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Pbhyd1 and Pbhyd2: two mycelium-specific hydrophobin genes from the dimorphic fungus Paracoccidioides brasiliensis
Fungal Genetics and Biology 41 (2004) 510–520 www.elsevier.com/locate/yfgbi
Pbhyd1 and Pbhyd2: two mycelium-specific hydrophobin genes from the dimorphic fungus Paracoccidioides brasiliensis P. Albuquerque, C.M. Kyaw, R.R. Saldanha, M.M. Brigido, M.S.S. Felipe, and I. Silva-Pereira* Laboratorio de Biologia Molecular, CEL/IB, Universidade de Brasılia, Brasılia-DF, 70910-900, Brazil Received 12 October 2003; accepted 1 January 2004
Abstract Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 °C and as yeasts (Y) at 37 °C, or after the invasion of host tissues. Although the dimorphic transition in P. brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood. Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P. brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2). Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence. These two hydrophobin genes are present as single copy in P. brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Hydrophobin; Paracoccidioides brasiliensis; Dimorphic fungus; Differentially expressed genes; DDRT-PCR; 50 RACE; RT-PCR
1. Introduction Paracoccidioides brasiliensis is a dimorphic fungus, which causes paracoccidioidomycosis (PCM), an endemic human disease (San-Blas and Ni~ no-Vega, 2001). Geographically, it is confined to Latin America, with its areas of endemicity extending from Central America to Argentina, representing one of the most prevalent deep mycoses in this region (Restrepo, 1985). Over 10 million people are estimated to be infected with P. brasiliensis, but only up to 2% develop the disease (McEwen et al., 1995). This fungus, previously considered a member of the phylum Deuteromycota, since sexual structures were not found, was recently classified as belonging to the phylum Ascomycota, order Onygenales, family Onygenaceae (San-Blas et al., 2002). Although known for several years, the ecology of this microorganism still remains unclear, especially with respect to its environ-
* Corresponding author. Fax: +55-61-349-8411. E-mail address: [email protected] (I. Silva-Pereira).
1087-1845/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.01.001
mental niche (McEwen et al., 1995; Restrepo et al., 2001). This fungus is found as mycelia (M) at about 26 °C and as yeasts (Y) at 37 °C. Mycelia and/or conidia are supposed to be the infective form found in nature, probably in the soil and/or plants, while yeasts are the form usually found in infected tissues. The mycelium to yeast transition, triggered by the temperature shift, is easily reproduced in vitro and seems to be essential to the establishment of the infective process (San-Blas et al., 2002). Regardless the importance of dimorphic transition, the biochemical events regulating this process in P. brasiliensis are poorly defined and several important questions remain unanswered. Although the temperature shift, the cAMP (Paris and Duran, 1985), and Ca2þ / calmodulin (Carvalho et al., 2003) pathways seem to be important in the control of transition, the signaling cascades and regulating factors involved in differential gene expression and subsequent morphological changes of this fungus, are still a puzzle. One approach that allows a better understanding of the molecular
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mechanisms involved in dimorphism corresponds to the characterization of genes differentially expressed during the fungal transition (San-Blas et al., 2002). It has been shown that several genes related to cell wall structure, metabolism, and signaling cascades undergo significant changes in their expression profile, probably playing an important role in P. brasiliensis dimorphism. Among the several approaches currently employed to characterize differentially expressed genes, by using differential display reverse transcriptase PCR (DDRTPCR, Liang and Pardee, 1992; Liang et al., 1993) and Northern blot analysis, we were able to detect several phase-specific cDNA fragments corresponding to genes differentially expressed in one of the two forms of P. brasiliensis (Venancio et al., 2002). Here, we report the isolation and characterization of the mycelium-specific full-length Pbhyd1 cDNA, previously named M73 cDNA (Venancio et al., 2002). The predicted protein sequence is composed of 100 amino acid residues, showing similarity with hydrophobins, a group of small hydrophobic proteins of filamentous fungi (Wessels, 1999). Hydrophobins are small secreted proteins, of about 100 amino acid residues, which, although do not share significant sequence similarity, are all characterized by the presence of eight cysteine residues in conserved positions, and a typical hydropathy profile (Wessels, 1997). These proteins are secreted as monomers that self-assemble into an amphipathic film at hydrophilic/hydrophobic interfaces covering fungal structures (W€ osten and de Vocht, 2000; W€ osten et al., 1994). For the last two years, our group has been developing the ‘‘P. brasiliensis Functional and Differential Genome Project—Brazilian Middle-West Network’’ (https:// www.biomol.unb.br/Pb), in order to map the yeast and mycelium transcriptomes of this microorganism, as described by Felipe et al. (2003). By this approach, a second putative hydrophobin sequence, named Pbhyd2 (this work), was identified. Herein, two P. brasiliensis hydrophobin cDNAs were characterized and shown to be specifically expressed in the mycelium phase of this fungus.
2. Materials and methods 2.1. Paracoccidioides brasiliensis growth conditions and nucleic acid extraction The clinical isolate of P. brasiliensis, strain Pb01 (ATCC-MYA-826), was grown on semi-solid FavaNetoÕs medium as mycelium (M), at 26 °C; yeast (Y), at 36 °C; and in the M to Y transition experiments, as previously described (Silva et al., 1994). Genomic DNA from P. brasiliensis was obtained as described by Bainbridge et al. (1990). Total RNA was
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extracted using Trizol reagent (Gibco-BRL), according to suppliersÕ recommendations, as previously described (Silva et al., 1999; Venancio et al., 2002). To remove any genomic DNA contamination, RNA was precipitated twice with LiCl, followed by RNase-free-DNaseI treatment (Venancio et al., 2002). 2.2. Identification and isolation of full-length Pbhyd1 and Pbhyd2 cDNAs After DDRT-PCR and Northern blot assays confirming the differentially expressed nature of M73 cDNA fragment (Venancio et al., 2002), 50 RACE (rapid amplification of cDNA ends) and RT-PCR experiments were performed, in other to obtain the full-length Pbhyd1 cDNA. A 50 RACE experiment, using DNaseI treated mycelium total RNA, was performed to isolate the corresponding 50 end missing region of this cDNA. The oligonucleotides GSP1 (50 -GGAAACAGGATCGAGAA GTCCCACC-30 ) and GSP2 (50 -CCACCTCCTCATCC ATTAACTCTCCGC-30 ) were designed to prime the cDNA synthesis and to perform PCR, respectively, according to the instructions of the suppliers of the ‘‘50 RACE, Version 2.0’’ (Gibco-BRL). After purification and cloning, a DNA fragment of approximately 750 bp, named 50 cDNA M73, was sequenced (data not shown). The primers 50 Hyd (50 -ATCATCAACAAGCATCAGT AC-30 ) and 30 Hyd (50 -CTAAAGGAAAGTTAAGAA GC-30 ) used in the RT-PCR experiment were synthesized based on the nucleotide sequence of the two cDNA fragments obtained from DDRT-PCR (30 cDNA M73, Venancio et al., 2002) and 50 RACE (50 cDNA M73). Following denaturation (80 °C/5 min), 1 lg of mycelial total RNA was used in cDNA synthesis, at 37 °C/1 h, in a 25 ll reaction consisting of: 0.5 mM of each dNTP, 0.2 mM of primer 30 Hyd, 200 U of Reverse Transcriptase (RT, Superscript II, Gibco-BRL), 5 mM of DTT, and 1 RT-buffer. Four ll of the cDNA first strand were amplified in a final volume of 25 ll (2 U of Taq DNA polymerase (Cenbiot-RS/Brazil), 0.2 mM of each dNTP, 0.4 lM of each primer (30 Hyd and 50 Hyd) and 1 PCRbuffer containing 1.5 mM MgCl2 ). In order to isolate the genomic sequence corresponding to Pbhyd1 cDNA, a PCR using 20 ng of P. brasiliensis genomic DNA as template were done (data not shown). The amplified DNA fragment was cloned into pGEM-T easy vector (Promega). Pbhyd2 cDNA was identified by the EST genome project, as described by Felipe et al. (2003). 2.3. Northern blot analysis About 15 lg of total RNA from Y and M cells of P. brasiliensis, and from M cells undergoing the dimorphic transition for 0.5, 1, 2, 6, 12, and 24 h, after a temperature shift from 26 to 37 °C, were run on denaturing 1% agarose gel and blotted onto a Hybond Nþ membrane
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(Amersham Biosciences), according to standard protocols (Sambrook and Russell, 2001). The Pbhyd1 genomic DNA fragment and Pbhyd2 cDNA were labeled using [a-32 P]dATP by random priming procedure (MegaPrime DNA labeling System, Amersham Biosciences), purified and used in overnight hybridization, at 42 °C (Silva-Pereira et al., 1992; Venancio et al., 2002). The membranes were then washed to a final stringency of 0.1 SSPE-0.1% SDS at 65 °C for 30 min and exposed either to a X-ray film at )80 °C for 48 h or to a Phosphor Screen (Molecular Dynamics, Amersham Biosciences) for 2 h. 2.4. Southern blot analysis About 10 lg of P. brasiliensis genomic DNA were digested with BamHI, BglII, HindIII, and PstI restriction endonucleases, separated on 1% agarose gel, and transferred to a Hybond Nþ membrane, as specified by the manufacturer (Amersham Biosciences). Radioactive probes, hybridization, and washing conditions were performed as described for the Northern blot experiment. 2.5. Production of the hydrophobin PbHYD1 in Escherichia coli To produce the hydrophobin PbHYD1, we amplified the corresponding ORF from the plasmid containing the Pbhyd1 cDNA, using 2 U of Taq DNA polymerase (Cenbiot-RS/Brazil) and the primers BamATG (50 -CG GGATCCATGCAGCTCTCCAACATCGTC-30 ) and TAAXho (50 -CCGCTCGAGGTCTCGATTGGGCAT TTAC-30 ). The PCR-amplified Pbhyd1-ORF was cloned into pGEM-T easy vector (Promega). The BamHI and XhoI insert was released by double digestion and cloned into the expression vector pGEX-4T-3 (Amersham Biosciences). The resulting vector, containing the Pbhyd1-ORF fused to the 26 kDa domain of glutathione S-transferase (GST), was transformed into E. coli Ori gami B pLacI (F ompT hsdSB (r B mB ) gal dcm lacY1 r aphC gor522::Tn10 (Tc ) trxB::kan (DE3) pLacI (Cmr )Novagen) (Bessette et al., 1999; Stewart et al., 1998). In order to obtain the fusion protein (GST-Pbhyd1ORF), its expression was induced by adding isopropyl-b-D -thiogalactopyranoside (IPTG; Sigma) to a final concentration of 1 mM to an early-exponential-phase culture of the recombinant E. coli Origami B cells. After 3 h of induction, bacterial cells were harvested by centrifugation and washed with PBS (140 mM NaCl; 2.7 mM KCl; 10 mM Na2 HPO4 ; and 1.8 mM KH2 PO4 , pH 7.3). Cell suspensions were sonicated, centrifuged, and the resulting supernatants were further purified by affinity chromatography using Glutathione–Sepharose 4B (Amersham Biosciences), according to supplierÕs instructions. The recombinant protein was eluted with glutathione and analyzed by SDS–PAGE (Sambrook and Russell, 2001).
