, the undigested substrate was precipitated by trichloroacetic acid (5 % final concentration) and separated from the supernatant by centrifugation. Five hundred microlitres of Tris/HCl buffer (500 mM; pH 9·4) was added to the collected supernatant (neutralization step) and the A574 of the mixture (1 ml) was measured. For practical purposes, one arbitrary unit (U) of proteolytic activity was defined as that producing an absorbance change of 0·001 min-1.

Construction of genomic and cDNA libraries.
Genomic DNA libraries were prepared using DNA isolated from freshly growing mycelium (Yelton et al., 1984) of T. rubrum, T. mentagrophytes and M. canis. The DNA was partially digested with Sau3A, and DNA fragments ranging from 12 to 20 kb were isolated from low-melting-point agarose (Bio-Rad) with agarase (Roche Diagnostics). These fragments were inserted into the bacteriophage {lambda}EMBL3 cloning system (Promega). A T. rubrum cDNA library was prepared in pSPORT6 plasmid (Invitrogen Life Technologies) using the micro quantity mRNA system and 500 µl of total RNA. The RNA was prepared from 10-day-old cultures in soy protein liquid medium (10x100 ml). The mycelium was ground under liquid nitrogen to a fine powder using a mortar and pestle, and the total RNA was isolated using a RNeasy total RNA purification kit (Qiagen).

MEP gene cloning.
Recombinant plaques (2x104) of the genomic libraries of T. rubrum and T. mentagrophytes were immobilized on GeneScreen nylon membranes (NEN Life Science Products). The filters were hybridized with a 32P-labelled PCR probe under low-stringency conditions (Monod et al., 1994). The probe was obtained by PCR amplification of partial MEP genes from T. rubrum, T. mentagrophytes or M. canis genomic DNA (see Results). PCR amplifications were performed in a total volume of 100 µl consisting of 5 mM MgCl2, 2·5 µl of each primer at a concentration of 40 µM, 3 µl deoxynucleotide mix (containing 10 mM of each dNTP), 2·5 units Taq DNA polymerase (Roche Diagnostics) and 1 µl DNA. The amplification profile consisted of 35 cycles of 30 s at 94 °C, 30 s at 52 °C and 120 s at 72 °C, followed by 5 min at 72 °C for final extension. All positive plaques were purified and the associated bacteriophage DNAs were isolated as described previously (Grossberger, 1987). Agarose gel electrophoresis of restricted recombinant bacteriophage {lambda}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 (Balgach, Switzerland).

T. rubrum cDNAs were obtained by PCR using 200 ng DNA prepared from 106 clones of the cDNA library. PCRs were performed with homologous primers designed on DNA sequences of the different MEP genes.

Phylogenetic analyses.
Nucleotide and amino acid MEP sequences were aligned using CLUSTAL W (Thompson et al., 1994) as implemented in the BioEdit Sequence Alignment Editor software (Hall, 1999). Phylogenetic analyses of MEP genes and proteins were performed in PAUP* v4.0b10 (Swofford, 1998) using orthologous fungalysins from A. fumigatus (accession no. Z30424) and A. oryzae (accession no. AF099904) as an outgroup. DNA and protein sequences were analysed using the maximum-parsimony (MP) method, the neighbour-joining (NJ) method and the maximum-likelihood (ML) method. Introns were excluded from the analyses. MODELTEST 3.06 (Posada & Crandall, 1998) was used to select the appropriate model of substitution for ML and NJ analyses of nucleotide sequences. The Dayhoff PAM model of protein evolution was used to compute the distances between the amino acid sequences using the PROTDIST program implemented in BioEdit. All analyses were performed using heuristic search with TBR branch swapping algorithm and random addition of taxa (10 replicates). The reliability of internal branches was assessed using the bootstrap method (Felsenstein, 1988), with 500 replicates. Phylogenetic trees were edited using TreeView (Page, 1996).

