Trichophyton rubrum secreted and membrane-associated carboxypeptidases

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International Journal of Medical Microbiology 298 (2008) 669–682 www.elsevier.de/ijmm

Trichophyton rubrum secreted and membrane-associated carboxypeptidases Christophe Zaugga,1, Olivier Joussonb,1, Barbara Le´chennea, Peter Staiba, Michel Monoda, a

Service de Dermatologie et Ve´ne´re´ologie, Laboratoire de Mycologie, BT422, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland b Centre for Integrative Biology, University of Trento, I-38100 Trento, Italy Received 11 July 2007; received in revised form 8 October 2007; accepted 8 November 2007

Abstract Dermatophytes are the most common agents of superficial mycoses, and exclusively infect stratum corneum, nails or hair. Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA) of the M14 family according to the MEROPS proteolytic enzyme database. TruMcpA is homologous to human pancreatic carboxypeptidase A, and is synthesized as a precursor in a preproprotein form. The propeptide is removed to generate the mature active enzyme alternatively by either one of two subtilisins which are concomitantly secreted by the fungus. In addition, T. rubrum was shown to possess two genes (TruSCPA and TruSCPB) encoding serine carboxypeptidases of the S10 family which are homologues of the previously characterized Aspergillus and Penicillium secreted acid carboxypeptidases. However, in contrast to the Aspergillus and Penicillium homologues, TruScpA and TruScpB enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (GPI) anchor. During infection, T. rubrum secreted and GPI-anchored carboxypeptidases may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides. r 2007 Elsevier GmbH. All rights reserved. Keywords: Dermatophytes; Trichophyton rubrum; Aspergillus fumigatus; Carboxypeptidases

Introduction Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections (Kwong-Chung and Bennet, 1992; Weitzman and Summerbell, 1995). Corresponding author. Tel.: +41 21 314 0376; fax: +41 21 314 0378. E-mail address: [email protected] (M. Monod). 1 These authors contributed equally.

1438-4221/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2007.11.005

These fungi grow exclusively in the stratum corneum, nails or hair, and produce hydrolytic enzymes that degrade compact keratinized tissues. Like many other ascomycete fungi, dermatophytes secrete substantial proteolytic activity into a medium containing protein as a sole nitrogen and carbon source (Jousson et al., 2004a, b; Monod et al., 2005). Endoproteases and aminopeptidases secreted by dermatophytes show homology to those secreted by species of the genus Aspergillus. However, dermatophytes differ by secreting

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multiple endoproteases of the subtilisin subfamily (serine proteases S8A) and of the fungalysin family (metalloproteases M36) according to the MEROPS proteolytic enzyme database (http://merops.sanger. ac.uk) (Barrett et al., 2004). Specificity of individual endoproteases of the subtilisin family towards keratin substrate, and the multiplicity of these proteases reflect the high degree of specialization of dermatophytes (Jousson et al., 2004b). Secreted exoproteases so far characterized in Trichophyton rubrum include two leucine aminopeptidases (MEROPS M28), and two dipeptidyl-peptidases (MEROPS S9B and MEROPS S9C) (Monod et al., 2005). The purpose of the present investigation was to further characterize the proteases secreted by dermatophytes. While several carboxypeptidases have been isolated from Aspergillus culture supernatants (Nakadai et al., 1972a, b; Svendsen and Dal Degan, 1998; Blinkovsky et al., 1999), there are no existing data for carboxypeptidase activity in dermatophytes, with the exception of a negative report (Danew et al., 1971). We show herein that T. rubrum secretes a metallocarboxypeptidase A (MEROPS M14A) at the same time as endoproteases and aminopeptidases in a protein medium. In addition, we demonstrate the presence of two serine membrane-associated carboxypeptidases (MEROPS S10), which were predicted to be glycosylphosphatidylinositol (GPI)-anchored according to the amino acid sequence of their gene translation products.

7 (TruSub7), metalloprotease 1 (TruMep1), and metalloprotease 3 (TruMep3) were produced in our laboratory (Jousson et al., 2004b; unpublished results). Proteinase K, subtilisin Carlsberg, Aspergillus oryzae alkaline protease (protease XXIII), and trypsin were purchased from Sigma (St. Louis, MO, USA).

Dermatophyte growth media All dermatophytes were routinely grown on Sabouraud agar and liquid medium (Bio-Rad, Hercules, CA, USA) or, to promote production of proteolytic activity, in soy protein liquid medium (SP) and keratin liquid medium (KSP) as previously described (Jousson et al., 2004a; Monod et al., 2005).

T. rubrum cDNA partial sequence database Three thousand eight hundred and four clones of the T. rubrum cDNA library (Jousson et al., 2004a) were sequenced using an SP6 primer generating an EST collection. The sequencing was done by Synergene Biotech GmbH (Schlieren, Switzerland) on an ABI Prism 3100 DNA sequencer using BigDye Terminator chemistry (Applied Biosystems, Foster City, CA, USA). Pregap4 from Staden package version 1.5 was used to remove vector sequences, to evaluate sequence quality, and for conversion of data formats of the EST sequences. Filtered sequences were assembled by Gap version 4.10 in a database including 514 contigs and 1631 singletons for a total of 2145 clusters.

Materials and methods Microbial strains and gene libraries Trichophyton rubrum CHUV1673-05, Arthroderma benhamiae CBS112371 (Fumeaux et al., 2004) and clinical isolates of Trichophyton mentagrophytes, Trichophyton soudanense, Trichophyton verrucosum, Trichophyton violaceum, Microsporum canis, and Microsporum gypseum were used in this study. Escherichia coli LE392 was used for the propagation of the bacteriophage lEMBL3 (Promega, Madison, WI, USA). All plasmid subcloning experiments were performed in E. coli XL1 blue. Pichia pastoris GS115 and KM71 (Invitrogen, Carlsbad, CA, USA) were used for transformation and production of recombinant proteins. A lEMBL3 genomic library of T. rubrum and cDNA libraries of T. rubrum and A. fumigatus (Jousson et al., 2004a; Denikus et al., 2005) were previously constructed.