2.6. SDS–PAGE and Western blot analysis SDS–PAGE was performed using 15% acrylamide gels according to standard protocols. Prior to electrophoresis, all samples were dried, dissolved in sample loading buffer, and heat denatured (100 °C/5 min). Proteins were visualized by staining with Coomassie brilliant blue R-250. After SDS–PAGE, proteins were transferred to a nitrocellulose membrane (Amersham Biosciences), saturated with 3% BSA and incubated for 1 h with an antiGST antibody conjugated to alcaline phosphatase. Following incubation, bound antibody was detected by using alkaline phosphatase colorimetric detection (Sambrook and Russell, 2001). 2.7. Hydropathy profile of PbHYD1 and PbHYD2 Hydrophobicity plots were generated by DNA Strider 1.2, based on the Kyte–Doolittle hydrophatic table. 2.8. Phylogenetic analysis PbHYD1 and PbHYD2 sequences, as well as related hydrophobins from other fungi, deposited at GenBank, were aligned with ClustalW (Higgins et al., 1996). Multisequence alignment was phylogenetically analyzed using neighbor-joining trees, constructed as described by Saitou and Nei (1987). 2.9. Sequences accession numbers The Pbhyd1 and Pbhyd2 accession numbers are AF526275 and AY427793, respectively. The accession number of related hydrophobins from other fungi are: Mpg1 (AAA20128.1, Magnaporthe grisea); Ccg2 (CAA47754.1, Neurospora crassa); RodA (AAA33321.1, Aspergillus nidulans); Hyp1 (AAB60712.1, Aspergillus fumigatus); DewA (AAC13762.1, Emericella nidulans); SsgA (AAA33418.1, Metarhizium anisopliae); XEH1 (CAC86096.1, Xanthoria ectaneoides); XEP1 (CAC8 6092.1, Xanthoria parietina); HCF1 (CAC27408.1, Cladosporium fulvum); HCF2 (CAD97454.1, C. fulvum); HCF3 (CAB39310.1, C. fulvum); HCF4 (CAB39311.1, C. fulvum); and EAS (AAB24462, N. crassa).
3. Results 3.1. Isolation and characterization of Pbhyd1 and Pbhyd2 cDNAs Using DDRT-PCR we have isolated several cDNA fragments, differentially expressed in M or Y cells of P. brasiliensis (Venancio et al., 2002). Among them, we have found a 281 bp cDNA fragment (M73.4) that was
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cloned and sequenced. Northern blot analysis confirmed that M73.4 was specific for the mycelial phase of this fungus, corresponding to an mRNA of about 1100 nucleotides. To obtain the 50 missing region of this cDNA, we have employed the 50 RACE methodology, which generated a 772 bp cDNA fragment (50 M73) showing the expected overlapping sequence with the 30 M73 cDNA previously obtained by DDRT-PCR (Venancio et al., 2002). Subsequently, in order to obtain the M73 full-
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length cDNA by RT-PCR, we designed primers corresponding to the 50 and 30 ends of 50 M73 and M73.4 nucleotide sequences, respectively. A 968 bp cDNA fragment was amplified and sequenced, confirming the isolation of the complete M73 cDNA (Fig. 1A), as predicted by previous Northern blot results. Using P. brasiliensis total genomic DNA as template, and the same oligonucleotides used in RT-PCR experiment, we have also amplified a DNA fragment slightly larger, suggesting the presence of an intron in the genomic M73
Fig. 1. Nucleotide and deduced amino acid sequences of Pbhyd1 (A) and Pbhyd2 (B) genes. The Pbhyd1 and Pbhyd2 coding sequences (Accession numbers AF526275 and AY427793, respectively) are shown in uppercase letters, and the deduced amino acid sequences are shown below the nucleotide sequences. The 50 UTR and 30 UTR are indicated by lowercase letters and the intron sequence in Pbhyd1 gene is underlined. Primers used in RT-PCR are indicated by arrows. Cysteine residues are displayed in black boxes.
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sequence. Sequence comparisons of M73 cDNA and its corresponding genomic DNA fragment, confirmed the presence of a small intron of 79 bp, located within the M73 coding region. The intron nucleotide sequence displays the splicing site consensus (50 GT-AG 30 ) and the canonical internal sequence required for lariat formation (Mount, 1982). A detailed analysis of the predicted M73 amino acids sequence indicated features common to the hydrophobins family (Wessels, 1997). Noteworthy, the presence of eight cysteine residues with their characteristic spacing (Fig. 1, panel A, black boxes), as well as the typical hydropathy profile (Fig. 2B), which constitute the signature of this group of proteins. Therefore, the corresponding gene was designated Pbhyd1 (P. brasiliensis
hydrophobin 1). The deduced amino acid sequence of PbHYD1 exhibits a strongly hydrophobic putative signal sequence of 19 amino acids in the N-terminus (Fig. 2B), which is characteristically observed in other hydrophobins (Wessels, 1997; W€ osten, 2001). Consequently, the mature PbHYD1 should have 81 amino acid residues, with a predicted molecular mass of about 8 kDa. The P. brasiliensis Functional and Differential Genome Project (Brazilian Middle-West Network), based on the generation of gene expressed sequence tags (ESTs) from mycelium and yeast cDNA libraries of P. brasiliensis (Felipe et al., 2003), revealed 56 ESTs similar to Pbhyd1 cDNA. These data suggest that Pbhyd1 mRNA is highly abundant, as confirmed by the
Fig. 2. Major characteristics observed in PbHYD1 and PbHYD2 deduced amino acid sequences. (A) Schematic representation of the putative PbHYD1 and PbHYD2 primary sequences, based on disulfide bonds determined for the hydrophobin cerato-ulmin (Yaguchi et al., 1993). Hydrophilic and hydrophobic amino acid residues are shown in black and in gray, respectively. The eight cysteine residues are shown in red. (B) Hydropathy profile of P. brasiliensis PbHYD1 and PbHYD2 deduced amino acid sequences. The hydropathy plots were generated using the Kite and Doolittle algorithm (window of six amino acids), by the program DNA Strider 1.2 (Marck, 1988). The +/) values in the ordinate axis indicate the hydrophobic/hydrophilic nature of the amino acid residues, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
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Northern blot results (Fig. 5B). Further analyses of the P. brasiliensis EST project have revealed a second hydrophobin, represented by 9 ESTs, that was named Pbhyd2. A similar nucleotide sequence was also identified by Goldman et al. (2003). The nucleotide and the deduced amino acid sequences of Pbhyd2 are depicted in Fig. 1 (panel B, cysteine residues are shown in black boxes). The deduced protein sequence, of 133 amino acid residues, contains a putative signal sequence of 18 amino acid residues at its amino-terminal end (Fig. 2B). The resulting mature protein is composed of 115 amino acid residues, with a predicted molecular mass of about 11 kDa. The amino acid sequence similarities between PbHYD1 and PbHYD2 are as low as 18.6%, a value usually observed for different hydrophobins produced in a single organism (W€ osten, 2001). A schematic representation of the primary structure of PbHYD1 and PbHYD2, and their corresponding hydropathy plots, are represented in Fig. 2. The cysteine spacing of both P. brasiliensis predicted hydrophobins is almost identical to the class I hydrophobins consensus, as proposed by Wessels (1997). The PbHYD1 and PbHYD2 deduced amino acid sequences, and related hydrophobins from other filamentous fungi deposited at GenBank, were aligned with ClustalW (Higgins et al., 1996). The multi-sequence alignment analysis of the two P. brasiliensis hydrophobins clearly positioned PbHYD1 and PbHYD2 in two separate clusters (Fig. 3). The results reveal that PbHYD1 is more related to EAS (ccg-2) hydrophobin from N. crassa (Bell-Pedersen et al., 1996), while PbHYD2 clusterizes with RODA of A. nidulans (Stringer et al., 1991) and HYP1 from A. fumigatus (Paris et al., 2003). To determine the copy number of Pbhyd1 and Pbhyd2 genes in the P. brasiliensis genome, a Southern blot analysis was performed. The resulting hybridization profiles, depicted in Fig. 4, suggest that both genes are encoded as single copies in P. brasiliensis genome, which is in accordance to other hydrophobin genes such as fvh1 from Flammulina velutipes (Ando et al., 2001) and hyp1 from A. fumigatus (Parta et al., 1994). 3.2. Analysis of the expression levels of Pbhyd1 and Pbhyd2 genes Fig. 5 illustrates a Northern blot experiment, showing the expression of Pbhyd1 and Pbhyd2 genes, using total RNA from mycelium and yeast cells of P. brasiliensis, and from mycelium cells induced to dimorphic transition at different time intervals, after 26–37 °C temperature shift (0.5, 1, 2, 6, 12, and 24 h). The Pbhyd1 and Pbhyd2 transcripts were not detected in yeast cells, whereas high levels of these transcripts were observed in mycelium cells. Although the accumulation of both mRNAs occurs during the first 24 h after the tempera-
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Fig. 3. Phylogenetic neighbor-joining tree of PbHYD1 and PbHYD2 deduced amino acid sequences. PbHYD1 and PbHYD2 and related hydrophobins from other fungi were analyzed, as described in Section 2. The tree was confirmed by one thousand bootstrap re-sampling.
ture shift, the relative abundance of Pbhyd1 and Pbhyd2 transcripts seems to increase at 0.5 and 6 h after the temperature shift. This apparently complex profile of Pbhyd1 and Pbhyd2 is in accordance to the expression profiles described for other hydrophobins in different fungi (Bell-Pedersen et al., 1996; Kershaw and Talbot, 1998; Paris et al., 2003). 3.3. Heterologous expression of Pbhyd1 gene in E. coli To produce the PbHYD1 hydrophobin in bacterial cells, the corresponding ORF was cloned into the expression vector pGEX-4T-3 (Amersham Biosciences). The resulting vector, bearing the Pbhyd1-ORF fused to the 26 kDa domain of glutathione S-transferase (GST), was used to transform cells of E. coli Origami B, which is genetically manipulated to improve the disulfide bond formation in the cytoplasm (Bessette et al., 1999; Stewart et al., 1998). After induction of protein expression, the fusion protein (GST-HYD1) could be detected, revealing a low level of expression, as described in the literature for other hydrophobins (Scholtmeijer et al., 2001). After purification by glutathione–Sepharose
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Fig. 4. Southern blot analysis of P. brasiliensis genomic DNA, using the probes Pbhyd1 and Pbhyd2. Equal amounts of genomic DNA (10 lg) were digested with the restriction enzymes BglII, HindIII, PstI, XhoI, and BamHI, separated on 1% agarose gel stained with ethidium bromide (not shown), and blotted onto a nylon membrane (HybondN, Amersham Biosciences). Hybridization was performed using the homologous Pbhyd1 (Panel A) and Pbhyd2 (Panel B) radiolabeled probes. The kDNA/HindIII fragments used as molecular marker, are depicted on the left (in kb).
chromatography and elution using 1, 5, and 10 mM of reduced glutathione, the samples were analyzed by SDS–PAGE and Western blot (Figs. 6A and B). Fig. 6A shows the SDS–PAGE analysis of the eluted fusion protein, where a weak band with the expected size of approximately 36 kDa can be observed. It is important to notice a relative abundance of a same-size protein in the fraction corresponding to the insoluble pellet, obtained after centrifugation of the sonicated cell suspension (Fig. 6A, lane 4). Subsequently, a Western blot using antibodies raised against the GST domain of the fusion protein was performed, confirming the expression of the expected fusion protein (Fig. 6B). We can also observe that most of the fusion protein GST-HYD1 was present in the insoluble protein pellet, which was solubilized in 8 M urea (Fig. 6B, lane 4), probably confirming the highly insoluble nature described for hydrophobins in several heterologous expression systems, as well as in the fungus (Scholtmeijer et al., 2001). The heterologous expression optimization and the purification of significant amounts of the fusion protein
Fig. 5. Northern blot analysis showing the differential expression of Pbhyd1 and Pbhyd2 genes. Pbhyd1 and Pbhyd2 cDNA probes were prepared using MegaPrime labeling kit (Amersham Biosciences) and used to probe a membrane containing 15 lg of Y and M total RNA, and from M cells induced to dimorphic transition at different times after temperature shift (0.5, 1, 2, 6, 12, and 24 h). Panels A and B: Pbhyd1; Panels C and D: Pbhyd2. (A, C) Ethidium bromide stained 1.0% denaturing agarose gels, showing P. brasiliensis major and minor ribosomal RNAs, of about 3.2 and 1.6 kb, respectively. (B, D) Resulting hybridization profile, showing the mycelium-specific Pbhyd1 and Pbhyd2 mRNAs, respectively.
will be necessary in order to produce antibodies against P. brasiliensis recombinant hydrophobins, as an important reagent for later biochemical characterization of P. brasiliensis hydrophobins and immunocytolocalization of these proteins during the dimorphic process.
