Comparative analysis of the cDNA encoding a ClpA homologue of Paracoccidioides brasiliensis

Mycol. Res. 109 (6): 707–716 (June 2005). f The British Mycological Society

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doi:10.1017/S0953756205002789 Printed in the United Kingdom.

Comparative analysis of the cDNA encoding a ClpA homologue of Paracoccidioides brasiliensis

Juliana CAMARGOS OLIVEIRA1, Nadya DA SILVA CASTRO1, Maria Sueli SOARES FELIPE2, Maristela PEREIRA1 and Ce´lia Maria DE ALMEIDA SOARES1* 1

Laborato´rio de Biologia Molecular, ICB, Universidade Federal de Goia´s, 74.001-970, Goiaˆnia, Goia´s, Brazil. Universidade de Brası´lia, 70910-900, Brası´lia, DF, Brazil. E-mail : [email protected]

2

Received 21 June 2004; accepted 17 February 2005.

A cDNA encoding a chaperone ClpA homologue of Paracoccidioides brasiliensis was isolated and characterized. The ClpA belongs to a group of ClpATPAses proteins, which are highly conserved, and include several heat inducible molecular chaperones. In this study, a 2879 bp cDNA designated as Pbclpa was obtained which encodes a predicted protein of 927 amino acids. Characteristic consensus motifs of the ClpATPases family are present. The PbClpA middle region was compared to other related ClpA and ClpB proteins from fungi and bacteria. Comparative analysis demonstrated in the middle region the presence of a heptad repeat sequence, characteristic of ClpBs from prokaryotes and fungi, which are absent in ClpAs from prokaryotes but were present in all described fungal ClpAs. Our comparative analysis reveals that one of the criteria typically used to distinguish the prokaryotic subfamilies ClpA and ClpB, the size of the middle sequence, may not be useful in fungi. Phylogenetic analyses were performed with the complete sequences of ClpAs from fungi and bacteria and with the middle regions of those ClpAs present at NCBI and Pfam databases. Our results indicated that both types of analysis can be useful as a tool in the determination of phylogenetic relationships.

INTRODUCTION Hsp100/ClpATPases, a subfamily of AAA proteins, are represented in all kingdoms of life and show a high degree of conservation. Several members of these proteins have been described as molecular chaperones, which are inducible in stress conditions. ClpA and ClpB are typical members of the class I family of AAA proteins, containing two highly conserved nucleotide binding domains (NBD) or AAA-domains (Neuwald et al. 1999). The variable middle region links the two AAA-domains and has been described as one criterion for the classification of ClpATPases into the A and B subfamilies in prokaryotic cells. The linker region is larger in ClpB (approximately 130–172 residues) when compared to ClpA proteins from prokaryotic organisms (approximately 5–54 residues) (Gottesman, Clark & Maurizi 1990, Squires & Squires 1992, Schirmer et al. 1996).

* Corresponding author.

The middle region sequence of ClpB proteins from prokaryotes and fungi contains periodic repeats of seven amino acids residues, heptad repeats sequences, which are found in proteins with coiled-coil domains (Celerin et al. 1998, Kedzierska et al. 2003). These heptad repeats, form in secondary structure, complexes of a-helices whose amino acids heptad repeats are named a-b-c-d-e-f-g. The residues a and d are often hydrophobic residues whereas e and g are charged residues. The a and d form the inter-helical hydrophobic core and the residues e and g form inter-helical ionic interactions which are associated with the stability of the protein (Yu 2002). In prokaryotes, the ClpA was discovered as a specific factor, which stimulates the peptidase activity of the ClpP protease (Hwang et al. 1988, Katayama et al. 1988, Wickner et al. 1994). ClpA can also function as a molecular chaperone preventing the irreversible inactivation of proteins during heat treatment (Wickner et al. 1994) and also in remodelling inactive proteins to the active form (Wickner et al. 1994, Pak & Wickner 1997, Singh et al. 2000). ClpAs have

