YeastYeast 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 ofParacoccidioides brasiliensis

Rosalia S. A. Jesuino,1 Maristela O. Azevedo,2 M. Sueli S. Felipe,2 Maristela Pereira1 and Celia M. deAlmeida Soares1*1 Laboratorio de Biologia Molecular, Instituto de Ciencias Biologicas, Universidade Federal de Goias, ICB II, Campus II, 74001-970, Goiania,Goias, Brazil2 Laboratorio de Biologia Molecular, Instituto de Biologia, Universidade de Brası lia, 70910-900 Brası lia, D.F., Brazil

*Correspondence to:Celia M. de Almeida Soares,Laboratorio de BiologiaMolecular, ICBII, Campus II,Universidade Federal de Goias,74001-970, Goiania, Goias,Brazil.E-mail: celia@icb2.ufg.br

Received: 16 January 2002Accepted: 10 May 2002

AbstractWe report the cloning and sequence analysis of a genomic clone encoding aParacoccidioides brasiliensis ClpB chaperone homologue (PbClpB). The clpb genewas identified in a λ Dash II library. Sequencing of Pbclpb revealed a long openreading 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, chaperoninsClpA/B signatures and highly conserved casein kinase phosphorylation domains arepresent. ClpB is 69% to 49% identical to members of the ClpB family from severalorganisms from prokaryotes to eukaryotes. The transcript of PbclpB was detectedas a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phaseof 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 submittedto 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 aetiologicalagent of paracoccidioidomycosis (PCM), a fungaldisease that affects many individuals in LatinAmerica (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 onPCM will occur in a given patient, depending onfactors such as fungal virulence and fluctuations inthe 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, issubjected to heat stress as a regular feature of itslife cycle. When the infectious propagules of thefungus mycelium phase reach the host pulmonaryalveoli, they face a temperature upshift that may

trigger adaptative mechanisms to resist the newenvironmental conditions.

Changing environmental conditions elicit theexpression of new types of cellular proteins inall organisms. In particular, a group of ATP-dependent proteases is overexpressed during heatshock in prokaryotic and eukaryotic organisms.This new group of conserved proteins includesClp (caseinolytic protease)/HSP100, initially iden-tified as a heat shock-inducible, multicomponent,ATP-dependent protease (Schirmer et al., 1996).The Clp proteins have acquired great interest forresearchers because of their links to both thechaperonin and proteolytic activities (Gottesmanet al., 1997; Schirmer et al., 1996). They includerepresentatives within different intracellular com-partments of prokaryotes and eukaryotes (Squireset al., 1991; Leonhardt et al., 1993; Krobitschet al., 1998) and can be divided into subfamilies

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964 R. S. A. Jesuino et al.

according to conserved structural characteristics(Schimmer et al., 1996). The ClpB family are heatshock-inducible representatives of the Clp familyof ATPases that function as molecular chaperones,preventing protein denaturation and aggregation athigh temperatures and also promoting the refoldingof aggregated polypeptides (Leonhardt et al., 1993;Parsell et al., 1994; Schmitt et al., 1995).

The fact that P. brasiliensis is confronted by aheat stress after the invasion of the human host andthat the morphogenetic conversion from myceliumto yeast is a crucial step in the establishmentof infection has led our group to investigate thestress response in the fungus. Early work from ourlaboratory using metabolically labelled cells has ledto a group of heat-induced proteins (Silva et al.,1994). Our group also isolated and characterizedthe genes encoding the heat-induced proteins 70and 60 (Silva et al., 1999; Salem-Izaac et al.,2001). Recently the cloning and characterization ofthe gene encoding the heat-inducible Lon proteaseof P. brasiliensis has been described by Barros andPuccia (2001).

In this paper, we describe the cloning, sequenc-ing and characterization of a clpb-encoding geneof P. brasiliensis. To our knowledge, the PbClpBprotein is the first described ClpB in a pathogenicfungus. This work was performed in anticipationthat the obtained data could give new insights tothe fungus dimorphic transition and to the eventsthat led to the establishment of infection.

Materials and methods

Microrganism and growth conditions

The P. brasiliensis isolate Pb01 (ATCC, MYA826) has been previously investigated by our group(Pereira et al., 2000; Fonseca et al., 2001). Itwas cultivated in Fava-Neto’s medium (Fava-Neto,1961) at 23 ◦C in the mycelial form and at 37 ◦Cfor its yeast form.

