Characterization of a chaperone ClpB homologue of Paracoccidioides brasiliensis

Yeast Yeast 2002; 19: 963–972. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.888

Yeast Sequencing Report

Characterization of a chaperone ClpB homologue of Paracoccidioides brasiliensis Ros´alia S. A. Jesuino,1 Maristela O. Azevedo,2 M. Sueli S. Felipe,2 Maristela Pereira1 and C´elia M. de Almeida Soares1 * 1 Laborat´ orio de Biologia Molecular, Instituto de Ciˆencias Biol´ogicas, Universidade Federal de Goi´as, ICB II, Campus II, 74001-970, Goiˆania, Goi´as, Brazil 2 Laborat´ orio de Biologia Molecular, Instituto de Biologia, Universidade de Bras´ılia, 70910-900 Bras´ılia, D.F., Brazil

*Correspondence to: C´elia M. de Almeida Soares, Laborat´orio de Biologia Molecular, ICBII, Campus II, Universidade Federal de Goi´as, 74001-970, Goiˆania, Goi´as, Brazil. E-mail: [email protected]

Received: 16 January 2002 Accepted: 10 May 2002

Abstract We report the cloning and sequence analysis of a genomic clone encoding a Paracoccidioides brasiliensis ClpB chaperone homologue (PbClpB). The clpb gene was identified in a λ Dash II library. Sequencing of Pbclpb revealed a long open reading frame capable of encoding a 792 amino acid, 87.9 kDa protein, pI of 5.34. The predicted polypeptide contains several consensus motifs of the ClpB proteins. Canonical sequences such as two putative nucleotide-binding sites, chaperonins ClpA/B signatures and highly conserved casein kinase phosphorylation domains are present. ClpB is 69% to 49% identical to members of the ClpB family from several organisms from prokaryotes to eukaryotes. The transcript of PbclpB was detected as a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phase of the fungus. A 89 kDa protein was also detected in yeast cells of P. brasiliensis. The sequence of the clpb gene and the deduced ClpB protein have been submitted to GenBank under Accession No. AF449501. Copyright  2002 John Wiley & Sons, Ltd. Keywords: Paracoccidioides brasiliensis; dimorphism; infection; ClpB chaperone; ATP-dependent proteases

Introduction Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM), a fungal disease that affects many individuals in Latin America (Lacaz et al., 1991). Once established, the disease may present several clinical forms (Brummer et al., 1993). Variations in intensity, dissemination and characteristics of the lesions on PCM will occur in a given patient, depending on factors such as fungal virulence and fluctuations in the host defence mechanisms (Franco et al., 1993). The fungus grows as budding yeast or mycelia, depending on the temperature (Restrepo et al., 1985). P. brasiliensis, as a dimorphic organism, is subjected to heat stress as a regular feature of its life cycle. When the infectious propagules of the fungus mycelium phase reach the host pulmonary alveoli, they face a temperature upshift that may Copyright  2002 John Wiley & Sons, Ltd.

trigger adaptative mechanisms to resist the new environmental conditions. Changing environmental conditions elicit the expression of new types of cellular proteins in all organisms. In particular, a group of ATPdependent proteases is overexpressed during heat shock in prokaryotic and eukaryotic organisms. This new group of conserved proteins includes Clp (caseinolytic protease)/HSP100, initially identified as a heat shock-inducible, multicomponent, ATP-dependent protease (Schirmer et al., 1996). The Clp proteins have acquired great interest for researchers because of their links to both the chaperonin and proteolytic activities (Gottesman et al., 1997; Schirmer et al., 1996). They include representatives within different intracellular compartments of prokaryotes and eukaryotes (Squires et al., 1991; Leonhardt et al., 1993; Krobitsch et al., 1998) and can be divided into subfamilies

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according to conserved structural characteristics (Schimmer et al., 1996). The ClpB family are heat shock-inducible representatives of the Clp family of ATPases that function as molecular chaperones, preventing protein denaturation and aggregation at high temperatures and also promoting the refolding of aggregated polypeptides (Leonhardt et al., 1993; Parsell et al., 1994; Schmitt et al., 1995). The fact that P. brasiliensis is confronted by a heat stress after the invasion of the human host and that the morphogenetic conversion from mycelium to yeast is a crucial step in the establishment of infection has led our group to investigate the stress response in the fungus. Early work from our laboratory using metabolically labelled cells has led to a group of heat-induced proteins (Silva et al., 1994). Our group also isolated and characterized the genes encoding the heat-induced proteins 70 and 60 (Silva et al., 1999; Salem-Izaac et al., 2001). Recently the cloning and characterization of the gene encoding the heat-inducible Lon protease of P. brasiliensis has been described by Barros and Puccia (2001). In this paper, we describe the cloning, sequencing and characterization of a clpb-encoding gene of P. brasiliensis. To our knowledge, the PbClpB protein is the first described ClpB in a pathogenic fungus. This work was performed in anticipation that the obtained data could give new insights to the fungus dimorphic transition and to the events that led to the establishment of infection.

Materials and methods Microrganism and growth conditions The P. brasiliensis isolate Pb01 (ATCC, MYA 826) has been previously investigated by our group (Pereira et al., 2000; Fonseca et al., 2001). It was cultivated in Fava-Neto’s medium (Fava-Neto, 1961) at 23 ◦ C in the mycelial form and at 37 ◦ C for its yeast form.

Screening of P. brasiliensis genomic library The λ Dash II genomic library, isolate Pb01, was prepared as described by Pereira et al. (2000). The library (approximately 106 plaques) was screened by using a PCR fragment of 660 bp of the chitinase gene of Coccidioides immitis, cts2 (Pishko et al., 1995). Replicate filters were hybridized with the Copyright  2002 John Wiley & Sons, Ltd.