The ClpA encoding cDNA of Paracoccidioides brasiliensis 1 M N G T Q L T D R A N Q A L V D A H -45 atctatcgtttccaatctccagagatttaattatagactgccatcATGAACGGAACACAGTTGACAGACAGAGCTAATCAAGCTCTTGTCGATGCTCACG 19 A L A E Q H A H P Q L L P I H L A V S L L D P P V D E S K D Q Q V T 56 CACTCGCAGAGCAGCATGCTCACCCACAGCTCCTCCCCATTCATCTCGCAGTTTCGCTCCTGGACCCCCCTGTGGATGAGTCCAAGGACCAGCAAGTTAC 53 T H P S H Q A S S G S L F K R V V E K A H G D P Q Q L R R A L N K 156 CACACATCCCTCACACCAAGCCAGTTCAGGCTCTCTCTTCAAACGAGTTGTGGAGAAAGCCCATGGCGATCCTCAGCAGCTACGCAGGGCCCTCAACAAG 86 S L V R L A S Q D P P P E T I S P S P A F A K V L R A A S N L S K 256 TCTTTGGTCCGGCTAGCTTCGCAAGATCCGCCTCCAGAGACCATCTCCCCGTCACCGGCGTTTGCAAAGGTTCTCCGGGCGGCTTCCAATTTATCAAAGA 119 T Q K D T Y V A I D H L I A A L A Q D P T I Q R A L A D A N I P N V 356 CCCAGAAGGATACTTATGTCGCAATTGATCATTTAATAGCTGCGCTGGCGCAGGATCCTACTATCCAGCGGGCGCTTGCGGACGCAAATATCCCCAATGT 153 K M I D S A I Q Q I R G M K R V D S K T A D T E E E S E N L K K F 456 CAAGATGATCGATTCTGCTATCCAGCAAATCCGCGGGATGAAACGCGTGGATTCAAAAACGGCAGATACAGAAGAAGAAAGTGAAAACTTAAAAAAGTTT 186 T V D M T A M A R E G K I D P V I G R E E E I R R V I R I L S R R 556 ACAGTTGACATGACTGCCATGGCGAGAGAAGGGAAGATTGATCCCGTGATCGGCCGAGAAGAGGAGATTAGAAGAGTGATTCGCATTCTCAGCCGACGGA 219 T K N N P V L I G E P G V G K T T V V E G L A R R I V N A D V P A N 656 CAAAGAACAATCCAGTGCTTATTGGCGAACCTGGCGTGGGAAAGACAACGGTCGTCGAAGGCCTAGCACGACGGATTGTCAATGCCGATGTTCCCGCCAA 256 L A N C K L L S L D V G S L V A G S K Y R G E F E E R M K G V L K. 756 TCTGGCTAATTGCAAGCTTCTATCCTTGGATGTCGGATCACTTGTTGCTGGCAGCAAGTATCGTGGTGAGTTCGAGGAGAGGATGAAGGGCGTTTTGAAA 286 E I E E S K E T I V L F V D E I H L L M G A G S S G E G G M D A A . 856 GAGATTGAAGAATCAAAGGAGACCATCGTTCTGTTTGTGGATGAAATCCATCTTCTTATGGGAGCTGGCTCCAGTGGAGAAGGTGGCATGGATGCCGCAA 319 N L L K P M L A R G Q L H C I G A T T L G E Y R K Y I E K D Q A F E 956 ATCTACTCAAGCCCATGCTTGCAAGAGGTCAGCTACATTGCATTGGCGCAACCACTCTTGGCGAATACAGGAAGTACATCGAGAAAGACCAAGCTTTCGA 353 R R F Q Q V L V K E P T V G E T I S I L R G L K E R Y E V H H G V 1056 GCGTCGATTCCAACAGGTCTTGGTCAAGGAGCCTACTGTTGGCGAGACCATCTCTATTCTCAGGGGTCTGAAAGAACGATACGAAGTTCACCATGGTGTT 386 N I L D G A I V S A A N L A S R Y L T A R R L P D S A V D L I D E 1156 AACATCCTCGATGGAGCCATTGTATCCGCAGCCAACCTTGCTTCTCGTTACCTTACTGCCCGAAGACTTCCAGATTCTGCAGTTGATCTAATTGACGAAG 419 A A A A V R V T R E S Q P E A L D T L E R R A R Q L Q I E I H A L A 1256 CAGCCGCCGCTGTTCGCGTCACGAGAGAATCTCAACCTGAAGCCCTGGATACTTTGGAACGCCGTGCCCGACAACTCCAGATCGAAATCCATGCTCTGGC 453 R E K D A A S K A R L E A A K Q E A A N V N E E L R P L R E K Y E . 1356 TCGCGAAAAGGATGCGGCCTCGAAGGCTCGACTCGAGGCTGCCAAACAGGAAGCAGCTAACGTCAACGAAGAGCTTCGCCCTCTACGAGAAAAATATGAA 486 S E K Q R S K D I Q D A K I K L D L L K V K R D E A T R S G D T Q . 1456 AGTGAGAAACAACGGAGCAAGGATATTCAAGATGCTAAAATCAAATTGGACCTTTTGAAAGTGAAAAGGGACGAGGCAACCCGGTCTGGAGATACCCAAA 519 T A S D L I Y Y A I P D V E K R I E Q L E A E R A R Q D A E L S A Q 1556 CTGCTTCGGATCTGATTTACTATGCCATCCCGGATGTCGAGAAGCGCATCGAACAATTGGAGGCTGAAAGAGCTCGACAAGATGCGGAACTATCAGCCCA 553 P G A G E T L M A D A V G P E Q I N E I V A R W T G I P V T R L R . 1656 GCCAGGTGCAGGAGAAACCCTTATGGCAGATGCTGTTGGCCCTGAGCAAATCAATGAAATTGTGGCCAGATGGACTGGCATTCCCGTTACCAGACTGAGG 586 T T E K D R L L H M E S H L S K I V V G Q K E A V Q S V S N A I R 1756 ACCACCGAAAAGGACAGGCTTCTGCACATGGAATCGCACCTTTCCAAAATTGTTGTTGGCCAGAAGGAAGCCGTCCAATCAGTGTCGAACGCAATTCGAC 619 L Q R S G L S N P N S P P S F L F C G P S G T G K T L L T K A L A E 1856 TCCAACGGTCTGGCCTGAGCAACCCTAACTCCCCGCCAAGCTTCCTCTTCTGTGGGCCATCAGGCACGGGTAAAACCCTGCTTACTAAGGCACTGGCGGA 653 F L F D D P K A M I R F D M S E Y Q E R H S L S R M I G A P P G Y 1956 ATTCCTGTTCGATGACCCCAAGGCTATGATCCGATTTGACATGTCTGAATATCAGGAACGGCATTCTTTGAGTCGAATGATCGGAGCGCCACCTGGCTAC 686 V G H D A G G Q L T E S L R R R P F S I L L F D E V E K A A K E V 2056 GTTGGGCATGATGCTGGTGGCCAGCTAACAGAATCACTTCGCCGGCGACCATTCTCCATTCTCCTCTTCGATGAGGTGGAAAAAGCTGCGAAGGAAGTTT 719 L T V L L Q L M D D G R I T D G Q G R I V D A K N C I V V M T S N L 2156 TAACCGTCCTCCTTCAGCTGATGGATGACGGAAGAATCACAGACGGTCAGGGAAGAATTGTCGATGCAAAGAATTGCATCGTTGTCATGACTTCCAACCT * * * 753 G A E F L Q R P T A S N G Q I D P T T K E L V M G A L R N Y F L P 2256 TGGCGCCGAGTTTCTCCAACGGCCCACAGCTTCCAATGGCCAGATTGACCCCACCACTAAAGAGCTTGTTATGGGCGCACTGCGGAACTACTTCCTGCCC * * * * * * * * * * * * * * * 786 E F L N R I S S I I I F N R L T R R E I R K I V D V R L Q E I Q R 2356 GAATTCCTTAATCGAATCTCTAGCATCATTATCTTCAATCGCCTCACAAGGCGGGAGATACGGAAGATCGTGGATGTTCGGTTACAAGAGATCCAACGGA 819 R L E Q N D R T V T I D C T D E V K D Y L G N A G Y S P V Y G A R P 2456 GGCTAGAGCAGAACGACCGCACGGTGACGATAGATTGTACAGACGAAGTCAAGGATTACTTGGGTAATGCCGGATACTCGCCAGTCTATGGCGCCAGGCC 853 L S R L I E K E V L N R L A V L I L R G A I K D G E T A R V V M Q 2556 ACTCTCGCGACTCATTGAGAAAGAGGTCTTGAACCGCCTTGCGGTCTTGATCTTAAGAGGGGCCATCAAAGACGGCGAGACTGCACGTGTTGTGATGCAG 886 E G R I T V L P N H V E T E S E D E E M I D E S D T L A E M E E D 2656 GAAGGAAGGATCACTGTCCTACCAAACCACGTTGAAACGGAAAGTGAAGACGAGGAGATGATTGATGAATCTGACACACTTGCGGAGATGGAAGAGGATA 919 M G E R D L Y E # 2756 TGGGAGAACGGGACCTTTATGAGTGAgcctggcagagaacaggatatatatgtattacgatgctaaaaaaaaaaaaaa