4. Discussion The successful establishment of P. brasiliensis in the host tissues depends on its ability to respond to the different environmental signals, which is determined by changes in the expression of specific genes involved in differentiation and adaptation. According to this scenario, we have been studying the differentially expressed genes of P. brasiliensis (Felipe et al., 2003; Venancio et al., 2002). Here, we describe the isolation and characterization of two hydrophobin genes (Pbhyd1 and Pbhyd2) from P. brasiliensis, which are expressed by the mycelial form of this microorganism. Hydrophobins are small hydrophobic proteins secreted exclusively by filamentous fungi, which contain eight cysteine residues arranged in a conserved pattern,
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Fig. 6. Heterologous expression of Pbhyd1 ORF in E. coli Origami B pLacI cells. Protein extracts of IPTG-induced recombinant E. coli were purified by affinity chromatography using Glutathione–Sepharose 4B (Amersham Biosciences). The GST-PbHYD1 fusion protein is indicated by an arrow. (A) SDS–PAGE analysis of purified GST-PbHYD1 protein. Following elution with glutathione, the purified protein was submitted to SDS–PAGE and Coomassie blue staining. (Lanes 1–3) GST-PbHYD1 protein eluted with 10, 5, and 1 mM of reduced glutathione, respectively; (lanes 4 and 5) insoluble (soluble in 8 M urea) and soluble protein fractions after cell lysis, respectively. (B) Western blot analysis of the purified GST-PbHYD1 protein. After SDS–PAGE, proteins were transferred to a nitrocellulose membrane (Amersham Biosciences) and incubated with an anti-GST antibody conjugated to alkaline phosphatase, following colorimetric detection. Lanes 1–4 as described above; lane 5 corresponds to the total extract of IPTG-induced recombinant E. coli. The molecular-mass marker (Bench Marck; Invitrogen, Life and Technologies) is indicated on the left (in kDa).
and share a typical hydropathy profile and the ability to self-assemble into amphipathic films, at hydrophilic– hydrophobic interfaces (Wessels, 1999, 2000; W€ osten, 2001; W€ osten et al., 1994). Most hydrophobins have been discovered by the analysis of mRNAs abundantly expressed during fungal development. Hydrophobins are linked to a broad range of processes, playing an important role in the formation of aerial structures (Wessels, 2000), probably involved in cell wall construction (van Wetter et al., 2000) and in the attachment of hyphae to hydrophobic surfaces—which are of strong relevance in the establishment of infection of pathogenic fungi (Ebbole, 1997; Kershaw and Talbot, 1998; Talbot et al., 1996). Based on the great relevance of hydrophobins in fungi, we believe that a careful study of these proteins would provide some new important insights concerning the molecular biology of P. brasiliensis.
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Several fungi possess multiple hydrophobin genes. Four hydrophobins were described in Schizophyllum commune (Schuren and Wessels, 1990), six in C. fulvum (Nielsen et al., 2001; Segers et al., 1999), and three in Pleurotus ostreatus (Asgeirsdottir et al., 1998). Different hydrophobins produced by the same organism usually shares very low sequence identity, not being detected by cross-hybridization (Wessels, 1997). The deduced amino acid sequences of PbHYD1 and PbHYD2 are only 18.6% identical. Despite this very low similarity between hydrophobins, they seem to assume the same general structure. In this sense, a differential regulation of these proteins or the fulfill of distinct roles in fungi have been suggested (W€ osten, 2001). The amino acid sequence of PbHYD2 (X21 -Cys-X6 Cys-Cys-X36 -Cys-X18 -Cys-X5 -Cys-Cys-X15- Cys-X6 ) matches the consensus for class I hydrophobins, as proposed by Wessels (1997). The pattern observed in PbHYD1 (X22 -Cys-X6 -Cys-Cys-X9 -Cys-X9 -Cys-X5 -Cys-Cys-X9 -CysX13 ) is almost identical to the consensus spacing of class I hydrophobins, except for the fewer number of amino acid residues between the third and fourth cysteines, than those described for both class I and class II hydrophobin consensus. Therefore, only after the isolation and characterization of their protein products we will be able to make a more precise classification of these P. brasiliensis hydrophobins. Since only few hydrophobins are yet well characterized, it cannot be discarded the finding of new hydrophobins, showing intermediate features between the two classes proposed by Wessels (1997). To obtain the protein product encoded by Pbhyd1 cDNA, its putative ORF was expressed in E. coli, using the pGEX system as described in Section 2. The production and purification of hydrophobins has already been shown to be difficult (Scholtmeijer et al., 2001). The remarkable features of hydrophobins, mainly linked to their insolubility, hidden this protein class from science until the last decade of the past century (Wessels, 1999). The high number of disulfide bonds, which are very important for the monomers solubility in water (de Vocht et al., 2000), could not be properly formed in the reducing bacterial cytoplasmic environment, which does not make bacterial systems the best choice for hydrophobin production. In spite of that, three class II hydrophobins, the P. ostreatus POH1 (Pe~ nas et al., 1998), the Ophiostoma ulmi cerato-ulmin (Bolyard and Sticklen, 1992) and the Trichoderma reesei HFBI (Nakari-S€etal€a et al., 1996) have been expressed in E. coli. Although the production levels were low (micrograms quantities per liter), limiting the use of such bacterial systems for bulk hydrophobin production, it seemed to be sufficient for antibody production. In our attempt of produce PbHYD1 in E. coli, the yield of the purified protein was low, as observed for the other hydrophobins described above, probably because most of
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the fusion protein GST-HYD1 was highly insoluble, as observed in Figs. 6A and B (lane 4). Southern blot analyses have shown that Pbhyd1 and Pbhyd2 genes are found as single copies in P. brasiliensis genome. Since the similarity among the members of these group of genes is very low and cross-hybridization can be ruled out, we cannot exclude the existence of other hydrophobins in P. brasiliensis. The P. brasiliensis hydrophobins described in this work have revealed, by Northern blot analysis, to be mycelium-specific, which is in accordance to the literature describing that hydrophobins are specifically produced only by filamentous fungi (Wessels, 1999). The increase in the Pbhyd1 and Pbhyd2 mRNA accumulation at 0.5 and 6 h after 26–36 °C temperature shifting during M to Y transition (Fig. 5, panels B and D, respectively), suggests a complex pattern of expression, as described for other hydrophobin genes (Ando et al., 2001; Asgeirsdottir et al., 1995). Of particular interest are the findings of Bell-Pedersen et al. (1996), with the N. crassa hydrophobin eas gene (or ccg2—clock controlled gene). In this work, the authors demonstrated that the ccg2 mRNA cycles with periods, showing a rhythmic accumulation pattern. In this sense, the Pbhyd1 mRNA accumulation and sequence clusterization results (Fig. 3) could be interpreted in a similar way. Different hydrophobins produced by the same organism could have their expression associated to a specific fungal structure or specific fungal developmental phase, and sometimes are found playing a slightly different function. As an example, of the four hydrophobin-encoding genes of Agaricus bisporus, ABH3 transcript was found to be present in the vegetative mycelium of primary and secondary mycelium, but not in the fruiting bodies (Lugones et al., 1998), whereas the reverse was observed for ABH1, which is specifically accumulated at the outer surface of fruiting bodies (Lugones et al., 1996). Northern blot analysis and in situ hybridization have showed that hypA mRNA was specific of tissue fractions consisting of undifferentiated white hyphae (de Groot et al., 1999). The highest hypA mRNA accumulation level was found in tissue adjacent to the outermost cell layers. The highest level of expression of hypB occurs early in development, during the primordium differentiation, with a strong accumulation of hypB transcripts in mature mushrooms only in the transitional zone, between cap and stipe tissue (de Groot et al., 1999). Similar complex profiles are also demonstrated in hydrophobins produced by S. commune, with the expression of hydrophobin genes affecting hyphal wall composition (van Wetter et al., 2000). The biological significance of our findings concerning the hydrophobin complex expression pattern are still unclear, since the biochemistry and molecular biology of P. brasiliensis dimorphism are yet poorly understood. It is well established that during the dimorphic transition,
P. brasiliensis cell wall composition and structure undergoes remarkable changes, revealing important differences in the two morphological cell types (San-Blas, 1985; San-Blas and Ni~ no-Vega, 2001). However, at this moment, we cannot assume any direct correlation between hydrophobin mRNA accumulation and morphological events in P. brasiliensis. The detection of Pbhyd1 and Pbhyd2 mRNAs throughout the first 24 h after temperature shift could be explained by previous results, showing that the complete dimorphic process from M to Y cells, in vitro, takes place in about 15 days (Silva et al., 1994). In these experiments, it was observed that even 4 days after the temperature shift, the percentage of yeast cells in the culture corresponded to less than 20%, suggesting a delay of morphological changes with respect to the start of alterations in gene expression. Hence, the use of RNA from cells in later stages of the dimorphic transition could provide a better evaluation of the expression kinetics of Pbhyd1 and Pbhyd2 genes. In a similar way, Goldman et al. (2003), using real-time quantitative reverse transcription-PCR, have shown that Pbhyd2 mRNA was typically accumulated in the mycelium phase of P. brasiliensis, as well as during the first 24 h of M to Y dimorphic transition. However, curiously in the same publication, the authors have described about 1000 ESTs, corresponding to Pbhyd2, generated from the yeast phase cDNA library of P. brasiliensis. At the moment we cannot explain these apparently contradictory findings. Finally, the characterization of the promoter sequences of these genes (work in progress) will help us to better understand the mechanisms involved in gene regulation underlying the dimorphic transition of P. brasiliensis.