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P-labelled 660 bp DNA fragment. Filters were pre-hybridized with 6× sodium saline citrate (SSC), 5× Denhardt’s solution, 0.1% sodium dodecyl sulphate (SDS), for 1 h at 42 ◦ C and hybridized with the labelled DNA under the same conditions for 24 h. Filters were then rinsed three times with 1× SSC, 0.1% SDS at room temperature for 5 min and washed in 0.1× SSC, 0.1% SDS at 42 ◦ C for 15 min, three times for each rinse. After three rounds of plaque purification, the genomic clones were analysed by restriction mapping. A 6.5 kb EcoRI fragment was subcloned into the vector pUC18, EcoRI restricted, for sequence determination.

Sequence determination and analysis DNA sequencing was performed on both strands by the dideoxynucleotide method, according to Sanger et al. (1977). The obtained DNA sequence was translated and compared to all non-redundant polypeptides in the translated NCBI database using the BLAST program (Altschul et al., 1990).

Identity analysis Pairwise comparisons of amino acids between ClpB homologues were calculated with the Genetics Computer Group (GCG) (Devereux et al., 1984). Alignments of ClpB amino acid sequences were carried out with the Clustal W program, version 1.7 (Jones et al., 1992; Thompson et al., 1994).

RT–PCR Reverse transcriptase (RT)–PCR was performed according to standard procedures (Sambrook and Russell, 2001). Total RNA was isolated from mycelium and yeast cells with Trizol, according to the manufacturer’s instructions (Gibco, BRL, Carlsbad, CA, USA). The cells were washed with Tris–buffer (20 mM Tris–HCl, pH 8.8, 2 mM CaCl2 ) and disrupted by maceration until a fine powder was obtained. The RNA samples were treated with, RNasefree DNase I at 37 ◦ C, followed by phenol–chloroform extraction and ethanol precipitation (Sambrook and Russell, 2001). Complementary DNA was synthesized from 1 µg total RNA in the presence of the synthetic oligonucleotide primer antisense 1R derived from nucleotides Yeast 2002; 19: 963–972.

ClpB of Paracoccidioides brasiliensis

513–535 in the Pbclpb sequence (see Figure 1) (5 TGGCACTTCACCATTCACAATAC-3 ). A quarter of the RT reaction was amplified by PCR using the sense primer (1F) derived from nucleotides 277–299 in the clpb sequence: (5 -CGAGGTGATGAAAAAGTGATGAG-3 ) and the antisense primer (3R) based on nucleotides 395–413 (5 -CGGCGGATTTCATCATCAC-3 ). The cDNA synthesis reaction was performed at 48 ◦ C for 45 min. The PCR was subjected to an initial denaturation at 94 ◦ C for 2 min followed by 40 cycles at 94 ◦ C (45 s), 40 ◦ C (2 min), 72 ◦ C (2 min) and a final extension at 74 ◦ C (7 min). The RT–PCR product was gel-purified, subcloned into plasmid pGEM-T-easy (Promega, Madison, WI, USA) and sequenced as described above.

Southern blot analysis Southern blot analysis was performed according to standard procedures (Sambrook and Russell, 2001). The RT–PCR product was fractionated on a 1.0% (w/v) agarose gel and transferred to membrane after denaturation for 15 min in 0.5 M NaOH. A probe encompassing 300 bp of the Pbclpb gene obtained by PCR of the genomic DNA of P. brasiliensis (see Figure 1) was labelled with (α-32 P) dATP using a random primer DNA labelling Kit RPN 1604 (Amersham Biosciences AB, Uppsala, Sweden). Pre-hybridization and hybridization reactions were performed at 65 ◦ C in a blocking reagent containing 50% (v/v) formamide. The blots were washed at 65 ◦ C with 0.1× sodium saline citrate (SSC 0.015 M, NaCl 0.0015 M), 0.1% (w/v) sodium dodecyl sulphate (SDS).

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nitrogen. The cells were frozen in the same buffer containing the protease inhibitors: 50 µg/ml N-α-ptosyl-L-lysine (TLCK) and N-tosyl-L-phenylalanine (TPCK), 5 mM iodoacetamide, 1 mM EDTA, 20 mM leupeptin and 4-chloromercuribenzoic acid (PCMB). The cells were disrupted by mechanical maceration, and the proteins were prepared as described by Fonseca et al. (2001). Debris was removed by centrifugation at 12 000× g for 5 min and the proteins were precipitated by 10% (w/v) trichloroacetic acid (TCA) addition. The pellets were washed in 10% (v/v) cold acetone and the samples were resuspended in lysis buffer containing 9.5 M urea, 2% (v/v) Nonidet P-40 (NP-40), 5% (v/v) β-mercaptoethanol, ampholytes 5.0–8.0 and 3.5–10.0 (ratio 4 : 1). The proteins were fractionated by two-dimensional electrophoresis according to O’Farrel (1975). Onedimensional protein analysis and separation of the second dimension were performed in a 13% SDS–polyacrylamide gel according to Laemmli (1970). The proteins were electrophoretically transferred to a nylon membrane. The ClpB protein was detected with a polyclonal antibody raised to the cyanobacterium Synechococcus sp. (Porankiewicz and Clarke, 1997). After reaction with alkaline phosphatase anti-rabbit IgG, the reaction was developed with 5-bromo-4-chloro3-indolylphosphate/nitro-blue-tetrazolium (BCIP/ NBT).