Proteases Recombinant T. rubrum subtilisin 3 (TruSub3), subtilisin 4 (TruSub4), subtilisin 5 (TruSub5), subtilisin

T. rubrum and A. fumigatus carboxypeptidase cDNAs T. rubrum and A. fumigatus carboxypeptidase cDNAs were obtained by PCR with a standard protocol (Jousson et al., 2004a, b) using homologous sense and antisense primers (P1–P18, Tables 1 and 2) and 200 ng of DNA prepared from 106 clones of the cDNA libraries as a template.

Screening of the genomic library Recombinant bacteriophage plaques (2  104) of a T. rubrum genomic DNA library (Jousson et al., 2004a) were immobilized on GeneScreen nylon membranes (Perkin-Elmer, Waltham, MA, USA). The filters were hybridized with 32P-labeled DNA fragments under lowstringency conditions (Monod et al., 1994). The probes were amplified DNA of five A. fumigatus genes (AfuCP1–AfuCP5) which were obtained by PCR with the primers P15/P16, P17/P18, P19/P20, P21/P22, P23/P24, respectively (Table 1), and A. fumigatus genomic DNA as a template. Alternatively, the filters were

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Table 1.

671

Primers used in this study and PCR products

Primer

Oligonucleotide sequence

Location

PCR product size (bp) with cloning sites

SP6 P1

50 -CCTATTTAGGTGACACTATAG-30 50 -GAAGTCTCGTTTCTGATAGAC-30

pSPORT6 Complement of TruMCPA

X326 bp

SP6 P2

50 -CCTATTTAGGTGACACTATAG-30 50 -AGCAGGTTCGTTACCGTG-30

pSPORT6 Complement of TruMCPB

X426 bp

P3 P4

50 -GTTCTCGAGCTGTAGTTTCCCCCTTTG-30 50 -CTTGGATCCTCATCTATCCCTGAATCACAG-30

TruMCPA Complement of TruMCPA

1239 bp XhoI–BamHI

P5 P6

50 -GTTCTCGAGTGCAGTATGGCTACAACCAG-30 50 -GTTAGATCTTATTTTAACCTGAAAATAGGAT-30

TruMCPB Complement of TruMCPB

1570 bp XhoI–BglII

P7 P8

50 -GTTGTCGACTTCAAGGCTTCCCTCCACCCGTT-30 50 -CTTGTCGACGCGGCCGCCTACAAGAAGAAAGCAAG-30

TruSCPA Complement of TruSCPA

1930 bp SalI–NotI

P9 P10

50 -GTTGTCGACTTCAAGGCTTCCCTCCACCCGTT-30 50 -CTTGCGGCCGCCTACTTCGACGCAGCAGGGCTCTTAAT-30

TruSCPA Complement of TruSCPA

1801 bp SalI–NotI

P11 P12

50 -CTTCTCGAGCTCAGTTCCCACCAAAACCGG-30 50 -CTTGGATCCTTACATTGCCAGCTCTATAAC-30

TruSCPB Complement of TruSCPB

1946 bp XhoI–BamHI

P13 P14

50 -CTTCTCGAGCTCAGTTCCCACCAAAACCGG-30 50 -CTTGGATCCTTAAGAGGTCGAGTTGGAGTCGAC-30

TruSCPB Complement of TruSCPB

1805 bp XhoI–BamHI

P15 P16

50 -CTTCTCGAGCCATTTGGGCACCAGCGATAC-30 50 -CTTAGATCTTCAGTGGACACTGGTGCTAGG-30

AfuCP2 Complement of AfuCP2

1637 bp XhoI–BglII

P17 P18

50 -GTTCTCGAGAAAAGATCGGCACAGTATTTCCCTCCC-30 50 -CTTGTCGACGCGGCCGCCTAGTAAATGTCCACTTTCTCCACC-30

AfuCP1 Complement of AfuCP1

1820 bp XhoI–NotI

P19 P20

50 -CTTGTCGACTCGCTCCTCCCTTCCAGCAGATCCTC-30 50 -CTTAGATCTTTAGAACCATTCACCACCAAG-30

AfuCP3 Complement of AfuCP3

1657 bp XhoI–BglII

P21 P22

50 -GTTCTCGAGTGGCTCTGGAAAATCCGCATC-30 50 -GTTGGATCCCTATGCAGTATAGCTTCCTGGCGACG-30

AfuCP4 Complement of AfuCP4

1743 bp XhoI–BamHI

P23 P24

50 -CTTCTCGAGCAAAACATGGTCGGTTCGGC-30 50 -CTTAGATCTTCACAGAGACTCAATGTGGCCC-30

AfuCP5 Complement of AfuCP5

1750 bp XhoI–BglII

P25 P26

50 -GCTTCATGATCCCGACATCAACTGGTTC-30 50 -CTTGGATCCTCATCTATCCCTGATCACAG-30

TruMCPA Complement of TruMCPA

958 bp RcaI–BamHI

P27 P28

50 -GTTTCATGAGTGGGATCCATACATCACTCTGGG-30 50 -CTTAGATCTCTATTTTAACCTGAAAATAGGATATTG-30

TruMCPB Complement of TruMCPB

796 bp RcaI–BglII

P29 P30

50 -GTTCCATGGGCAAGGCTTCCCTCCACCCGTT-30 50 -CTTGGATCCATTAGATATTTGTCGC-30

TruSCPA Complement of TruSCPA

1059 bp NcoI–BamHI

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Table 1. (continued ) Primer

Oligonucleotide sequence

Location

PCR product size (bp) with cloning sites

P31 P32

50 -CTGCCATGGGGCTCTCTGGCACTTCGCCC-30 50 -GTTGGATCCCTACGAAACCTTGATGCCATTCTCC-30

TruSCPB Complement of TruSCPB

745 bp NcoI–BamHI

P33 P34

50 -ATGAAGGGCCTTCTCTCAC-30 50 -TCAACGTTGAGTACTGGAAG-30

TruSCPD Complement of TruSCPD

1638 bp

Primers P1 and P2 were used as antisense primers to amplify cDNA fragments encoding the N-terminus of TruMcpA and TruMcpB. Primers P3–P18 were used for carboxypeptidase gene expression in P. pastoris. Primers P15–P24 were used for A. fumigatus carboxypeptidase gene amplification. Primers P25–P32 were used for antigen production in E. coli. Primers P33–P34 were used for TruSCPD amplification from different T. rubrum isolates.

