mycological research 113 (2009) 1091–1096
journal homepage: www.elsevier.com/locate/mycres
Transcription levels of CHS5 and CHS4 genes in Paracoccidioides brasiliensis mycelial phase, respond to alterations in external osmolarity, oxidative stress and glucose concentration Gustavo A. NIN˜O-VEGA*, Franc¸oise SORAIS, Gioconda SAN-BLAS Instituto Venezolano de Investigaciones Cientı´ficas (IVIC), Centro de Microbiologı´a y Biologı´a Celular, Apartado 20632, Caracas 1020A, Venezuela
article info
abstract
Article history:
The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin
Received 24 March 2009
synthase revealed a 5583 nt open reading frame, interrupted by three introns of 82, 87 and
Received in revised form
97 bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids
7 July 2009
with a predicted molecular weight of 206.9 kDa. Both its large size and the presence of
Accepted 9 July 2009
a N-terminal region of approx. 800 residues with a characteristic putative myosin motor-
Available online 17 July 2009
like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence
Corresponding Editor:
analysis of over 4 kb from the 50 UTR region in CHS5, revealed the presence of a previously
Daniel C. Eastwood
reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements
Keywords:
(STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated
CHS5
by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative
Glucose repression
RT-PCR analysis pointed to changes in transcription levels for both genes following oxida-
Hypo-osmotic stress
tive stress, alteration of external osmolarity and under glucose-repressible conditions, sug-
Myosin motor domain
gesting a common regulatory mechanism of transcription. ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction The fungal wall is a structure that provides protection to the cell, acting as an initial barrier against hostile environments while, at the same time, holding up the cell integrity against its internal turgor pressure. It is a dynamic structure, which could change in composition and structural organization as the cell grows and/or modifies its morphology. These changes are tightly regulated during the cell cycle and in response to changing environmental conditions, stress and mutations in cell wall biosynthetic processes (Klis et al. 2006; Ruiz-Herrera et al. 2006). In yeasts, any stress on the cell wall leads to
compensatory responses (Popolo et al. 2001) such as the upregulation of chitin synthesis, among others. Chitin is one major structural component of the fungal cell wall. It has important functions in wall integrity (Fujiwara et al. 1997; Wang & Szaniszlo 2000), morphogenesis (Cabib et al. 1988) and conidiophore development (Aufauvre-Brown et al. 1997; Fujiwara et al. 2000). Exposure of Candida albicans to cell wall stress by Calcofluor White or CaCl2 induces an increase in both the in vitro chitin synthase activity and the amount of chitin in the cell wall (Munro et al. 2007). Chitin synthesis in fungi is regulated by multigene families encoding chitin synthase isoenzymes, some of them
* Corresponding author. Tel.: 58 212 504 1364; fax: 58 212 504 1382. E-mail address:
[email protected] 0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2009.07.005
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redundant, whose activities may be spatially and strictly regulated to fulfill the several roles ascribed to them (Horiuchi & Takagi 1999). Based on differences in regions of high sequence conservation, chitin synthases have been organized according to their amino acid sequences into seven classes (Bowen et al. 1992; Bulawa 1993; Mandel et al. 2006; Munro & Gow 2001; Nin˜o-Vega et al. 2004a; Specht et al. 1996) whose functional implications are not always clear. Following homology analyses of sixty fungal chitin synthases, such classes have been grouped into two divisions (Ruiz-Herrera et al. 2002). Classes V and VII chitin synthases are both characterized by long sequences, usually over 1700 amino acids, clearly distinct from other chitin synthases whose lengths are less than 1300 amino acids. Both classes V and VII have chitin synthase domains at their C-termini; instead, class V chitin synthase hosts a N-terminus with a myosin motor-like domain, characterized by signatures like P-loop or switch I and switch II, that is absent in class VII chitin synthases; such characteristic is determinant in their separate classification (Amnuaykanjanasin & Epstein 2003; Mandel et al. 2006; Martı´n-Urdı´roz et al. 2008; Nin˜o-Vega et al. 2004a, b; Park et al. 1999). Class V chitin synthases have been shown to be subject to transcriptional regulation in Aspergillus nidulans, Fusarium oxysporum and Wangiella dermatitidis (Takeshita et al. 2002; Madrid et al. 2003; Liu & Szaniszlo 2007; Martı´n-Urdı´roz et al. 2008; Abrahamczyk et al. 2009). A characteristic head-to-head configuration of genes belonging to classes V and VII chitin synthases has been reported in A. nidulans (Takeshita et al. 2006), Coccidioides posadasii (Mandel et al. 2006) and F. oxysporum (Martı´nUrdı´roz et al. 2008). In A. nidulans, this configuration has been related to a common transcriptional regulation for csmA and csmB genes (Takeshita et al. 2006), while in F. oxysporum, chsV regulation is independent from that of chsVb (Martı´n-Urdı´roz et al. 2008). In Paracoccidioides brasiliensis, a dimorphic fungal pathogen, chitin is one of the major components of its cell wall (San-Blas 1985); in it, six different chitin synthase genes have been identified (Nin˜o-Vega et al. 1998, 2000; Tomazett et al. 2005) and the full sequences of two of them reported (Nin˜o-Vega et al. 1998, 2004a). From the deduced amino acid sequences of the encoded products, P. brasiliensis chitin synthases have been classified into six of the seven proposed chitin synthase classes (Nin˜o-Vega et al. 2000, 2004a; Tomazett et al. 2005), the exception being class III chitin synthase. In this study, we describe the cloning and complete sequence of CHS5, a gene encoding a class V chitin synthase, from the pathogenic fungus P. brasiliensis, and its arrangement within the genome in a head-to-head configuration with CHS4 (Nin˜o-Vega et al. 2004a, b), both genes sharing a common 50 UTR. Changes in transcript levels for both genes, following oxidative stress, alterations of external osmolarity and glucose concentration, suggest a common regulatory mechanism of transcription.