Acknowledgments We thank Dra. Patrıcia Cisalpino for providing us with the antibody anti-GST, Mauro Xavier and Alessandra Ericsson Xavier for helpful experimental support in the heterologous expression and glutathione–Sepharose chromatography purification, and Maria F atima L. Cesario, Josefa Ivanildes S. de Santana, and Celso A. Tavares for laboratory assistance. This work was supported by CNPq/MCT and PADCT/CNPq. PA was supported by CAPES fellowship.
References Ando, A., Harada, A., Miura, K., Tamai, Y., 2001. A gene encoding a hydrophobin, fvh1, is specifically expressed after the induction of fruiting in the edible mushroom Flammulina velutipes. Curr. Genet. 39, 190–197.
P. Albuquerque et al. / Fungal Genetics and Biology 41 (2004) 510–520 Asgeirsdottir, S.A., de Vries, O.M., Wessels, J.G., 1998. Identification of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom). Microbiology 144, 2961–2969. Asgeirsdottir, S.A., van Wetter, M., Wessels, J.G.H., 1995. Differential expression of genes under control of the mating-type genes in the secondary mycelium of Schizophyllum commune. Microbiology 141, 1281–1288. Bainbridge, B.W., Spreadbury, C.L., Scalise, F.G., Cohen, J., 1990. Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi. FEMS Microbiol. Lett. 54, 113–117. Bell-Pedersen, D., Dunlap, J.C., Loros, J.J., 1996. Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene. Mol. Cell. Biol. 16, 513–521. Bessette, P.H., Aslund, F., Beckwith, J., Georgiou, G., 1999. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. USA 96, 13703–13708. Bolyard, M.G., Sticklen, M.B., 1992. Expression of a modified Dutch elm disease toxin in Escherichia coli. Mol. Plant Microbe Interact. 5, 520–524. Carvalho, M.J.A., Jesuino, R.S.A., Daher, B.S., Silva-Pereira, I., Freitas, S.M., Soares, C.M.A., Felipe, M.S.S., 2003. Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis. Fungal Genet. Biol. 39, 204–210. de Groot, P.W., Roeven, R.T., Van Griensven, L.J., Visser, J., Schaap, P.J., 1999. Different temporal and spatial expression of two hydrophobin-encoding genes of the edible mushroom Agaricus bisporus. Microbiology 145, 1105–1113. de Vocht, M.L., Reviakine, I., Wosten, H.A., Brisson, A., Wessels, J.G., Robillard, G.T., 2000. Structural and functional role of the disulfide bridges in the hydrophobin SC3. J. Biol. Chem. 275, 28428–28432. Ebbole, D.J., 1997. Hydrophobins and fungal infection of plants and animals. Trends Microbiol. 5, 405–408, Review. Felipe, M.S., Andrade, R.V., Petrofeza, S.S., Maranh~ao, A.Q., Torres, F.A., Albuquerque, P., Arraes, F.B., Arruda, M., Azevedo, M.O., Baptista, A.J., Bataus, L.A., Borges, C.L., Campos, E.G., Cruz, M.R., Daher, B.S., Dantas, A., Ferreira, M.A., Ghil, G.V., Jesuino, R.S., Kyaw, C.M., Leit~ao, L., Martins, C.R., Moraes, L.M., Neves, E.O., Nicola, A.M., Alves, E.S., Parente, J.A., Pereira, M., Pocas-Fonseca, M.J., Resende, R., Ribeiro, B.M., Saldanha, R.R., Santos, S.C., Silva-Pereira, I., Silva, M.A., Silveira, E., Sim~ oes, I.C., Soares, R.B., Souza, D.P., De-Souza, M.T., Andrade, E.V., Xavier, M.A., Veiga, H.P., Venancio, E.J., Carvalho, M.J., Oliveira, A.G., Inoue, M.K., Almeida, N.F., Walter, M.E., Soares, C.M., Brigido, M.M., 2003. Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis. Yeast 20, 263–271. Goldman, G.H., Marques, E.R., Ribeiro, D.C.D., Bernardes, L.A.S., Quiapin, A.C., Vitorelli, P.M., Savoldi, M., Semighini, C.P., Oliveira, R.C., Nunes, L.R., Travassos, L.R., Puccia, R., Batista, W.L., Ferreira, L.E., Moreira, J.C., Bogossian, A.P., Tekaia, F., Nobrega, M.P., Nobrega, F.G., Goldman, M.H.S., 2003. Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes. Eukaryot. Cell 2, 34–48. Higgins, D.G., Thompson, J.D., Gibson, T.J., 1996. Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 266, 383–402. Kershaw, M.J., Talbot, N.J., 1998. Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis. Fungal Genet. Biol. 23, 18–33. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.
519
Liang, P., Averboukh, L., Pardee, A.B., 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res. 21, 3269–3275. Lugones, L.G., Wosten, H.A., Wessels, J.G., 1998. A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom). Microbiology 144, 2345–2353. Lugones, L.G., Bosscher, J.S., Scholtmeyer, K., de Vries, O.M., Wessels, J.G., 1996. An abundant hydrophobin (ABH1) forms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies. Microbiology 142, 1321–1329. Marck, C., 1988. DNA Strider: a ‘‘C’’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 6, 1829–1836. McEwen, J.G., Garcia, A.M., Ortiz, B.L., Botero, S., Restrepo, A., 1995. In search of the natural habitat of Paracoccidioides brasiliensis. Arch. Med. Res. 26, 305–306. Mount, S.M., 1982. A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459–472. Nakari-S€etal€a, T., Aro, N., Kalkkinen, N., Alatalo, E., Penttila, M., 1996. Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI. Eur. J. Biochem. 235, 248–255. Nielsen, P.S., Clark, A.J., Oliver, R.P., Huber, M., Spanu, P.D., 2001. HCf-6, a novel class II hydrophobin from Cladosporium fulvum. Microbiol. Res. 156, 59–63. Paris, S., Duran, S., 1985. Cyclic adenosine 30 ,50 monophosphate (cAMP) and dimorphism in the pathogenic fungus Paracoccidioides brasiliensis. Mycopathologia 92, 115–120. Paris, S., Debeaupuis, J.P., Crameri, R., Carey, M., Charles, F., Prevost, M.C., Schmitt, C., Philippe, B., Latge, J.P., 2003. Conidial hydrophobins of Aspergillus fumigatus. Appl. Environ. Microbiol. 69, 1581–1588. Parta, M., Chang, Y., Rulong, S., Pinto-DaSilva, P., Kwon-Chung, K.J., 1994. HYP1, a hydrophobin gene from Aspergillus fumigatus, complements the rodletless phenotype in Aspergillus nidulans. Infect. Immun. 62, 4389–4395. Pe~ nas, M.M., Asgeirsdottir, S.A., Lasa, I., Culianez-Macia, F.A., Pisabarro, A.G., Wessels, J.G., Ramirez, L., 1998. Identification, characterization, and in situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus. Appl. Environ. Microbiol. 64, 4028–4034. Restrepo, A., 1985. The ecology of P. brasiliensis: a puzzle still unsolved. J. Med. Vet. Mycol. 23, 323–334. Restrepo, A., McEwen, J.G., Castaneda, E., 2001. The habitat of Paracoccidioides brasiliensis: how far from solving the riddle? Med. Mycol. 39, 233–241. Saitou, N., Nei, M., 1987. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. San-Blas, G., 1985. Paracoccidioides brasiliensis: cell wall glucans, pathogenicity, and dimorphism. Curr. Top. Med. Mycol. 1, 235–257. San-Blas, G., Ni~ no-Vega, G., 2001. Paracoccidioides brasiliensis: virulence and host response. In: Cihlar, R.L., Calderone, R.A. (Eds.), Fungal Pathogenesis: Principles and Clinical Applications. Marcel Dekker, New York, pp. 205–226. San-Blas, G., Nino-Vega, G., Iturriaga, T., 2002. Paracoccidioides brasiliensis and paracoccidioidomycosis: molecular approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics. Med. Mycol. 40, 225–242. Scholtmeijer, K., Wessels, J.G., Wosten, H.A., 2001. Fungal hydrophobins in medical and technical applications. Appl. Microbiol. Biotechnol. 56, 1–8, Review. Schuren, F.H., Wessels, J.G., 1990. Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes. Gene 90, 199–205.
520
P. Albuquerque et al. / Fungal Genetics and Biology 41 (2004) 510–520
Segers, G.C., Hamada, W., Oliver, R.P., Spanu, P.D., 1999. Isolation and characterisation of five different hydrophobin-encoding cDNAs from the fungal tomato pathogen Cladosporium fulvum. Mol. Gen. Genet. 261, 644–652. Silva, S.P., Felipe, M.S.S., Pereira, M., Azevedo, M.O., Soares, C.M.A., 1994. Phase transition and stage-specific protein synthesis in the dimorphic fungus Paracoccidioides brasiliensis. Exp. Mycol. 18, 294–299. Silva, S.P., Borges-Walmsley, M.I., Pereira, I.S., Soares, C.M., Walmsley, A.R., Felipe, M.S., 1999. Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis. Mol. Microbiol. 31, 1039–1050. Silva-Pereira, I., Bey, F., Coux, O., Scherrer, K., 1992. Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes. Gene 120, 235–242. Stewart, E.J., Aslund, F., Beckwith, J., 1998. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J. 17, 5543–5550. Stringer, M.A., Dean, R.A., Sewall, T.C., Timberlake, W.E., 1991. Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation. Genes Dev. 5, 1161–1171. Talbot, N.J., Kershaw, M.J., Wakley, G.E., De Vries, O., Wessels, J., Hamer, J.E., 1996. MPG1 encodes a fungal hydrophobin involved in surface interactions during infectionrelated development of Magnaporthe grisea. Plant Cell 8, 985–999.
van Wetter, M.A., Wosten, H.A., Sietsma, J.H., Wessels, J.G., 2000. Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune. Fungal Genet. Biol. 31, 99–104. Venancio, E.J., Kyaw, C.M., Mello, C.V., Silva, S.P., Soares, C.M.A., Felipe, M.S.S., Silva-Pereira, I., 2002. Identification of differentially expressed cDNAs in the human pathogenic fungus Paracoccidioides brasiliensis by differential display. Med. Mycol. 40, 45–51. Wessels, J.G.H., 1997. Hydrophobins: proteins that change the nature of the fungal surface. Adv. Microb. Physiol. 38, 1–45. Wessels, J.G.H., 1999. Fungi in their own right. Fungal Genet. Biol. 27, 134–145. Wessels, J.G.H., 2000. Hydrophobins, unique fungal proteins. Mycologist 14, 153–159. W€ osten, H.A., 2001. Hydrophobins: multipurpose proteins. Annu. Rev. Microbiol. 55, 625–646. W€ osten, H.A., de Vocht, M.L., 2000. Hydrophobins, the fungal coat unravelled. Biochim. Biophys. Acta 1469, 79–86. W€ osten, H.A., Schuren, F.H., Wessels, J.G.H., 1994. Interfacial selfassembly of a hydrophobin into an amphipathic protein membrane mediates fungal attachment to hydrophobic surfaces. EMBO J. 13, 5848–5854. Yaguchi, M., Pusztai-Carey, M., Roy, C., Surewicz, W.K., Carey, P.R., Stevenson, K.J., Richards, W.C., Takai, S., 1993. Amino acid sequence and spectroscopic studies of Dutch elm disease toxin, cerato-ulmin. In: Sticklen, M.B., Sherald, J.L. (Eds.), Dutch Elm Disease Research, Cellular and Molecular Approaches. SpringerVerlag, New York, pp. 152–170.