Fig. 1. For legend see opposite page.

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J. C. Oliveira and others been described in Saccharomyces cerevisiae, such as Hsp104 protein (Parsell et al. 1991) which presents a complete ClpA domain ; other fungal ClpA sequences are described in the databases such as Candida albicans (GenBank accession no. AAK60625), Neurospora crassa (EAA27992), Schizosaccharomyces pombe (CAB38512), and Pleurotus sajor-caju (AAF01451). Paracoccidioidomycosis (PCM), the major human systemic mycosis in Latin America, is caused by the dimorphic fungus Paracoccidioides brasiliensis (Brummer, Castan˜eda, & Restrepo 1993). The infection is acquired by inhalation of airborne propagules produced by the fungal mycelium, which change into pathogenic yeast-like cells at the host temperature (McEwen et al. 1987). During the infection process, the sudden increase in temperature experienced by P. brasiliensis could lead to the denaturation and aggregation of several proteins, as well to the induction of several heat shock proteins (HSPs) (Silva et al. 1994, 1999, Izacc et al. 2001, Cunha et al. 2002). In particular, an ATP-dependent protease denominated PbClpB is over expressed during heat shock in P. brasiliensis (Jesuino et al. 2002). Here, we report for the first time, the identification of a cDNA encoding a ClpA of P. brasiliensis, as well as the characterization and comparative analysis of the deduced ClpA protein of this fungus. We verified that the middle and coiled-coil regions in ClpA of P. brasiliensis and other ClpAs of fungi have similar sizes, which are comparable to the described P. brasiliensis ClpB (Jesuino et al. 2002). In prokaryotes these regions are of different sizes in ClpAs and ClpBs (Gottesman et al. 1990, Squires & Squires 1992, Schirmer et al. 1996). This finding could be useful in the elucidation of the unknown role of the ClpA proteins in fungi, since the middle region is described as important for the stability of the ClpB protein (Kedzierska et al. 2003) and for its chaperone activity (Mogk et al. 2003).