Nucleotide sequence Accession No. The sequence of the clpb gene and the deduced ClpB protein have been filed in the GenBank database under Accession No. AF449501.

Northern blot analysis Northern hybridization was performed on a 1.2% (w/v) agarose-formaldehyde gel; 300 bp of the clpb gene (see above) was used as a probe. After hybridization the membrane was washed and probed to a sequence encoding a L35 ribosomal protein of P. brasiliensis (GenBank Accession No. AF 416509).

Protein electrophoresis and immunodetection of P. brasiliensis ClpB The P. brasiliensis yeast cells were harvested by centrifugation (5000× g for 10 min at 4 ◦ C), washed in Tris–buffer and frozen in liquid Copyright  2002 John Wiley & Sons, Ltd.

Results and discussion Molecular cloning and sequencing of the clpb gene Because we were interested in searching for a chitinase gene of P. brasiliensis we had used a genomic PCR fragment of 660 bp encoding the cts2 gene of C. immitis (Pishko et al., 1995). Six clones were isolated and the same restriction profile was obtained for the positive plaques. The selected λ Dash II recombinant clone contained a 6.5 kb EcoRI fragment that hybridized to the cts2 gene Yeast 2002; 19: 963–972.

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-121 cttaagcctagaactgcctatcgaaatgtagaagggttaaatttgttagcaaacacgct tata agctattcctagtcttactgttagaagactacccaaa 1 M L L K T T S -21 gttacgaagcgacctccacgaATGTTGgttccttaacttccactgtccggaatagccagtacggcagagcaacttgttctttagCTTAAGACAACCTCTT 8 L T H T I T I M D K H L L H T L T G V Q V N A I D K A L A V G P D W 80 TGACTCACACCATAACCATTATGGACAAGCACCTTCTACACACCCTAACTGGGGTACAGGTCAATGCCATAGATAAAGCTTTAGCGGTTGGTCCCGACTG 42 V L L G L S E V G A T K S L L N S I G V S A D S L R Q V I L K I R 180 GGTATTATTGGGCCTTAGCGAAGTTGGGGCCACCAAAAGCTTATTGAACAGCATCGGTGTCAGTGCAGACAGTCTGCGTCAGGTAATTTTAAAAATTCGA 75 G D E K V M S N N H E D Q R D S L N K Y T V D L T E R A L A G K L 280 GGTGATGAAAAAGTGATGAGTAATAATCATGAAGATCAACGTGATTCACTCAATAAATACACTGTAGATTTAACCGAACGTGCGCTGGCCGGCAAGCTCG 1F

108 D P V I G R D D E I R R T V Q V L S R R T K N N P V L I G E P G V G 380 ATCCGGTGATTGGCCGTGATGATGAAATCCGCCGTACCGTACAGGTCTTGTCACGTCGGACCAAAAATAACCCGGTACTGATTGGTGAGCCTGGGGTCGG 3R

142 K T A I V E G L A Q R I V N G E V P E S L K G K R V L S L D L G S 480 TAAAACCGCAATTGTAGAAGGTCTGGCACAGCGTATTGTGAATGGTGAAGTGCCAGAAAGCTTAAAAGGCAAACGCGTGTTATCGCTGGACCTGGGTTCA 1R

175 L L A G A K Y R G E F E E R L K A V L K D L A K H E G E I I L F I 580 TTGCTGGCCGGTGCCAAATATCGCGGCGAGTTTGAGGAGCGTTTAAAAGCGGTCCTGAAAGATCTGGCCAAGCATGAAGGCGAAATCATCCTGTTTATTG 208 D E L H T L V G A G K G D G A M D A G N M L K P A L A R G E L R C V 680 ATGAGTTGCATACCCTGGTTGGCGCAGGTAAAGGTGATGGCGCGATGGATGCCGGCAACATGCTGAAACCGGCGCTGGCACGCGGTGAATTACGCTGCGT 242 G A T T L D E Y R Q Y I E K D A A L E R R F Q K V L V D E P S V E 780 GGGGGCAACCACACTCGATGAATATCGTCAGTACATTGAAAAAGATGCCGCCCTTGAGCGCCGTTTCCAGAAAGTGCTGGTCGATGAGCCAAGTGTCGAA 275 D T I A I L R G L K E R Y E V H H G V K I L D S A I I A A A K M S 880 GACACCATTGCCATTTTACGTGGTCTGAAAGAGCGTTATGAAGTCCACCACGGGGTGAAAATTCTCGACTCAGCGATTATTGCTGCAGCGAAAATGTCAC 308 H R Y I T D R Q L P D K A I D L I D E A A S R I K M E L D S K P E A 980 ATCGTTATATCACTGACCGTCAGTTGCCCGATAAAGCCATTGACCTGATTGATGAAGCAGCATCGCGCATCAAGATGGAGCTGGACTCTAAACCGGAAGC 342 L D K L E R R L I Q L K L Q L E A V K K D E D A G S R A E V S H L 1080 GCTGGATAAGCTGGAACGCCGTCTGATTCAGTTAAAGTTGCAACTTGAAGCTGTGAAAAAAGATGAAGATGCCGGCAGCCGTGCAGAAGTCAGTCATCTG 375 E K Q I E D V Q K D Y N D L E E V W K S E K T L V E G T K Q I Q A 1180 GAAAAACAGATCGAAGACGTCCAAAAAGACTATAACGATCTGGAAGAAGTGTGGAAGTCTGAAAAAACTCTGGTCGAAGGCACTAAGCAGATTCAGGCAC * * * * * * * * * * * * * * * * * 408 Q L D Q A R I A L Q K A Q R E N D L G E M S R L Q Y G V I P E L E K 1280 AGCTGGATCAGGCGCGTATTGCCTTGCAAAAAGCACAGCGTGAAAATGATCTGGGCGAGATGTCGCGTCTGCAATATGGCGTGATTCCGGAACTGGAAAA 442 Q L A Q D E L V E E K E E P K L L R N K V T D N E I A E V V S A A 1380 ACAGCTGGCCCAAGATGAGCTGGTGGAAGAGAAAGAAGAACCGAAGTTATTGCGCAATAAAGTCACCGATAACGAAATTGCGGAAGTGGTCAGCGCAGCC * * * * * * * * 475 T G I P V A K M L Q G E R E K L L Q M E S F L H Q R V V G Q D E A 1480 ACCGGTATTCCGGTTGCGAAAATGCTGCAAGGCGAGCGTGAAAAACTGCTGCAAATGGAAAGCTTCCTGCATCAGCGTGTGGTAGGGCAGGATGAAGCGG 508 V I A V S N A V R R S R A G L S D P N R P S G S F L F L G P T G V G 1580 TGATTGCGGTATCCAATGCGGTGCGCCGTTCACGTGCCGGGTTGTCGGATCCGAACCGTCCAAGTGGTTCTTTCCTGTTCCTGGGTCCAACCGGTGTCGG 542 K T E L T K A L A N F L F D S D D A M I R I D M S E F M E K H S V 1680 TAAGACCGAGTTAACCAAAGCCTTGGCCAATTTCCTGTTTGACAGCGATGACGCCATGATCCGGATTGATATGTCCGAATTTATGGAAAAACACTCAGTC 575 S R L V G A P P G Y V G Y E E G G V L T E A V R R K P Y S V V L F 1780 AGCCGTCTGGTCGGTGCGCCTCCGGGCTATGTCGGCTATGAAGAAGGCGGGGTATTAACCGAAGCCGTACGCCGTAAACCATATAGCGTGGTGCTGTTTG