Pbhyd1 and Pbhyd2: two mycelium-specific hydrophobin genes from the dimorphic fungus Paracoccidioides brasiliensis P. Albuquerque, C.M. Kyaw, R.R. Saldanha, M.M. Brigido, M.S.S. Felipe, and I. Silva-Pereira* Laboratorio de Biologia Molecular, CEL/IB, Universidade de Brasılia, Brasılia-DF, 70910-900, Brazil Received 12 October 2003; accepted 1 January 2004
Abstract Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 °C and as yeasts (Y) at 37 °C, or after the invasion of host tissues. Although the dimorphic transition in P. brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood. Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P. brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2). Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence. These two hydrophobin genes are present as single copy in P. brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Hydrophobin; Paracoccidioides brasiliensis; Dimorphic fungus; Differentially expressed genes; DDRT-PCR; 50 RACE; RT-PCR
1. Introduction Paracoccidioides brasiliensis is a dimorphic fungus, which causes paracoccidioidomycosis (PCM), an endemic human disease (San-Blas and Ni~ no-Vega, 2001). Geographically, it is confined to Latin America, with its areas of endemicity extending from Central America to Argentina, representing one of the most prevalent deep mycoses in this region (Restrepo, 1985). Over 10 million people are estimated to be infected with P. brasiliensis, but only up to 2% develop the disease (McEwen et al., 1995). This fungus, previously considered a member of the phylum Deuteromycota, since sexual structures were not found, was recently classified as belonging to the phylum Ascomycota, order Onygenales, family Onygenaceae (San-Blas et al., 2002). Although known for several years, the ecology of this microorganism still remains unclear, especially with respect to its environ-
* Corresponding author. Fax: +55-61-349-8411. E-mail address: [email protected] (I. Silva-Pereira).
1087-1845/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.01.001
mental niche (McEwen et al., 1995; Restrepo et al., 2001). This fungus is found as mycelia (M) at about 26 °C and as yeasts (Y) at 37 °C. Mycelia and/or conidia are supposed to be the infective form found in nature, probably in the soil and/or plants, while yeasts are the form usually found in infected tissues. The mycelium to yeast transition, triggered by the temperature shift, is easily reproduced in vitro and seems to be essential to the establishment of the infective process (San-Blas et al., 2002). Regardless the importance of dimorphic transition, the biochemical events regulating this process in P. brasiliensis are poorly defined and several important questions remain unanswered. Although the temperature shift, the cAMP (Paris and Duran, 1985), and Ca2þ / calmodulin (Carvalho et al., 2003) pathways seem to be important in the control of transition, the signaling cascades and regulating factors involved in differential gene expression and subsequent morphological changes of this fungus, are still a puzzle. One approach that allows a better understanding of the molecular
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mechanisms involved in dimorphism corresponds to the characterization of genes differentially expressed during the fungal transition (San-Blas et al., 2002). It has been shown that several genes related to cell wall structure, metabolism, and signaling cascades undergo significant changes in their expression profile, probably playing an important role in P. brasiliensis dimorphism. Among the several approaches currently employed to characterize differentially expressed genes, by using differential display reverse transcriptase PCR (DDRTPCR, Liang and Pardee, 1992; Liang et al., 1993) and Northern blot analysis, we were able to detect several phase-specific cDNA fragments corresponding to genes differentially expressed in one of the two forms of P. brasiliensis (Venancio et al., 2002). Here, we report the isolation and characterization of the mycelium-specific full-length Pbhyd1 cDNA, previously named M73 cDNA (Venancio et al., 2002). The predicted protein sequence is composed of 100 amino acid residues, showing similarity with hydrophobins, a group of small hydrophobic proteins of filamentous fungi (Wessels, 1999). Hydrophobins are small secreted proteins, of about 100 amino acid residues, which, although do not share significant sequence similarity, are all characterized by the presence of eight cysteine residues in conserved positions, and a typical hydropathy profile (Wessels, 1997). These proteins are secreted as monomers that self-assemble into an amphipathic film at hydrophilic/hydrophobic interfaces covering fungal structures (W€ osten and de Vocht, 2000; W€ osten et al., 1994). For the last two years, our group has been developing the ‘‘P. brasiliensis Functional and Differential Genome Project—Brazilian Middle-West Network’’ (https:// www.biomol.unb.br/Pb), in order to map the yeast and mycelium transcriptomes of this microorganism, as described by Felipe et al. (2003). By this approach, a second putative hydrophobin sequence, named Pbhyd2 (this work), was identified. Herein, two P. brasiliensis hydrophobin cDNAs were characterized and shown to be specifically expressed in the mycelium phase of this fungus.
2. Materials and methods 2.1. Paracoccidioides brasiliensis growth conditions and nucleic acid extraction The clinical isolate of P. brasiliensis, strain Pb01 (ATCC-MYA-826), was grown on semi-solid FavaNetoÕs medium as mycelium (M), at 26 °C; yeast (Y), at 36 °C; and in the M to Y transition experiments, as previously described (Silva et al., 1994). Genomic DNA from P. brasiliensis was obtained as described by Bainbridge et al. (1990). Total RNA was
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extracted using Trizol reagent (Gibco-BRL), according to suppliersÕ recommendations, as previously described (Silva et al., 1999; Venancio et al., 2002). To remove any genomic DNA contamination, RNA was precipitated twice with LiCl, followed by RNase-free-DNaseI treatment (Venancio et al., 2002). 2.2. Identification and isolation of full-length Pbhyd1 and Pbhyd2 cDNAs After DDRT-PCR and Northern blot assays confirming the differentially expressed nature of M73 cDNA fragment (Venancio et al., 2002), 50 RACE (rapid amplification of cDNA ends) and RT-PCR experiments were performed, in other to obtain the full-length Pbhyd1 cDNA. A 50 RACE experiment, using DNaseI treated mycelium total RNA, was performed to isolate the corresponding 50 end missing region of this cDNA. The oligonucleotides GSP1 (50 -GGAAACAGGATCGAGAA GTCCCACC-30 ) and GSP2 (50 -CCACCTCCTCATCC ATTAACTCTCCGC-30 ) were designed to prime the cDNA synthesis and to perform PCR, respectively, according to the instructions of the suppliers of the ‘‘50 RACE, Version 2.0’’ (Gibco-BRL). After purification and cloning, a DNA fragment of approximately 750 bp, named 50 cDNA M73, was sequenced (data not shown). The primers 50 Hyd (50 -ATCATCAACAAGCATCAGT AC-30 ) and 30 Hyd (50 -CTAAAGGAAAGTTAAGAA GC-30 ) used in the RT-PCR experiment were synthesized based on the nucleotide sequence of the two cDNA fragments obtained from DDRT-PCR (30 cDNA M73, Venancio et al., 2002) and 50 RACE (50 cDNA M73). Following denaturation (80 °C/5 min), 1 lg of mycelial total RNA was used in cDNA synthesis, at 37 °C/1 h, in a 25 ll reaction consisting of: 0.5 mM of each dNTP, 0.2 mM of primer 30 Hyd, 200 U of Reverse Transcriptase (RT, Superscript II, Gibco-BRL), 5 mM of DTT, and 1 RT-buffer. Four ll of the cDNA first strand were amplified in a final volume of 25 ll (2 U of Taq DNA polymerase (Cenbiot-RS/Brazil), 0.2 mM of each dNTP, 0.4 lM of each primer (30 Hyd and 50 Hyd) and 1 PCRbuffer containing 1.5 mM MgCl2 ). In order to isolate the genomic sequence corresponding to Pbhyd1 cDNA, a PCR using 20 ng of P. brasiliensis genomic DNA as template were done (data not shown). The amplified DNA fragment was cloned into pGEM-T easy vector (Promega). Pbhyd2 cDNA was identified by the EST genome project, as described by Felipe et al. (2003). 2.3. Northern blot analysis About 15 lg of total RNA from Y and M cells of P. brasiliensis, and from M cells undergoing the dimorphic transition for 0.5, 1, 2, 6, 12, and 24 h, after a temperature shift from 26 to 37 °C, were run on denaturing 1% agarose gel and blotted onto a Hybond Nþ membrane
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(Amersham Biosciences), according to standard protocols (Sambrook and Russell, 2001). The Pbhyd1 genomic DNA fragment and Pbhyd2 cDNA were labeled using [a-32 P]dATP by random priming procedure (MegaPrime DNA labeling System, Amersham Biosciences), purified and used in overnight hybridization, at 42 °C (Silva-Pereira et al., 1992; Venancio et al., 2002). The membranes were then washed to a final stringency of 0.1 SSPE-0.1% SDS at 65 °C for 30 min and exposed either to a X-ray film at )80 °C for 48 h or to a Phosphor Screen (Molecular Dynamics, Amersham Biosciences) for 2 h. 2.4. Southern blot analysis About 10 lg of P. brasiliensis genomic DNA were digested with BamHI, BglII, HindIII, and PstI restriction endonucleases, separated on 1% agarose gel, and transferred to a Hybond Nþ membrane, as specified by the manufacturer (Amersham Biosciences). Radioactive probes, hybridization, and washing conditions were performed as described for the Northern blot experiment. 2.5. Production of the hydrophobin PbHYD1 in Escherichia coli To produce the hydrophobin PbHYD1, we amplified the corresponding ORF from the plasmid containing the Pbhyd1 cDNA, using 2 U of Taq DNA polymerase (Cenbiot-RS/Brazil) and the primers BamATG (50 -CG GGATCCATGCAGCTCTCCAACATCGTC-30 ) and TAAXho (50 -CCGCTCGAGGTCTCGATTGGGCAT TTAC-30 ). The PCR-amplified Pbhyd1-ORF was cloned into pGEM-T easy vector (Promega). The BamHI and XhoI insert was released by double digestion and cloned into the expression vector pGEX-4T-3 (Amersham Biosciences). The resulting vector, containing the Pbhyd1-ORF fused to the 26 kDa domain of glutathione S-transferase (GST), was transformed into E. coli Ori gami B pLacI (F ompT hsdSB (r B mB ) gal dcm lacY1 r aphC gor522::Tn10 (Tc ) trxB::kan (DE3) pLacI (Cmr )Novagen) (Bessette et al., 1999; Stewart et al., 1998). In order to obtain the fusion protein (GST-Pbhyd1ORF), its expression was induced by adding isopropyl-b-D -thiogalactopyranoside (IPTG; Sigma) to a final concentration of 1 mM to an early-exponential-phase culture of the recombinant E. coli Origami B cells. After 3 h of induction, bacterial cells were harvested by centrifugation and washed with PBS (140 mM NaCl; 2.7 mM KCl; 10 mM Na2 HPO4 ; and 1.8 mM KH2 PO4 , pH 7.3). Cell suspensions were sonicated, centrifuged, and the resulting supernatants were further purified by affinity chromatography using Glutathione–Sepharose 4B (Amersham Biosciences), according to supplierÕs instructions. The recombinant protein was eluted with glutathione and analyzed by SDS–PAGE (Sambrook and Russell, 2001).