MATERIALS AND METHODS Microorganism growth and cultivation Yeast cells of Paracoccidioides brasiliensis isolate Pb01 (ATCC MYA 826) were grown in Fava Neto’s medium (Fava-Netto 1961) at 36 xC and was utilized throughout this study. The fungus was subcultured every 7 d in the fresh medium.

709 cDNA cloning A cDNA library from the yeast phase of Paracoccidioides brasiliensis, isolate Pb01, was constructed in lZAP II (Stratagene, La Jolla, CA). The cDNA library was screened by plaque hybridization using a PCR fragment of 650-bp encoding a described endopeptidase (GenBank accession no. AF441251) of P. brasiliensis. 5r104 pfu were plated into NZY (0.5 % w/v NZ amine (casein hydrolysate), 1.5 % w/v agar, pH 7.5), with 600 ml of Escherichia coli XL-1 Blue MRFk cells (OD600=0.5) and 8 ml of top agarose (NZY broth, 0.7 % w/v agarose) and incubated at 37 x overnight. The plates were chilled for 2 h. The filters were laid on to the plates for 2 min. Filters were soaked in a denaturation buffer (1.5 M NaCl, 0.5 M Tris-HCl pH 8.0) for 10 min and rinsed in washing buffer (0.2 M Tris-HCl pH 7.5, 2r sodium saline citrate SSC 0.03 M sodium citrate, 0.3 M NaCl) for 30 s. Filters were pre- hybridized with 6rSSC (0.09 M sodium citrate, 0.9 M NaCl), 5rDenhardt’s solution (0.0025 M ficoll, 0.0028 M polyvinylpyrrolidone, 1 % w/v bovine serum albumin), 0.5 % (w/v) sodium dodecyl sulfate (SDS), 100 mg mlx1 salmon sperm DNA, 45 % v/v formamide, for 2 h at 42 x. Hybridization was performed for 12 h with the 650 bp DNA encoding the endopeptidase, radiolabelled with [a-32P]dCTP. The filters were then rinsed three times with 1rSSC (0.015 M sodium citrate, 0.15 M NaCl), 0.1 % w/v SDS at 42 x for 15 min ; three times for each rinse. After three rounds of plaque purification, five positive clones were obtained and three of them were sequenced. The in vivo excision of the pBluescript phagemids (Stratagene) in E. coli XL1-Blue MRFk cells was performed.

Sequencing and computer-based sequence analysis DNA sequencing was performed on both strands according to Sanger, Nicklen & Coulson (1977). The sequencing of the cDNA used standard fluorescence labelling dye-terminator protocols. Automated sequencing of the products was performed using a MegaBACE 1000 sequencer (Amersham Biosciences, Little Chalfont). Nucleotide sequence analysis was performed with the Wisconsin Genetics Computer Group (GCG) analysis software package, version 7.0 (Devereux, Haeberli & Smithies 1984). The DNA sequences obtained were translated and compared to all non-redundant polypeptides in the translated NCBI

Fig. 1. Nucleotide sequence of PbClpA and the deduced amino acid sequence. The start, stop codons and poly-A tail are in bold. The amino acid sequence is shown above the nucleotide sequence by a single letter code. The putative NBD1 Walker-type consensus motifs A1, B1 and B2 are enclosed in rectangles. The NBD2 Walker-type consensus motifs A2 and B3 are enclosed by dotted rectangles. Underlined amino acids indicate the middle sequence that separates the domains NBD1 and NBD2. The C-terminal signature is marked by upper asterisks. The SSD domain is double boxed. Black blocks with white letters represent the two chaperonin signatures of ClpA/ClpB.

NBD 1 a

NBD 2

Walker consensus distinctions

Pb ClpA / organism sequence

Chaperonin ClpA/ClpB signature

Walker consensus distinctions

similarity (%) identity (%)

I

II

A1

B1

B2

A2

Pb ClpA/ Neurospora crassa

GEPGVGKT GEPGVGKT ********

KEIEESKETIVLFVD KEISESKEMIILFID ***.**** *:**:*

RRLPDSAVDLID RRLPDSAIDLID *******:****

GPSGTGKT GPSGTGKT ********

RRRPFSILLFD GARPLSRLI DAANLLKPMLARG DMSEYQERHSLSRMIGA RRKPFSILLFD GARPLQRVL DAANLLKPMLARG DMSEYQERHSLSRMIGA **:******** *****.*:: ************* *****************

93

b

77

Pb ClpA/ Schizosaccharomyces pombe

GEPGVGKT GEPGVGKT ********

KEIEESKETIVLFVD KEVEESETPIILFVD **:***: .*:****

RRLPDSAVDLID RRLPDSAIDLVD *******:**:*

GPSGTGKT GPSGTGKT ********

RRRPFSILLFD GARPLSRLI DAANLLKPMLARG DMSEYQERHSLSRMIGA RRRPYSVILFD GARPLNRVI DAANLLKPMLARG DMSEYMEKHSVSRLIGA ****:*::*** *****.*:* ************* ***** *:**:**:***