Figure 1. Nucleotide sequence of PbclpB and the predicted amino acid sequence of the gene product. The putative TATA box is double-boxed. The three pentamers nGAAn, corresponding to the putative heat shock consensus element, are marked with brackets in the 5 non-coding region. The putative 3 polyadenylation site is in italic and boxed. The intron sequence is represented in lower case. The 5 /3 of the intron is double underlined. The presumptive start and stop codons are in bold. The amino-acid sequence is shown above the nucleotide sequence (single letter code). The two putative nucleotide-binding motifs are boxed. The middle signatures are indicated by asterisks. The chaperonins ClpA/B signature motifs are in bold and italic. The putative phosphorylation sites for casein kinase II (cK2) are underlined. The SSD domain is dot boxed. The C-terminal signatures are indicated by superior dots. The sequence related to the probe used for the Northern blot and Southern blot is underlined by dashes. The primers used in the RT–PCR reactions are in bold, underlined and marked by arrows

Copyright  2002 John Wiley & Sons, Ltd.

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608 D E V E K A H P D V F N I L L Q V L D D G R L T D S Q G R V V D F K 1880 ATGAGGTGGAAAAAGCCCATCCGGATGTATTTAACATCTTGCTGCAGGTACTGGATGATGGTCGCTTGACCGACTCGCAGGGCCGTGTAGTGGACTTTAA 642 N T V I V M T S N L G S S D V R E L G D N P S R D D V R N V V M A 1980 AAACACCGTGATTGTAATGACCTCGAACCTGGGTTCAAGCGATGTGCGTGAACTGGGCGATAATCCAAGCCGTGATGATGTACGCAATGTAGTGATGGCA . . . . . . . . . . . . . . . . . . 675 A V S E H F R P E F I N R I D E L V V F H A L E K A Q I R G I A D 2080 GCCGTGTCTGAGCATTTCCGTCCGGAGTTTATCAACCGTATTGATGAGCTGGTAGTATTCCATGCCCTGGAAAAAGCGCAGATTCGTGGTATTGCCGATA 708 I Q L D R L R A R L A D R D I K L T V D D S A F D Q L I E A G F D P 2180 TCCAGCTGGACCGTTTACGTGCCCGTCTGGCCGACCGCGATATCAAGTTAACGGTAGATGATTCTGCCTTTGACCAGCTGATTGAAGCCGGTTTTGACCC . . . . . . . . . 742 V Y G A R P L K R A I Q Q Q V E N N L A Q K I L A G D F Q P G D T 2280 AGTTTACGGTGCGCGTCCATTAAAACGTGCCATCCAGCAACAGGTAGAAAATAATCTGGCGCAGAAAATTCTGGCCGGTGACTTCCAGCCGGGAGATACC 775 I V I T A D A G Q L D F Q K L K L N @ 2380 ATTGTGATTACGGCAGATGCAGGCCAACTGGATTTCCAGAAGCTGAAACTGAACTAAtccgcacacggttcatctaaactgtttaatcaaagacctggct 2480 2580 2680 2780 2880 2980 3080

tcggccgggtttttttatgggtatatctgcagcaaaaagccgggtagatgtcgtgattcactctgcaaagtaagtccacgctggatgatgagtgacagcg taaagtttccgatgtaaaaaatcctgctatagggtagcaggctttttattgctaatcgaatgaactgtgaatagaaaaggatcaaatcatcctttgccat tcaccgtccttgccaatcacattttagatttgtggatgctggattttccaggcgcggtgaatcttctggttacgcttgaaatctggaccaatggtttcat ggctgatttcctgaatatcaaacagcgctggcaaggtttcatccatttcaaaaccgcgatagttattcgagaagtacagggtgccttctgtggttaaacg gttcatggcgcgtttaattagcgaaacatggtcacgttgtacatcaaaagtgccgtagaattttttcgagttggagaaggtgggcggatc aataaa gatc aggtcatattgctcatgaccttcttttagccactcaaaacagtcgctggcaaagaactggtgttgttcatcggcatggtcgacggttaagccgtttaaga caaagttttctttggaccagttcaaataggtgttggacaagtccaca