Analysis of protein extracts.
Secreted proteins were precipitated from T. rubrum, T. mentagrophytes and M. canis culture supernatants using trichloroacetic acid and purified using PlusOne SDS-PAGE Clean-Up Kit (Amersham). Extracts were analysed by SDS-PAGE (Laemmli, 1970) with a separation gel of 8 % polyacrylamide. Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad). Measures of band molecular mass and optical density were performed using the TotalLab software (Nonlinear Dynamics). Immunoblots were performed using rabbit antisera and peroxidase-conjugated goat anti-rabbit IgG (Amersham Pharmacia) as secondary labelled antibodies. Rabbit antisera to A. oryzae secreted neutral protease (NpI) of the fungalysin family (Doumas et al., 1999) were made by Eurogentec (Liège, Belgium) using purified recombinant enzyme. Protein concentrations were measured by the Bradford method using a commercial reagent (Bio-Rad).

Treatment with N-glycosidase F.
Culture supernatants were 20-fold concentrated by ultrafiltration on a Centricon-30 (Amicon). Ten microlitres of the concentrate containing approximately 5 µg protein was diluted in a solution of 25 mM sodium phosphate (pH 7·0), 25 mM EDTA, 0·15 % SDS and then heated at 100 °C for 5 min. After cooling, 10 % Nonidet P-40 and 0·6 U N-glycosidase F (Boehringer) were added (final detergent concentrations: 0·1 % SDS, 0·5 % Nonidet P-40), and the mixture was incubated at 37 °C for 20 h. The reaction was stopped by adding SDS-PAGE sample loading buffer, followed by incubation at 100 °C for 5 min.

Protein identification by liquid chromatography (LC)-MS/MS.
Coomassie-blue-stained bands were excised from the SDS-PAGE gels and transferred to special 96-well plates (Perkin Elmer Life Sciences). In-gel proteolytic cleavage with sequencing-grade trypsin (Promega) was performed automatically in the robotic workstation Investigator ProGest (Perkin Elmer Life Sciences) according to the protocol of Wilm et al. (1996). Supernatants containing proteolytic peptides were analysed by LC-MS/MS on a SCIEX QSTAR Pulsar (Concord, Ontario, Canada) hybrid quadrupole-time of flight instrument equipped with a nanoelectrospray source and interfaced to an LC-Packings Ultimate (Amsterdam, Netherlands) HPLC system. Peptides were separated on a PepMap reversed-phase capillary C18 (75 µm i.d.x15 cm) column at a flow rate of 200 nl min-1 along a 52 min gradient of acetonitrile (0–40 %). The instrument-controlling Analyst software was used to perform peak detection and automatically select peptides for collision-induced fragmentation. Collections of non-interpreted collision-induced fragmentation spectra were bundled and used for searching an in-house built database of T. rubrum, T. mentagrophytes and M. canis fungalysin sequences (see Table 1) with the Mascot software (www.matrixscience.com) (Perkins et al., 1999). Only peptide sequences ranked as statistically significant were taken into account, and all peptide hits were manually examined for validation.


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Table 1. Main characteristics of MEP genes and deduced translation products from A. fumigatus (Afum), A. oryzae (Aory), T. rubrum (ru), T. mentagrophytes (me) and M. canis (ca)

The theoretical molecular mass of the mature domain and the pI were calculated using VectorNTI suite 8 (InforMax). The putative glycosylation sites correspond to the NXT/S pattern (X: any amino acid except for P).

 

   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Proteolytic activities
All T. rubrum, T. mentagrophytes and M. canis isolates grew well at 30 °C in a medium containing 0·2 % soy protein as a sole carbon and nitrogen source. After 10 days of growth, clarification of the culture medium was observed. At this time, the amount of protein was 20–50 µg ml-1 in T. rubrum and T. mentagrophytes and 20–25 µg ml-1 in M. canis culture supernatants. Concomitantly, a substantial proteolytic activity measured using resorufin-labelled casein as substrate (200–250, 250–450 and 50–100 U ml-1 for T. rubrum, T. mentagrophytes and M. canis isolates, respectively). This proteolytic activity was reduced more than by half by o-phenanthroline and totally inhibited by o-phenanthroline and PMSF. These results attested that serine and metalloproteases were secreted by the three dermatophyte species and that the metalloprotease activity was rather dominant.

