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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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
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
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
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.
|
|
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 min1). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 5070 % 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
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 1519 aa signal sequence (Table 1). The intronexon 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, 10100 µg ml1 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
.
|
|
|
|
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
).
|
|
|
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beauvais, A., Monod, M., Wyniger, J., Debeaupuis, J. P., Grouzmann, E., Brakch, N., Svab, J., Hovanessian, A. G. & Latgé, J. P. (1997b). Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans. Infect Immun 65, 30423047.[Abstract]
Becker, J. M. & Naider, F. (1995). Fungal peptide transport as a drug delivery system. In Peptide-Based Drug Design: Controlling Transport and Metabolism, pp. 369384. Edited by M. Taylor & G. Amidon. Washington, DC: American Chemical Society.
Beggah, S., Léchenne, B., Reichard, U., Foundling, S. & Monod, M. (2000). Intra and intermolecular events direct the propeptide-mediated maturation of the Candida albicans secreted aspartic proteinase Sap1p. Microbiology 146, 27652773.[Medline]
Ben-Meir, D., Spungin, A., Ashkenazi, R. & Blumberg, S. (1993). Specificity of Streptomyces griseus dinuclear aminopeptidase and modulation of activity by divalent metal ion binding and substitution. Eur J Biochem 212, 107112.[Abstract]
Blinkovsky, A. M., Byun, T., Brown, K. M., Golightly, E. J. & Klotz, A. V. (2000). A non-specific aminopeptidase from Aspergillus. Biochim Biophys Acta 1480, 171181.[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.
Byun, T., Kofod, L. & Blinkovsky, A. (2001). Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis. J Food Chem 49, 20612063.[CrossRef]
Chambers, S. P., Prior, S. E., Barstow, D. A. & Minton, N. P. (1988). The pMTL nic cloning vectors. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68, 139149.[CrossRef][Medline]
Chien, H. C., Lin, S. H., Chao, S. H., Chen, C. C., Wang, W. C., Shaw, C. Y., Tsai, Y. C., Hu, H. Y. & Hsu, W. H. (2002). Purification, characterisation, and genetic analysis of a leucine aminopepeptidase from Aspergillus sojae. Biochim Biophys Acta 1576, 119126.[Medline]
Danew, P. & Friedrich, E. (1980). Untersuchung zur Peptidaseaktivität hautpathogener Pilze. IV. Aminopeptidaseaktivität bei Microsporum gypseum und Trichophyton rubrum nach Wachstum in einer Glukose-Salzlösung mit Zusatz von Lysinmonochlorid als N-Quelle. Mycosen 23, 502511.
De Bersaques, J. & Dockx, P. (1973). Proteolytic and leucylnaphthylamidase activity in some dermatophytes. Arch Belg Dermatol Syphiligr 29, 135140.[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 70, 830836.
Doumas, A., Van den Broek, P., Affolter, M. & Monod, M. (1998). Characterization of the prolyl dipeptidyl peptidase gene (dppIV) from the koji mold Aspergillus oryzae. Appl Environ Microbiol 64, 48094815.
Ellis, K. J. & Morrison, J. F. (1982). Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol 87, 405426.[Medline]
Greenblatt, H. M., Almog, O., Maras, B., Spungin-Bialik, A., Barra, D., Blumberg, S. & Shoham, G. (1997). Streptomyces griseus dinuclear aminopeptidase: X-ray crystallographic structure at 1·75 Å resolution. J Mol Biol 212, 620636.[CrossRef]
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.