Figure 1. Continued

product. The entire fragment was subcloned into pUC18 and sequenced on both strands. Although chitinase genes were not detected, we obtained by chance a ClpB homologue of P. brasiliensis. Some similar regions in cts2 and clpb, as detected by Clustal W analysis (data not shown), could explain that cross-hybridization. The fragment contained the entire coding region for a gene homologue to clpb. The DNA and the predicted amino acid sequence of the clpb gene of P. brasiliensis are presented in Figure 1. The entire clpb gene is consisted of 2436 bp from the start to the stop codons and is organized in two exons, interrupted by a small intron at the 5 terminus, position 7–63. The intron sequence is flanked by 5 GT and 3 AG, which correspond to the consensus sequence of known splicing sites (Ballance, 1986). The P. brasiliensis clpb gene consists of a single open reading-frame (ORF) capable of encoding a 792 amino acid polypeptide with a pI of 5.34 and a calculated molecular mass of 87.9 kDa. Although the promotor region is not complete, analysis at the 5 non-coding region in the genomic clone revealed structural features typical of regulatory regions in eukaryotic genes. One TATA box-like sequence is present at position 62 in the 5 upstream sequence (Struhl, 1987). Highly expressed eukaryotic genes usually have multiple short sequence elements that function as binding sites for one or more Copyright  2002 John Wiley & Sons, Ltd.

regulatory proteins (Pelham and Bienz, 1982). A typical example is the heat-shock consensus element (HSE), which is found in multiple copies upstream from the transcription start codon of heatshock genes in a wide variety of organisms (Nover and Scharf, 1997). The PbclpB gene presents three putative heat shock consensus elements, NGAAN, that were present at positions 112, 100 and 92 in the 5’upstream region. One polyadenylation signal AATAAA is present in the downstream sequence, at position 2970 of the gene (Proudfoot, 1991). The putative poly(A) site is followed by a T/GT-rich element, as described by McLauchlan et al. (1985). The function of T/GT-rich elements remains to be determined in all the organisms. The deduced protein presented several canonical motifs characteristic of the Clp family of proteins, as shown in Figure 1. The ClpB of P. brasiliensis presents two well-conserved ATP-binding domains (NBD) at positions 104–337 and 463–655 (Walker et al., 1982). These NBDs are separated by a middle region (spacer) that can be of variable size in organisms (Schrimer et al., 1996). The presence of the two NDBs places this protein in the class I Clp/HSP100 family (Squires and Squires, 1992; Schrimer et al., 1996). Two ClpA/B chaperonin signature motifs were detected at amino acids 224–238 and 563–580; two carboxyl-terminal signatures at positions 680–697 and 744–752 Yeast 2002; 19: 963–972.

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(Schirmer et al., 1996) and a substrate recognition domain (SSD), GARPL, in the C-terminal region at position 744–748, which is conserved in the Clp family of proteases (Smith et al., 1999). Another feature in PbClpB is the presence of putative casein kinase II phosphorylation sites, S/T–X–X–D/E, as described for other ClpBs (Celerin et al., 1998). These phosphorylation targets were spread over 11 positions in the deduced PbClpB protein, as shown in Figure 1. A diagrammatic scheme was constructed for the ClpB of P. brasiliensis and is presented in Figure 2. From this, we can see that the Walker consensus distinctions for ATP binding sites 1 and 2 are separated by a large spacer region (middle), a characteristic found only in the ClpB proteins (Squires and Squires, 1992). The ATP-1 and 2 domains consisting of the conserved A, B, B1 and B2 consensus sequences (Walker) are also shown. The consensus A is a rich conserved glycine motif (Schirmer et al., 1998). The motifs B, B1 and B2 are hydrophobic segments and comprise different regions of ATP-binding domains (Walker et al., 1982; Chin et al., 1988). In the PbClpB protein, the ATP-binding domain 1 presents one A segment and the two hydrophobic consensus, B and B1 (Figure 2), while the ATP-binding domain 2 presents one A region and only one hydrophobic conserved segment, B2 . The two carboxyterminal signatures, a characteristic of ClpBs, are also shown. The N-terminal region is less well conserved in P. brasiliensis as in all Clp ATPases and its role in the protein function is not clear (Schirmer

middle ATP-2

ATP-1 A B

B1

I II A B2

C-terminal III

IV

Figure 2. Schematic diagram of PbClpB. The diagram was constructed on basis of the consensus motifs of the ClpB sub-family. ATP1 and ATP2 represent nucleotide-binding sites, A is a glycine-rich segment and B, B1 and B2 represent hydrophobic segment motifs. I and II represent middle signature consensus sequences. III and IV represent C-terminal consensus signatures

et al., 1996). We could only detect a ALA conserved sequence in the middle of the N-terminal, as found for some ClpBs (Schirmer et al., 1996).