Screening of P. brasiliensis genomic library

The λ Dash II genomic library, isolate Pb01, wasprepared as described by Pereira et al. (2000). Thelibrary (approximately 106 plaques) was screenedby using a PCR fragment of 660 bp of the chitinasegene of Coccidioides immitis, cts2 (Pishko et al.,1995). Replicate filters were hybridized with the

32P-labelled 660 bp DNA fragment. Filters werepre-hybridized with 6× sodium saline citrate(SSC), 5× Denhardt’s solution, 0.1% sodium dode-cyl sulphate (SDS), for 1 h at 42 ◦C and hybridizedwith the labelled DNA under the same condi-tions for 24 h. Filters were then rinsed threetimes with 1× SSC, 0.1% SDS at room temper-ature for 5 min and washed in 0.1× SSC, 0.1%SDS at 42 ◦C for 15 min, three times for eachrinse. After three rounds of plaque purification, thegenomic clones were analysed by restriction map-ping. A 6.5 kb EcoRI fragment was subcloned intothe vector pUC18, EcoRI restricted, for sequencedetermination.

Sequence determination and analysis

DNA sequencing was performed on both strandsby the dideoxynucleotide method, according toSanger et al. (1977). The obtained DNA sequencewas translated and compared to all non-redundantpolypeptides in the translated NCBI database usingthe BLAST program (Altschul et al., 1990).

Identity analysis

Pairwise comparisons of amino acids betweenClpB homologues were calculated with the Genet-ics Computer Group (GCG) (Devereux et al.,1984). Alignments of ClpB amino acid sequenceswere carried out with the Clustal W program, ver-sion 1.7 (Jones et al., 1992; Thompson et al., 1994).

RT–PCR

Reverse transcriptase (RT)–PCR was performedaccording to standard procedures (Sambrook andRussell, 2001). Total RNA was isolated frommycelium and yeast cells with Trizol, accord-ing to the manufacturer’s instructions (Gibco,BRL, Carlsbad, CA, USA). The cells werewashed with Tris–buffer (20 mM Tris–HCl,pH 8.8, 2 mM CaCl2) and disrupted by mac-eration until a fine powder was obtained.The RNA samples were treated with, RNase-free DNase I at 37 ◦C, followed by phe-nol–chloroform extraction and ethanol precipita-tion (Sambrook and Russell, 2001). Complemen-tary DNA was synthesized from 1 µg total RNAin the presence of the synthetic oligonucleotideprimer antisense 1R derived from nucleotides

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ClpB of Paracoccidioides brasiliensis 965

513–535 in the Pbclpb sequence (see Figure 1) (5′-TGGCACTTCACCATTCACAATAC-3′). A quar-ter of the RT reaction was amplified byPCR using the sense primer (1F) derived fromnucleotides 277–299 in the clpb sequence:(5′-CGAGGTGATGAAAAAGTGATGAG-3′) andthe antisense primer (3R) based on nucleotides395–413 (5′-CGGCGGATTTCATCATCAC-3′).The cDNA synthesis reaction was performed at48 ◦C for 45 min. The PCR was subjected to aninitial denaturation at 94 ◦C for 2 min followedby 40 cycles at 94 ◦C (45 s), 40 ◦C (2 min), 72 ◦C(2 min) and a final extension at 74 ◦C (7 min). TheRT–PCR product was gel-purified, subcloned intoplasmid pGEM-T-easy (Promega, Madison, WI,USA) and sequenced as described above.

Southern blot analysis

Southern blot analysis was performed according tostandard procedures (Sambrook and Russell, 2001).The RT–PCR product was fractionated on a 1.0%(w/v) agarose gel and transferred to membrane afterdenaturation for 15 min in 0.5 M NaOH. A probeencompassing 300 bp of the Pbclpb gene obtainedby PCR of the genomic DNA of P. brasiliensis(see Figure 1) was labelled with (α-32P) dATPusing a random primer DNA labelling Kit RPN1604 (Amersham Biosciences AB, Uppsala, Swe-den). Pre-hybridization and hybridization reactionswere performed at 65 ◦C in a blocking reagentcontaining 50% (v/v) formamide. The blots werewashed at 65 ◦C with 0.1× sodium saline citrate(SSC 0.015 M, NaCl 0.0015 M), 0.1% (w/v) sodiumdodecyl sulphate (SDS).