88

63

GEPGVGKT Pb ClpA/ Saccharomyces cerevisiae GEPGIGKT ****:***

KEIEESKETIVLFVD KEIEESKTLIVLFID ******* ****:*

RRLPDSAVDLID RRLPDSALDLVD *******:**:*

GPSGTGKT GLSGSGKT * **:***

RRRPFSILLFD GARPLSRLI DAANLLKPMLARG DMSEYQERHSLSRMIGA QYKPYSVLLFD GARPLNRLI DAANILKPALSRG DCSELSEKYAVSKLLGT : :*:*:**** *****.*** ****:*** *:** * ** .*::::*:::*:

83

52

GEPGVGKT GDAGVGKT *:.*****

KEIEESKETIVLFVD NEIEKSKEFIILFID :***:*** *:**:*

RRLPDSAVDLID RALPDSAVDLVD * ********:*

GPSGTGKT GLSGSGKT * **:***

RRRPFSILLFD GARPLSRLI DAANLLKPMLARG DMSEYQERHSLSRMIGA IRRPYSVVLLD GARPLNRLI DAANLLKPMLARG DCSELGDKWSASKLLGA ***:*::*:* *****.*** ************* * ** :: * *:::**

82

70

GEPGVGKT KEIEESKE----TIVLFVD RRLPDSAVDLID GEPGVGKT NEVEKASEDGGPGVILFVD IHALEAPRAREG ******** :*:*::.* ::**** : ::.

GPSGTGKT GPSGTGKT *******:

RRRPFSILLFD GARPLSRLI DAANLLKPMLARG DMSEYQERHSLSRMIGA RRKPYSIILID GARPLNRAI DAANLFKPLLARG DGSEYSEKHSIARLIGA **:*:**:*:* *****.* * *****:**:**** **.*:**::*:******

77

50

Pb ClpA/ Candida albicans

Pb ClpA/ Pleurotus sajor-caju

B3

SSD

b

The ClpA encoding cDNA of Paracoccidioides brasiliensis

Table 1. Comparison of Pb ClpA conserved regions which define the protein as a member of the ClpATPase family with the regions of ClpAs from fungi.

a The sequences used were: Paracoccidioides brasiliensis [ClpA protein, AAO73810], Neurospora crassa [hypothetical protein, EAA27992], Schizosaccharomyces pombe [heat shock protein, CAB38512], Saccharomyces cerevisiae [Hsp104, AAA50477], Candida albicans [Hsp104-a, AAK60625], Pleurotus sajor-caju [Hsp 104, AAF01451]. b The values were calculated based in Clustal X alignment.

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(A)

(B)

Fig. 2. Segment of the alignment of the middle regions of the ClpAs and ClpBs described in NCBI database. (A) Alignment of the middle regions of the ClpAs of Candida albicans (AAK 60625), Saccharomyces cerevisiae (AAA 50477), Paracoccidioides brasiliensis (AAO 73810), Neurospora crassa (EAA 27992), Schizosaccharomyces pombe (CAB 38512), and Pleurotus sajorcaju (AAF 01451). The apparent coiled-coil heptad repeats a and d, and e and g are indicated by bold letters. (B) Segment of the alignment of amino acids of the middle sequences of the deduced PbClpB (AAL 47016) and PbClpA (AAO 73810). The apparent coiled-coil heptad repeats a and d, and e and g are indicated by bold letters.

database using the BLAST program (http:// www.ncbi.nlm.nih.gov/BLAST/ ; Altschul et al. 1997). Comparative analysis of PbClpA and related sequences of prokaryotes and eukaryotes Multiple sequence alignment of PbClpA and related sequences was carried out with CLUSTAL X version 1.8 (Thompson et al. 1997). The Prosite database allowed the identification of the ClpA/B signature sequences (http://www.us.expasy.org/prosite ; Bairoche, Bucher & Hoffmann, 1997). The ATP binding sites 1 and 2 with their respective Walker type consensus motifs, SSD (Sensor and Substrate Discrimination) domain and middle region were determined by the classification of Gottesman et al. (1990) and Schimer et al. (1996). Schematic representation of ClpAs and ClpBs described at databases were performed based on the relative sizes and structural organization of the proteins from eukaryotes and prokaryotes.

Phylogenetic analysis The Pfam (http://www.sanger.ac.uk/Software/Pfam/; Bateman et al. 2004) and NCBI (http://www. ncbi.nlm.nih.gov) databases were used to search for complete protein sequences of ClpA. Alignments of ClpA amino acid sequences or middle regions were carried out with the Clustal X program, version 1.8 using the Neighbour Joining method (Thompson et al. 1997). The TreeVIEW software was used to visualize alignments. The robustness of branches was estimated using 100-boot-strapped replicates.

Nucleotide sequence accession number The nucleotide sequence of the cDNA encoding the PbClpA of Paracoccidioides brasiliensis and the deduced protein were submitted to GenBank database under accession nos AY229978 and AAO73810, respectively.

