Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum

Michel Monod1, Barbara Léchenne1, Olivier Jousson1, Daniela Grand2, Christophe Zaugg1, Reto Stöcklin3 and Eric Grouzmann2

1 Service de Dermatologie et Vénéréologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
2 Division de Pharmacologie et Toxicologies Cliniques, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
3 Atheris Laboratories, case postale 314, Bernex-Genève, Switzerland

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58–65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.


Abbreviations: AMC, 7-amido-4-methylcoumarin; E-64, L-trans-epoxysuccinyl-Leu-4-guanidinobutylamide; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AY496930, AY436356, AY496929, AY436357, AY497021, U87950, AF407232 and L48074.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Dermatophytes are the most common agents of superficial mycoses (Kwong-Chung & Bennet, 1992; Weitzman & Summerbell, 1995). These highly specialized pathogenic fungi are found exclusively in the stratum corneum, nails or hair, and it is evident that secreted proteolytic activity is important for their virulence. Recent investigations have shown that proteases secreted by dermatophytes are similar to those of other fungi such as Aspergillus spp. (Brouta et al., 2002; Descamps et al., 2002; Jousson et al., 2004a, b), and are members of large protein families. In particular, two gene families encoding endoproteases of the S8 (subtilisins) and M36 (fungalysins) families (see the MEROPS proteolytic enzyme database, http://merops.sanger.ac.uk/) were found in Trichophyton rubrum, Trichophyton mentagrophytes, Arthroderma benhamiae and Microsporum canis. Individual subtilisins and fungalysins have been shown to be keratinolytic and produced during infections (Mignon et al., 1998; Brouta et al., 2001).

Until now, no amino- and carboxypeptidases have been isolated and characterized from dermatophytes. However, aminopeptidase activity was detected in the mycelium and culture supernatant of different species of this group of fungi (De Bersaques & Dockx, 1973; Danew & Friedrich, 1980), and this exoproteolytic activity could play an important role in the development of the fungus during infection. It is indeed likely that only amino acids or short peptides from digested cornified cell envelope and from digested keratin can be used by dermatophytes as nutrients for growth in vivo. Bacteria, yeasts and filamentous fungi, as well as specialized cells of plants and animals, express membrane proteins for uptake of amino acids, dipeptides and tripeptides (Payne & Smith, 1994; Becker & Naider, 1995; Hauser et al., 2001; Stacey et al., 2002; Rubio-Aliaga & Daniel, 2002). Transporters that also accept oligopeptides of four or five amino acid residues are known in yeasts and plants (Lubkowitz et al., 1997; Hauser et al., 2001; Stacey et al., 2002).

A fundamental objective of our research on dermatophytes is to obtain a comprehensive view of the enzymes that allow the digestion of an insoluble protein structure, such as the cornified cell envelope, into oligopeptides and free amino acids. The present study was performed on T. rubrum, the most frequent dermatophyte found in man in European countries (Monod et al., 2002). We have shown herein that, in addition to endoproteases of the S8 and M36 families, T. rubrum secretes leucine aminopeptidases (Lap) of the M28 family, and dipeptidyl-peptidases (Dpp) of the S10 family. Using recombinant proteins, aminopeptidases secreted by T. rubrum were compared to their orthologues from the opportunist Aspergillus fumigatus.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and plasmids.
A clinical isolate, T. rubrum CHUV 862-00 (Jousson et al., 2004a, b), was used in this study. 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 using plasmids pMTL21 (Chambers et al., 1988) and pUC19. Pichia pastoris GS115 and KM71, and the expression vectors pKJ111 (Monod et al., 1999), pKJ113 (Borg-von Zepelin et al., 1998) and pPICZ{alpha}A (Invitrogen), were used to express recombinant peptidases.

T. rubrum growth media.
T. rubrum was grown on Sabouraud agar and liquid medium (Bio-Rad) or, to promote production of proteolytic activity, in soy protein liquid medium (SP) (Jousson et al., 2004a) and keratin liquid medium (KSP). SP was prepared by dissolving 2 g soy protein (Supro 1711, Protein Technologies International) in 1 l distilled water. KSP aliquots of 100 ml were prepared by adding 0·2 g keratin (Merck 5201) and 5 ml SP to 95 ml distilled water. Both media were sterilized by autoclaving at 120 °C for 15 min. Volumes (100 ml) of each medium poured into 800 ml tissue culture flasks were inoculated with a plug of freshly growing mycelium on Sabouraud agar. T. rubrum cultures in SP and KSP were incubated for 10 and 28 days, respectively, at 30 °C without shaking.