2.6. SDS–PAGE and Western blot analysis SDS–PAGE was performed using 15% acrylamide gels according to standard protocols. Prior to electrophoresis, all samples were dried, dissolved in sample loading buffer, and heat denatured (100 °C/5 min). Proteins were visualized by staining with Coomassie brilliant blue R-250. After SDS–PAGE, proteins were transferred to a nitrocellulose membrane (Amersham Biosciences), saturated with 3% BSA and incubated for 1 h with an antiGST antibody conjugated to alcaline phosphatase. Following incubation, bound antibody was detected by using alkaline phosphatase colorimetric detection (Sambrook and Russell, 2001). 2.7. Hydropathy profile of PbHYD1 and PbHYD2 Hydrophobicity plots were generated by DNA Strider 1.2, based on the Kyte–Doolittle hydrophatic table. 2.8. Phylogenetic analysis PbHYD1 and PbHYD2 sequences, as well as related hydrophobins from other fungi, deposited at GenBank, were aligned with ClustalW (Higgins et al., 1996). Multisequence alignment was phylogenetically analyzed using neighbor-joining trees, constructed as described by Saitou and Nei (1987). 2.9. Sequences accession numbers The Pbhyd1 and Pbhyd2 accession numbers are AF526275 and AY427793, respectively. The accession number of related hydrophobins from other fungi are: Mpg1 (AAA20128.1, Magnaporthe grisea); Ccg2 (CAA47754.1, Neurospora crassa); RodA (AAA33321.1, Aspergillus nidulans); Hyp1 (AAB60712.1, Aspergillus fumigatus); DewA (AAC13762.1, Emericella nidulans); SsgA (AAA33418.1, Metarhizium anisopliae); XEH1 (CAC86096.1, Xanthoria ectaneoides); XEP1 (CAC8 6092.1, Xanthoria parietina); HCF1 (CAC27408.1, Cladosporium fulvum); HCF2 (CAD97454.1, C. fulvum); HCF3 (CAB39310.1, C. fulvum); HCF4 (CAB39311.1, C. fulvum); and EAS (AAB24462, N. crassa).
3. Results 3.1. Isolation and characterization of Pbhyd1 and Pbhyd2 cDNAs Using DDRT-PCR we have isolated several cDNA fragments, differentially expressed in M or Y cells of P. brasiliensis (Venancio et al., 2002). Among them, we have found a 281 bp cDNA fragment (M73.4) that was
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cloned and sequenced. Northern blot analysis confirmed that M73.4 was specific for the mycelial phase of this fungus, corresponding to an mRNA of about 1100 nucleotides. To obtain the 50 missing region of this cDNA, we have employed the 50 RACE methodology, which generated a 772 bp cDNA fragment (50 M73) showing the expected overlapping sequence with the 30 M73 cDNA previously obtained by DDRT-PCR (Venancio et al., 2002). Subsequently, in order to obtain the M73 full-
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length cDNA by RT-PCR, we designed primers corresponding to the 50 and 30 ends of 50 M73 and M73.4 nucleotide sequences, respectively. A 968 bp cDNA fragment was amplified and sequenced, confirming the isolation of the complete M73 cDNA (Fig. 1A), as predicted by previous Northern blot results. Using P. brasiliensis total genomic DNA as template, and the same oligonucleotides used in RT-PCR experiment, we have also amplified a DNA fragment slightly larger, suggesting the presence of an intron in the genomic M73
Fig. 1. Nucleotide and deduced amino acid sequences of Pbhyd1 (A) and Pbhyd2 (B) genes. The Pbhyd1 and Pbhyd2 coding sequences (Accession numbers AF526275 and AY427793, respectively) are shown in uppercase letters, and the deduced amino acid sequences are shown below the nucleotide sequences. The 50 UTR and 30 UTR are indicated by lowercase letters and the intron sequence in Pbhyd1 gene is underlined. Primers used in RT-PCR are indicated by arrows. Cysteine residues are displayed in black boxes.
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sequence. Sequence comparisons of M73 cDNA and its corresponding genomic DNA fragment, confirmed the presence of a small intron of 79 bp, located within the M73 coding region. The intron nucleotide sequence displays the splicing site consensus (50 GT-AG 30 ) and the canonical internal sequence required for lariat formation (Mount, 1982). A detailed analysis of the predicted M73 amino acids sequence indicated features common to the hydrophobins family (Wessels, 1997). Noteworthy, the presence of eight cysteine residues with their characteristic spacing (Fig. 1, panel A, black boxes), as well as the typical hydropathy profile (Fig. 2B), which constitute the signature of this group of proteins. Therefore, the corresponding gene was designated Pbhyd1 (P. brasiliensis
hydrophobin 1). The deduced amino acid sequence of PbHYD1 exhibits a strongly hydrophobic putative signal sequence of 19 amino acids in the N-terminus (Fig. 2B), which is characteristically observed in other hydrophobins (Wessels, 1997; W€ osten, 2001). Consequently, the mature PbHYD1 should have 81 amino acid residues, with a predicted molecular mass of about 8 kDa. The P. brasiliensis Functional and Differential Genome Project (Brazilian Middle-West Network), based on the generation of gene expressed sequence tags (ESTs) from mycelium and yeast cDNA libraries of P. brasiliensis (Felipe et al., 2003), revealed 56 ESTs similar to Pbhyd1 cDNA. These data suggest that Pbhyd1 mRNA is highly abundant, as confirmed by the
Fig. 2. Major characteristics observed in PbHYD1 and PbHYD2 deduced amino acid sequences. (A) Schematic representation of the putative PbHYD1 and PbHYD2 primary sequences, based on disulfide bonds determined for the hydrophobin cerato-ulmin (Yaguchi et al., 1993). Hydrophilic and hydrophobic amino acid residues are shown in black and in gray, respectively. The eight cysteine residues are shown in red. (B) Hydropathy profile of P. brasiliensis PbHYD1 and PbHYD2 deduced amino acid sequences. The hydropathy plots were generated using the Kite and Doolittle algorithm (window of six amino acids), by the program DNA Strider 1.2 (Marck, 1988). The +/) values in the ordinate axis indicate the hydrophobic/hydrophilic nature of the amino acid residues, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
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Northern blot results (Fig. 5B). Further analyses of the P. brasiliensis EST project have revealed a second hydrophobin, represented by 9 ESTs, that was named Pbhyd2. A similar nucleotide sequence was also identified by Goldman et al. (2003). The nucleotide and the deduced amino acid sequences of Pbhyd2 are depicted in Fig. 1 (panel B, cysteine residues are shown in black boxes). The deduced protein sequence, of 133 amino acid residues, contains a putative signal sequence of 18 amino acid residues at its amino-terminal end (Fig. 2B). The resulting mature protein is composed of 115 amino acid residues, with a predicted molecular mass of about 11 kDa. The amino acid sequence similarities between PbHYD1 and PbHYD2 are as low as 18.6%, a value usually observed for different hydrophobins produced in a single organism (W€ osten, 2001). A schematic representation of the primary structure of PbHYD1 and PbHYD2, and their corresponding hydropathy plots, are represented in Fig. 2. The cysteine spacing of both P. brasiliensis predicted hydrophobins is almost identical to the class I hydrophobins consensus, as proposed by Wessels (1997). The PbHYD1 and PbHYD2 deduced amino acid sequences, and related hydrophobins from other filamentous fungi deposited at GenBank, were aligned with ClustalW (Higgins et al., 1996). The multi-sequence alignment analysis of the two P. brasiliensis hydrophobins clearly positioned PbHYD1 and PbHYD2 in two separate clusters (Fig. 3). The results reveal that PbHYD1 is more related to EAS (ccg-2) hydrophobin from N. crassa (Bell-Pedersen et al., 1996), while PbHYD2 clusterizes with RODA of A. nidulans (Stringer et al., 1991) and HYP1 from A. fumigatus (Paris et al., 2003). To determine the copy number of Pbhyd1 and Pbhyd2 genes in the P. brasiliensis genome, a Southern blot analysis was performed. The resulting hybridization profiles, depicted in Fig. 4, suggest that both genes are encoded as single copies in P. brasiliensis genome, which is in accordance to other hydrophobin genes such as fvh1 from Flammulina velutipes (Ando et al., 2001) and hyp1 from A. fumigatus (Parta et al., 1994). 3.2. Analysis of the expression levels of Pbhyd1 and Pbhyd2 genes Fig. 5 illustrates a Northern blot experiment, showing the expression of Pbhyd1 and Pbhyd2 genes, using total RNA from mycelium and yeast cells of P. brasiliensis, and from mycelium cells induced to dimorphic transition at different time intervals, after 26–37 °C temperature shift (0.5, 1, 2, 6, 12, and 24 h). The Pbhyd1 and Pbhyd2 transcripts were not detected in yeast cells, whereas high levels of these transcripts were observed in mycelium cells. Although the accumulation of both mRNAs occurs during the first 24 h after the tempera-
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Fig. 3. Phylogenetic neighbor-joining tree of PbHYD1 and PbHYD2 deduced amino acid sequences. PbHYD1 and PbHYD2 and related hydrophobins from other fungi were analyzed, as described in Section 2. The tree was confirmed by one thousand bootstrap re-sampling.
ture shift, the relative abundance of Pbhyd1 and Pbhyd2 transcripts seems to increase at 0.5 and 6 h after the temperature shift. This apparently complex profile of Pbhyd1 and Pbhyd2 is in accordance to the expression profiles described for other hydrophobins in different fungi (Bell-Pedersen et al., 1996; Kershaw and Talbot, 1998; Paris et al., 2003). 3.3. Heterologous expression of Pbhyd1 gene in E. coli To produce the PbHYD1 hydrophobin in bacterial cells, the corresponding ORF was cloned into the expression vector pGEX-4T-3 (Amersham Biosciences). The resulting vector, bearing the Pbhyd1-ORF fused to the 26 kDa domain of glutathione S-transferase (GST), was used to transform cells of E. coli Origami B, which is genetically manipulated to improve the disulfide bond formation in the cytoplasm (Bessette et al., 1999; Stewart et al., 1998). After induction of protein expression, the fusion protein (GST-HYD1) could be detected, revealing a low level of expression, as described in the literature for other hydrophobins (Scholtmeijer et al., 2001). After purification by glutathione–Sepharose
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Fig. 4. Southern blot analysis of P. brasiliensis genomic DNA, using the probes Pbhyd1 and Pbhyd2. Equal amounts of genomic DNA (10 lg) were digested with the restriction enzymes BglII, HindIII, PstI, XhoI, and BamHI, separated on 1% agarose gel stained with ethidium bromide (not shown), and blotted onto a nylon membrane (HybondN, Amersham Biosciences). Hybridization was performed using the homologous Pbhyd1 (Panel A) and Pbhyd2 (Panel B) radiolabeled probes. The kDNA/HindIII fragments used as molecular marker, are depicted on the left (in kb).
chromatography and elution using 1, 5, and 10 mM of reduced glutathione, the samples were analyzed by SDS–PAGE and Western blot (Figs. 6A and B). Fig. 6A shows the SDS–PAGE analysis of the eluted fusion protein, where a weak band with the expected size of approximately 36 kDa can be observed. It is important to notice a relative abundance of a same-size protein in the fraction corresponding to the insoluble pellet, obtained after centrifugation of the sonicated cell suspension (Fig. 6A, lane 4). Subsequently, a Western blot using antibodies raised against the GST domain of the fusion protein was performed, confirming the expression of the expected fusion protein (Fig. 6B). We can also observe that most of the fusion protein GST-HYD1 was present in the insoluble protein pellet, which was solubilized in 8 M urea (Fig. 6B, lane 4), probably confirming the highly insoluble nature described for hydrophobins in several heterologous expression systems, as well as in the fungus (Scholtmeijer et al., 2001). The heterologous expression optimization and the purification of significant amounts of the fusion protein
Fig. 5. Northern blot analysis showing the differential expression of Pbhyd1 and Pbhyd2 genes. Pbhyd1 and Pbhyd2 cDNA probes were prepared using MegaPrime labeling kit (Amersham Biosciences) and used to probe a membrane containing 15 lg of Y and M total RNA, and from M cells induced to dimorphic transition at different times after temperature shift (0.5, 1, 2, 6, 12, and 24 h). Panels A and B: Pbhyd1; Panels C and D: Pbhyd2. (A, C) Ethidium bromide stained 1.0% denaturing agarose gels, showing P. brasiliensis major and minor ribosomal RNAs, of about 3.2 and 1.6 kb, respectively. (B, D) Resulting hybridization profile, showing the mycelium-specific Pbhyd1 and Pbhyd2 mRNAs, respectively.
will be necessary in order to produce antibodies against P. brasiliensis recombinant hydrophobins, as an important reagent for later biochemical characterization of P. brasiliensis hydrophobins and immunocytolocalization of these proteins during the dimorphic process.