The ClpA encoding cDNA of Paracoccidioides brasiliensis

Fig. 3. Schematic diagram comparing the ClpAs and ClpBs from fungi and bacteria. The relative sizes and organization of the consensus motifs are shown: NBD1 and NBD2 represented by diamonds ; Middle regions are represented by thick black lines ; thick grey lines in the extremes are Nand C-terminus regions, respectively. The sequences are : CaClpA (Candida albicans ClpA, AAK 60625), ScClpA (Saccharomyces cerevisiae ClpA, AAA 50477), PbClpA (paracoccidioides brasiliensis ClpA, AA O73810), NcClpA (Neurospora crassa ClpA, EAA 27992), SpClpA (Schizosaccharomyces pombe ClpA, CAB 38512), PscClpA (Pleurotus sajor-caju ClpA, AAF 01451), PbClpB (paracoccidioides brasiliensis ClpB, AAL 47016), EcClpB (Escherichia coli ClpB, M 29364), DnClpB (Dichelobacter nodosus ClpB, M 32229), EcClpA (E. coli ClpA, BAA 35601), BsClpA (Brucella suis ClpA, NP_698174), and AtClpA (Agrobacterium tumefaciens ClpA, NP_532054).

RESULTS AND DISCUSSION Sequence analysis of the cDNA encoding PbClpA and comparative analysis of the deduced protein Because we were interested in searching for the endopeptidases of Psendococcoidiodes brasiliensis, we used a fragment of 650-bp encoding an endopeptidase of this fungus. Five positive clones were obtained, and three of them were sequenced on both strands. The selected l Zap II recombinant clones contained fragments that hybridized to the 650-bp fragment encoding the P. brasiliensis endopeptidase. Although, endopeptidase cDNAs were not detected we obtained by chance a full-length cDNA encoding a chaperone ClpA homologue of P. brasiliensis. A homologous region present in the endopeptidase and in the cDNA encoding PbClpA, was observed (45 % of identity at

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nucleotide level ; data not shown) could explain the cross- hybridization. The entire sequence of the cDNA was 2879 nucleotides in length and encoded a single open reading frame (ORF) of 927 amino acids, as presented in Fig. 1. The presumed initiating methionine and the stop codon, were located at positions 46 and 2779, respectively in the obtained sequence. In the 3kunstranslated region (UTR), the poly-A tail was initiated at position 2820. The deduced protein has a theoretical isoelectric point of 5.77 and a calculated molecular mass of 102 kDa. PbClpA belongs to the class I of ClpATPases which present the two highly conserved domains NBD1 and NBD2. The characteristic Walker-type consensus motifs, A and B from NBD1 (A1, B1 and B2), are at the positions 227 to 234, 285 to 299 and 406 to 417, respectively (Fig. 1). The NBD2 motifs (A2 and B3) are marked in Fig. 1 at positions 637 to 644 and 699 to 709, respectively. The C-terminal signature sequence, well-conserved in other ClpA molecules (Schirmer et al. 1996, Ekaza et al. 2000), is also present in PbClpA at positions 783 to 800. The middle region is present at the positions 431 to 596 (Fig. 1) and also detached in the Fig. 2A. Another characteristic region present in PbClpA is the SSD motif, which seems to play a critical role in the mechanism by which ClpA recognizes the correct substrate (Smith, Baker & Sauer 1999). The putative SSD domain, GARPLSRLI, was located at positions 849 to 857 in the deduced protein. Two characteristic internal signature sequences for chaperonins ClpA/ClpB were found, DA ANLLKPMLARG and DMSEYQERHSLRMIGA at positions 316 to 328 and 665 to 681, respectively (Fig. 1). The deduced ClpA of P. brasiliensis was highly homologous over its amino acid sequence to other fungi ClpA-like proteins previously described, such as these of Neurospora crassa, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Candida albicans, Pleurotus sajor-caju. Table 1 presents the alignment of the predicted conserved motifs in PbClpA and in the cited ClpA proteins, which represent fungal ClpA sequences present at databases. High conservation at the amino acid level was observed in the NBD 1 and 2 domains (identity values of 100 to 46 % and similarity values of 100 to 73 %), in the SSD domain (identity values of 88 to 77 % and similarity values of 100 to 88 %) and in the ClpA/ClpB signature regions I and II (identity values of 100 to 35% and similarity values of 100 to 70%) (data not shown). When considering the entire amino acid sequences of the ClpAs of the cited fungi, identity values were of 77 to 50 %. The segment of the alignment in the conserved middle regions from fungal ClpAs is shown in Fig. 2A. The alignment shows four regions which represent the periodic seven-residue repeats, a-b-c-d-e-f-g present in the middle regions. The hydrophobic residues often occupy the positions a and d (the most common are Leu, Ile, Val) and charged residues in the positions