Genomic and cDNA libraries.
T. rubrum {lambda}EMBL3 genomic and pSPORT6 cDNA libraries were prepared using DNA and RNA isolated from freshly growing mycelium in SP (Jousson et al., 2004a). An A. fumigatus {lambda}gt11 cDNA library was previously constructed with the CHUV192-88 strain grown 40 h at 30 °C in liquid medium containing collagen as a sole nitrogen and carbon source (Monod et al., 1991). Total RNA was extracted as described previously (Monod & Applegate, 1994), and the mRNA was purified using oligo(dT) cellulose (Sigma), according to standard protocols (Sambrook et al., 1989). A library was prepared with this mRNA using phage {lambda}gt11 (Promega), according to the protocols of the manufacturer.

Gene cloning.
Recombinant plaques (2x104) of the genomic libraries of T. rubrum were immobilized on GeneScreen nylon membranes (NEN Life Science Products). The filters were hybridized with 32P-labelled DNA fragments under low-stringency conditions (Monod et al., 1994). All positive plaques were purified, and the bacteriophage DNAs were isolated as described by Grossberger (1987). Agarose gel electrophoresis of enzyme-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).

cDNA amplification by standard PCR.
T. rubrum and A. fumigatus cDNAs were obtained by PCR using DNA prepared from 106 clones of the cDNA libraries as a target. PCR was performed according to standard conditions using homologous primers derived from genomic DNA sequences of the different peptidase genes (Tables 1 and 2). Target DNA (200 ng), 10 µl of each sense and antisense oligonucleotides at a concentration of 42 mM, and 8 µl deoxynucleotide mix (containing 10 mM of each dNTP) were dissolved in 100 µl PCR buffer (10 mM Tris/HCl pH 8·3, 50 mM KCl and 1·5 mM MgCl2). To each reaction, 2·5 U AmpliTAQ DNA polymerase (Perkin Elmer) was added. The reaction mixture was incubated for 5 min at 94 °C, subjected to 25 cycles of 0·5 min at 94 °C, 0·5 min at 55 °C and 0·5 min at 72 °C, and finally incubated for 10 min at 72 °C.


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Table 1. T. rubrum (ru) and A. fumigatus (fu) genes encoding aminopeptidases, and main characteristics of deduced translation products

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 P). NI, Not investigated.

 
Production of recombinant peptidases.
Expression plasmids were constructed by cloning cDNA PCR products in P. pastoris expression vectors. The PCR products were purified using a PCR purification kit (Roche Diagnostics), and digested by restriction enzymes for which a site was previously included at the 5' extremity of the primers (Table 2). P. pastoris transformation, selection of transformants, and production of recombinant enzymes in methanol medium, were performed as previously described (Borg-von Zepelin et al., 1998; Beggah et al., 2000).


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Table 2. Materials used for the expression of the different Laps in P. pastoris, and yields of recombinant proteins

 
Purification of recombinant aminopeptidases.
The secreted proteins from 400 ml P. pastoris culture supernatant were concentrated by ultrafiltration using an Amicon cell and an Ultracel Amicon YM30 membrane (30 kDa cut-off) (Millipore). The concentrate was washed with 50 mM Tris/HCl or sodium acetate buffer at a pH value one unit below the theoretical isoelectric point (pI) of the protein to be purified, and applied to a Mono Q-Sepharose (Amersham Pharmacia) column equilibrated with the same buffer. After washing the column with the corresponding buffer, the recombinant enzyme was eluted with a linear gradient of 0–0·5 M NaCl at a flow rate of 1 ml min–1, and active fractions were pooled. The enzyme extract was concentrated using an Amicon ultrafiltration cell with an Ultracel Amicon YM30 membrane, washed with 50 mM Tris/HCl, and loaded on a size-exclusion Superose 6 FPLC column (Amersham Pharmacia). Elution was performed with 50 mM Tris/HCl at a flowrate of 0·2 ml min–1, and active fractions were pooled. The recombinant enzymes were subjected to further characterization after concentration to a final volume of 0·4–1·0 ml in a Centricon concentrator with a 30 kDa cut-off (Millipore) at 4 °C.