Table 2. Materials used for the expression of T. rubrum and A. fumigatus carboxypeptidases in P. pastoris, and yields of heterologous proteins Gene

Oligonucleotide primers (Table 1)

Encoded amino acid sequencea

PCR product and cloning sitesb

Expression vector and used cloning sites

Yield of heterologous protein (mg/ml)

TruMCPA

P3 P4

(R)AVVSPF AVIQG

1239 bp XhoI–BamHI

pKJ113 XhoI–BamHI

2

TruMCPB

P5 P6

(R)(V)QYGYNQ PIFRLK

1570 bp XhoI–BglII

pKJ113 XhoI–BamHI

Tracesc

TruSCPA

P7 P8

(RL)QGFPPPV LAFFL

1930 bp SalI–NotI

pKJ113 XhoI–NotI

NDd

TruSCPA

P9 P10

(RL)QGFPPPV IKSPAASK

1801 bp SalI–NotI

pKJ113 XhoI–NotI

2e

TruSCPB

P11 P12

(R)AQFPPKP VIELAM

1946 bp XhoI–BamHI

pKJ113 XhoI–BamHI

NDd

TruSCPB

P13 P14

(R)AQFPPKP VDSNSTS

1805 bp XhoI–BamHI

pKJ113 XhoI–BamHI

NDe

AfuCP2

P15 P16

(R)AIWAPAI PSTSVH

1637 bp XhoI–BglII

pKJ113 XhoI–BamHI

200

AfuCP1

P17 P18

SAQYFPP VEKVDIY

1820 bp XhoI–NotI

pPICZaA XhoI–NotI

30

ND: not detected. a Amino acids shown in parentheses are encoded by the restriction site sequences and added to the N-terminal end of heterologously expressed enzymes. b Target DNA was prepared from 106 clones of the cDNA library (see Material and method section). c Detection by Western blotting. d Full-length protein. e Truncated form.

hybridized with 32P-labeled oligonucleotides as previously described (Monod et al., 1994). Oligonucleotides were labeled with polynucleotide kinase and [g-32P]ATP (Amersham Pharmacia, Buckinghamshire, UK). All positive plaques were purified and the bacteriophage DNAs were isolated as described previously (Grossberger, 1987). Agarose gel electrophoresis of

restriction enzyme-digested recombinant bacteriophage lEMBL3 DNA, Southern blotting and subcloning of hybridizing fragments from bacteriophages into pUC18 and pCL1920 (Lerner and Inouye, 1990) were performed using standard protocols (Sambrook et al., 1989). DNA sequencing was performed by Microsynth (Balgach, Switzerland).

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Production of recombinant carboxypeptidases

Polyclonal antibodies

Expression plasmids were constructed by cloning a cDNA PCR product in the P. pastoris expression vectors pKJ113 (Borg-von Zepelin et al., 1998) or pPICZaA (Invitrogen, Carlsbad, CA, USA) (Table 2). The PCR products were purified using a High Pure PCR Purification Kit (Roche Diagnostics, Rotkreuz, Switzerland), and digested by restriction enzymes for which a site was previously introduced at the 50 extremity of the primers. P. pastoris GS115 and KM71 were transformed with 10 mg of linearized plasmid DNA as previously described. Selected transformants were grown to near saturation (OD600 of 20) at 30 1C in 10 ml of glycerolbased yeast medium (0.1 M potassium phosphate buffer at pH 6.0, containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB without amino acids, 1% (v/v) glycerol and 4  105% (w/v) biotin). Cells were harvested and resuspended in 2 ml of the same medium with 0.5% (v/v) methanol instead of glycerol and incubated for 2 days. The supernatant was then harvested and tested for protein production on SDSPAGE gels. Salts and small-molecular-weight solutes were removed from 2.5 ml of P. pastoris culture supernatant by passing through a PD10 column (Amersham Pharmacia) using 20 mM Tris–HCl buffer, pH 7.5, before testing for proteolytic activity. Active fractions were concentrated by ultrafiltration using Centricon centrifugal filter devices with a YM-30 membrane (Amicon, Millipore, Bedford, MA, USA). Protein concentrations were measured using the method of Bradford (1976) using a commercial reagent (Bio-Rad, Hercules, CA, USA). Supernatants of P. pastoris GS115 and KM71 which were grown in the same conditions were used as negative controls for comparison.

To raise polyclonal antibodies against T. rubrum carboxypeptidases, large peptides (241–348 aa) corresponding to sequences from TruMcpA (res 112–422), TruMcpB (res 281–536), TruScpA (res 20–368), and TruScpB (res 211–452) were produced in E. coli BL21 using the pET expression system from Novagen (Darmstadt, Germany) with the modified pET11a plasmid pET-11aH6 (Reichard et al., 2006). The following pairs of sense/antisense primers P25/P26, P27/P28, P29/P30, P31/P32 (Table 1) were used to amplify DNA from the corresponding pKJ113 constructs (Table 2) which were generated for expression of carboxypeptidase genes in P. pastoris. The PCR products were digested with appropriate restriction enzymes (Table 1) and cloned into the NcoI and BamHI sites of pET-11aH6. The resulting plasmids were used to transform E. coli BL21. E. coli transformants were grown in Luria broth (LB) liquid medium at 37 1C to an OD600 of 0.6, and 6  His tagged peptide expression was induced by adding IPTG to a 0.1 mM final concentration. Incubation was continued for an additional 4-h period at 37 1C. Cells were collected by centrifugation (4500g, 4 1C, 15 min), and 6  His-tagged peptides were extracted with guanidine hydrochloride buffer and Ni-NTA resin (Qiagen, Hilden, Germany) columns according to the instructions of the manufacturer. Rabbit antisera were made by Eurogentec (Lie`ge, Belgium) by using the purified carboxypeptidase polypeptide chains as antigens.