4. Discussion The successful establishment of P. brasiliensis in the host tissues depends on its ability to respond to the different environmental signals, which is determined by changes in the expression of specific genes involved in differentiation and adaptation. According to this scenario, we have been studying the differentially expressed genes of P. brasiliensis (Felipe et al., 2003; Venancio et al., 2002). Here, we describe the isolation and characterization of two hydrophobin genes (Pbhyd1 and Pbhyd2) from P. brasiliensis, which are expressed by the mycelial form of this microorganism. Hydrophobins are small hydrophobic proteins secreted exclusively by filamentous fungi, which contain eight cysteine residues arranged in a conserved pattern,
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Fig. 6. Heterologous expression of Pbhyd1 ORF in E. coli Origami B pLacI cells. Protein extracts of IPTG-induced recombinant E. coli were purified by affinity chromatography using Glutathione–Sepharose 4B (Amersham Biosciences). The GST-PbHYD1 fusion protein is indicated by an arrow. (A) SDS–PAGE analysis of purified GST-PbHYD1 protein. Following elution with glutathione, the purified protein was submitted to SDS–PAGE and Coomassie blue staining. (Lanes 1–3) GST-PbHYD1 protein eluted with 10, 5, and 1 mM of reduced glutathione, respectively; (lanes 4 and 5) insoluble (soluble in 8 M urea) and soluble protein fractions after cell lysis, respectively. (B) Western blot analysis of the purified GST-PbHYD1 protein. After SDS–PAGE, proteins were transferred to a nitrocellulose membrane (Amersham Biosciences) and incubated with an anti-GST antibody conjugated to alkaline phosphatase, following colorimetric detection. Lanes 1–4 as described above; lane 5 corresponds to the total extract of IPTG-induced recombinant E. coli. The molecular-mass marker (Bench Marck; Invitrogen, Life and Technologies) is indicated on the left (in kDa).
and share a typical hydropathy profile and the ability to self-assemble into amphipathic films, at hydrophilic– hydrophobic interfaces (Wessels, 1999, 2000; W€ osten, 2001; W€ osten et al., 1994). Most hydrophobins have been discovered by the analysis of mRNAs abundantly expressed during fungal development. Hydrophobins are linked to a broad range of processes, playing an important role in the formation of aerial structures (Wessels, 2000), probably involved in cell wall construction (van Wetter et al., 2000) and in the attachment of hyphae to hydrophobic surfaces—which are of strong relevance in the establishment of infection of pathogenic fungi (Ebbole, 1997; Kershaw and Talbot, 1998; Talbot et al., 1996). Based on the great relevance of hydrophobins in fungi, we believe that a careful study of these proteins would provide some new important insights concerning the molecular biology of P. brasiliensis.
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Several fungi possess multiple hydrophobin genes. Four hydrophobins were described in Schizophyllum commune (Schuren and Wessels, 1990), six in C. fulvum (Nielsen et al., 2001; Segers et al., 1999), and three in Pleurotus ostreatus (Asgeirsdottir et al., 1998). Different hydrophobins produced by the same organism usually shares very low sequence identity, not being detected by cross-hybridization (Wessels, 1997). The deduced amino acid sequences of PbHYD1 and PbHYD2 are only 18.6% identical. Despite this very low similarity between hydrophobins, they seem to assume the same general structure. In this sense, a differential regulation of these proteins or the fulfill of distinct roles in fungi have been suggested (W€ osten, 2001). The amino acid sequence of PbHYD2 (X21 -Cys-X6 Cys-Cys-X36 -Cys-X18 -Cys-X5 -Cys-Cys-X15- Cys-X6 ) matches the consensus for class I hydrophobins, as proposed by Wessels (1997). The pattern observed in PbHYD1 (X22 -Cys-X6 -Cys-Cys-X9 -Cys-X9 -Cys-X5 -Cys-Cys-X9 -CysX13 ) is almost identical to the consensus spacing of class I hydrophobins, except for the fewer number of amino acid residues between the third and fourth cysteines, than those described for both class I and class II hydrophobin consensus. Therefore, only after the isolation and characterization of their protein products we will be able to make a more precise classification of these P. brasiliensis hydrophobins. Since only few hydrophobins are yet well characterized, it cannot be discarded the finding of new hydrophobins, showing intermediate features between the two classes proposed by Wessels (1997). To obtain the protein product encoded by Pbhyd1 cDNA, its putative ORF was expressed in E. coli, using the pGEX system as described in Section 2. The production and purification of hydrophobins has already been shown to be difficult (Scholtmeijer et al., 2001). The remarkable features of hydrophobins, mainly linked to their insolubility, hidden this protein class from science until the last decade of the past century (Wessels, 1999). The high number of disulfide bonds, which are very important for the monomers solubility in water (de Vocht et al., 2000), could not be properly formed in the reducing bacterial cytoplasmic environment, which does not make bacterial systems the best choice for hydrophobin production. In spite of that, three class II hydrophobins, the P. ostreatus POH1 (Pe~ nas et al., 1998), the Ophiostoma ulmi cerato-ulmin (Bolyard and Sticklen, 1992) and the Trichoderma reesei HFBI (Nakari-S€etal€a et al., 1996) have been expressed in E. coli. Although the production levels were low (micrograms quantities per liter), limiting the use of such bacterial systems for bulk hydrophobin production, it seemed to be sufficient for antibody production. In our attempt of produce PbHYD1 in E. coli, the yield of the purified protein was low, as observed for the other hydrophobins described above, probably because most of
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the fusion protein GST-HYD1 was highly insoluble, as observed in Figs. 6A and B (lane 4). Southern blot analyses have shown that Pbhyd1 and Pbhyd2 genes are found as single copies in P. brasiliensis genome. Since the similarity among the members of these group of genes is very low and cross-hybridization can be ruled out, we cannot exclude the existence of other hydrophobins in P. brasiliensis. The P. brasiliensis hydrophobins described in this work have revealed, by Northern blot analysis, to be mycelium-specific, which is in accordance to the literature describing that hydrophobins are specifically produced only by filamentous fungi (Wessels, 1999). The increase in the Pbhyd1 and Pbhyd2 mRNA accumulation at 0.5 and 6 h after 26–36 °C temperature shifting during M to Y transition (Fig. 5, panels B and D, respectively), suggests a complex pattern of expression, as described for other hydrophobin genes (Ando et al., 2001; Asgeirsdottir et al., 1995). Of particular interest are the findings of Bell-Pedersen et al. (1996), with the N. crassa hydrophobin eas gene (or ccg2—clock controlled gene). In this work, the authors demonstrated that the ccg2 mRNA cycles with periods, showing a rhythmic accumulation pattern. In this sense, the Pbhyd1 mRNA accumulation and sequence clusterization results (Fig. 3) could be interpreted in a similar way. Different hydrophobins produced by the same organism could have their expression associated to a specific fungal structure or specific fungal developmental phase, and sometimes are found playing a slightly different function. As an example, of the four hydrophobin-encoding genes of Agaricus bisporus, ABH3 transcript was found to be present in the vegetative mycelium of primary and secondary mycelium, but not in the fruiting bodies (Lugones et al., 1998), whereas the reverse was observed for ABH1, which is specifically accumulated at the outer surface of fruiting bodies (Lugones et al., 1996). Northern blot analysis and in situ hybridization have showed that hypA mRNA was specific of tissue fractions consisting of undifferentiated white hyphae (de Groot et al., 1999). The highest hypA mRNA accumulation level was found in tissue adjacent to the outermost cell layers. The highest level of expression of hypB occurs early in development, during the primordium differentiation, with a strong accumulation of hypB transcripts in mature mushrooms only in the transitional zone, between cap and stipe tissue (de Groot et al., 1999). Similar complex profiles are also demonstrated in hydrophobins produced by S. commune, with the expression of hydrophobin genes affecting hyphal wall composition (van Wetter et al., 2000). The biological significance of our findings concerning the hydrophobin complex expression pattern are still unclear, since the biochemistry and molecular biology of P. brasiliensis dimorphism are yet poorly understood. It is well established that during the dimorphic transition,
P. brasiliensis cell wall composition and structure undergoes remarkable changes, revealing important differences in the two morphological cell types (San-Blas, 1985; San-Blas and Ni~ no-Vega, 2001). However, at this moment, we cannot assume any direct correlation between hydrophobin mRNA accumulation and morphological events in P. brasiliensis. The detection of Pbhyd1 and Pbhyd2 mRNAs throughout the first 24 h after temperature shift could be explained by previous results, showing that the complete dimorphic process from M to Y cells, in vitro, takes place in about 15 days (Silva et al., 1994). In these experiments, it was observed that even 4 days after the temperature shift, the percentage of yeast cells in the culture corresponded to less than 20%, suggesting a delay of morphological changes with respect to the start of alterations in gene expression. Hence, the use of RNA from cells in later stages of the dimorphic transition could provide a better evaluation of the expression kinetics of Pbhyd1 and Pbhyd2 genes. In a similar way, Goldman et al. (2003), using real-time quantitative reverse transcription-PCR, have shown that Pbhyd2 mRNA was typically accumulated in the mycelium phase of P. brasiliensis, as well as during the first 24 h of M to Y dimorphic transition. However, curiously in the same publication, the authors have described about 1000 ESTs, corresponding to Pbhyd2, generated from the yeast phase cDNA library of P. brasiliensis. At the moment we cannot explain these apparently contradictory findings. Finally, the characterization of the promoter sequences of these genes (work in progress) will help us to better understand the mechanisms involved in gene regulation underlying the dimorphic transition of P. brasiliensis.
Acknowledgments We thank Dra. Patrıcia Cisalpino for providing us with the antibody anti-GST, Mauro Xavier and Alessandra Ericsson Xavier for helpful experimental support in the heterologous expression and glutathione–Sepharose chromatography purification, and Maria F atima L. Cesario, Josefa Ivanildes S. de Santana, and Celso A. Tavares for laboratory assistance. This work was supported by CNPq/MCT and PADCT/CNPq. PA was supported by CAPES fellowship.