Protein extract analysis.
Protein extracts were analysed in SDS-PAGE gels stained with Coomassie brilliant blue R-250 (Bio-Rad). N-Glycosidase F digestion was performed as described by Doumas et al. (1998). Western blots were revealed using rabbit antisera and alkaline-phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) as secondary labelled antibodies. Rabbit antisera were made by Eurogentec (Liège, Belgium) using purified recombinant enzyme.

Enzymic activities.
Microsomal porcine kidney aminopeptidase (pkLap) was from Sigma. Lap activities were measured with different fluorogenic aminoacyl-4-methylcoumaryl-7-amide derivatives as substrates. Gly-Pro-7-amido-4-methylcoumarin (Gly-Pro-AMC) and Lys-Ala-AMC were used for dipeptidyl-peptidase activities. Lys(Abz)-Pro-Pro-pNA, as a substrate for aminopeptidase P activity, was also tested. All substrates were from Bachem (Bubendorf, Switzerland). Substrate stock solutions were prepared at 0·1 M concentration and stored at –20 °C. The reaction mixture contained a concentration of 5 mM substrate and enzyme preparation (between 56 and 2·7 ng per assay, depending on the cleavage activity of each enzyme for the substrates) in 25 µl 50 mM Tris/HCl buffer adjusted at the optimal pH for each Lap (between 7 and 8·5). After incubation at 37 °C for 60 min, the reaction was terminated by adding 5 µl glacial acetic acid and 3·5 ml water. The released AMC was measured using a spectrofluorophotometer (Perkin Elmer LS-5 fluorometer) at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. A standard curve established with synthetic AMC was used to quantify the released product. The enzymic activities were expressed in mU (1 mU represents 1 nmol AMC released min–1). The released Lys(Abz) product was measured at an excitation wavelength of 310 nm and an emission wavelength of 410 nm. In the absence of a standard curve, the enzyme activity of aminopeptidase P was reported as arbitrary units of fluorescence.

Effect of various chemical reagents on Laps.
Inhibitors and metallic cations were pre-incubated with the enzymes for 15 min at 37 °C. Then Leu-AMC, at a 5 mM final concentration, was added. After further incubation for 60 min, enzyme activity was measured as described above. The inhibitors and their concentrations tested on purified Laps were: 500 µM amastatin (Bachem), 40 µM benzamidine (Sigma), 500 µM bestatin (Bachem), 5 mM or 1 mM EDTA (Sigma), 100 µM E-64 (L-trans-epoxysuccinyl-Leu-4-guanidinobutylamide) (Bachem), 100 µM leupeptin (Sigma), 5 mM/1 mM o-phenanthroline (Sigma), 500 µM p-chloromercuribenzoic acid (Sigma), 100 µM pepstatin A (Sigma), 40 µM PMSF (Sigma), 20 µM N-p-tosyl-L-lysine chloromethyl ketone (TLCK; Roche Diagnostics), 20 µM N-tosyl-L-phenylalanine chloromethyl ketone (TPCK; Roche Diagnostics). CaCl2, MgCl2, MnCl2, CoCl2, ZnCl2, NiCl2 and CuCl2 were tested at concentrations of 0·5 and 1 mM.

Optimal pH of LAPs.
The optimal pH for enzymic activities was determined using the Ellis and Morrison buffer system (Ellis & Morrison, 1982). The buffer contained three components with different pKa values, while the ionic strength of buffer remained constant throughout the entire pH range chosen for study. The pH of the buffer was adjusted from 6 to 11 in half-pH unit increments with 1 M HCl or 1 M NaOH. The assay conditions for activity on the Leu-AMC substrate was the same as that described above, except that Ellis and Morrison buffer at different pH values was used instead of Tris/HCl buffer.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Secreted proteolytic activity of T. rubrum
T. rubrum was grown for 10 and 28 days in SP and KSP, respectively. A maximum of Lap (0·8 mU ml–1) and DppIV (0·6 mU ml–1 in SP, and 4·0 mU ml–1 in KSP) activity was measured in the culture supernatants using Leu-AMC and Gly-Pro-AMC as substrates, respectively. At these times, a substantial endoproteolytic activity was recorded, with a concomitant clarification of the culture medium (Jousson et al., 2004a, b). For comparison, maximum Lap activity of A. fumigatus in SP and KSP was about 0·02 mU ml–1, while DppIV activity was similar to that of T. rubrum. In Sabouraud liquid medium, no aminopeptidase activity was detected for T. rubrum or A. fumigatus.