Enzymatic activities Carboxypeptidase activities were measured with N-(2furanacryloyl)-L-phenylalanyl-L-phenylalanine (FAPP) (Sigma) as a substrate (Peterson et al., 1982). Substrate stock solutions were prepared at 5  102 M concentration in ethanol and stored at 20 1C. The reaction mixture contained a concentration of 0.1 mM substrate and enzyme preparation (0.5 mg per assay) in 1.0 ml buffer at different pH values. For metallocarboxypeptidase, 50 mM Tris–HCl buffer was used. For serine carboxypeptidases, 50 mM sodium acetate buffer was used. FAPP hydrolysis at 30 1C was monitored by following the decrease in absorbance at 330 nm with a thermoregulated spectrophotometer (Beckman, Fullerton, CA, USA). For each assay, a control with buffer instead of enzyme was performed in parallel. The enzymatic activities were expressed in mU (nmoles of hydrolyzed FAPP min1).

Pichia pastoris cell protein extracts Pichia pastoris cell protein extracts of 100 ml culture were prepared following the method of Yaffe and Schatz (1984). The pelleted cells were resuspended in 1.8 M NaOH, 1.2 M b-mercaptoethanol, and incubated for 5 min on ice. After addition of 200 ml 10% TCA, the mixture was incubated for 5 min, centrifuged and the pellet was resuspended in 50 ml 2  SDS-PAGE loading buffer and neutralized at pH 7.0 by adding 5–10 ml 1 M Tris base. The samples were heated to 95 1C for 3 min before loading onto 12% SDS-PAGE gels.

T. rubrum membrane preparations T. rubrum was grown in Sabouraud, SP and KSP liquid medium (100 ml) (Monod et al., 2005) for 8, 12 and 30 days, respectively. The mycelium was disrupted using a mortar and a pestle in liquid nitrogen. The broken mycelium was resuspended in 1 ml Tris–HCl, pH 7.5, containing 0.25 M sucrose, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride. The homogenate was then centrifuged for 10 min at 4000g to remove the cell

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walls. The supernatant was then centrifuged for 1 h at 36,000g, and the membrane pellets were suspended in 400 ml of the same buffer, divided into 20-ml aliquots, and stored at 20 1C. Ten microliters of membrane preparation and 10 ml of SDS-PAGE loading buffer were mixed before loading on gels.

Protein extract analysis SDS-PAGE of the different protein extracts was performed on a 10% separating gel. The gels were stained with Coomassie brilliant blue R-250 (Bio-Rad). N-glycosidase F digestion was performed as previously described (Doumas et al., 1998). Western blots were revealed using rabbit antisera and alkaline phosphataseconjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA, USA). Protein concentrations were measured by the method of Bradford (1976), and by densitometry on SDS-PAGE gels using different amounts of bovine serum albumin as standards.

Results Trichophyton rubrum secreted carboxypeptidase activity T. rubrum secreted carboxypeptidase activity was detected in SP and KSP medium culture supernatants using FAPP as a substrate at pH 7.0. The activity was maximal (0.8 mU/ml) when the culture media were clarified and a substantial endoproteolytic activity was recorded after 10 and 30 days of growth in SP and KSP medium, respectively. No carboxypeptidase activity was detected at acidic pH. No neutral and acidic carboxypeptidase activities were detected in supernatants of cultures in Sabouraud liquid medium (data not shown).

Cloning of genes encoding T. rubrum metallocarboxypeptidases In a screen of a collection of 3804 ESTs, two sequences were detected coding for partial amino acid sequences of carboxypeptidases, which were homologous to human carboxypeptidase A of the M14 family. These proteases were named TruMcpA and TruMcpB. Other secreted proteases encoded by ESTs were previously identified fungalysins, subtilisins, dipeptidyl-peptidases and leucine aminopeptidases (Jousson et al., 2004a, b; Monod et al., 2005). A partial cDNA encoding the N-terminus of both proteases was obtained by PCR using an SP6 promoter primer as a sense primer, an antisense primer (P1 for TruMCPA cDNA and P2 for TruMCPB cDNA) which

was based on the identified EST sequence (Table 1), and DNA prepared from 106 clones of the cDNA library as a target. The sequence of the obtained cDNA fragment was used to complete the sequence of the whole insert of the cDNA plasmid from which the EST was obtained. The full-length TruMCPA cDNA contained an open reading frame of 1266 bp encoding a polypeptide chain of 422 amino acids (Table 2 and Fig. 1). The full length TruMCPB cDNA contained an open reading frame of 1614 bp encoding a polypeptide chain of 538 amino acids. The analysis of the amino acid sequences of both polypeptide chains suggested the existence of a signal peptide with a putative signal peptidase cleavage site (von Heijne, 1986). Trichophyton rubrum genomic DNA was amplified using primers P3/P4 and P5/P6. Sequencing of the PCR products revealed the presence of 5 and 2 introns in TruMCPA and TruMCPB, respectively.

Cloning of genes encoding T. rubrum serine carboxypeptidases Secreted acidic serine carboxypeptidases of the S10 family were previously characterized in Aspergillus niger and Aspergillus oryzae (Dal Degan et al., 1992; Svendsen and Dal Degan, 1998; Blinkovsky et al., 1999). However, no sequence indicating a carboxypeptidase of the S10 family was identified among the 3804 ESTs. Therefore, we investigated the presence of homologous peptidases in dermatophytes using a reverse genetic approach (from genes to proteins). Dermatophytes endoprotease and aminopeptidase activities were previously investigated using data available from Aspergillus fumigatus (Brouta et al., 2002; Descamps et al., 2002; Monod et al., 2005). In the present work, the DNA sequences of five A. fumigatus S10 carboxypeptidase genes (AfuCP1 to AfuCP5) identified in the A. fumigatus genome (www.tigr.org/tdb/e2k1/afu1) were used to design probes for screening a T. rubrum genomic DNA library. Hybridizing clones were obtained with AfuCP1, AfuCP2 and AfuCP3 as probes, but not with AfuCP4 and AfuCP5. The bacteriophage DNA of the positive clones was isolated, and restriction fragments identified by Southern blotting were subcloned and sequenced. Translated BLAST searches were performed for all sequences obtained and revealed three different sequences encoding parts of putative carboxypeptidase proteins in T. rubrum. The corresponding putative T. rubrum genes were designated TruSCPA, TruSCPB and TruSCPC. The cDNA of these genes was obtained by PCR using 50 -sense and 30 -antisense primers designed on isolated genomic DNA (Table 2), and total DNA from a pool of 106 clones of the T. rubrum cDNA library was used as a template. Comparison of the