References Ando, A., Harada, A., Miura, K., Tamai, Y., 2001. A gene encoding a hydrophobin, fvh1, is specifically expressed after the induction of fruiting in the edible mushroom Flammulina velutipes. Curr. Genet. 39, 190–197.
P. Albuquerque et al. / Fungal Genetics and Biology 41 (2004) 510–520 Asgeirsdottir, S.A., de Vries, O.M., Wessels, J.G., 1998. Identification of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom). Microbiology 144, 2961–2969. Asgeirsdottir, S.A., van Wetter, M., Wessels, J.G.H., 1995. Differential expression of genes under control of the mating-type genes in the secondary mycelium of Schizophyllum commune. Microbiology 141, 1281–1288. Bainbridge, B.W., Spreadbury, C.L., Scalise, F.G., Cohen, J., 1990. Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi. FEMS Microbiol. Lett. 54, 113–117. Bell-Pedersen, D., Dunlap, J.C., Loros, J.J., 1996. Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene. Mol. Cell. Biol. 16, 513–521. Bessette, P.H., Aslund, F., Beckwith, J., Georgiou, G., 1999. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. USA 96, 13703–13708. Bolyard, M.G., Sticklen, M.B., 1992. Expression of a modified Dutch elm disease toxin in Escherichia coli. Mol. Plant Microbe Interact. 5, 520–524. Carvalho, M.J.A., Jesuino, R.S.A., Daher, B.S., Silva-Pereira, I., Freitas, S.M., Soares, C.M.A., Felipe, M.S.S., 2003. Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis. Fungal Genet. Biol. 39, 204–210. de Groot, P.W., Roeven, R.T., Van Griensven, L.J., Visser, J., Schaap, P.J., 1999. Different temporal and spatial expression of two hydrophobin-encoding genes of the edible mushroom Agaricus bisporus. Microbiology 145, 1105–1113. de Vocht, M.L., Reviakine, I., Wosten, H.A., Brisson, A., Wessels, J.G., Robillard, G.T., 2000. Structural and functional role of the disulfide bridges in the hydrophobin SC3. J. Biol. Chem. 275, 28428–28432. Ebbole, D.J., 1997. Hydrophobins and fungal infection of plants and animals. Trends Microbiol. 5, 405–408, Review. Felipe, M.S., Andrade, R.V., Petrofeza, S.S., Maranh~ao, A.Q., Torres, F.A., Albuquerque, P., Arraes, F.B., Arruda, M., Azevedo, M.O., Baptista, A.J., Bataus, L.A., Borges, C.L., Campos, E.G., Cruz, M.R., Daher, B.S., Dantas, A., Ferreira, M.A., Ghil, G.V., Jesuino, R.S., Kyaw, C.M., Leit~ao, L., Martins, C.R., Moraes, L.M., Neves, E.O., Nicola, A.M., Alves, E.S., Parente, J.A., Pereira, M., Pocas-Fonseca, M.J., Resende, R., Ribeiro, B.M., Saldanha, R.R., Santos, S.C., Silva-Pereira, I., Silva, M.A., Silveira, E., Sim~ oes, I.C., Soares, R.B., Souza, D.P., De-Souza, M.T., Andrade, E.V., Xavier, M.A., Veiga, H.P., Venancio, E.J., Carvalho, M.J., Oliveira, A.G., Inoue, M.K., Almeida, N.F., Walter, M.E., Soares, C.M., Brigido, M.M., 2003. Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis. Yeast 20, 263–271. Goldman, G.H., Marques, E.R., Ribeiro, D.C.D., Bernardes, L.A.S., Quiapin, A.C., Vitorelli, P.M., Savoldi, M., Semighini, C.P., Oliveira, R.C., Nunes, L.R., Travassos, L.R., Puccia, R., Batista, W.L., Ferreira, L.E., Moreira, J.C., Bogossian, A.P., Tekaia, F., Nobrega, M.P., Nobrega, F.G., Goldman, M.H.S., 2003. Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes. Eukaryot. Cell 2, 34–48. Higgins, D.G., Thompson, J.D., Gibson, T.J., 1996. Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 266, 383–402. Kershaw, M.J., Talbot, N.J., 1998. Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis. Fungal Genet. Biol. 23, 18–33. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.
519
Liang, P., Averboukh, L., Pardee, A.B., 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res. 21, 3269–3275. Lugones, L.G., Wosten, H.A., Wessels, J.G., 1998. A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom). Microbiology 144, 2345–2353. Lugones, L.G., Bosscher, J.S., Scholtmeyer, K., de Vries, O.M., Wessels, J.G., 1996. An abundant hydrophobin (ABH1) forms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies. Microbiology 142, 1321–1329. Marck, C., 1988. DNA Strider: a ‘‘C’’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 6, 1829–1836. McEwen, J.G., Garcia, A.M., Ortiz, B.L., Botero, S., Restrepo, A., 1995. In search of the natural habitat of Paracoccidioides brasiliensis. Arch. Med. Res. 26, 305–306. Mount, S.M., 1982. A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459–472. Nakari-S€etal€a, T., Aro, N., Kalkkinen, N., Alatalo, E., Penttila, M., 1996. Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI. Eur. J. Biochem. 235, 248–255. Nielsen, P.S., Clark, A.J., Oliver, R.P., Huber, M., Spanu, P.D., 2001. HCf-6, a novel class II hydrophobin from Cladosporium fulvum. Microbiol. Res. 156, 59–63. Paris, S., Duran, S., 1985. Cyclic adenosine 30 ,50 monophosphate (cAMP) and dimorphism in the pathogenic fungus Paracoccidioides brasiliensis. Mycopathologia 92, 115–120. Paris, S., Debeaupuis, J.P., Crameri, R., Carey, M., Charles, F., Prevost, M.C., Schmitt, C., Philippe, B., Latge, J.P., 2003. Conidial hydrophobins of Aspergillus fumigatus. Appl. Environ. Microbiol. 69, 1581–1588. Parta, M., Chang, Y., Rulong, S., Pinto-DaSilva, P., Kwon-Chung, K.J., 1994. HYP1, a hydrophobin gene from Aspergillus fumigatus, complements the rodletless phenotype in Aspergillus nidulans. Infect. Immun. 62, 4389–4395. Pe~ nas, M.M., Asgeirsdottir, S.A., Lasa, I., Culianez-Macia, F.A., Pisabarro, A.G., Wessels, J.G., Ramirez, L., 1998. Identification, characterization, and in situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus. Appl. Environ. Microbiol. 64, 4028–4034. Restrepo, A., 1985. The ecology of P. brasiliensis: a puzzle still unsolved. J. Med. Vet. Mycol. 23, 323–334. Restrepo, A., McEwen, J.G., Castaneda, E., 2001. The habitat of Paracoccidioides brasiliensis: how far from solving the riddle? Med. Mycol. 39, 233–241. Saitou, N., Nei, M., 1987. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. San-Blas, G., 1985. Paracoccidioides brasiliensis: cell wall glucans, pathogenicity, and dimorphism. Curr. Top. Med. Mycol. 1, 235–257. San-Blas, G., Ni~ no-Vega, G., 2001. Paracoccidioides brasiliensis: virulence and host response. In: Cihlar, R.L., Calderone, R.A. (Eds.), Fungal Pathogenesis: Principles and Clinical Applications. Marcel Dekker, New York, pp. 205–226. San-Blas, G., Nino-Vega, G., Iturriaga, T., 2002. Paracoccidioides brasiliensis and paracoccidioidomycosis: molecular approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics. Med. Mycol. 40, 225–242. Scholtmeijer, K., Wessels, J.G., Wosten, H.A., 2001. Fungal hydrophobins in medical and technical applications. Appl. Microbiol. Biotechnol. 56, 1–8, Review. Schuren, F.H., Wessels, J.G., 1990. Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes. Gene 90, 199–205.
520
P. Albuquerque et al. / Fungal Genetics and Biology 41 (2004) 510–520
Segers, G.C., Hamada, W., Oliver, R.P., Spanu, P.D., 1999. Isolation and characterisation of five different hydrophobin-encoding cDNAs from the fungal tomato pathogen Cladosporium fulvum. Mol. Gen. Genet. 261, 644–652. Silva, S.P., Felipe, M.S.S., Pereira, M., Azevedo, M.O., Soares, C.M.A., 1994. Phase transition and stage-specific protein synthesis in the dimorphic fungus Paracoccidioides brasiliensis. Exp. Mycol. 18, 294–299. Silva, S.P., Borges-Walmsley, M.I., Pereira, I.S., Soares, C.M., Walmsley, A.R., Felipe, M.S., 1999. Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis. Mol. Microbiol. 31, 1039–1050. Silva-Pereira, I., Bey, F., Coux, O., Scherrer, K., 1992. Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes. Gene 120, 235–242. Stewart, E.J., Aslund, F., Beckwith, J., 1998. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J. 17, 5543–5550. Stringer, M.A., Dean, R.A., Sewall, T.C., Timberlake, W.E., 1991. Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation. Genes Dev. 5, 1161–1171. Talbot, N.J., Kershaw, M.J., Wakley, G.E., De Vries, O., Wessels, J., Hamer, J.E., 1996. MPG1 encodes a fungal hydrophobin involved in surface interactions during infectionrelated development of Magnaporthe grisea. Plant Cell 8, 985–999.
van Wetter, M.A., Wosten, H.A., Sietsma, J.H., Wessels, J.G., 2000. Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune. Fungal Genet. Biol. 31, 99–104. Venancio, E.J., Kyaw, C.M., Mello, C.V., Silva, S.P., Soares, C.M.A., Felipe, M.S.S., Silva-Pereira, I., 2002. Identification of differentially expressed cDNAs in the human pathogenic fungus Paracoccidioides brasiliensis by differential display. Med. Mycol. 40, 45–51. Wessels, J.G.H., 1997. Hydrophobins: proteins that change the nature of the fungal surface. Adv. Microb. Physiol. 38, 1–45. Wessels, J.G.H., 1999. Fungi in their own right. Fungal Genet. Biol. 27, 134–145. Wessels, J.G.H., 2000. Hydrophobins, unique fungal proteins. Mycologist 14, 153–159. W€ osten, H.A., 2001. Hydrophobins: multipurpose proteins. Annu. Rev. Microbiol. 55, 625–646. W€ osten, H.A., de Vocht, M.L., 2000. Hydrophobins, the fungal coat unravelled. Biochim. Biophys. Acta 1469, 79–86. W€ osten, H.A., Schuren, F.H., Wessels, J.G.H., 1994. Interfacial selfassembly of a hydrophobin into an amphipathic protein membrane mediates fungal attachment to hydrophobic surfaces. EMBO J. 13, 5848–5854. Yaguchi, M., Pusztai-Carey, M., Roy, C., Surewicz, W.K., Carey, P.R., Stevenson, K.J., Richards, W.C., Takai, S., 1993. Amino acid sequence and spectroscopic studies of Dutch elm disease toxin, cerato-ulmin. In: Sticklen, M.B., Sherald, J.L. (Eds.), Dutch Elm Disease Research, Cellular and Molecular Approaches. SpringerVerlag, New York, pp. 152–170.