Cloning of genes encoding T. rubrum and A. fumigatus Laps
The nucleotide sequences of dermatophyte endoprotease genes (Descamps et al., 2002; Brouta et al., 2002; Jousson et al., 2004a, b) exhibited 50–70 % identity to homologous genes in Aspergillus spp. Therefore, we investigated the aminopeptidase activity of T. rubrum by a reverse genetic approach (from genes to proteins). DNA sequences available for Aspergillus spp. and Saccharomyces cerevisiae genes encoding aminopeptidases were used to design oligonucleotide probes for screening a T. rubrum genomic DNA library.

A 1200 bp fragment containing the nucleotide sequence of the gene encoding an Aspergillus oryzae/Aspergillus sojae 31 kDa Lap (orLap1) (Nakadai et al., 1973; Chien et al., 2002; US Patent no. 5,994,113) was obtained by PCR of A. oryzae genomic DNA using the oligonucleotides 5'-ATGCGTTTCCTCCCCTGCATCGCG-3' (sense) and 5'-CAGCGAATCTGCGAAGGCAAGCTC-3' (antisense). This fragment was used as a probe for screening a {lambda}EMBL3 phage T. rubrum genomic DNA library. Isolated clones contained a nucleotide sequence (GenBank accession no. AY496930, Table 1) encoding a putative Lap. This enzyme displayed about 50 % amino acid sequence identity with orLap1 and was called ruLap1.

A. oryzae DNA encoding a second 52 kDa aminopeptidase (orLap2) (US patent no. 6,127,161; Blinkovsky et al., 2000) was also used to screen the T. rubrum genomic DNA library; however, no hybridizing sequences were detected. The 52 kDa A. oryzae aminopeptidase is homologous to the S. cerevisiae aminopeptidase Y. Therefore, another attempt at cloning a T. rubrum aminopeptidase DNA was performed using oligonucleotide probes (GGXATXAAYGAYGAYGGXTCXGG and TTXGGXGAXGCXATCATRTC) based on a consensus of the nucleotide sequences encoding two conserved amino acid sequences in the A. oryzae aminopeptidase and S. cerevisiae aminopeptidase Y [GPGINDDGSG and DM(I/M)ASPN, respectively]. These two oligonucleotides were used as sense and antisense to amplify DNA from T. rubrum. A 220 bp PCR product was obtained and sequenced. The deduced amino acid sequence of one ORF showed high similarity to the amino acid sequence of the A. oryzae and S. cerevisiae aminopeptidases. This 220 bp PCR fragment was used as a probe for screening the T. rubrum genomic DNA library. Hybridizing DNA contained a nucleotide sequence (GenBank accession no. AY496929, Table 1) encoding a second putative T. rubrum secreted Lap, which displayed 50 % amino acid sequence identity with orLap2. This enzyme was called ruLap2. No further new LAP paralogues were found in a third screening performed with ruLap1- and ruLap2-encoding DNA probes.

Nucleotide sequences (GenBank accession nos AY436356 and AY436357) encoding putative orthologues of orLap1 and orLap2 were found in the A. fumigatus genome sequence (http://www.tigr.org/tdb/e2k1/afu1/). These aminopeptidases were called fuLap1 and fuLap2, respectively. From their identified nucleotide sequences, ruLap1, ruLap2, fuLap1 and fuLap2 predicted a 15–19 aa signal sequence (Table 1). The intron–exon structure of the T. rubrum and A. fumigatus genes was verified by sequencing a PCR product, using 5'-sense and 3'-antisense primers based on isolated genomic DNA (Table 2), and total DNA from a pool of 106 clones of the T. rubrum or A. fumigatus cDNA libraries as a target. The genes ruLAP1 and fuLAP1 revealed similar collinear structures with two introns and three exons (positions designated in GenBank). The first of the three introns in ruLAP2 was in a position similar to that of the unique intron of fuLAP2.