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Fig. 1. Alignment of the deduced amino acid sequences of carboxypeptidases from the M14A subfamily, including TruMcpA, Metarhizium anisopliae carboxypeptidase A (MeCpA, MEROPS identifier MER03908) and human carboxypeptidase A1 (CpA1, MER01190). The alignment was optimized by introducing gaps using the CLUSTALW multiple sequence alignment program (Thompson et al., 1994). Identical and similar residues are black and gray shaded, respectively. The arrow indicates a potential TruMcpA signal peptidase cleavage site. Mature Metarhizium anisopliae and human carboxypeptidases A start at residue Gly100 (Joshi and St Leger, 1999) and Ala111 (Auld, 2004), respectively. The zinc-binding residues are indicated by an asterisk. The activesite residues are indicated by a rhombus. Conserved residues involved in substrate binding are indicated by solid triangles. The conserved Cys residues forming disulfide bridges are indicated by solid circles.

sequences of cDNA with genomic DNA revealed one intron in TruSCPA, two in TruSCPB, and one in TruSCPC. In an independent approach we screened the T. rubrum genomic library with an oligonucleotide probe (50 -TGG YTX AAY GGX GGX CCX GG-30 ; X ¼ inosine, Y ¼ C/T) encoding an amino acid sequence (WLNGGPG) conserved in T. rubrum and Aspergillus spp. serine carboxypeptidases. In addition to the previously characterized TruSCPA, TruSCPB and TruSCPC, a fourth DNA sequence (TruSCPD) with homology to serine carboxypeptidase genes could be identified. However, the frame encoding an amino acid sequence which was 66% similar and 45% identical to TruScpC was interrupted by four stop codons. The occurrence of this pseudogene with four stop codons in three different T. rubrum strains was confirmed by sequencing PCR products which were obtained using specific primers (P33/P34, Table 1) and fungal genomic DNA as a template.

TruSCPA, TruSCPB and TruSCPC contained open reading frames of 1956, 1986, and 1608 bp encoding proteins of 652, 662, and 536 amino acids, respectively (Table 3 and Fig. 2). TruScpA and TruScpB were closely related to AfuCp1 (53% and 52% aa sequence identity, respectively), whereas TruScpC was more related to AfuCp3 (69% identity). The two latter proteases were closely related to Saccharomyces cerevisiae vacuolar carboxypeptidase Y (52% and 53% identity, respectively) (Mortensen et al., 2004), and were therefore putatively vacuolar. The analysis of the amino acid sequences of TruScpA, TruScpB and TruScpC suggested the existence of a signal peptide with putative signal peptidase cleavage sites (Fig. 2). Interestingly, the deduced translation products of TruSCPA and TruSCPB exhibited a C-terminal extremity of 41 and 47 amino acids, respectively, which was absent in the A. fumigatus homologues. These C-terminal sequences were serine/threonine-rich, suggesting that TruScpA and TruScpB were GPI-anchored proteins. Using a

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GPI-modification site prediction algorithm (Eisenhaber et al., 1999), potential GPI anchors were detected in both protein sequences, with an o site located at Ser630 for TruScpA (score 12.53, P-value 6.28  103) and at Gly633 for TruScpB (score 5.83, P-value 9.33  103).

Production of recombinant T. rubrum and A. fumigatus carboxypeptidases Trichophyton rubrum cDNAs encoding TruMcpA, TruMcpB, TruScpA and TruScpB were independently cloned in P. pastoris expression vectors and expressed in P. pastoris. The corresponding culture supernatants of P. pastoris grown in methanol inducing medium contained about 2 mg/ml of a 42-kDa recombinant TruMcpA, which could be detected on SDS-PAGE gels stained with Coomassie brilliant blue (Fig. 3A, lane 1). In contrast, secreted recombinant TruMcpB was only revealed by Western blotting with antibodies raised against a TruMcpB 257 residue long peptide (residues 295–551) (data not shown). No recombinant full-length TruScpA and TruScpB could be detected in the corresponding P. pastoris culture supernatants. However, a 90-kDa protein was secreted (2 mg/ml) by expression of a cDNA encoding TruScpA of which the C-terminus was truncated (Table 2 and Fig. 4, lane 5; Fig. 5, lane 2). Similar amounts of full-length and truncated TruScpA were detected in cell protein extracts (Fig. 5, lanes 5 and 6). These results were in agreement with the prediction that TruScpA is GPI-anchored and not released from the cells. In the case of truncated and the full-length TruSCPB expression (Table 2), Western blotting experiments revealed recombinant protein in yeast cell extract, but not in culture supernatant (Fig. 5, lanes 8–9 and 11–12). As controls full-length AfuCp1 and AfuCp2 which are closely related to TruScpA and TruScpB were also made in P. pastoris (Table 2). Recombinant active AfuCp1 and AfuCp2 were obtained with a yield higher than that of truncated TruScpA (30 and 200 mg/ml of culture supernatant, respectively). Recombinant TruScpA, AfuCp1 and AfuCp2 were glycoproteins, as attested by a reduction in their molecular weights following treatment with N-glycosidase F (Fig. 4). The apparent molecular mass of each deglycosylated protein was close to that of the calculated molecular mass of the polypeptide chain deduced from the nucleotide sequence of the protease genes. Characteristics of the primary

Fig. 3. (A) SDS-PAGE (9% gel) of recombinant TruMcpA (zymogen) secreted by P. pastoris (lane 1) stained with Coomassie blue. The proteins of 100 ml P. pastoris culture supernatant (10 ml, 10  concentrated by ultrafiltration) containing approximately 0.2 mg of recombinant TruMcpA, were loaded on the gel. In lane 2, recombinant TruMcpA was incubated for 30 min at 30 1C with 1 ml of culture supernatant of T. rubrum grown in KSP medium, and then loaded on the gel. (B) Western blotting detection of TruMcpA in T. rubrum culture supernatant and identification of T. rubrum secreted proteases cleaving the TruMcpA zymogene. Lane 1: TruMcpA secreted by T. rubrum in KSP medium. T. rubrum KSP culture supernatant was 50  concentrated by ultrafiltration, and 10 ml of concentrate approximately containing 15 ng TruMcpA were loaded on the gel. Lane 2: recombinant 42-kDa TruMcpA (zymogen) secreted by P. pastoris (10 ml). Lane 3: recombinant 42-kDa TruMcpA from P. pastoris culture supernatant (10 ml) incubated for 30 min at 30 1C with 1 ml of culture supernatant of T. rubrum grown in KSP medium. In lanes 4–9, 10 ml of P. pastoris culture supernatant were incubated with 10 ng of recombinant TruSub3 (lane 4), TruSub4 (lane 5), TruSub5 (lane 6), TruSub7 (lane 7), TruMep1 (lane 8), and TruMep3 (lane 9), respectively, before loading on the SDS-PAGE gel (9%). TruMcpA was revealed by immunolabeling using antiTruMcpA.

structure of each recombinant carboxypeptidase are summarized in Table 3.

Detection of carboxypeptidases in T. rubrum culture supernatant and membrane extracts Western blot analysis of culture supernatants of T. rubrum grown in SP and KSP medium revealed a secreted TruMcpA of 34 kDa which had an electrophoretic mobility higher than that of the recombinant enzyme (42 kDa) produced in P. pastoris. (Fig. 3B, lanes 1 and 2). However, recombinant enzyme pre-incubated (30 1C, 30 min) with either T. rubrum culture supernatant, or recombinant TruSub3, or recombinant TruSub4, was cleaved and had the same electrophoretic mobility than the native enzyme found in T. rubrum culture supernatant (Fig. 3B, lanes 3–5). Other T. rubrum secreted proteases such as TruSub5, TruSub7,

Fig. 2. Alignment of the deduced amino acid sequence of carboxypeptidases from the S10.016 subfamily, including TruScpA and TruScpB, AfuCp1 (MER79359) and AfuCp2 (MER79360), and A. oryzae AoryS1 (MER16549). The alignment was optimized by introducing gaps using the CLUSTALW multiple sequence alignment program (Thompson et al., 1994). Identical and similar residues are black and gray shaded, respectively. The arrow indicates the potential cleavage site for the signal sequence of TruScpA and TruScpB. Residues that form the catalytic triad are indicated by a rhombus. The Trp, Asn and Glu residues interacting with the carboxylate group of the substrate in yeast carboxypeptidase Y are indicated by solid triangles. The omega site of potential GPI anchors predicted by the GPI-modification site algorithm in TruScpA and TruScpB is indicated by an asterisk.

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Fig. 4. N-glycosidase F treatment of recombinant AfuCp2, AfuCp1 and TruScpA. The proteins of 2, 12 and 200 ml (20 ml, 10  concentrated by ultrafiltration) P. pastoris culture supernatant containing approximately 0.4 mg of recombinant AfuCp2, AfuCp1, and TruScpA, respectively, were loaded on a 10% SDS-PAGE gel (lanes 1, 3, and 5). Lanes 2, 4 and 6 show the proteins deglycosylated by N-glycosidase F treatment (N-GF +). The gel was stained with Coomassie brilliant blue R-250. M: Molecular mass markers.

Fig. 5. Western blot detection of recombinant C-terminally truncated (*) or full-length (#) TruScpA and TruScpB in P. pastoris cells (CE) and culture supernatant (SN). Five microliters of P. pastoris culture supernatant and proteins from 106 P. pastoris cells were loaded per lane. Immunodetection was performed using specific anti-TruScpA and antiTruScpB antibodies. Samples of strain GS115 were used as negative controls (lanes 1, 4, 7, and 10).

TruMep1 and TruMep3 were inactive on the secreted recombinant TruMcpA product (Fig. 3B, lanes 6–9). Recombinant carboxypeptidase cleavage could also be achieved using other subtilisins such as proteinase K, subtilisin Carlsberg and Aspergillus oryzae alkaline protease, but not by trypsin (data not shown). In Western blotting experiments with anti-TruMcpA antibodies, a 34-kDa carboxypeptidase was also detected in culture supernatants of other dermatophyte species, e.g. Arthroderma benhamiae, Trichophyton mentagrophytes, T. soudanense, T. verrucosum, Microsporum canis, and M. gypseum, but not in culture supernatant of T. violaceum (data not shown).

In contrast to TruMcpA, a TruMcpB-specific protein band could not be detected by Western blotting using anti-TruMcpB antibodies in culture supernatants of all dermatophyte species tested. Western blot analysis revealed both TruScpA and TruScpB in membrane extracts of T. rubrum grown in Sabouraud and protein medium (Fig. 6). TruSCPB appeared to be induced in the latter medium, while comparable amounts of TruScpA were observed in the aforementioned media. We have verified that antiTruScpA serum did not cross-react with TruScpB. Absence of cross reactivity between anti-TruScpB serum and TruScpA was also confirmed (data not shown). None of these serine carboxypeptidases was detected in T. rubrum culture supernatants.

Activities of T. rubrum and A. fumigatus recombinant carboxypeptidases The recombinant mature 34-kDa TruMcpA very efficiently hydrolyzed the substrate FAPP at 30 1C between pH 4.0 and 10.0 with an optimum at pH 7.5. The specific activity was measured as 23 mU/mg protein, while that of the 42-kDa proenzyme, which was secreted by P. pastoris, was measured as 2.6 mU/mg protein. The activating subtilisins TruSub3 and TruSub4 did not hydrolyze FAPP (data not shown). Under identical culture conditions P. pastoris strains GS115 and KM71 used for transformation did not secrete any carboxypeptidase activity into the culture medium. Recombinant truncated TruScpA, AfuCp1 and AfuCp2, very efficiently hydrolyzed FAPP at 30 1C between pH 4.0 and 8.0 with an optimum at pH 4.5. The specific activity of TruScp1, AfuCp1 and AfuCp2 for FAPP as a substrate, was measured as 10, 4.3 and 13 mU/mg protein, respectively.

Discussion In a medium containing protein as sole nitrogen and carbon source, T. rubrum and other dermatophyte species secrete a carboxypeptidase of the M14A subfamily. To our knowledge, this is the first time that secreted carboxypeptidase activity is reported in dermatophytes. The amount of TruMcpA secreted by T. rubrum in protein medium is low in comparison to those of TruSub3, TruSub4 and TruLap2, which are in the range of 1 mg/ml (Jousson et al., 2004b; Monod et al., 2005). When the proteins of T. rubrum culture supernatant were separated by SDS-PAGE, gels stained with Coomassie brilliant blue revealed TruSub3, TruSub4 and TruLap2 as dominant bands (Jousson et al., 2004b; Monod et al., 2005), while in the same experimental conditions, TruMcpA was not detected.

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Table 3.

679

T. rubrum and A. fumigatus genes encoding carboxypeptidases, and main characteristics of deduced translation products

Gene name

TruMCPA TruMCPB TruSCPA TruSCPB

TruSCPC TruSCPD AfuCP2

AfuCP1

Gene length (nt) with intron(s) and stop codon Number of introns ORF length (nt) in cDNA Pre(pro)-protein (aa) Signal peptide (aa) Catalytic domain of the protein (aa) Calculated molecular mass of the polypeptide chain of the preproprotein (kDa) Calculated molecular mass of the polypeptide chain of the mature protein (kDa) Molecular mass of recombinant enzyme (kDa; SDS-PAGE) Molecular mass of heterologous enzyme after deglycosylation (kDa; SDS-PAGE) Number of putative Nglycosylation sites (mature domain) Calculated pI (mature domain) GenBank accession number

1604

1732

2030

2106

1680

1638

2253

1886

5 1266 422 18 E327

2 1614 538 21 517

1 1956 652 19 592a

2 1986 662 21 594a

1 1608 536 17 519

– – – –

9 1659 553 15 538

1 1839 613 18 595

47.1

59.9

71.8

73

60.1

–

61.4

68.3

ND

57.6

66.2a

66.4a

58.4

–

59.9

66.6

42 (33)b

60

90

85

ND

88

110

42 (33)b

ND

70

ND

ND

–

65

70

0

4

13

14

2

–

11

15

8.5 8.7 5.1a 6.7a 5.3 – 5.0 4.7 DQ778058 DQ786567 AY497023 AY497022 AY497024 AY497020 AY436352 AY433801 EU024297 EU024296

The theoretical molecular mass of the mature domain and the pI were calculated using VectorNTI suite 8 (InforMax, Inc.). The putative glycosylation sites correspond to the NXT/S pattern (X: any amino acid except for P). Signal peptide predictions were based on the method of von Heijne (1986). ND: not determined. a Without GPI. b After secreted subtilisin treatment, molecular mass similar to that of active enzyme in T. rubrum culture supernatant.

Fig. 6. Detection of TruScpA and TruScpB in T. rubrum membrane extracts. TruScpA and TruScpB were revealed by immunolabeling using specific polyclonal antibodies. Proteins of 10 ml membrane preparation (see Materials and methods) were separated in each lane. T. rubrum was grown in Sabouraud liquid medium (lanes 1, 3) and in protein liquid medium (KSP) (lanes 2, 4). No TruScpA and TruScpB was detected in T. rubrum culture supernatant (not shown).

TruMcpA is homologous to Metarhizium anisopliae secreted carboxypeptidase (48% and 67% aa sequence identity and similarity, respectively) (Joshi and St Leger, 1999) and to human carboxypeptidase A (34% and 54% aa sequence identity and similarity, respectively) which was extensively studied and is considered the type enzyme of the M14A family in the MEROPS proteolytic enzyme data base. The TruMcpA amino acid sequence shows residues which are conserved in all carboxypeptidases of the M14A subfamily (Auld, 2004). These residues are His179, Glu182 and His309 which are ligands for the catalytic zinc, cysteine residues that form disulfide bridges, and other amino acids, e.g. Arg237, Arg255, Tyr311, Tyr362, and Glu385, which are important for substrate binding and catalysis in the M14A family (Fig. 1). A Blast analysis revealed that a gene encoding a carboxypeptidase of the M14A subfamily is also present in the genomes of Magnaporthe grisea, Giberella spp. and Coccidioides spp. In contrast, an orthologue of TruMcpA was found neither in the genome of yeasts nor in that of filamentous ascomycetes such as Aspergillus spp. Only the preproprotein form of TruMcpA was obtained as recombinant secreted protein by expression