J. C. Oliveira and others

713 S. flexneri 301 E. coli O157:H7 S. flexneri 2457T E. coli K12 S. enterica E. coli CFT073 87 100 S. typhimurium 71

100

s icu P. luminescens yt 87 ol Y. pestis 100 71 m S. oneidensis e 100 ha ra X. fastidiosa pa V. vulnificus . X. campestris 100 V 100 X. axonopodis V. cholerae

N. meningitidis MC58

100 100 100

100

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N. meningitidis Z2491

P. aeruginosa P. syringae P. putida

100 100

R. solanacearum

100

C. burnetii

98 94

100

N. europaea

98 92

B. suis 100 B. melitensis

D. radiodurans

60 83

L. interrogans 96

90 53 100

A. tumefaciens

41 96

B. japonicum

94 75

90

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C. crescentus

100 83 100 100

100

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C. acetobutylicum

P. brasiliensis T. pallidum

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H. pylori 2695 A. pyrophilus H. pylori J99 T. tengcongesis S. coelicolor S. agalactiae 1 S. agalactiae 2

B. burgdorferi

Fig. 4. Phylogenetic tree illustrating the relationship of PbClpA and related sequences. The phylogenetic analysis was based on the complete amino acid sequences of 47 ClpA proteins. The tree was calculated by the neighbour-joining method implement in the program Clustal X and drawn by using the program TreeVIEW software. The clade of Fungi is detached by a grey circle. Prokaryotes are clustered into others clades. The following ClpA amino acids sequences were used to construct the tree : Pleurotus sajor-caju (AAF 01451), Schizosaccharomyces pombe (CAB 38512), Neurospora crassa (EAA 27992), paracoccidioides brasiliensis (AAO 73810), Saccharomyces cerevisiae (AAA 50477), Candida albicans (AAK 60625); Clades from prokaryotes organisms Streptococcus agalactiae NEM316 (CAD 46362), Streptococcus agalactiae NEM316 (CAD 46650), Streptomyces coelicolor A3(2) (CAA 19619), Thermoanaerobacter tengcongensis (AAM 25468), Aquifex pyrophilus (AAD 25872), Borrelia burgdorferi B31 (AAC 66745), Helicobacter pylori J99 (NP_222751), Helicobacter pylori 26695 (NP_206835), Treponema pallidum subsp. pallidum str. ‘Nichols’ (NP_219238), Clostridium acetobutylicum ATCC 824 (NP_348449); Caulobacter crescentus CB15 (NP_421271), Bradyrhizobium japonicum USDA 110 (AAG 17282), Agrobacterium tumefaciens str. C58 (NP_532054), Brucella melitensis 16M (NP_539733), Brucella suis 1330 (NP_698174); Nitrosomonas europaea ATCC 19718 (CAD 85644), Ralstonia solanacearum (CAD 16171), Neisseria meningitidis Z2491 (NP_283818), Neisseria meningitidis MC58 (NP_273877); Xanthomonas axonopodis pv. citri str. 306 (AAM 36863), X. campestris pv. campestris str. ATCC 33913 (AAM 41256), Xylella fastidiosa Temecula1 (NP_778887); Shewanella oneidensis MR-1 (NP_718211), Photorhabdus luminescens subsp. laumondii TTO1 (CAE 13887), Escherichia coli CFT073 (NP_752948), E. coli O157:H7 EDL933 (AAG 55264), E. coli K12 (BAA 35601), Shigella flexneri 2a str. 301 (AAN 42475), S. flexneri 2a str. 2457T (AAP 16347), Salmonella enterica subsp. enterica serovar. Typhi (CAD 05347), S. typhimurium LT2 (AAL 19881), Yersinia pestis KIM (AAM 86360), Vibrio vulnificus YJ016 (NP_935115), V. parahaemolyticus RIMD 2210633 (NP_797393), V. cholerae O1 biovar. eltor str. N16961 (AAF 94303), Pseudomonas aeruginosa PAO1 (AAG 06008), P. syringae pv. tomato str. DC3000 (AAO 56831), P. putida KT2440 (AAN 69602); Coxiella burnetii RSA 493 (NP_820191), Deinococcus radiodurans R1 (AAF 10168), and Leptospira interrogans serovar. lai str. 56601 (NP_712285).

The ClpA encoding cDNA of Paracoccidioides brasiliensis

714 T. tengcongensis

A. pyrophilus

P. putida P. aeruginosa P. syringae

82

99

P. brasiliensis

H. pylori J99 H. pylori 26959 64

92

75

73 72 65

92

Y. pestis 55 15

18

72

40 19

S. flexneri 301 :H726 42 157 li O E. coli K12 o c . E 12 25 11 S. enterica S. oneidesis

E. coli CFT073

60 T. pallidum

14 P. luminescens S. flexneri 2457T S. typhimurium

46

S. coelicolor

R. solanacearum

34

S. agalactiae 1 55

S. agalactiae 2 17

32 97

15

B. burgdorferi

37

N. meningitidis Z2491 N. meningitidis MC58

34

C. burnetti

43

74

C. acetobutylicum

D. radiodurans

38

N. europaea

90 B. suis 94 B. melitensis

98 V. cholerae

A. tumefaciens V. vulnificus V. parahaemolyticus

C. crescentus

B. japonicum

X. fastidiosa

100 X. campestris X. axonopodis

Fig. 5. Phylogenetic tree constructed using the middle regions of ClpAs of the same organisms described in Fig. 4. The clade of Fungi is indicated by a grey circle.