Cloning of genes encoding T. rubrum Dpps
A nucleotide sequence (GenBank accession no. AY497021, Table 1) similar to those encoding the A. oryzae and the A. fumigatus 94 kDa DppIV (Beauvais et al., 1997b; Doumas et al., 1998) was found in the T. rubrum genomic library, using the consensus oligonucleotide 5'-CAYGGIACIGGIGAYGAXAAYGTICAYTTYCA-3' as a probe. This oligonucleotide encodes the amino acid sequence HGTGDDNVHFQ, which was found to be conserved in Aspergillus, human and mouse DppIVs (Beauvais et al., 1997b). The cloned ruDPPIV gene contained no introns and encoded a putative protein with an amino acid sequence 61 % identical to that of A. fumigatus DppIV (fuDppIV).

The cDNA encoding a T. rubrum DppV (ruDppV) was reported by Woodfolk et al. (1998). RuDppV has an amino acid sequence 57 % identical to that of A. fumigatus DppV (fuDppV) (Beauvais et al., 1997a). The corresponding T. rubrum genomic DNA (GenBank accession no. AF407232, Table 1) was cloned using cDNA obtained by PCR as a probe (Table 2). The genomic sequence of this gene contained four introns in positions similar to four of the seven introns of fuDPPV (positions designated in GenBank).

Production of recombinant T. rubrum and A. fumigatus aminopeptidases
The T. rubrum and A. fumigatus cDNAs obtained by PCR were cloned in P. pastoris expression vectors, and expressed in P. pastoris grown in methanol inducing medium. Under identical culture conditions, wild-type P. pastoris did not secrete any Lap, DppIV and DppV activities into the culture medium. Depending on the peptidase produced, 10–100 µg ml–1 active enzyme was obtained (Table 2). Recombinant A. fumigatus DppIV, and DppV from P. pastoris, have been previously produced and characterized (Beauvais et al., 1997a, b).

In contrast to recombinant ruLAP1, recombinant ruLAP2, fuLAP1 and fuLAP2 were glycoproteins, as attested by a reduction in their molecular masses following treatment with N-glycosidase F (Fig. 1). RuDppIV and ruDppV were also glycosylated. The apparent molecular mass of each deglycosylated recombinant Lap and Dpp was close to that of the calculated molecular mass of the polypeptide chain deduced from the nucleotide sequence of the genes encoding the protease. Characteristics of the primary structure of each recombinant enzyme are summarized in Table 1.



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Fig. 1. Protein profile of recombinant ruLap1 (lanes 1 and 2), fuLap1 (lanes 3 and 4), ruLap2 (lanes 5 and 6), fuLap2 (lanes 7 and 8), ruDppIV (lanes 9 and 10) and ruDppV (lanes 11 and 12) produced in P. pastoris. A 1 µg quantity of each purified recombinant LAP was loaded onto a 10 % SDS-PAGE gel. Lanes 2, 4, 6, 8, 10 and 12 show the proteins deglycosylated by N-glycosidase F treatment (+). The gel was stained with Coomassie brilliant blue R-250. M, molecular mass markers.

 
Detection of T. rubrum aminopeptidases in culture supernatant
Using specific antisera, Western blot analysis of culture supernatant of T. rubrum grown in SP and KSP revealed secreted glycosylated ruLap2, DppIV and DppV with the same electrophoretic mobility as that of the recombinant enzyme from P. pastoris (Fig. 2). Using anti-ruLap1 antiserum, an accumulation of enzyme with an electrophoretic mobility higher than recombinant ruLap1 was detected in the culture supernatant. The 33 kDa size of native ruLap1 is compatible with a cleavage at a KR site of the polypeptide chain (positions 64–65 in Fig. 4) by a Kex2-like enzyme (Julius et al., 1984) in T. rubrum. ruLap2 corresponded to the major 58 kDa protein secreted by T. rubrum (Fig. 2). All other major bands corresponded to endoproteases of the subtilisin and fungalysin families (Jousson et al., 2004a, b).



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Fig. 2. Detection of T. rubrum aminopeptidases in culture supernatant. Lanes 1 and 2, electrophoretic profile of T. rubrum proteins secreted in SP and KSP, respectively. The proteins from 1 ml dermatophyte culture supernatant were precipitated using TCA. A 1 µg quantity of purified recombinant ruLap2 (lane 3) was loaded for comparison. The 9 % SDS-PAGE gel was stained with Coomassie brilliant blue R-250. Arrows indicate metalloproteases of the fungalysin family, and asterisks indicate serine proteases of the subtilisin family (Jousson et al., 2004a, b). Lanes 4–15, Western blots of T. rubrum culture supernatant and recombinant enzymes. Aminopeptidases were revealed with anti-ruLap1 (lanes 4–6), anti-ruLap2 (lanes 7–9), anti DppIV (lanes 10–12) and anti-DppV (lanes 13–15) antisera. In lanes 4 and 5, 7 and 8, 10 and 11, and 13 and 14, the proteins of 0·5, 0·1, 1·5 and 0·5 ml T. rubrum culture supernatant, respectively, were precipitated with TCA before loading onto the SDS-PAGE gel (9 %). Purified recombinant ruLap1 (lane 6), ruLap2 (lane 9), ruDppIV (lane 12) and ruDppV (15) (0·1 µg quantities) were loaded as controls.

 


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Fig. 4. Comparison of deduced amino acid sequences of aminopeptidases of the M28E subfamily. Putative signal sequence cleavage sites are underlined. A putative KR processing site in ruLap1 is indicated by a solid triangle (positions 64–65). The amino acids of the two Zn2+ binding sites conserved in aminopeptidases of the M28 family are indicated as in Fig. 3. The alignment was performed with the PILEUP algorithm implemented in the GCG package of the University of Wisconsin, and reformatted with Boxshade 3.2. The GenBank accession number of the Vibrio LAP is D84215.

 
Properties of recombinant LAPs
The aminopeptidases ruLap1, ruLap2, fuLap1 and fuLap2, as well as the microsomal porcine kidney aminopeptidase (pkLap) used for comparison, hydrolysed Leu-AMC very efficiently. This substrate was used to determine the optimum temperature and pH of activity, and to further characterize the enzymes by measuring the effect of: (i) various known peptidase inhibitors (Table 3) and (ii) different divalent cations (Table 4). Each Lap was capable of cleaving Leu-AMC at 20 °C, and had an optimum temperature ranging from 40 to 50 °C. A 10 min pre-treatment at 80 °C totally and irreversibly inactivated the enzymes. The optimum pH was 8·5 for fuLap1, and 7·0 for the other Laps. The specific activity of ruLap1, fuLap1, ruLap2, fuLap2 and pkLap for Leu-AMC, as a substrate, was measured as 2·76, 0·12, 0·56, 0·03 and 0·75 mU (µg protein)–1, respectively.


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Table 3. Hydrolytic activity of different Laps in the presence of various protease inhibitors using Leu-AMC as a substrate

Activity is given as a percentage of the activity of the control enzymic reaction without the inhibitor.

 

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Table 4. Hydrolytic activity of different Laps in presence of various cations using Leu-AMC as a substrate

Activity is given as a percentage of the activity of the control enzymic reaction without the cation. All cations were added as their chloride salts.

 
All Laps tested were strongly or totally inhibited by amastatin at a concentration of 500 µM (Table 3). ruLap1, fuLap1 and pkLap were also inhibited by bestatin, but this inhibitor had only a partial inhibitory effect on both ruLap2 and fuLap2. Of the chelating agents tested, o-phenanthroline totally inhibited the five enzymes at concentrations of 1 and 5 mM. fuLap1, ruLap1 and ruLap2 were more sensitive to EDTA than the two other Laps. E-64 and p-chloromercuribenzoate (cysteine protease inhibitors) affected the activity of ruLap2 only, indicating the presence of critical thiol residues for activity on the amino acid sequence of this enzyme. Leupeptin (serine/cysteine protease inhibitor), PMSF (serine protease inhibitor), benzamidine, TLCK and TPCK had no clear inhibitory effects on any of the Laps tested. Surprisingly, ruLap2 and fuLap1 exhibited a moderate sensitivity to 0·1 mM pepstatin (aspartic acid protease inhibitor).

With the exception of fuLap1, which was generally inhibited by divalent cations, Co2+ increased the activity of the Laps from 200 to 900 % at concentrations up to 1 mM (Table 4). The four fungal Laps showed variable sensitivities to other divalent cations. The microsomal pkLap, highly activated by Zn2+, Ni2+ and Cu2+, differed from the four fungal Laps of the M28 family.