Northern blot analysis

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

Protein electrophoresis and immunodetectionof P. brasiliensis ClpB

The P. brasiliensis yeast cells were harvestedby centrifugation (5000× g for 10 min at 4 ◦C),washed in Tris–buffer and frozen in liquid

nitrogen. The cells were frozen in the same buffercontaining the protease inhibitors: 50 µg/ml N-α-p-tosyl-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 mechanicalmaceration, and the proteins were prepared asdescribed by Fonseca et al. (2001). Debris wasremoved by centrifugation at 12 000× g for5 min and the proteins were precipitated by10% (w/v) trichloroacetic acid (TCA) addition.The pellets were washed in 10% (v/v) coldacetone and the samples were resuspended in lysisbuffer containing 9.5 M urea, 2% (v/v) NonidetP-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-dimensionalelectrophoresis according to O’Farrel (1975). One-dimensional protein analysis and separation ofthe second dimension were performed in a 13%SDS–polyacrylamide gel according to Laemmli(1970). The proteins were electrophoreticallytransferred to a nylon membrane. The ClpBprotein was detected with a polyclonal antibodyraised to the cyanobacterium Synechococcus sp.(Porankiewicz and Clarke, 1997). After reactionwith alkaline phosphatase anti-rabbit IgG, thereaction was developed with 5-bromo-4-chloro-3-indolylphosphate/nitro-blue-tetrazolium (BCIP/NBT).

Nucleotide sequence Accession No.

The sequence of the clpb gene and the deducedClpB protein have been filed in the GenBankdatabase under Accession No. AF449501.

Results and discussion

Molecular cloning and sequencing of the clpbgene

Because we were interested in searching for achitinase gene of P. brasiliensis we had used agenomic PCR fragment of 660 bp encoding thects2 gene of C. immitis (Pishko et al., 1995). Sixclones were isolated and the same restriction profilewas obtained for the positive plaques. The selectedλ Dash II recombinant clone contained a 6.5 kbEcoRI fragment that hybridized to the cts2 gene

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966 R. S. A. Jesuino et al.

-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

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 G380 ATCCGGTGATTGGCCGTGATGATGAAATCCGCCGTACCGTACAGGTCTTGTCACGTCGGACCAAAAATAACCCGGTACTGATTGGTGAGCCTGGGGTCGG

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

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 I580 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 E780 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 L1080 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 G1580 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 V1680 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 F1780 AGCCGTCTGGTCGGTGCGCCTCCGGGCTATGTCGGCTATGAAGAAGGCGGGGTATTAACCGAAGCCGTACGCCGTAAACCATATAGCGTGGTGCTGTTTG

1F

1R

3R

Figure 1. Nucleotide sequence of PbclpB and the predicted amino acid sequence of the gene product. The putativeTATA 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. Theintron sequence is represented in lower case. The 5′/3′ of the intron is double underlined. The presumptive start andstop codons are in bold. The amino-acid sequence is shown above the nucleotide sequence (single letter code). The twoputative nucleotide-binding motifs are boxed. The middle signatures are indicated by asterisks. The chaperonins ClpA/Bsignature motifs are in bold and italic. The putative phosphorylation sites for casein kinase II (cK2) are underlined. The SSDdomain is dot boxed. The C-terminal signatures are indicated by superior dots. The sequence related to the probe usedfor 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

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ClpB of Paracoccidioides brasiliensis 967

775 I V I T A D A G Q L D F Q K L K L N @2380 ATTGTGATTACGGCAGATGCAGGCCAACTGGATTTCCAGAAGCTGAAACTGAACTAAtccgcacacggttcatctaaactgtttaatcaaagacctggct

2480 tcggccgggtttttttatgggtatatctgcagcaaaaagccgggtagatgtcgtgattcactctgcaaagtaagtccacgctggatgatgagtgacagcg2580 taaagtttccgatgtaaaaaatcctgctatagggtagcaggctttttattgctaatcgaatgaactgtgaatagaaaaggatcaaatcatcctttgccat2680 tcaccgtccttgccaatcacattttagatttgtggatgctggattttccaggcgcggtgaatcttctggttacgcttgaaatctggaccaatggtttcat2780 ggctgatttcctgaatatcaaacagcgctggcaaggtttcatccatttcaaaaccgcgatagttattcgagaagtacagggtgccttctgtggttaaacg2880 gttcatggcgcgtttaattagcgaaacatggtcacgttgtacatcaaaagtgccgtagaattttttcgagttggagaaggtgggcggatcaataaagatc2980 aggtcatattgctcatgaccttcttttagccactcaaaacagtcgctggcaaagaactggtgttgttcatcggcatggtcgacggttaagccgtttaaga3080 caaagttttctttggaccagttcaaataggtgttggacaagtccaca