Comparison of the P. brasiliensis ClpB and related sequences The alignment of the deduced PbClpB with other sequences is shown in Figure 3. It is shown a high number of identical and conserved amino acids among the considered sequences of the ClpB from Escherichia coli (Gottesman et al., 1990), Synechococcus sp. (Eriksson and Clarke, 1996), Schizosaccharomyces pombe (Sanches et al., 2001) and Saccharomyces cerevisiae (Parsell et al., 1991). Of special note is the conservation of the ATPbinding motifs 1 and 2, which is typical of ClpB sequences. The chaperonin signatures, as well as the sensor and substrate discrimination domain (SSD; Smith et al., 1999), are also highly conserved. The values of identity and similarity of PbClpB and related sequences were calculated. The amino acid sequence of PbClpB manifests, for example, a 69% identity and a 77% similarity, respectively, to ClpB from E. coli. Identity of 59%, 53% and 46% were observed with ClpB from Synechococcus sp., Sz. pombe and S. cerevisiae, respectively, which reflects the phylogenetic proximity of these genes among prokaryotes and eukaryotes.

Expression of the P. brasiliensis clpb The clpb expression was analysed by RT–PCR and Northern blot. RT–PCR assays provided a product of 138 bp (Figure 4A). Southern blot analysis showed strong hybridization of the 138 bp product to the 300 bp clpb probe (Figure 4B). The 138 bp product was cloned into plasmid pGEMT-easy and sequenced, showing 100% identity to PbclpB (data not shown). A PbclpB transcript of 3.0 kb was identified by Northern blot analysis of total RNA from P. brasiliensis mycelium and yeast cells, as shown in Figure 5A. The 3.0 kb mRNA species is preferentially expressed in the yeast

Figure 3. Alignment of the deduced PbClpB and related sequences. The sequences were: Escherichia coli (Gottesman et al., 1990), Synechococcus sp. (Eriksson and Clarke, 1996), Schizosaccharomyces pombe (Sanches et al., 2001) and Saccharomyces cerevisiae (Parsell et al.,1991). Asterisks indicate amino acid identity and dots represent conserved substitutions. The marked regions are the ATP-binding domains 1 and 2 (boxed), the chaperonin signatures (brackets) and the sensor and substrate domain (GARPL) Copyright  2002 John Wiley & Sons, Ltd.

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PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

ALAGKLDPVIGRDDEIRRTVQVLSRRTKNNPVLIGEPGVGKTAIVEGLAQRIVNGEVPES AEQGKLDPVIGRDEEIRRTIQVLQRRTKNNPVLIGEPGVGKTAIVEGLAQRIINGEVPEG AREGKLDPVIGRDEEVRRTIQILSRRTKNNPVLIGEPGVGKTAIAEGLAQRIINHDVPES ARNGQLDPVIGREDEIRRTIRVLSRRTKNNPVLIGEPGVGKTSIAEGLARRIIDDDVPAN ARQGKLDPVIGREEEIRSTIRVLARRIKSNPCLIGEPGIGKTAIIEGVAQRIIDDDVPTI * *:*******::*:* *:::* ** *.** ******:***:* **:*:**:: :**

161 231 233 234 237

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

LKGKRVLSLDLGSLLAGAKYRGEFEERLKAVLKDLAKHEGEIILFIDELHTLVGAGKG-D LKGRRVLALDMGALVAGAKYRGEFEERLKGVLNDLAKQEGNVILFIDELHTMVGAGKA-D LRDRKLISLDMGALIAGAKYRGEFEERLKAVLKEVTDSQGQIILFIDEIHTVVGAGAT-Q LSNCKLLSLDVGSLVAGSKFRGEFEERIKSVLKEVEESETPIILFVDEMHLLMGAGSGGE LQGAKLFSLDLAALTAGAKYKGDFEERFKGVLKEIEESKTLIVLFIDEIHMLMGNGKD-* . ::::**:.:* **:*::*:****:*.**::: . : ::**:**:* ::* *

220 290 292 294 295

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

GAMDAGNMLKPALARGELRCVGATTLDEYRQYIEKDAALERRFQKVLVDEPSVEDTIAIL GAMDAGNMLKPALARGELHCVGATTLDEYRQYIEKDAALERRFQKVFVAEPSVEDTIAIL GAMDAGNLLKPMLARGALRCIGATTLDEYRKYIEKDAALERRFQEVLVDEPNVLDTISIL GGMDAANLLKPMLARGKLHCIGATTLAEYKKYIEKDAAFERRFQIILVKEPSIEDTISIL ---DAANILKPALSRGQLKVIGATTNNEYRSIVEKDGAFERRFQKIEVAEPSVRQTVAIL **.*:*** *:** *: :**** **:. :***.*:***** : * **.: :*::**

280 350 352 354 352

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

RGLKERYEVHHGVKILDSAIIAAAKMSHRYITDRQLPDKAIDLIDEAASRIKMELDSKPE RGLKERYELHHHVQITDPAIVAAATLSHRYIADRQLPDKAIDLIDEAASSIRMQIDSKPE RGLKERYEVHHGVKIADSALVAAAMLSNRYISDRFLPDKAIDLVDEAAAKLKMEITSKPE RGLKEKYEVHHGVTISDRALVTAAHLASRYLTSRRLPDSAIDLVDEAAAAVRVTRESQPE RGLQPKYEIHHGVRILDSALVTAAQLAKRYLPYRRLPDSALDLVDISCAGVAVARDSKPE ***: :**:** * * * *:::** :: **:. * ***.*:**:* :.: : : *:**