The hydrolytic activity of the enzymes toward different aminoacyl-AMC substrates was compared to Leu-AMC used as a reference (Table 5). Depending on the Lap tested, various preferences for the different aminoacyl residues were detected. The aminopeptidase pkLap differs from the four fungal Laps by an extremely high efficiency with Ala-AMC, Arg-AMC and Phe-AMC. ruLap1 was clearly the most selective for Leu-AMC. Other preferential cleavage activities were observed for ruLap2, fuLap1 and fuLap2. For instance, Ser- and Pro-AMC were more efficiently cleaved by ruLap2, whereas fuLap1 showed preference for Arg-, Val- and Phe-AMC. Only ruLap2 efficiently cleaved Asp- and Glu-AMC. The tested Laps were not capable of cleaving the Gly-Pro-AMC substrate, indicating that the presence of a Pro residue in position p'1 affects the efficacy of these enzymes. In addition to a lack of DppIV activity, no Laps exhibited an aminopeptidase P activity tested with Lys(Abz)-Pro-Pro-pNA as a substrate.


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Table 5. Hydrolytic activity of different Laps toward various aminoacyl-AMC substrates

Activity is given as a percentage in comparison to Leu-AMC used as a standard.

 
Properties of recombinant ruDppIV and ruDppV
Recombinant ruDppIV and ruDppV were active between pH 6·5 and 10·5 with a broad optimum peak between pH 7·0 and 9·0. Recombinant ruDppIV hydrolysed Gly-Pro-AMC and Lys-Ala-AMC with a specific activity of 16 and 5 mU (µg protein)–1, respectively at the optimal pH of activity. Like CD26 and A. fumigatus DppIV, ruDppIV was inhibited by Lys-[Z(NO2)]-pyrrolidide and Lys-[Z(NO2)]-thiozolidide. Recombinant ruDppV hydrolysed Lys-Ala-AMC with a specific activity of 35 mU (µg protein)–1 at the optimal pH of activity, but was not capable of digesting Gly-Pro-AMC. Mono- and tripeptides were not hydrolysed by any of the DppIV or DppV peptidases tested (data not shown). For comparison at the optimal pH of activity, fuDppIV (Beauvais et al., 1997b) hydrolysed Gly-Pro-AMC with a specific activity of 5·4 mU (µg protein)–1, and fuDppV (Beauvais et al., 1997a) hydrolysed Lys-Ala-AMC with a specific activity of 46 mU (µg protein)–1.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Among several tested protein sources, soy proteins were found to be the best for growing T. rubrum and other dermatophyte species in liquid medium, and promoting proteolytic activity (Jousson et al., 2004a, b). No to poor growth was obtained in liquid medium containing 0·2 % keratin as the sole nitrogen source (data not shown). A low amount of soy protein in KSP medium was necessary to initiate the growth of the fungus with keratin as the substrate. The present investigation on aminopeptidase activity in T. rubrum, and previous studies on endoproteases (Jousson et al., 2004a, b), show that dermatophytes secrete a battery of proteases similar to that of Aspergillus spp. In contrast to subtilisin and fungalysin endoproteases, Laps and Dpps are not members of large families of secreted proteins, since no other paralogues were found in further gene library screenings using T. rubrum genes as probes. The intron–exon structures of the T. rubrum genes encoding Laps and Dpps are similar to those of the genes encoding orthologues in A. fumigatus and A. oryzae.

The growth of dermatophytes is rather slow, and it is difficult to get enough material for the purification of native proteins in sufficient quantities for further characterization. Therefore, a reverse genetic approach (from genes to proteins) was chosen to investigate aminopeptidase activity of T. rubrum. This approach also avoids the problem of purification without contamination from individual proteases from culture supernatant, when numerous proteases were secreted by T. rubrum at the same time in a protein medium. In contrast to the other aminopeptidases investigated here, ruLap2 appeared as a dominant protein secreted in SP (Fig. 2).