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 K1880 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 P2180 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

Figure 1. Continued

product. The entire fragment was subcloned intopUC18 and sequenced on both strands. Althoughchitinase genes were not detected, we obtained bychance a ClpB homologue of P. brasiliensis. Somesimilar regions in cts2 and clpb, as detected byClustal W analysis (data not shown), could explainthat cross-hybridization. The fragment containedthe entire coding region for a gene homologueto clpb. The DNA and the predicted amino acidsequence of the clpb gene of P. brasiliensis arepresented in Figure 1. The entire clpb gene isconsisted of 2436 bp from the start to the stopcodons and is organized in two exons, interruptedby a small intron at the 5′ terminus, position7–63. The intron sequence is flanked by 5′GTand 3′AG, which correspond to the consensussequence of known splicing sites (Ballance, 1986).The P. brasiliensis clpb gene consists of a singleopen reading-frame (ORF) capable of encoding a792 amino acid polypeptide with a pI of 5.34 and acalculated molecular mass of 87.9 kDa. Althoughthe promotor region is not complete, analysis at the5′ non-coding region in the genomic clone revealedstructural features typical of regulatory regions ineukaryotic genes. One TATA box-like sequence ispresent at position 62 in the 5′ upstream sequence(Struhl, 1987). Highly expressed eukaryotic genesusually have multiple short sequence elementsthat function as binding sites for one or more

regulatory proteins (Pelham and Bienz, 1982).A typical example is the heat-shock consensuselement (HSE), which is found in multiple copiesupstream from the transcription start codon of heat-shock genes in a wide variety of organisms (Noverand Scharf, 1997). The PbclpB gene presents threeputative heat shock consensus elements, NGAAN,that were present at positions 112, 100 and 92 inthe 5’upstream region. One polyadenylation signalAATAAA is present in the downstream sequence,at position 2970 of the gene (Proudfoot, 1991). Theputative poly(A) site is followed by a T/GT-richelement, as described by McLauchlan et al. (1985).The function of T/GT-rich elements remains to bedetermined in all the organisms.

The deduced protein presented several canonicalmotifs characteristic of the Clp family of proteins,as shown in Figure 1. The ClpB of P. brasiliensispresents two well-conserved ATP-binding domains(NBD) at positions 104–337 and 463–655 (Walkeret al., 1982). These NBDs are separated by a mid-dle region (spacer) that can be of variable sizein organisms (Schrimer et al., 1996). The pres-ence of the two NDBs places this protein in theclass I Clp/HSP100 family (Squires and Squires,1992; Schrimer et al., 1996). Two ClpA/B chaper-onin signature motifs were detected at amino acids224–238 and 563–580; two carboxyl-terminalsignatures at positions 680–697 and 744–752

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(Schirmer et al., 1996) and a substrate recognitiondomain (SSD), GARPL, in the C-terminal region atposition 744–748, which is conserved in the Clpfamily of proteases (Smith et al., 1999). Anotherfeature in PbClpB is the presence of putative caseinkinase II phosphorylation sites, S/T–X–X–D/E, asdescribed for other ClpBs (Celerin et al., 1998).These phosphorylation targets were spread over 11positions in the deduced PbClpB protein, as shownin Figure 1.

A diagrammatic scheme was constructed forthe ClpB of P. brasiliensis and is presented inFigure 2. From this, we can see that the Walkerconsensus distinctions for ATP binding sites 1 and2 are separated by a large spacer region (middle),a characteristic found only in the ClpB proteins(Squires and Squires, 1992). The ATP-1 and 2domains consisting of the conserved A, B, B1 andB2 consensus sequences (Walker) are also shown.The consensus A is a rich conserved glycine motif(Schirmer et al., 1998). The motifs B, B1 and B2are hydrophobic segments and comprise differentregions of ATP-binding domains (Walker et al.,1982; Chin et al., 1988). In the PbClpB protein,the ATP-binding domain 1 presents one A seg-ment and the two hydrophobic consensus, B andB1 (Figure 2), while the ATP-binding domain 2presents one A region and only one hydropho-bic conserved segment, B2. The two carboxy-terminal signatures, a characteristic of ClpBs, arealso shown. The N-terminal region is less well con-served in P. brasiliensis as in all Clp ATPases andits role in the protein function is not clear (Schirmer