340 410 412 414 412

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

ALDKLERRLIQLKLQLEAVKKDEDAGS--RAEVSHLEKQIEDVQKDYNDLEEVWKSEKTL ELDRLDRRIIQLKLEQQALMKESDEAS--KKRLDMLNEELSDKERQYSELEEEWKAEKAS ELDEVDRKILQLEMERLSLQRENDSAS--KERLEKLEKELADFKEEQSKLNGQWQSEKTV VLDNLERKLRQLRVEIRALEREKDEAS--KERLKAARKEAEQVEEETRPIREKYELEKSR ELDSKERQLQLIQVEIKALERDEDADSTTKDRLKLARQKEASLQEELEPLRQRYNEEKHG ** :*:: :.:: :: ::.* * : .:. .:: . :.: :. :: **

398 468 470 472 472

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

VEGTKQIQAQLDQARIALQKAQRENDLGEMSRLQYGVIPELEKQLAQDE----------L LSGTQTIKAELEQAKIAIEQARRVGDLARMSELQYGKIPELEKQLEAAT----------Q IDQIRTVKETIDQVNLEIQQAQRDYDYNKAAELQYGKLTDLQRQVEALETQ--------L GSELQDAKRRLDELKAKAEDAERRNDFTLAADLKYYGIPDLQKRIEYLEQQKRKADAEAI HEELTQAKKKLDELENKALDAERRYDTATAADLRYFAIPDIKKQIEKLEDQ------VAE . : ::: . .*.* * : *:* :.::::::

448 518 522 532 526

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

VEEKEE-PKLLRNKVTDNEIAEVVSAATGIPVAKMLQGEREKLLQMESFLHQRVVGQDEA LEGKT--MRLLRNKVTDAEIAEVLARWTGIPVSRMMESEREKLLRMEQELHHRVIGQNEA AEQQTSGKSLLREEVLESDIAEIISKWTGIPISKLVESEKEKLLHLEDELHSRVIGQDEA ANAQPGSEPLLIDVVGPDQINEIVARWTGIPVTRLKTTEKERLLNMEKVLSKQVIGQNEA EERRAGANSMIQNVVDSDTISETAARLTGIPVKKLSESENEKLIHMERDLSSEVVGQMDA : : :: : * * * : ****: :: *.*:*:.:* * .*:** :*

507 576 582 592 586

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

VIAVSNAVRRSRAGLSDPNRPSGSFLFLGPTGVGKTELTKALANFLFDSDDAMIRIDMSE VDAVSNAIRRSRAGLADPNRPIGSFLFLGPTGVGKTELCKALANFMFDSDEAMVRIDMSE VTAVAEAIQRSRAGLSDPNRPTASFIFLGPTGVGKTELAKALAKNLFDTEEALVRIDMSE VTAVANAIRLSRAGLSDPNQPIASFLFCGPSGTGKTLLTKALASFMFDDENAMIRIDMSE IKAVSNAVRLSRSGLANPRQP-ASFLFLGLSGSGKTELAKKVAGFLFNDEDMMIRVDCSE : **::*:: **:**::*.:* .**:* * :* *** * * :* :*: :: ::*:* **

567 636 642 652 645

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

FMEKHSVSRLVGAPPGYVGYEEGGVLTEAVRRKPYSVVLFDEVEKAHPDVFNILLQVLDD FMEKHSVSRLVGAPPGYVGYEEGGYLTEAVRRRPYSVILLDEVEKAHPDVFNILLQVLDD YMEKHAVSRLMGAPPGYVGYEEGGQLTEAIRRRPYSVILFDEIEKAHGDVFNVMLQILDD YMEKHSVSRLIGAPPGYVGHEAGGQLTEQLRRRPYSVILFDEIEKAAPEVLTVLLQVLDD LSEKYAVSKLLGTTAGYVGYDEGGFLTNQLQYKPYSVLLFDEVEKAHPDVLTVMLQMLDD **::**:*:*:..****:: ** **: :: :****:*:**:*** :*:.::**:***

627 696 702 712 705

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

GRLTDSQGRVVDFKNTVIVMTSNLGSSDVRELGDNPS---RDDVRNVVMAAVSEHFRPEF GRLTDGQGRTVDFRNTVVIMTSNLGSDLIQERFGELD---YAHMKELVLGVVSHNFRPEF GRLTDAQGHVVDFKNTIIIMTSNLGSQYILDVAGDDSR--YEEMRSRVMDVMRENFRPEF GRITSGQGQVVDAKNAVIIMTSNLGAEYLTTDNESDDGKIDSTTREMVMNSIRGFFRPEF GRITSGQGKTIDCSNCIVIMTSNLGAEFINSQQGSKIQ---ESTKNLVMGAVRQHFRPEF **:*..**:.:* * :::******:. : . :. *: : *****

684 753 760 772 762

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

INRIDELVVFHALEKAQIRGIADIQLDRLRARLAD--RDIKLTVDDSAFDQLIEAGFDPV INRIDEVVVFHPLGEQHIASIAQIQLKRLYKRLEE--RGYEIHISDEALKLLSENGYDPV LNRVDETIIFHGLQKSELRSIVQIQIQSLATRLEE--QKLTLKLTDKALDFLAAVGYDPV LNRISSIVIFNRLRRVDIRNIVENRILEVQKRLQSNHRSIKIEVSDEAKDLLGSAGYSPA LNRISSIVIFNKLSRKAIHKIVDIRLKEIEERFEQNDKHYKLNLTQEAKDFLAKYGYSDD :**:.. ::*: * . : *.: :: : *: . : : : :.* . * *:.