The four fungal enzymes (ruLap1, fuLap1, ruLap2 and fuLap2) and pkLap, which share a common preference for Leu-AMC as a substrate, were considered as leucine aminopeptidases in this study, although the aminopeptidase pkLap, which has a high efficiency for Ala-AMC, is also called alanine aminopeptidase (MEROPS>M01·001). The specific activity of ruLap1 and ruLap2 was more than 10 times higher than that of A. fumigatus orthologues. ruLap2, fuLap2 and orLap2 structurally belong to the same subfamily M28A as the vacuolar protease Y of S. cerevisiae (Nishizawa et al., 1994; MEROPS>M28·001) and the Streptomyces griseus secreted aminopeptidase (MEROPS>M28·003) (Fig. 3, Table 6). ruLap1, fulap1 and A. oryzae 31 kDa Lap (Chien et al., 2002) structurally belong to the same subfamily M28E as Vibrio and Aeromonas leucyl aminopeptidases (Toma & Honma, 1996) (MEROPS>M28·002 and MEROPS>M28·004, respectively) (Fig. 4, Table 7). The members of the M28A and M28E subfamilies share low sequence similarity. However, the amino acid sequence of the two Zn2+-binding sites in these aminopeptidases are conserved, and they could be identified in the fungal Laps characterized in this study (Figs 3 and 4). In S. griseus and Aeromonas proteolytica secreted aminopeptidases, two residues, His and Asp, bind a first Zn2+ ion, two additional residues His and Glu bind a second Zn2+ ion, while a second Asp residue bridges the two Zn2+ ions (Greenblatt et al., 1997; Hasselgren et al., 2001). Substitution of Zn2+ by different divalent ions in S. griseus secreted aminopeptidase is affected by Ca2+, and has variable effects (Ben-Meir et al., 1993; Lin et al., 1997; Hasselgren et al., 2001). The aminopeptidases tested in this study were found to be sensitive to different ions. Like S. griseus aminopeptidase, ruLap2 and fuLap2 are highly activated by Co2+. In contrast to fuLap2, ruLap2 is able to efficiently hydrolyse Asp- and Glu-AMC (Table 5).



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Fig. 3. Comparison of deduced amino acid sequences of aminopeptidases of the M28A subfamily. ScerY, S. cerevisiae aminopeptidase Y. Putative signal sequence cleavage sites are underlined. Two residues, His and Asp, conserved in aminopeptidases of the M28 family, and binding a first Zn2+ ion, are indicated by open triangles; two additional residues, His and Glu, binding a second Zn2+ ion, are indicated by solid diamonds, while the Asp residue bridging the two Zn2+ ions is indicated by an open arrow. Met residues found only in ruLap2 are indicated by asterisks. The alignment was performed with the PILEUP algorithm implemented in the GCG package of the University of Wisconsin, and reformatted with Boxshade 3.2.

 

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Table 6. Pairwise amino acid sequence comparisons between Laps of the M28A subfamily

 

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Table 7. Pairwise amino acid sequence comparisons between Laps of the M28E subfamily

 
T. rubrum DppIV and DppV are 57–61 % and 54–57 % identical to A. fumigatus orthologues, respectively. The putative catalytic triad of these serine proteases of the Ser family were deduced/determined as Ser613, Asp690, His725 (DppIV) and Ser558, Asp641 and His673 (DppV) using comparative alignments with other Dpps. Comparable activities towards Gly-Pro-pNA were found with ruDppIV and fuDppIV, and towards Lys-Ala-AMC with ruDppV and fuDppV.

Apparently, the T. rubrum genes encoding Laps and Dpps, like genes encoding the secreted subtilisins and fungalysins, are repressed by small molecules such as ammonium and amino acids. It is evident that the dermatophyte secreted proteases use keratin and the different cross-linked proteins of the cornified cell envelope as a substrate, since these fungi grow exclusively in the stratum corneum, nails or hair as sole nitrogen and carbon sources. It is reasonable to postulate that during infection, the dermatophytes are under catabolic repression to secrete a complete battery of endo- and exoproteases, allowing the degradation of the keratinized tissues. Protein digestion into amino acids has been thoroughly investigated in micro-organisms used in the food fermentation industry. Bacteria of the genus Lactobacillus (O'Cuinn et al., 1999) and fungi of the genus Aspergillus (Byun et al., 2001; Doumas et al., 1998) secrete endo- and exoproteases, which cooperate efficiently in protein digestion. The main function of the former is to produce a large number of free ends on which the latter may act. Synergism of endoproteases of the subtilisin and fungalysin families, and the exopeptidases characterized in the present study, is probably essential for dermatophyte virulence.


   ACKNOWLEDGEMENTS
 
We thank Dr Lee Applegate and Dr Phillip Shaw for critical review of the manuscript and assistance with the English. This work was supported by the Swiss National Foundation for Scientific Research, grants 3100-043193 and 3100-105313/1.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 14 July 2004; revised 6 October 2004; accepted 12 October 2004.



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