Analysis of protein extracts
SDS-PAGE electrophoretic profiles of proteins extracted from T. rubrum, T. mentagrophytes and M. canis consisted of 13–19 bands ranging from 31 to 111 kDa (Fig. 1). The protein profile was similar for 10 isolates of T. rubrum, T. mentagrophytes and M. canis and appeared to be species-specific (data not shown). Western blotting using A. oryzae anti-NpI antibody (Doumas et al., 1999) revealed four proteins of 42, 43, 45 and 46 kDa in T. rubrum and T. mentagrophytes, and three proteins of 42, 44 and 46 kDa in M. canis (Figs 1 and 2a). The optical density of these protein bands was 25·6 %, 36·5 % and 19·2 % of that of the total proteins secreted by T. rubrum, T. mentagrophytes and M. canis, respectively. These proteins cross-reacting with NpI were glycosylated as attested by an apparent reduction of their molecular mass following N-glycosidase F treatment (Fig. 2b).



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Fig. 1. Protein electrophoretic profiles from culture supernatant of M. canis (ca), T. rubrum (ru) and T. mentagrophytes (me). The proteins of 20 ml of dermatophyte culture supernatant were precipitated using trichloroacetic acid and purified using PlusOne SDS-PAGE Clean-Up Kit (Amersham). The proteins were resuspended in a total volume of 40 µl of loading buffer and separated by SDS-PAGE. The 8 % polyacrylamide SDS-PAGE gel was stained with Coomassie brilliant blue. Positions of molecular mass markers are shown on the left (phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; bovine carbonic anhydrase, 31 kDa). Bands marked by a circle were those revealed by anti-Mep Western blotting (see Fig. 2): open circles indicate a dominance of Mep4; solid circles indicate a dominance of Mep3 (see Table 3).

 


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Fig. 2. Western blot analyses of M. canis (ca), T. rubrum (ru) and T. mentagrophytes (me) extracts using A. oryzae anti-NpI antisera. (a) Untreated extracts; (b) extracts treated with N-glycosidase F as described in Methods. Approximately 0·5 µg protein was loaded per lane.

 
MEP gene cloning
A sequence alignment of all available genes encoding fungalysins (A. fumigatus MEP, A. oryzae NPI, M. canis MEP1, MEP2 and MEP3) (Jaton-Ogay et al., 1994; Doumas et al., 1999; Brouta et al., 2002) was performed. Consensus forward primer M1 (5'-GCCAAITTTGCIACICCICCIGATGG-3') (I=inosine) and reverse primer M3 (5'-CCGTGITTITCIATIARGTTCCA-3') were designed at positions 1278 and 1766, respectively, of the A. fumigatus MEP gene and were used to amplify T. rubrum and T. mentagrophytes genomic DNA. To estimate the number of genes encoding metalloproteases in each genome, the PCR product was first used as a probe in a Southern blot of SalI- and BamHI-digested genomic DNA of T. rubrum, T. mentagrophytes and M. canis, which revealed between three and five hybridizing bands (data not shown).

The PCR product was subsequently used as a probe to screen 2x104 individual recombinant bacteriophages of the {lambda}EMBL3 genomic libraries corresponding to about 10 genomic equivalents of the fungi. Twenty-four and 30 hybridizing clones were obtained for T. rubrum and T. mentagrophytes, respectively. All hybridizing clones were purified and bacteriophage DNA was isolated. PCR amplifications using the consensus metalloprotease primers M1 and M3 were performed on each bacteriophage DNA. Sequencing of all PCR products and translated BLAST searches (BLASTX) (http://www.ncbi.nlm.nih.gov/blast/) revealed four different sequences encoding parts of Mep proteins for both Trichophyton species. Restriction fragments hybridizing with the MEP probe were identified by Southern blotting, subcloned in pUC19 or pMTL21 and sequenced. Nucleotide sequences encoding putative full-length MEP preproproteins subsequently designated as MEP1, MEP2, MEP4 and MEP5 for T. rubrum, and as MEP1, MEP3, MEP4 and MEP5 for T. mentagrophytes, were obtained. The alignment of dermatophytes and Aspergillus Mep proteins showed that all sequences contained an HEXXH motif characteristic of zinc-containing metalloproteases of the M36 fungalysin family.

A preliminary phylogenetic tree including three previously known MEP sequences from M. canis (MEP1, MEP2 and MEP3), and the four new MEP sequences obtained from T. rubrum (MEP1, MEP2, MEP4 and MEP5) and from T. mentagrophytes (MEP1, MEP3, MEP4 and MEP5) revealed five robust clusters containing sequences belonging to two or three of the analysed species. To find the putatively missing MEP genes, consensus primers were designed on sequences included in each of the five clusters and PCR amplifications were performed on genomic DNA of each of the three species. PCR products were subsequently used as probes for screening of genomic libraries of T. rubrum, T. mentagrophytes and M. canis. Two additional genes encoding metalloproteases were found in M. canis (MEP4 and MEP5), one gene in T. rubrum (MEP3), and one gene in T. mentagrophytes (MEP2). As a result, a total of five genes encoding metalloproteases (MEP1–5) were isolated from T. rubrum and T. mentagrophytes, and two new genes (MEP4–5) were isolated from M. canis. MEP gene properties and GenBank accession numbers are given in Table 1.

Phylogenetic analyses
The analysed MEP nucleotide dataset comprised 1977 sites, among which 1296 are variable (65·6 %) and 1163 are informative (58·8 %). The amino acid dataset consisted of 665 sites, among which 404 are variable (60·8 %) and 355 are informative (53·4 %). Hierarchical likelihood ratio tests indicated that TrN (Tamura–Nei)+G (gamma distribution shape parameter)+I (proportion of invariable sites) was the most appropriate model of nucleotide substitution for subsequent analyses. Settings for the TrN+G+I model were as follows: base frequencies of 0·2437 (A), 0·3127 (C), 0·2354 (G), 0·2083 (T); substitution rates of 1·0000 (A–C), 2·3415 (A–G), 1·0000 (A–T), 1·0000 (C–G), 5·0882 (C–T), 1·0000 (G–T); a gamma distribution shape parameter ({alpha}) of 2·0279; 24·9 % of invariable sites. The analyses of MEP nucleotide and amino acid sequences using NJ, MP and ML phylogenetic methods produced the same tree topology, with strong bootstrap support (Fig. 3). MEP genes isolated from dermatophytes appeared to be monophyletic using A. fumigatus MEP and A. oryzae NPI as an outgroup. The tree consisted of five main clades, each of them including a MEP sequence type from each dermatophyte species. Within each MEP type cluster, the branching order of sequences reflected the taxonomic relationships of species, with Microsporum branching basally to Trichophyton. On the basis of the tree topology, MEP genes from T. rubrum and T. mentagrophytes were named following the nomenclature of their orthologues from M. canis (MEP1, MEP2, MEP3) (Brouta et al., 2002). The new MEP genes from the three species were named MEP4 and MEP5 (Tables 1 and 2).



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Fig. 3. Maximum-likelihood (ML) phylogenetic tree inferred from MEP nucleotide sequences. Orthologous sequences of each MEP type are shown in shaded boxes. Solid circles: sequences from M. canis (ca); open triangles: sequences from T. rubrum (ru); open squares: sequences from T. mentagrophytes (me). Bootstrap values (500 replicates) are given for amino acid (above branches) and nucleotide (below branches) sequences. Arrows at nodes indicate the gene duplication events. Scale bar: number of substitutions per site.

 

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Table 2. Pairwise amino acid sequence comparisons between A. fumigatus (Afum), A. oryzae (Aory), T. rubrum (ru), T. mentagrophytes (me) and M. canis (ca) Mep preproproteins

Above diagonal, amino acid identity; below diagonal, amino acid similarity. Bold values correspond to intra-type identity/similarity.

 
Characterization of dermatophyte MEP genes
The G+C content of dermatophyte MEP genes ranged from 48·8 to 56·3 mol% (Table 1). The comparison of open reading frames predicted from nucleotide sequences revealed a collinear intron–exon structure between T. rubrum, T. mentagrophytes and M. canis MEP genes. The PCR amplifications of MEP1–5 from the T. rubrum cDNA library gave a single product of about 1900 bp (data not shown) and the subsequent sequencing of PCR products confirmed the intron–exon structure. All MEP genes have four introns located at conserved positions in the three species, with the exception of MEP2 genes, which lack intron numbers 2, 3 and 4. Phases of intron numbers 1–4 are of type 0, 2, 1 and 2, respectively (Fig. 4), and are conserved among MEP types isolated from the three species. Among the deduced translation products, there was a high degree of intra-type amino acid sequence identity, ranging from 72 % (in Mep4) to 92 % (in Mep3). The inter-type amino acid sequence identity ranged from 57 % to 80 %. The percentage identity of dermatophyte Mep proteins with A. fumigatus Mep and A. oryzae NpI ranged from 59 % to 64 % (Table 2).