Hasselgren, C., Park, H. I. & Ming, L. J. (2001). Metal ion binding and activation of Streptomyces griseus dinuclear aminopeptidase: cadmium(II) binding as a model. J Biol Inorg Chem 6, 120127.[CrossRef][Medline]
Hauser, M., Narita, V., Donhardt, A. M., Naider, F. & Becker, J. M. (2001). Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae. Mol Membr Biol 18, 105112.[CrossRef][Medline]
Jousson, O., Léchenne, B., Bontems, O., Capoccia, S., Mignon, B., Barblan, J., Quadroni, M. & Monod, M. (2004a). Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum. Microbiology 150, 301310.[CrossRef][Medline]
Jousson, O., Léchenne, B., Bontems, O., Mignon, B., Reichard, U., Barblan, J., Quadroni, M. & Monod, M. (2004b). Secreted subtilisin gene family in Trichophyton rubrum. Gene 339, 7988.[CrossRef][Medline]
Julius, D., Brake, A., Blair, L., Kunisawa, R. & Thorner, J. (1984). Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro--factor. Cell 37, 10751089.[Medline]
Kwong-Chung, K. J. & Bennet, J. E. (1992). Medical Mycology. Philadelphia & London: Lea & Febiger.
Lin, L.-Y., Park, H. I. & Ming, L. J. (1997). Metal-binding and active-site structure of di-zinc Streptomyces griseus aminopeptidase. J Biol Inorg Chem 2, 744749.[CrossRef]
Lubkowitz, M. A., Hauser, L., Breslav, M., Naider, F. & Becker, J. M. (1997). An oligopeptide transport gene from Candida albicans. Microbiology 143, 387396.[Medline]
Mignon, B., Swinnen, M., Bouchara, J., 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. & Applegate, L. A. (1994). RNA extractions and Northern hybridisation of Aspergillus fumigatus for the detection of alkaline and metalloprotease gene expression. In Molecular Biology of Pathogenic Fungi: a Laboratory Manual, pp. 2932. Edited by B. Maresca & G. S. Kobayashi. New York: Telos Press.
Monod, M., Togni, G., Rahalison, L. & Frenk, E. (1991). Isolation and characterisation of an extracellular alkaline protease of Aspergillus fumigatus. J Med Microbiol 35, 2328.[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., Jaton-Ogay, K. & Reichard, U. (1999). Aspergillus fumigatus secreted proteases as antigenic molecules and virulence factors. Contrib Microbiol 2, 182192.[Medline]
Monod, M., Jaccoud, S., Zaugg, C., Léchenne, B., Baudraz, F. & Panizzon, R. (2002). Survey of dermatophyte infections in Lausanne area (Switzerland). Dermatology 205, 201203.[CrossRef][Medline]
Nakadai, T., Nasuno, S. & Iguchi, N. (1973). Purification and properties of leucine aminopeptidase I from Aspergillus oryzae. Agric Biol Chem 37, 757765.
Nishizawa, M., Yasuhara, T., Nakai, T., Fujiki, Y. & Ohashi, A. (1994). Molecular cloning of the aminopeptidase Y gene of Saccharomyces cerevisiae. J Biol Chem 269, 1365113655.
O'Cuinn, G., Fitzgerald, R., Bouchier, P. & McDonnell, M. (1999). Generation of non-bitter casein hydrolysates by using combinations of a proteinase and aminopeptidases. Biochem Soc Trans 27, 730734.[Medline]
Payne, J. W. & Smith, M. W. (1994). Peptide transport by microorganisms. Adv Microbiol Physiol 36, 180.[Medline]
Rubio-Aliaga, I. & Daniel, H. (2002). Mammalian peptide transporters as targets for drug delivery. Trends Pharmacol Sci 23, 434440.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Stacey, G., Koh, S., Granger, C. & Becker, J. M. (2002). Peptide transport in plants. Trends Plant Sci 80, 270279.[CrossRef]
Toma, C. & Honma, Y. (1996). Cloning and genetic analysis of the Vibrio cholerae aminopeptidase gene. Infect Immun 64, 44954500.[Abstract]
Weitzman, I. & Summerbell, R. C. (1995). The dermatophytes. Clin Microbiol Rev 8, 240259.[Abstract]
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.
Received 14 July 2004;
revised 6 October 2004;
accepted 12 October 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |