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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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Escherichia coli LE392 was used for the propagation of the bacteriophage 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
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
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 (040 %). 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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RESULTS |
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Analysis of protein extracts
SDS-PAGE electrophoretic profiles of proteins extracted from T. rubrum, T. mentagrophytes and M. canis consisted of 1319 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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The PCR product was subsequently used as a probe to screen 2x104 individual recombinant bacteriophages of the 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 (MEP15) were isolated from T. rubrum and T. mentagrophytes, and two new genes (MEP45) 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 (TamuraNei)+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 (AC), 2·3415 (AG), 1·0000 (AT), 1·0000 (CG), 5·0882 (CT), 1·0000 (GT); a gamma distribution shape parameter () 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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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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DISCUSSION |
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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 (7297 % 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Apodaca, G. & McKerrow, J. H. (1989b). Regulation of Trichophyton rubrum proteolytic activity. Infect Immun 57, 30813090.[Medline]
Asahi, M., Lindquist, R., Fukuyama, K., Apodaca, G., Epstein, W. L. & McKerrow, J. H. (1985). Purification and characterization of major extracellular proteinases from Trichophyton rubrum. Biochem J 232, 139144.[Medline]
Borg-von Zepelin, M., Beggah, S., Boggian, K., Sanglard, D. & Monod, M. (1998). The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages. Mol Microbiol 28, 543554.[CrossRef][Medline]
Brouta, F., Descamps, F., Fett, T., Losson, B., Gerday, C. & Mignon, B. (2001). Purification and characterization of a 43·5 kDa keratinolytic metalloprotease from Microsporum canis. Med Mycol 39, 269275.[Medline]
Brouta, F., Descamps, F., Monod, M., Vermout, S., Losson, B. & Mignon, B. (2002). Secreted metalloprotease gene family of Microsporum canis. Infect Immun 70, 56765683.
Chambers, S. P., Prior, S. E., Barstow, D. A. & Minton, N. P. (1988). The pMTL nic-cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68, 139149.[CrossRef][Medline]
Collins, J. P., Grappel, S. F. & Blank, F. (1973). Role of keratinases in dermatophytosis. II. Fluorescent antibody studies with keratinase II of Trichophyton mentagrophytes. Dermatologica 146, 95100.[Medline]
Descamps, F., Brouta, F., Monod, M., Zaugg, C., Baar, D., Losson, B. & Mignon, B. (2002). Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo. J Invest Dermatol 119, 830835.
Doumas, A., Crameri, R., Léchenne, B. & Monod, M. (1999). Cloning of the gene encoding neutral protease I of the koji mold Aspergillus oryzae and its expression in Pichia pastoris. J Food Mycol 2, 271279.
Felsenstein, J. (1988). Phylogenies from molecular sequences: inference and reliability. Annu Rev Genet 22, 521565.[CrossRef][Medline]
Fitch, W. M. (1970). Distinguishing homologous from analogous proteins. Syst Zool 19, 99113.[Medline]
Gogarten, J. P. & Olendzenski, L. (1999). Orthologs, paralogs and genome comparisons. Curr Opin Genet Dev 9, 630636.[CrossRef][Medline]
Grappel, S. F. & Blank, F. (1972). Role of keratinases in dermatophytosis. I. Immune responses of guinea pigs infected with Trichophyton mentagrophytes and guinea pigs immunized with keratinases. Dermatologica 145, 245255.[Medline]
Gräser, Y., El Fari, M., Vilgalys, R., Kuijpers, A. F., De Hoog, G. S., Presber, W. & Tietz, H. (1999). Phylogeny and taxonomy of the family Arthrodermataceae (dermatophytes) using sequence analysis of the ribosomal ITS region. Med Mycol 37, 105114.[CrossRef][Medline]
Grossberger, D. (1987). Minipreps of DNA from bacteriophage lambda. Nucleic Acids Res 15, 6737.[Medline]
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 9598.