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Fig. 4. Intron–exon structure of T. rubrum MEP genes. The structure of A. fumigatus MEP is shown for comparison. Arrows on ruMEP1 represent the position of the DNA encoding the putative signal sequence cleavage site (solid arrow) and the putative propeptide cleavage site (open arrow). Numbers above sequences correspond to the nucleotide positions. Numbers below sequences indicate the intron phase. Intron phases are defined as the relative positions of introns falling between codons (phase 0) or within a codon after the first (phase 1) or second (phase 2) nucleotide, respectively.

 
The previously characterized N-terminus of mature Mep3 from M. canis (Brouta et al., 2001) and the analysis of the Mep sequence alignment suggested that dermatophyte Meps would be synthesized as preproproteins with a propeptide of 224–242 amino acid residues. The mature domain generated after cleavage of the prosequences predicted a molecular mass ranging from 42·46 to 43·27 kDa. The pI of mature Meps varied from 6·9 to 9, with the exception of Mep2 proteins, whose pI ranged from 4·5 to 4·9. The number of putative glycosylation sites in mature Meps varied from 1 to 5 (Table 1).

Mep identification by protein digestion and mass spectrometry
The Mep protein bands previously identified by Western blotting were subsequently analysed using trypsin fingerprinting. The secreted metalloproteases essentially corresponded to Mep types 3 and 4 for the three species. Mep1 was unambiguously identified in T. rubrum extracts (Ru45 band in Table 3) but neither Mep2 nor Mep5 could be detected. Due to the presence of glycosylated and processed forms of all proteases, often the gel bands proved to contain both Mep3 and Mep4 proteins. However, the predominance of one Mep relative to the other was always evident for each band. The results of mass spectrometric analysis are summarized in Table 3. The MASCOT score (Perkins et al., 1999) can be taken as a very raw measure of the numbers and signal intensity of peptides recovered and therefore of the relative amount of proteins present in each band (Fig. 1, Table 3). None of the other bands in Fig. 1 corresponded to a translation product of the 15 MEP genes considered in the present work (data not shown).


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Table 3. Protein sequences matched to gel bands from Fig. 1 and their MASCOT scores

The MASCOT score is defined as -10.log10(P), where P is the absolute probability that the observed match is a random event. MASCOT also calculates a confidence threshold which corresponds to a 95 % probability of correctness (p=0·05). For all spectral matching performed in this study the threshold was at 12. All the matches reported were considerably above this value and all the peptide matches added up to calculate the score were manually validated.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We report the isolation and characterization of five genes (named MEP1–5) encoding metalloproteases of the fungalysin family in T. rubrum and T. mentagrophytes, as well as two new genes in M. canis (MEP4–5) in addition to the three (MEP1–3) previously isolated (Brouta et al., 2002). Among several tested protein sources, soy proteins were found to be the best for promoting proteolytic activity of dermatophytes (data not shown). Apparently, all proteins of the medium are digested by the fungus after growing for 10 days at 30 °C as attested by clarification of the medium. Identifying proteases secreted by dermatophytes using proteolysis and mass spectrometry saved a lot of work and energy since it was not necessary to first isolate and purify the proteins. In culture medium containing soy proteins as a sole nitrogen source, T. rubrum, T. mentagrophytes and M. canis secreted two major Meps (Mep3 and Mep4) encoded by orthologous genes, although full-length cDNA of all MEP types was found to be present in the T. rubrum cDNA library. Several putative glycosylation sites were identified in the Meps (Table 1). The multiplicity of Mep3 and Mep4 protein bands can be explained by different levels of glycosylation (Fig. 2).