C-terminal

III IV

middle

I II

ATP-2

A B2

ATP-1

A B B1

Figure 2. Schematic diagram of PbClpB. The diagram wasconstructed on basis of the consensus motifs of the ClpBsub-family. ATP1 and ATP2 represent nucleotide-bindingsites, A is a glycine-rich segment and B, B1 and B2represent hydrophobic segment motifs. I and II representmiddle signature consensus sequences. III and IV representC-terminal consensus signatures

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

Comparison of the P. brasiliensis ClpB andrelated sequences

The alignment of the deduced PbClpB with othersequences is shown in Figure 3. It is shown ahigh number of identical and conserved aminoacids among the considered sequences of the ClpBfrom 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 ATP-binding motifs 1 and 2, which is typical of ClpBsequences. The chaperonin signatures, as well asthe sensor and substrate discrimination domain(SSD; Smith et al., 1999), are also highly con-served. The values of identity and similarity ofPbClpB 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 Syne-chococcus sp., Sz. pombe and S. cerevisiae, respec-tively, which reflects the phylogenetic proximity ofthese genes among prokaryotes and eukaryotes.

Expression of the P. brasiliensis clpb

The clpb expression was analysed by RT–PCR andNorthern blot. RT–PCR assays provided a prod-uct of 138 bp (Figure 4A). Southern blot analysisshowed strong hybridization of the 138 bp prod-uct to the 300 bp clpb probe (Figure 4B). The138 bp product was cloned into plasmid pGEM-T-easy and sequenced, showing 100% identity toPbclpB (data not shown). A PbclpB transcript of3.0 kb was identified by Northern blot analysis oftotal RNA from P. brasiliensis mycelium and yeastcells, as shown in Figure 5A. The 3.0 kb mRNAspecies 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 Saccharomycescerevisiae (Parsell et al.,1991). Asterisks indicate amino acid identity and dots represent conserved substitutions. The markedregions are the ATP-binding domains 1 and 2 (boxed), the chaperonin signatures (brackets) and the sensor and substratedomain (GARPL)

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ClpB of Paracoccidioides brasiliensis 969