e and g (the most common are Lys and Glu) are present in most of the analysed sequences (Fig. 2A), as described by Yu (2002). The middle region of PbClpA and other fungal ClpA-like proteins, present the characteristic heptad repeats which could form a coiled-coil architecture, as observed in PbClpB (Fig. 2B). This arrangement is described as associated to the a-helice formation, which is important for the stability of molecules which present coiled-coil architeture (Yu 2002). The heptad repeats in the middle region of the Escherichia coli ClpB were related to the stabilization of intramolecular contacts (Kedzierska et al. 2003) and are essential for the chaperone activity (Mogk et al. 2003). Studies including deletion of different segments of the heptad repeats have demonstrated that the complete middle region is essential for the self-association of ClpB (Kedzierska et al. 2003). In ClpA proteins of prokaryotes, which have a short middle region (Gottesman et al. 1990, Squires & Squires 1992, Schirmer et al. 1996), structural analysis demonstrated that their internal stabilization is favored by the interaction with ClpP (Guo

et al. 2002). In the sequence of PbClpA and in the described fungi ClpA, the ClpP-interacting motif signature (LIV-G-FL) is absent, suggesting that ClpA stabilization in fungi could be performed by the middle region as described for ClpBs (Kedzierska et al. 2003, Mogk et al. 2003). The alignment between the middle sequences from PbClpA and the single fungi ClpB described until now (ClpB of P. brasiliensis) is shown in the Fig. 2B. The region which represents the heptad repeats was brought out. The alignment demonstrates the equivalent sizes of the middle regions between the ClpA and the ClpB of P. brasiliensis (Fig. 2B). The size of the middle regions has been described as a distinguishable characteristic between the sub-families ClpA and B. As shown in Fig. 2B the Clp A and B of P. brasiliensis display similar sizes at the middle region (166 and 156 amino acid residues, respectively). We also performed a comparative analysis of ClpAs and ClpBs of fungi and bacteria available into NCBI database. Fig. 3 presents a schematic diagram of ClpAs from C. albicans, S. cerevisiae, P. brasiliensis, N. crassa,

J. C. Oliveira and others Schizosaccharomycs pombe, and Pleurotus sajor-caju and of some bacteria. The middle regions of the fungal Clps presented similar sizes in A and B families. The ClpB and ClpA middle regions of bacteria were of different sizes, as shown in Fig. 3. Domains NBD1 and NBD2 are separated by a short middle region in prokaryotes ClpA proteins. This characteristic is used to separate the subfamilies of ClpATPases in prokaryotes (Gottesman et al. 1990, Squires & Squires 1992, Schirmer et al. 1996). Comparative analysis performed in this study suggested that these criteria might not be useful when considering ClpA proteins in fungi. The characteristics and sizes of the middle regions were very similar between fungal ClpAs and ClpB and all presented significant difference with ClpA proteins described to prokaryotes. Phylogenetic relationships The Pfam and NCBI databases were used to search for complete protein sequences of ClpAs. Forty-seven ClpA complete sequences were identified of both bacteria and fungal origin. The single plant ClpA of Brassica napus (GenBank accession no. CAA53077) was not considered in this analysis. The program described by Thompson et al. (1997), which employs a multiple sequence alignment method, was used to construct the phylogenetic tree (Fig. 4). The ClpAs of fungi formed a distinct cluster. The phylum Ascomycota presented 100% bootstrap confidence levels at branches and is distributed in four sub-clades. The subphyla Pezizomycotina (Paracoccidioides brasiliensis and Neurospora crassa), Saccharomycotina (Candida albicans and Saccharomyces cerevisiae) and Schizosaccharomycetes (Schizosaccharomyces pombe) were grouped separately. The only Basidiomycota, Pleurotus sajor-caju, presented 77 % of similarity and occupied a derived position inside the fungal clade. A phylogenetic tree using only the middle regions was performed using the same method and programs above (Fig. 5). Identical organization of the fungal subclades was obtained as noted in Fig. 4. The same clustering of fungal ClpAs pattern was observed in the phylogenetic trees using the complete sequences and the middle regions. In this way the complete sequences of ClpAs as well as the middle regions can be useful as a tool in the determination of phylogenetic relationships in fungi. The function performed by the ClpA in P. brasiliensis remains to be elucidated. The Hsp104 of Saccharomyces cerevisiae plays a critical role in induced thermotolerance and is very strongly induced by heat, playing a vital role in helping cells survive exposures to high temperatures (Lindquist & Kim 1996). A similar role could be proposed to the PbClpA during the temperature upshift that characterizes the infective process by P. brasiliensis ; further studies need to focus on this matter.

715 ACKNOWLEDGEMENTS This work at the Universidade Federal de Goia´s was supported by grants from Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico (CNPq).

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Corresponding Editor: M. Ramsdale