742 811 818 832 822

PbClpB EcF84.1 SnHSP100 SpHSP100 ScHSP104

YGARPLKRAIQQQVENNLAQKILAGDFQPGDTIVITADAGQ------------------YGARPLKRAIQQQIENPLAQQILSGELVPGKVIRLEVNEDR------------------YGARPLKRAVQKYLETAIAKGILRGDYKPGETIVVDETDER------------------YGARPLNRVIQNQVLNPMAVLILNGQLRDKETAHVVVQNGK-------IFVKPNHEANAN MGARPLNRLIQNEILNKLALRILKNEIKDKETVNVVLKKGKSRDENVPEEAEECLEVLPN *****:* :*: : . :* ** .: .. : :

783 852 859 885 882

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phase (Figure 5A). The membrane was washed and probed to a cDNA encoding a L35 ribosomal protein of P. brasiliensis (GenBank Accession No. AF416509; Figure 5B). The amount of mRNA, as detected by that internal control, was the same for yeast and mycelium. In Trypanosoma brucei the amount of mRNA encoding ClpB is higher in the mammalian stage when compared to the insect phase, similarly to our description (Glass et al., 1986). The low level of mycelial expression could characterize this gene as phase-specific, with possible function in mechanisms related to the prevalence of the yeast form.

Analysis of the expression of the ClpB in P. brasiliensis After one- or two-dimensional electrophoresis of the P. brasiliensis yeast cell lysates, the proteins were transferred to membrane and the blots were A Y

M 1

2

3

4

5

6

kb 0.200.150.10-

B M kb

1

2

Y 3

4

5

6

0.200.150.10-

Figure 4. RT–PCR analysis of the clpb transcript in yeast and mycelium of P. brasiliensis. Total RNA was obtained from mycelium (M) and yeast (Y) grown at 23 ◦ C and 37 ◦ C, respectively. The reactions were primed with oligonucleotides 1F, 1R and 3R, as shown schematically in Figure 1. The RT–PCR reaction products were separated on a 1.5% agarose gel and stained with ethidium bromide (A). The gel was transferred to membrane and probed to the clpb 300 bp fragment. Lanes: 1 and 4, control reaction without RNA and RT for mycelium and yeast; 2 and 5, control reaction without RT for mycelium and yeast, respectively; 3, RT–PCR reaction of the mycelium phase; 6, RT–PCR reaction of the yeast phase Copyright  2002 John Wiley & Sons, Ltd.

M

Y 3.0 kb Pb clpb

A

M

Y 0.7 kb Pb L35

B

Figure 5. Analysis of the clpb transcripts in P. brasiliensis. Total RNA (10 µg) from mycelium (M) and yeast cells (Y) was fractionated on a formaldehyde agarose gel (1.2%), and hybridized to the 300 bp clpb probe [GenBank Accession No. AY057112 (A)]. The membrane was washed and probed to a cDNA encoding a L35 ribosomal protein of P. brasiliensis [GenBank Accession No. AY057112 (B)]. The RNA sizes were calculated using the 0.24–9.5 marker RNA ladder (Gibco)

reacted against the polyclonal antibody directed to the C-terminal domain of the Synechococcus sp. ClpB (Porankiewicz and Clarke, 1997). A protein species of 89 kDa was detected in the yeast cell extracts by one-dimensional gel electrophoresis (Figure 6A). Two-dimensional analysis provided a protein species of 89 kDa, pI 5.3, reactive to the polyclonal antibody, as shown in Figure 6B. We were not able to detect the protein product in the mycelial phase grown at 23 ◦ C (data not shown). The preferential expression of the ClpB transcript and its putative protein in the yeast cells suggest that ClpB might be involved in P. brasiliensis survival in the host thermal conditions and that it could play a role in the host fungus interaction. In this vein, it was described that the ClpB of T . brucei (Glass et al., 1986) and of Leishmania major (H¨ubel et al., 1997) are preferentially induced in the intracellular stage. In addition, the ClpB of Helicobacter pylori and Yersinia enterocolitica are associated to the host invasion process and virulence (Allan et al., 1998; Badger et al., 2000). ClpB is a stress-induced protein in several microrganisms (Glass et al., 1986; H¨ubel et al., 1997). In P . brasiliensis it was detected the expression in the yeast phase. Such data in addition to the putative HSE motifs in the promoter region of PbClpB could suggest a role of ClpB during the temperature upshift that characterizes the physiological infective process by P. brasiliensis. Further studies are necessary to Yeast 2002; 19: 963–972.

ClpB of Paracoccidioides brasiliensis

A kDa 21217011676-

971

B ITF SDS

5.3

21217011676-

Figure 6. Analysis of PbClpB by one- and two-dimensional protein gels and Western blotting. The yeast cells were grown at 37 ◦ C for 7 days. Amounts of 50 µg and 200 µg protein were fractionated, respectively, by oneor two-dimensional gel electrophoresis, transferred to membranes and reacted to the polyclonal antibody to ClpB. Localization of the putative ClpB of P. brasiliensis is marked with arrows. (A) One-dimensional gel analysis; (B) two-dimensional gel analysis. The top number is related to the pI value of the ClpB in P. brasiliensis, as calculated by the pI markers at the first dimension, and those on the left refer to the molecular mass markers

clarify the role of ClpB in the morphogenetic events of P. brasiliensis. Our future projects will focus on these subjects.

Acknowledgements We thank Dr Garry T. Cole, Medical College of Ohio, Toledo, OH, USA, for providing the chitinase (cts2 ) probe. We also thank Dr Adrian K. Clarke, University of Umea, Sweden, for providing the ClpB polyclonal antibody. This work was supported by grants from CNPq (520679/99-7) and FUNAPE-UFG.

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