PbClpB ALAGKLDPVIGRDDEIRRTVQVLSRRTKNNPVLIGEPGVGKTAIVEGLAQRIVNGEVPES 161 EcF84.1 AEQGKLDPVIGRDEEIRRTIQVLQRRTKNNPVLIGEPGVGKTAIVEGLAQRIINGEVPEG 231 SnHSP100 AREGKLDPVIGRDEEVRRTIQILSRRTKNNPVLIGEPGVGKTAIAEGLAQRIINHDVPES 233 SpHSP100 ARNGQLDPVIGREDEIRRTIRVLSRRTKNNPVLIGEPGVGKTSIAEGLARRIIDDDVPAN 234 ScHSP104 ARQGKLDPVIGREEEIRSTIRVLARRIKSNPCLIGEPGIGKTAIIEGVAQRIIDDDVPTI 237 * *:*******::*:* *:::* ** *.** ******:***:* **:*:**:: :** PbClpB LKGKRVLSLDLGSLLAGAKYRGEFEERLKAVLKDLAKHEGEIILFIDELHTLVGAGKG-D 220 EcF84.1 LKGRRVLALDMGALVAGAKYRGEFEERLKGVLNDLAKQEGNVILFIDELHTMVGAGKA-D 290 SnHSP100 LRDRKLISLDMGALIAGAKYRGEFEERLKAVLKEVTDSQGQIILFIDEIHTVVGAGAT-Q 292 SpHSP100 LSNCKLLSLDVGSLVAGSKFRGEFEERIKSVLKEVEESETPIILFVDEMHLLMGAGSGGE 294 ScHSP104 LQGAKLFSLDLAALTAGAKYKGDFEERFKGVLKEIEESKTLIVLFIDEIHMLMGNGKD-- 295 * . ::::**:.:* **:*::*:****:*.**::: . : ::**:**:* ::* * PbClpB GAMDAGNMLKPALARGELRCVGATTLDEYRQYIEKDAALERRFQKVLVDEPSVEDTIAIL 280 EcF84.1 GAMDAGNMLKPALARGELHCVGATTLDEYRQYIEKDAALERRFQKVFVAEPSVEDTIAIL 350 SnHSP100 GAMDAGNLLKPMLARGALRCIGATTLDEYRKYIEKDAALERRFQEVLVDEPNVLDTISIL 352 SpHSP100 GGMDAANLLKPMLARGKLHCIGATTLAEYKKYIEKDAAFERRFQIILVKEPSIEDTISIL 354 ScHSP104 ---DAANILKPALSRGQLKVIGATTNNEYRSIVEKDGAFERRFQKIEVAEPSVRQTVAIL 352 **.*:*** *:** *: :**** **:. :***.*:***** : * **.: :*::** PbClpB RGLKERYEVHHGVKILDSAIIAAAKMSHRYITDRQLPDKAIDLIDEAASRIKMELDSKPE 340 EcF84.1 RGLKERYELHHHVQITDPAIVAAATLSHRYIADRQLPDKAIDLIDEAASSIRMQIDSKPE 410 SnHSP100 RGLKERYEVHHGVKIADSALVAAAMLSNRYISDRFLPDKAIDLVDEAAAKLKMEITSKPE 412 SpHSP100 RGLKEKYEVHHGVTISDRALVTAAHLASRYLTSRRLPDSAIDLVDEAAAAVRVTRESQPE 414 ScHSP104 RGLQPKYEIHHGVRILDSALVTAAQLAKRYLPYRRLPDSALDLVDISCAGVAVARDSKPE 412 ***: :**:** * * * *:::** :: **:. * ***.*:**:* :.: : : *:** PbClpB ALDKLERRLIQLKLQLEAVKKDEDAGS--RAEVSHLEKQIEDVQKDYNDLEEVWKSEKTL 398 EcF84.1 ELDRLDRRIIQLKLEQQALMKESDEAS--KKRLDMLNEELSDKERQYSELEEEWKAEKAS 468 SnHSP100 ELDEVDRKILQLEMERLSLQRENDSAS--KERLEKLEKELADFKEEQSKLNGQWQSEKTV 470 SpHSP100 VLDNLERKLRQLRVEIRALEREKDEAS--KERLKAARKEAEQVEEETRPIREKYELEKSR 472 ScHSP104 ELDSKERQLQLIQVEIKALERDEDADSTTKDRLKLARQKEASLQEELEPLRQRYNEEKHG 472 ** :*:: :.:: :: ::.* * : .:. .:: . :.: :. :: ** PbClpB VEGTKQIQAQLDQARIALQKAQRENDLGEMSRLQYGVIPELEKQLAQDE----------L 448 EcF84.1 LSGTQTIKAELEQAKIAIEQARRVGDLARMSELQYGKIPELEKQLEAAT----------Q 518 SnHSP100 IDQIRTVKETIDQVNLEIQQAQRDYDYNKAAELQYGKLTDLQRQVEALETQ--------L 522 SpHSP100 GSELQDAKRRLDELKAKAEDAERRNDFTLAADLKYYGIPDLQKRIEYLEQQKRKADAEAI 532 ScHSP104 HEELTQAKKKLDELENKALDAERRYDTATAADLRYFAIPDIKKQIEKLEDQ------VAE 526 . : ::: . .*.