Materials and methods Strains, media and growth conditions Paracoccidioides brasiliensis strain IVIC Pb73 (ATCC 32071) was grown as previously reported (Nin˜o-Vega et al. 2000).
G. A. Nin˜o-Vega et al.
Escherichia coli either XL1-Blue (Stratagene Ltd., Cambridge, UK) or TOP10 (Invitrogen, Carlsbad, USA), was used for propagation of plasmids and cloning experiments, and was grown in Luria-Bertani (LB) medium supplemented with 100 mg ml1 ampicillin (Sambrook et al. 1989). For analysis of gene expression under changes in osmotic pressure, P. brasiliensis in its mycelial form was grown in liquid YPD (0.5 % yeast extract, 0.5 % bactopeptone, 1.5 % glucose) supplemented with either 0.3, 0.6, or 1.2 M KCl (final concentrations) and incubated at 23 C for 3 d. RNA was extracted from the centrifuged mycelial culture. To study the effect of glucose on gene expression, the fungus was precultured in its mycelial phase at 23 C for 48 h in YPD liquid medium with a higher glucose concentration (3 %). To study eventual derepression of promoters, cells were collected by filtration, washed once with sterile water and then transferred to YPD medium containing a lower glucose concentration (0.05 %). Samples (100 ml) were taken at 2, 4, and 8 h after the shift to low glucose concentration, and total RNA extracted. For analysis of gene expression under oxidative stress, mycelial cells growing at 23 C for 3 d were subdivided in two aliquots, each aliquot collected by filtration and washed once with sterile water. Cells collected from one aliquot were transferred to 50 ml of fresh YPD medium, while those from the second aliquot were resuspended into 50 ml of fresh YPD medium supplemented with 5 mM H2O2, and both cultures incubated at 23 C for 20 min. Total RNA was extracted from each sample for expression analysis.
Nucleic acids isolation Genomic DNA was isolated as previously described (Nin˜oVega et al. 2000). Total RNA was prepared from ground cells (30 mg) using the AxyPrep multisource total RNA miniprep kit (Axygen Biosciences, Union City, CA, USA). Plasmid DNA extraction was done by the CONCERT mini kit (Life Technologies, Carlsbad, USA).
Isolation and sequencing of CHS5 For the isolation of CHS5, a previously reported partial EcoRI genomic library was screened with a radiolabelled PCR-generated CHS5 fragment (Nin˜o-Vega et al. 2000). For screening, the library was plated at a density of 250–300 colonies per plate, 10 plates per screening. Six positive clones were obtained and their plasmid inserts (about 3.7 kb) analysed by Southern hybridisation under high stringency conditions (Sambrook et al. 1989), using the labelled PCR fragment as probe. All of them showed positive signals and one was chosen for plasmid isolation and sequencing. The plasmid was designated pGAN8. Sequencing was automated by primer walking in both directions (CeSAAN, Centro de Microbiologı´a y Biologı´a Celular, IVIC, Caracas, Venezuela). Sequencing analysis of the pGAN8 insert revealed only 3425 bp up to the stop codon, but lacking the upstream region of the gene. For the isolation of the 50 region, a PCR-‘‘stepdown’’ technique (Zhang & Gurr 2000) was used. Primers GSP3 (50 -CGGCTGCCAGAAACGACAACATCGTCG-30 ) and GSP4 (50 -TCGGAGCGAGAGTCG ATTTCACGAGCG-30 ) were designed on the incomplete 50 region of the fragment obtained from the genomic library, towards the 50 region of the gene. From here, procedure was as follows: 1 mg
Up-regulation of P. brasiliensis CHS4 and CHS5
Paracoccidioides brasiliensis DNA was digested with NheI (New England Biolabs, Inc, Beverly, USA) and ligated to adaptors (Zhang & Gurr 2000). Amplification and reamplification were done with the Advantage 2 Polymerase Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA). A second round of amplifications was done with the specific primers GSP5 (50 -GGCGAGGAAGGAAGAAAGAAGATGGGAG-30 ) and GSP6 (50 -TCTGCTTCCCCCA TCGCACTCCCTTCTT-30 ), using NgoMIV (New England Biolabs, Inc, Beverly, USA) as the restriction enzyme. Amplified products were cloned into pCRII-TOPO (Invitrogen, Carlsbad, USA), and plasmids transformed into Escherichia coli TOP-10. Insert fragments were sequenced by primer walking in both directions (Macrogen Sequencing System, Seoul, South Korea). The complete sequence can be accessed at GenBank under accession number EF654132.