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of the TruMCPA gene in P. pastoris. The propeptide of the native mature T. rubrum enzyme is removed alternatively by either one of two endoproteases of the subtilisin family (TruSub3 or TruSub4) which are concomitantly secreted by the fungus. Sequencing of the 34-kDa TruMcpA N-terminus by Edman degradation did not yield usable results. However, the difference between the molecular mass of the proenzyme secreted by P. pastoris and that of the mature enzyme (8 kDa) indicates that the propeptide is about 90 amino acids long, and that the TruMcpA N-terminus is in a position close to that of the N-terminus of the M. anisopliae secreted carboxypeptidase. In an analogous manner, human carboxypeptidase A is secreted as a zymogen by acinar pancreatic cells and activated in the duodenum by trypsin which removes an N-terminal 94-amino-acid propeptide (Vendrell et al., 1992, 2000). It appears that the mode of propeptide removal by a concomitantly produced endoprotease is a general rule for the activation of M14A carboxypeptidases. Many other secreted proteases are synthesized with a propeptide that has been found to be essential and specific for assisting correct folding as well as secretion of the mature domain of the enzyme (for review, see Eder and Fersht, 1995). However, upon completion of folding, the propeptide of many other proteases was shown to be removed in order to generate the active enzyme through an autoproteolytic reaction or, like in the Candida albicans secreted aspartic proteases, through an exogenous proteolytic reaction in the Golgi apparatus via the membrane bound protease Kex2 (Togni et al., 1996; Newport and Agabian, 1997). The propeptides of pancreatic carboxypeptidases A like those of many other proteases behave as inhibitors of the enzymes from which they derive. The removal of the propeptide mediated by trypsin cleavage renders the active center of pancreatic carboxypeptidase A accessible to protein and peptide substrates (Vendrell et al., 2000). Like procarboxypeptidase A (Martinez et al., 1981), recombinant proTruMcpA which is secreted by P. pastoris had some activity which was about 10% of that of the mature TruMcpA. The activity which was measured for proTruMcpA cannot be attributed to a contamination with mature TruMcpA (Fig. 3). A gene called TruMCPB, encoding another secreted metalloprotease of the M14 family was found in T. rubrum. However, the protease was not detected in the T. rubrum culture supernatant under the tested conditions. In addition, only a small amount of secreted recombinant TruMcpB was revealed by Western blotting of samples of P. pastoris culture supernatant. A Blast analysis revealed that a gene encoding a similar protein is also present in the genome of many fungi such as Aspergillus spp., Magnaporthe grisea, Gibberella spp., and Coccidioides spp., as well as in bacteria such as Clostridium tetani (MER38686) and Bacillus licheniformis

(MER14747). All these proteins are still putative and unassigned proteases of the M14 family. It appears that the specialization of dermatophytes was accompanied by the evolution of particular carboxypeptidases of the S10 family. T. rubrum ScpA and ScpB differ from those of most other fungi by the presence of a GPI-modification site. In addition to TruScp1 and TruScp2, only a single homologous hypothetical protein from the ascomycete Gibberella zeae (EAA70910) was found with a potential GPI signal (data not shown). TruScpA and TruScpB possess the catalytic triad of the members which are in the SC clan from the MEROPS peptidase database (Ser238, Asp458, and His516 for TruScpA, and Ser240, Asp459, and His517 for TruScpB). Residues interacting with the carboxylate group of the substrate in yeast carboxypeptidase Y [Trp49, Asn51, Glu145, (Mortensen et al., 1994, 2004)] were found to be conserved (Fig. 2). Even after removal of the putative GPI anchor, recombinant TruScpB was not detected in the culture supernatant of P. pastoris. It is known that mature secreted enzymes can remain associated to yeast cell walls, e.g. Candida wickerlamii extracellular beta-glucosidase produced in S. cerevisiae (Skory et al., 1996) and laccase from Cryptococcus neoformans (Zhu et al., 2001). A phylogenetic analysis including T. rubrum and Aspergillus carboxypeptidases of the S10 family produced a robust tree consisting of three main clades (Fig. 7). The most derived clade included TruScpA, TruScpB, AfuCp1, AfuCp2, AfuCp6, Penicillium janthinellum and A. oryzae S1 carboxypeptidases. A second clade included all sequences homologous to S. cerevisiae CPY and other fungal putative vacuolar carboxypeptidases, among which are TruScpC and AfuCp3, and the amino acid sequence deduced from the pseudogene TruSCPD. The basal clade contained AfuCp4 and AfuCp5, and A. niger CPD1 and CPD2. Screening of the genomic library and the tree topology indicated that T. rubrum lacks AfuCp4 and AfuCp5 orthologues. Additional phylogenetic analyses including all putative S10 carboxypeptidases displayed by Blast in fungal genomes (data not shown) indicated that the clade clustering S10.008 and S10.016 sequences only contains secreted carboxypeptidases from filamentous fungi, and no yeast enzymes. In conclusion, T. rubrum carboxypeptidase activity differs in several aspects from that of Aspergillus spp. (i) T. rubrum McpA has no orthologue in the latter fungi. (ii) TruScpA and TruScpB are not secreted in the growth medium but are membrane associated. In contrast, secreted aminopeptidase activity is mediated by a similar battery of enzymes (Lap1, Lap2, DppIV and DppV) in dermatophytes and Aspergillus spp. (Monod et al., 2005). Aminopeptidases and carboxypeptidases could be used for the degradation of large peptides generated by endopeptidase digestion of

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der Naturforscher Leopoldina BMBF-LPD 9901/8-146).

681

(Fo¨rderkennzeichen

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Fig. 7. Neighbor-joining phylogenetic tree of fungal carboxypeptidases of the S10 family. Trichophyton rubrum sequences are boxed. Amino acid sequences were aligned using Clustal W as implemented in BioEdit software (Hall, 1999). The Dayhoff PAM model of protein evolution was used to compute the distances between sequences. Phylogenetic analyses were performed using DAMBE software (Xia and Xie, 2001). The tree was rooted with two plant carboxypeptidase D sequences of the S10.005 subfamily. The fungal carboxypeptidases analyzed belong to different S10 subfamilies indicated at the right. Bootstrap values (1000 replicates) are indicated at nodes. Scale bar: number of substitutions/site. Abbreviations are: Tru, Trichophyton rubrum; Afu, Aspergillus fumigatus; Aory, A. oryzae; Anid, A. nidulans; Anig, A. niger; Pja, Penicillium janthinellum; Scer, Saccharomyces cerevisiae. MEROPS identifiers are: AfuCp1, MER79359; AfuCp2, MER79360; AfuCp3, MER79361; AfuCp4, MER79362; AfuCp5, MER79363; AfuCp6, MER82516; AoryS1, MER16549; AnidCpyA, MER90176; AnigCPD1, MER27994; AnigCPD2, MER00415; PjaS1, MER00412; ScerCpY, MER02010; TruScpB, MER79400; TruScpC, MER79401. TruScpA and TruScpD are not registered in MEROPS.

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Acknowledgements We thank Olympia Bontems for technical assistance and Dr. Massimo Lurati for critical reading of the manuscript. This work was supported by the Swiss National Foundation for Scientific Research, Grant 3100-105313/1. Peter Staib is the recipient of a postdoctoral fellowship from the Deutsche Akademie

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