* * : *:* :.:::::: PbClpB VEEKEE-PKLLRNKVTDNEIAEVVSAATGIPVAKMLQGEREKLLQMESFLHQRVVGQDEA 507 EcF84.1 LEGKT--MRLLRNKVTDAEIAEVLARWTGIPVSRMMESEREKLLRMEQELHHRVIGQNEA 576 SnHSP100 AEQQTSGKSLLREEVLESDIAEIISKWTGIPISKLVESEKEKLLHLEDELHSRVIGQDEA 582 SpHSP100 ANAQPGSEPLLIDVVGPDQINEIVARWTGIPVTRLKTTEKERLLNMEKVLSKQVIGQNEA 592 ScHSP104 EERRAGANSMIQNVVDSDTISETAARLTGIPVKKLSESENEKLIHMERDLSSEVVGQMDA 586 : : :: : * * * : ****: :: *.*:*:.:* * .*:** :* PbClpB VIAVSNAVRRSRAGLSDPNRPSGSFLFLGPTGVGKTELTKALANFLFDSDDAMIRIDMSE 567 EcF84.1 VDAVSNAIRRSRAGLADPNRPIGSFLFLGPTGVGKTELCKALANFMFDSDEAMVRIDMSE 636 SnHSP100 VTAVAEAIQRSRAGLSDPNRPTASFIFLGPTGVGKTELAKALAKNLFDTEEALVRIDMSE 642 SpHSP100 VTAVANAIRLSRAGLSDPNQPIASFLFCGPSGTGKTLLTKALASFMFDDENAMIRIDMSE 652 ScHSP104 IKAVSNAVRLSRSGLANPRQP-ASFLFLGLSGSGKTELAKKVAGFLFNDEDMMIRVDCSE 645 : **::*:: **:**::*.:* .**:* * :* *** * * :* :*: :: ::*:* ** PbClpB FMEKHSVSRLVGAPPGYVGYEEGGVLTEAVRRKPYSVVLFDEVEKAHPDVFNILLQVLDD 627 EcF84.1 FMEKHSVSRLVGAPPGYVGYEEGGYLTEAVRRRPYSVILLDEVEKAHPDVFNILLQVLDD 696 SnHSP100 YMEKHAVSRLMGAPPGYVGYEEGGQLTEAIRRRPYSVILFDEIEKAHGDVFNVMLQILDD 702 SpHSP100 YMEKHSVSRLIGAPPGYVGHEAGGQLTEQLRRRPYSVILFDEIEKAAPEVLTVLLQVLDD 712 ScHSP104 LSEKYAVSKLLGTTAGYVGYDEGGFLTNQLQYKPYSVLLFDEVEKAHPDVLTVMLQMLDD 705 **::**:*:*:..****:: ** **: :: :****:*:**:*** :*:.::**:*** PbClpB GRLTDSQGRVVDFKNTVIVMTSNLGSSDVRELGDNPS---RDDVRNVVMAAVSEHFRPEF 684 EcF84.1 GRLTDGQGRTVDFRNTVVIMTSNLGSDLIQERFGELD---YAHMKELVLGVVSHNFRPEF 753 SnHSP100 GRLTDAQGHVVDFKNTIIIMTSNLGSQYILDVAGDDSR--YEEMRSRVMDVMRENFRPEF 760 SpHSP100 GRITSGQGQVVDAKNAVIIMTSNLGAEYLTTDNESDDGKIDSTTREMVMNSIRGFFRPEF 772 ScHSP104 GRITSGQGKTIDCSNCIVIMTSNLGAEFINSQQGSKIQ---ESTKNLVMGAVRQHFRPEF 762 **:*..**:.:* * :::******:. : . :. *: : ***** PbClpB INRIDELVVFHALEKAQIRGIADIQLDRLRARLAD--RDIKLTVDDSAFDQLIEAGFDPV 742 EcF84.1 INRIDEVVVFHPLGEQHIASIAQIQLKRLYKRLEE--RGYEIHISDEALKLLSENGYDPV 811 SnHSP100 LNRVDETIIFHGLQKSELRSIVQIQIQSLATRLEE--QKLTLKLTDKALDFLAAVGYDPV 818 SpHSP100 LNRISSIVIFNRLRRVDIRNIVENRILEVQKRLQSNHRSIKIEVSDEAKDLLGSAGYSPA 832 ScHSP104 LNRISSIVIFNKLSRKAIHKIVDIRLKEIEERFEQNDKHYKLNLTQEAKDFLAKYGYSDD 822 :**:.. ::*: * . : *.: :: : *: . : : : :.* . * *:. PbClpB YGARPLKRAIQQQVENNLAQKILAGDFQPGDTIVITADAGQ------------------- 783 EcF84.1 YGARPLKRAIQQQIENPLAQQILSGELVPGKVIRLEVNEDR------------------- 852 SnHSP100 YGARPLKRAVQKYLETAIAKGILRGDYKPGETIVVDETDER------------------- 859 SpHSP100 YGARPLNRVIQNQVLNPMAVLILNGQLRDKETAHVVVQNGK-------IFVKPNHEANAN 885 ScHSP104 MGARPLNRLIQNEILNKLALRILKNEIKDKETVNVVLKKGKSRDENVPEEAEECLEVLPN 882 *****:* :*: : . :* ** .: .. : :

