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Molecular cloning and characterization of a cDNA encoding the N-acetyl-b-D-glucosaminidase homologue of Paracoccidioides brasiliensis
NICA O. SANTOS*, MARISTELA PEREIRA*, MARIA SUELI S. FELIPE%, ROSALIA SANTOS A. JESUINO*, MO CIRANO J. ULHOA$, RENATA DE BASTOS A. SOARES* & CELIA MARIA DE A. SOARES* ncias Biolo rio de Biologia Molecular and $Laborato rio de Enzimologia, Instituto de Cie gicas, Universidade Federal *Laborato nia, Goia de Goia s, Goia s and %Laborato rio de Biologia Molecular, Universidade de Bras lia, Bras lia, Brazil

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A cDNA encoding the N-acetyl-b-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis , Pb NAG1, was cloned and characterized. The 2663nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with classic NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum , Aspergillus nidulans and Candida albicans . In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158. Keywords cell wall, human pathogen, N-acetyl-b-D-glucosaminidase, Paracoccidioides brasiliensis

Introduction
Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America [1]. The disease may occur as multiple clinical forms and is a major medical problem in the region, where it has been estimated that there are 10 million infected individuals [2]. The fungus shows thermal dimorphism. Inhaled airborne propagules derived from the mycelial phase

Received 24 September 2002; Final revision received 6 August 2003; Accepted 15 January 2003 Correspondence: Dr Celia Maria de Almeida Soares, Laborato rio de Biologia Molecular, Instituto de Cie ncias Biolo gicas, ICBII, Campus II, 74001-970, Goia nia, Goia s, Brazil. Tel/Fax: '/55 62 521 1110; E-mail: celia@icb2.ufg.br

are converted in the lung to the yeast form, thus establishing the infection [3]. The cell wall is a highly dynamic fungal structure. It is involved in the maintenance of cellular morphology and consequently in the dimorphic process, and also functions in matters such as the osmotic protection of the cell and the modulation of immunological responses against infection [4,5]. It forms an interface between pathogen and host and is believed to play a role in virulence [6,7]. The polysaccharide chitin is a major structural component of most fungal cell walls [4]. N-acetyl-b-D-glucosaminidase (NAG) (EC 3.2.1.30) is defined as a glycosyl hydrolase enzyme that, in concerted action with chitinase (EC 3.2.1.14), promotes efficient degradation of chitin by microorganisms [8,9]. Protein family 20 contains N-acetylglucosaminidases and chitobiases (EC.3.2.1.52) [10] that
DOI: 10.1080/13693780310001644671

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are thought to use an acid /base mechanism of catalysis involving a proton donor and a nucleophile [11]. Although chitinolitic enzymes have been detected and a number of chitinase genes have been isolated from a wide variety of organisms, up-to-date examples of cloning of the NAG genes are few. In particular, with regard to human pathogenic fungi, the cloning and characterization of NAG encoding genes has been reported in Candida albicans [12], Penicillium chrysogenum [5] and Aspergillus nidulans [13]. In P. brasiliensis the molecular architecture and the functional components of the cell wall differ between the yeast and mycelial growth forms [14]. Chitin is found in higher amounts in the yeast phase than in the mycelial phase. NAG may be involved in determining such differences, and thus may perform several roles during growth and differentiation of P. brasiliensis . Our group has been performing studies aimed at the isolation and characterization of genes and their products involved in cell wall metabolism in P. brasiliensis . We had previously described the cloning and characterization of the P. brasiliensis glucan synthase gene Pb FSK1 [15]. Also, the cloning and sequencing of a chitin synthase gene has been reported by Nin o-Vega et al. [16]. Here, we report the molecular cloning and characterization of a cDNA named Pb nag1, encoding a NAG from P. brasiliensis isolate Pb 01. This novel enzyme for this fungus is assigned to glycosyl hydrolase family 20 on the basis of cDNA sequence comparison and on the basis of comparison of the amino acid sequences of the catalytic domains [17,18]. This constitutes the first description of a gene or cDNA encoding an enzyme belonging to the chitinoltic system of P. brasiliensis.

modifications. The cell powder was added to 10 ml extraction buffer (2% w/v polyvinylpolypyrrolidone (PVP), 1.4 mol/l NaCl, 0.1 mol/l Tris HCl pH 8.0, 0.02 mol/l ethylenediamine tetraacetic acid (EDTA), 2% w/v CTAB. The mixture was incubated at 658C for 1 h, extracted with 50% chloroform/50% isoamyl alcohol [v/v] and precipitated with 100% ethanol. After treatment with RNASEI and ethanol precipitation, the DNA was resuspended in water.

Cloning of P. brasiliensis NAG homologue

Based on the alignment of coding regions of NAG sequences with those of Trichoderma harzianum [21] and C. albicans HEX1 [12], two oligonucleotides, one sense (5? GAYTCNCARTCNTGGCC 3?) and one antisense (5? CCRCARTCNAGRTA 3?), were designed and employed in PCR amplification of genomic DNA of P. brasiliensis . A yeast cDNA library, constructed in Eco RI and Xho I sites of Lambda ZAP II (Stratagene, La Jolla, CA, USA) was screened with a probe consisting of the 666 bp genomic fragment encoding the P. brasiliensis NAG homologue. Positive cDNA clones were plaque purified following three rounds of screening and subcloned by in vivo excision [22]. The nucleotide sequence for both strands was determined by the dideoxy-chain termination method according to Sanger et al . [23].

DNA sequence analyses

Sequence analyses were carried out with the Genetics Computer Group (GCG) package (Madison, WI, USA) [24] and PROSITE database [25]. Homology searches were conducted with the BLAST algorithm [26].

Materials and methods

Microorganism and growth conditions
P. brasiliensis isolate Pb 01 (ATCC MYA 826) is a clinical isolate. The fungus was grown in semi-solid medium as previously described [19]. The yeast cells were grown at 368C and subcultured every 7 days.

Protein homology and inferred phylogeny

The phylogenetic relationships of Pb NAG1 were studied using 21 NAG sequences available on the continuously updated carbohydrate-active enzymes (CAZy) web server (http://afmb.cnrs-mrs.fr/ /cazy/ CAZY/index.html) [27]. The entire amino acid sequences were compared using Tree View software [28]. The alignment was generated with Clustal V software [29].

Genomic DNA isolation

P. brasiliensis yeast cells were harvested after seven days of culturing at 368C, and were then washed and frozen in liquid nitrogen. The cells were broken by grinding with a mortar and pestle. The genomic DNA was prepared by the cationic hexadecyl trimethyl ammonium bromide (CTAB) method [20], with minor

Accession numbers
The genomic partial fragment encoding NAG and the entire cDNA were deposited in the GenBank database
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under accession numbers AF395815 and AF419158, respectively.

Comparison of the amino acid sequence of the cloned Pb NAG1 with related sequences
When BLAST alignments [26] of the translated Pb NAG1 were carried out, the full-length protein was shown to have strong sequence similarity to multiple NAG. The amino acid sequence of the enzyme showed significant homology with other NAG belonging to family 20, as described [32]. Fig. 2 shows Pb NAG1 and related proteins belonging to glycosyl hydrolase family 20 aligned over the region between Pb NAG1 amino acids 340 and 378. Of special note is the presence in Pb NAG1 of the canonical motif G X D E at the catalytic domain of family 20 [32]. The amino acids D and E at positions 375 and 376, in Pb NAG1 are the putative catalytic residues as ascribed to other family 20 members [33]. Paracoccidioides brasiliensis NAG homology and inferred phylogeny Amino acid sequences coding for members of glycosyl hydrolase family 20 in bacteria and in eukaryotes, including fungi and mammals, were aligned and a phylogenetic tree was constructed (Fig. 3). Bacterial and eukaryotic clades were well resolved. As expected, P. brasiliensis consistently clustered with other fungi. For example, Pb NAG1 and the sequence from T. harzianum showed an identity value of 45% [34]. Similarity scores with non-fungal organisms varied widely [33,35].

Results
Isolation and sequence analysis of the Pb nag1 cDNA
An alignment of amino acid sequences deduced for the NAG genes from T. harzianum (accession number GenBank S72211) and C. albicans (accession number GenBank P43077) was used to design primers that could be used to amplify comparable P. brasiliensis sequences. A P. brasiliensis genomic fragment of 666 bp was obtained and was utilized in the screening of the P. brasiliensis cDNA library. Four independent clones were isolated after three cycles of selection and were then submitted to in-vivo excision with pBluescript SK- (Stratagene). After partial sequencing it was confirmed that all the clones were identical. One clone was chosen for further analysis. It was sequenced for both strands. The analysis is shown in Fig. 1. The cloned Pb nag1 is 2663 nucleotides in length and has a 720-bp untranslated region (UTR) at the 5? end. The ATG at base 721 encoding the presumed initiating methionine was situated in the context of a consensus translation start codon [30]. The cDNA included a 209bp 3? UTR exclusive of the poly-A tail. The 2663-bp cDNA contained a single open reading frame (ORF) encoding a predicted protein of 64.73 kDa, pI 6.35. The correspondingly inferred protein contained the conserved amino acid sequence DSQSWP at positions 253 to 258 and another conserved sequence, YLDCG, at positions 473 to 477. We based the construction of our oligonucleotides on these regions. The predicted protein was 577 amino acids in length and contained a putative signal sequence, extending between amino acids 1 and 30, with a potential signal peptidase cleavage site between residues 17 and 18 [31]. Cleavage at this site would result in a mature protein with a calculated molecular mass of 62.97 kDa, pI 6.46. Also present were six potential N-glycosylation sites, N X S/ T X, at positions 29, 78, 117, 283, 343 and 450 in the deduced amino acid sequence. There are several regions related to the presence of residues mostly conserved in the family 20 of glycosyl hydrolases (Fig. 1, underlined). The amino acids possibly related to the active site are in positions 207 to 543 in the deduced protein. A search of the PROSITE database [25], showed the presence of a SGPG motif, potentially related to ligation to the glycosaminoglycan substrate at positions 194 to 197. The cDNA and the amino acid sequences were deposited in GenBank (accession number AF419158).
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Discussion
The putative catalytic domain of Pb NAG1 is conserved and is identical to those found in related sequences of T. harzianum [34], C. albicans [12] and U. maydis [36]. The enzyme as a whole has six potential N-glycosylation sites, suggesting a highly glycosylated protein. Other NAG have been described as extensively glycosylated molecules. For example, the A and B enzymes of C. albicans were described as isoforms with carbohydrate contents of 32 and 56%, respectively [37]. The deduced amino acid sequences of NAG from C. albicans and Caenorhabditis elegans contained, respectively, seven and four potential N-glycosylation sites [12,38]. The deduced NAG1 of P. brasiliensis includes 30 amino acid residues that comprise a putative signal peptide, suggesting that the protein is probably addressed to the cell wall of P. brasiliensis . A NAG from C. albicans has previously been shown to be deposited in the region of the cell wall [39] of yeast and mycelial cells.

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Fig. 1 The sequence of the cDNA encoding NAG1 of Paracoccidioides brasiliensis . Nucleotides are numbered in bold (left margin). Nucleotides 5? to the initiation codon are indicated by negative numbers. The putative start and stop codons are marked by asterisks. A possible polyadenylation signal at 3? end underlined with dots. The amino acid sequence is shown above the nucleotide sequence (single letter code). The putative signal peptide is double underlined. The putative peptidase signal cleavage site is marked by an arrow. The conserved amino acid residues related to the putative catalytic site are marked by a key. The most conserved amino acid residues in the catalytic site are marked by a dot. The most strongly conserved motifs related to glycosyl hydrolase family 20 are underlined. The putative glycosylation sites are boxed with a dashed line. The sequences of the region used in the design of the oligonucleotides are shown in bold italics. The SGPG motif is boxed.

We used the multiple sequence alignment of known NAG to construct an unrooted neighbour-joining tree. The P. brasiliensis NAG was clearly clustered with

fungal NAG. It is important to note that the database searched included NAG among several eukaryotic and prokaryotic protein sequences, and yet the NAG from
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Fig. 2 Comparison of the Paracoccidioides brasiliensis Pb NAG1 and sequences of other representatives of glycosyl hydrolase family 20. Alignment was performed using the Clustal V program. Black boxes indicate identical residues and grey indicates conserved amino acids. The putative glycosyl hydrolase family 20 catalytic domain is marked by a key. The D and E residues included in the putative catalytic region are marked by arrows. GenBank accession numbers of glycosyl hydrolases used in this analysis: P. brasiliensis (AF419158); Trichoderma harzianum (S80070); Aspergillus nidulans (AB039846); Penicillium chrysogenum (AAF00009.1); Candida albicans (L26488); Homo sapiens (M13519); Felis catus (S70340); Mus musculus (U07633); Sus scrofa (X92379); Vibrio harveyi (J05004); Serratia marcescens (L43594); Enterobacter sp. (AB022786); Alteromonas sp. (AB067646); Aeromonas hydrophila (AF196349); Aeromonas sp. (AB031330); Streptomyces olivaceoviridis (AJ310133); Streptomyces coelicolor (AL163641); Arthrobacter sp . (ASP250587); Vibrio furnissii (U41417); Agrobacterium tumefaciens (AE008173) and Ustilago maydis (AL603945).

Fig. 3 Phylogenetic analysis of Pb NAG1. Phylogenetic relationships were based on amino acid sequences of family 20 glycosyl hydrolases. The tree was calculated using the neighbourjoining option in the Clustal V program, and was drawn with TreeView software. Horizontal branch lengths are proportional to genetic distance. Downloaded sequences included are: Ustilago maydis ; P. brasiliensis ; Trichoderma harzianum ; Candida albicans ; Aspergillus nidulans ; Penicillium chrysogenum ; Sus scrofa ; Mus musculus ; Homo sapiens ; Felis catus ; Agrobacterium tumefaciens ; Vibrio furnissii ; Aeromonas sp.; Alteromonas sp.; Aeromonas hydrophila ; Vibrio harveyi ; Serratia marcescens ; Enterobacter sp.; Arthrobacter sp.; Streptomyces olivaceoviridis and Streptomyces coelicolor . The GenBank accession numbers are as in Fig. 2.

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fungi consistently clustered together. The bacterial and mammalian NAG were also clustered independently. As NAG are functional molecules, these data could in part reflect specialized functions of the NAG found in these organisms. The physiological role of Pb NAG1, although not investigated in this work, may be similar to that of other NAG. The P. brasiliensis cell wall contains different levels of chitin in yeasts and mycelium [14]. Because chitin levels decrease during the transition from yeast to mycelium it could be suggested that NAG are involved in the cell wall remodelling that occurs. The putative signal sequence for addressing Pb NAG1 to the cell surface may corroborate this suggestion. In C. albicans , NAG secretion occurs preferentially during hypha formation [39]. Also, in A . niger the degree of hyphal branching is related to the level of NAG activity [40]. The chitinolytic enzymes have been described as potential immunogens in fungi. The chitinase of the pathogenic fungus Coccidioides immitis is a major secreted antigen [41]. The NAG of P. chrysogenum has been described as an allergen [5]. In our own studies, we have detected an immunological reaction to purified P. brasiliensis NAG in serum of patients infected by this fungus (data not shown). The cloning and characterization of the present cDNA will allow the production of the recombinant NAG in order to investigate the role of NAG in the host response to P. brasiliensis . Finally, the mechanisms underlying alterations in cell wall constituents and architecture during growth and dimorphism of P. brasiliensis constitute a potential target for the development of new antifungal drugs. The characterization of the NAG1 encoding cDNA will facilitate the production of the recombinant protein in studies aimed at exploring potential new drugs.

Acknowledgements
The work at Laboratorio de Biologia Molecular, Universidade Federal de Goia s, was supported by grants form CNPq (520679/99-7) and FUNAPE-UFG.

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