Jaton-Ogay, K., Paris, S., Huerre, M., Quadroni, M., Falchetto, R., Togni, G., Latge, J. P. & Monod, M. (1994). Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus. Mol Microbiol 14, 917928.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lynch, M. & Force, A. (2000). The probability of duplicate gene preservation by subfunctionalization. Genetics 154, 459473.
Mignon, B., Swinnen, M., Bouchara, J. P., Hofinger, M., Nikkels, A., Pierard, G., Gerday, C. & Losson, B. (1998). Purification and characterization of a 31·5 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats. Med Mycol 36, 395404.[CrossRef][Medline]
Monod, M., Paris, S., Sanglard, D., Jaton-Ogay, K., Bille, J. & Latgé, J. P. (1993). Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus. Infect Immun 61, 40994104.[Abstract]
Monod, M., Togni, G., Hube, B. & Sanglard, D. (1994). Multiplicity of genes encoding secreted aspartic proteinases in Candida species. Mol Microbiol 13, 357368.[Medline]
Monod, M., Jaccoud, S., Zaugg, C., Léchenne, B., Baudraz, F. & Panizzon, R. (2002a). Survey of dermatophyte infections in the Lausanne area (Switzerland). Dermatology 205, 201203.[CrossRef][Medline]
Monod, M., Capoccia, S., Léchenne, B., Zaugg, C., Holdom, M. & Jousson, O. (2002b). Secreted proteases from pathogenic fungi. Int J Med Microbiol 292, 405419.[Medline]
Monod, M. & Borg-von Zepelin, M. (2002c). Secreted aspartic proteases as virulence factors of Candida species. Biol Chem 383, 10871093.[Medline]
Ninet, B., Jan, I., Bontems, O., Léchenne, B., Jousson, O., Panizzon, R., Lew, D. & Monod, M. (2003). Identification of dermatophyte species by 28S ribosomal DNA sequencing with a commercial kit. J Clin Microbiol 41, 826830.
Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357358.[Medline]
Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 35513567.[CrossRef][Medline]
Posada, D. & Crandall, K. A. (1998). Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817818.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sankoff, D. (2001). Gene and genome duplication. Curr Opin Genet Dev 11, 681684.[CrossRef][Medline]
Swofford, D. L. (1998). PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods). Sunderland, MA: Sinauer Associates.
Takiuchi, I., Higuchi, D., Sei, Y. & Koga, M. (1982). Isolation of an extracellular proteinase (keratinase) from Microsporum canis. Sabouraudia 20, 281288.[Medline]
Takiuchi, I., Sei, Y., Tagaki, H. & Negi, M. (1984). Partial characterization of the extracellular keratinase from Microsporum canis. Sabouraudia 22, 219224.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Thornton, J. W. & DeSalle, R. (2000). Gene family evolution and homology: genomics meets phylogenetics. Annu Rev Genomics Hum Genet 1, 4173.[CrossRef][Medline]
Tsuboi, R., Ko, I. J., Takamori, K. & Ogawa, H. (1989). Isolation of a keratinolytic proteinase from Trichophyton mentagrophytes with enzymatic activity at acidic pH. Infect Immun 57, 34793483.[Medline]
Weitzman, I. & Summerbell, R. C. (1995). The dermatophytes. Clin Microbiol Rev 8, 240259.[Abstract]
Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T. & Mann, M. (1996). Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466469.[CrossRef][Medline]
Woodfolk, J. A., Wheatley, L. M., Piyasena, R. V., Benjamin, D. C. & Platts-Mills, T. A. (1998). Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity. Sequence homology to two families of serine proteinases. J Biol Chem 273, 2948929496.
Yelton, M. M., Hamer, J. E. & Timberlake, W. E. (1984). Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci U S A 81, 14701474.[Abstract]
Yu, R. J., Harmon, S. R. & Blank, F. (1968). Isolation and purification of an extracellular keratinase from Trichophyton mentagrophytes. J Bacteriol 96, 14351436.[Medline]
Received 7 August 2003;
revised 14 November 2003;
accepted 26 November 2003.
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