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970 R. S. A. Jesuino et al.

phase (Figure 5A). The membrane was washed andprobed to a cDNA encoding a L35 ribosomal pro-tein of P. brasiliensis (GenBank Accession No.AF416509; Figure 5B). The amount of mRNA, asdetected by that internal control, was the samefor yeast and mycelium. In Trypanosoma bruceithe amount of mRNA encoding ClpB is higherin the mammalian stage when compared to theinsect phase, similarly to our description (Glasset al., 1986). The low level of mycelial expres-sion could characterize this gene as phase-specific,with possible function in mechanisms related to theprevalence of the yeast form.

Analysis of the expression of the ClpB inP. brasiliensis

After one- or two-dimensional electrophoresis ofthe P. brasiliensis yeast cell lysates, the proteinswere transferred to membrane and the blots were

0.20-

kb

1 2 3

1 2 3

4 5 6

4 5 6

0.10-

0.15-

0.20-

kb

0.10-

0.15-

AM

M

Y

YB

Figure 4. RT–PCR analysis of the clpb transcript inyeast and mycelium of P. brasiliensis. Total RNA wasobtained from mycelium (M) and yeast (Y) grown at 23 ◦Cand 37 ◦C, respectively. The reactions were primed witholigonucleotides 1F, 1R and 3R, as shown schematically inFigure 1. The RT–PCR reaction products were separatedon a 1.5% agarose gel and stained with ethidium bromide (A).The gel was transferred to membrane and probed to the clpb300 bp fragment. Lanes: 1 and 4, control reaction withoutRNA and RT for mycelium and yeast; 2 and 5, controlreaction without RT for mycelium and yeast, respectively;3, RT–PCR reaction of the mycelium phase; 6, RT–PCRreaction of the yeast phase

3.0 kb Pb clpb

M Y

YM

0.7 kb Pb L35

A

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%), andhybridized to the 300 bp clpb probe [GenBank AccessionNo. AY057112 (A)]. The membrane was washed and probedto a cDNA encoding a L35 ribosomal protein of P. brasiliensis[GenBank Accession No. AY057112 (B)]. The RNA sizeswere calculated using the 0.24–9.5 marker RNA ladder(Gibco)

reacted against the polyclonal antibody directed tothe C-terminal domain of the Synechococcus sp.ClpB (Porankiewicz and Clarke, 1997). A pro-tein species of 89 kDa was detected in the yeastcell extracts by one-dimensional gel electrophoresis(Figure 6A). Two-dimensional analysis provided aprotein species of 89 kDa, pI 5.3, reactive to thepolyclonal antibody, as shown in Figure 6B. Wewere not able to detect the protein product in themycelial phase grown at 23 ◦C (data not shown).The preferential expression of the ClpB transcriptand its putative protein in the yeast cells suggestthat ClpB might be involved in P. brasiliensis sur-vival in the host thermal conditions and that itcould play a role in the host fungus interaction.In this vein, it was described that the ClpB ofT . brucei (Glass et al., 1986) and of Leishma-nia major (Hubel et al., 1997) are preferentiallyinduced in the intracellular stage. In addition, theClpB of Helicobacter pylori and Yersinia entero-colitica are associated to the host invasion processand virulence (Allan et al., 1998; Badger et al.,2000).

ClpB is a stress-induced protein in severalmicrorganisms (Glass et al., 1986; Hubel et al.,1997). In P . brasiliensis it was detected theexpression in the yeast phase. Such data in addi-tion to the putative HSE motifs in the pro-moter region of PbClpB could suggest a role ofClpB during the temperature upshift that char-acterizes the physiological infective process byP. brasiliensis. Further studies are necessary to

Copyright 2002 John Wiley & Sons, Ltd. Yeast 2002; 19: 963–972.

ClpB of Paracoccidioides brasiliensis 971

A B

212-

170-

116-

76-

SDSITF 5.3

kDa212-

170-116-

76-

Figure 6. Analysis of PbClpB by one- and two-dimensionalprotein gels and Western blotting. The yeast cells weregrown at 37 ◦C for 7 days. Amounts of 50 µg and200 µg protein were fractionated, respectively, by one-or two-dimensional gel electrophoresis, transferred tomembranes and reacted to the polyclonal antibody toClpB. Localization of the putative ClpB of P. brasiliensisis marked with arrows. (A) One-dimensional gel analysis;(B) two-dimensional gel analysis. The top number is relatedto the pI value of the ClpB in P. brasiliensis, as calculated bythe pI markers at the first dimension, and those on the leftrefer to the molecular mass markers

clarify the role of ClpB in the morphogeneticevents of P. brasiliensis. Our future projects willfocus 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. Thiswork was supported by grants from CNPq (520679/99-7)and FUNAPE-UFG.

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