Most of the proteases isolated and characterized by different authors from dermatophyte culture supernatants have a molecular mass varying between 27 and 48 kDa under reducing conditions. A T. mentagrophytes 48 kDa protease (Yu et al., 1968) may be meMep4 and a 45 kDa M. canis protease (Takiuchi et al., 1982, 1984) is probably caMep3 or caMep4. In contrast, the T. rubrum keratinolytic protease active as a dimer with subunits of 44 kDa (Asahi et al., 1985) and the T. mentagrophytes 41 kDa protease (Tsuboi et al., 1989) are not fungalysins but serine proteases as attested by the effect of different inhibitors.

Drawing inferences about evolutionary history from a phylogeny of a gene family requires that the tree be correctly rooted. Since MEP genes are unique in Aspergillus (Monod et al., 1993; Doumas et al., 1999), and as Aspergillus and dermatophytes belong to the same taxonomic group of Ascomycetes producing prototunicate asci in cleistothecia (class Eurotiomycetes), fungalysins from Aspergillus spp. are potentially appropriate ancestral genes for rooting a dermatophyte gene phylogeny. The phylogenetic analyses of nucleotide and amino acid sequences demonstrated a monophyletic origin of dermatophyte MEP sequences when A. fumigatus MEP and A. oryzae NPI are used as an outgroup.

In the present MEP gene family phylogeny, the five MEP types from a given species represent paralogues, whereas sequences of a given MEP type from the three species correspond to orthologues. Orthologues can be defined as versions of the same gene in different genomes that have been created by the splitting of taxonomic lineages, and paralogues as genes in the same genome that have been created by gene duplication events (Fitch, 1970). A gene family phylogeny thus reflects the order in which gene duplication (producing paralogues) and speciation events (producing orthologues) occurred (Gogarten & Olendzenski, 1999; Thornton & DeSalle, 2000). Each of the five clades corresponding to the MEP types includes the entire species tree, with Microsporum branching basally to Trichophyton (Fig. 3). This branching pattern of orthologues and paralogues signifies that the multiplication of MEP genes occurred before any of the analysed dermatophyte taxa diverged from each other, suggesting that these genes have a conserved and essential function in dermatophytes. For each MEP type, the high degree of sequence identity observed among the three species (72–97 % amino acid sequence identity, Table 2) suggests that Trichophyton and Microsporum can be reduced to a single genus, as previously argued by Gräser et al. (1999) on the basis of ITS rDNA sequence analysis.

The occurrence of at least four gene duplication events in the putative ancestor of dermatophytes is required to explain the tree topology obtained (Fig. 3). A primary duplication produced the ancestral types of MEP1 and MEP5 on the one hand, and of MEP2, MEP3 and MEP4 on the other. Subsequent duplications produced MEP1 and MEP5, MEP4, and the ancestral type of MEP2 and MEP3. The duplication of the latter produced MEP2 and MEP3 and was accompanied by the loss of three introns in MEP2. Ancient gene duplications are recognized as one of the main forces in the generation of gene families and the creation of new functional capabilities (Gogarten & Olendzenski, 1999). New genes issuing from duplication events may have different kinds of fate, including the persistence of copies in the genome with perfect (or near-perfect) identity, the loss of one or several copies, or the acquisition of functional novelty through the accumulation of random mutations, also known as ‘subfunctionalization’ (Lynch & Force, 2000; Sankoff, 2001). A well-documented example of subfunctionalization in multigenic families from pathogenic fungi is given by the ten genes encoding secreted aspartic proteases (Saps) in Candida albicans. Several studies have shown the importance of Sap1, Sap2 and Sap3 in adherence (Monod et al., 2002c), and the role of Sap4, Sap5 and Sap6 in the establishment of deep-seated candidiasis (Borg-von Zepelin et al., 1998). An analogous situation may occur for the fungalysins secreted by dermatophytes. One could envisage that the regulation and secretion of Meps may be different depending on the culture conditions during fungal growth. The multiplication of an ancestral gene to generate a gene family encoding secreted proteases could reflect an evolutionary process allowing dermatophytes to invade keratinised tissues.


   ACKNOWLEDGEMENTS
 
We thank Dr Harold Pooley for critical review of the manuscript and assistance with the English. This work was supported by the Swiss National Foundation for Scientific Research, grant 3100-043193.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 7 August 2003; revised 14 November 2003; accepted 26 November 2003.



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