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subsequent experiments. Quantitative PCR was performed in triplicate on an iQ5 real time PCR detection system, using the iQ SYBR Green SuperMix (Bio-Rad Laboratories, Hercules, CA, USA), in a 25-ml volume (12.5 ml Supermix, 1.25 ml 5 mM of each forward and reverse primer, and 2.5 ml cDNA). Reaction conditions were 95 C for 3 min, followed by 40 cycles of 95 C for 10 s, 60 C for 30 s, and 72 C for 30 s, with dissociation conditions of 95 C for 1 min, 55 C for 1 min, and 81 cycles starting at 55 C, with temperature increases of 0.5 C every 10 s up to 95 C. PCRs with serial dilutions of Paracoccidioides brasiliensis DNA or cDNAs as template were used to calculate the amplification efficiency for each pair of primers. All Ct values were normalized to the Ct values of the standard genes and the relative expression levels were calculated using the Pfaffl method (Pfaffl 2001).
Intron confirmation This was done either with the SuperScript pre-amplification system or 30 RACE (Invitrogen, Carlsbad, USA) using specific primers CHS5-INT-F (50 -ATGACCCTCAATTCGTTGACG-30 ), CHS5-INT-R (50 -GCGAGGAA GGAAGAAAGA AG-30 ) and CHS5DOWN2 (50 -TTTCATGGGGGAACACCCGTATCGTCAC-30 ). In order to determine exon–intron boundaries, the amplified products were sequenced and compared with the corresponding genomic sequence.
Computational analysis Sequence assembly was done with the Contig (v 1.0.0.0) program, from the Vector NTI Suite (InforMax, Inc., USA). Deduced amino acid sequences were compared against the GenBank database, using Blast 2.0 (Altschul et al. 1997) and Clustal W (Thompson et al. 1994).
Quantitative RT-PCR Total RNA was treated with DNase by using the TURBO DNAfree kit (Ambion Inc., Austin, TX, USA). The RETROScript kit (Ambion Inc., Austin, TX, USA) was used for reverse transcription of mRNA. For real-time PCR of CHS4, primers CHS4 INT S5: 50 -ACCGGATGAGGCCACTATTACAGA-30 and CHS4 INT AS5: 50 -GTCTGCAATCGCTGCTCAACG-30 were used. For expression analysis of CHS5, sequence specific primers CHS5 INT S2: 50 -AGAGTATCAAGGCTGAGCTGGAACG-30 and CHS5 INT AS2 50 -CGGAAAGGACGGCTTCGGTT-30 were designed. To find the best internal control as normalizer for the expression experiments, two genes were used. Amplification of 18S rRNA was carried out, using the primer pair 18S S3: 50 -CGATTCCGGAGAGGGAGCC-30 and 18S AS3: 50 -CGTATCGGGATTGGGTAA TTTGC-30 . A second reference gene (ODC ) which has no changes in transcription levels during growth (Nin˜o-Vega et al. 2004b) was also used as follows: ODC S1: 50 -TGATTTGCGAGCGTATTGCCC-30 and ODC AS1: 50 -GCGTTGCGCTGCAC TTGGTAT-30 . In experiments aimed to evaluate changes induced by KCl hypo-osmolarity, similar results were obtained for both control genes. However, changes in expression levels of the ODC gene were observed in experiments designed to evaluate the effect of glucose concentration. Therefore, the 18S rRNA gene was chosen as the normalizer for all
Results CHS5 cloning was achieved by means of two strategies: (a) the 30 region is the result of scrutiny of the EcoRI plasmidic library, of approx. 3.7 kb size; (b) by using the Zhang & Gurr (2000) method, we were able to obtain a long 50 region of around 7 kb, including over 4 kb, well into the putative 50 untranslated region (50 UTR). The complete CHS5 sequence resulted into a 5583 bp long ORF, with three introns of 82 bp (nucleotides 114–195), 87 bp (nucleotides 385–471) and 97 bp (nucleotides 5724–5820), all confirmed by comparing the RT-PCR product sequences with the corresponding genomic sequences. From the deduced amino acid sequence of its translated product, Chs5p accounts for 1861 amino acids, with a molecular weight of 206.934 kDa. Sequence analysis of the 50 UTR resulted in overlapping with a previously reported sequence containing the CHS4 gene (Nin˜o-Vega et al. 2004a, b), arranged in a head-to-head configuration with CHS5 (Fig 1). The 5’ UTR sequence embraced 3795 bp between their respective translational start codons.
Fig 1 – Schematic representation of the head-to-head configuration of Paracoccidioides brasiliensis CHS5 and CHS4 genes. Black boxes represent the genes. White arrows represent direction of transcription and grey boxes represent untranslated regions. A projection of the intergenic region is represented, featuring possibles Rlm1p (hatched boxes) and clusters of Adr1p (white boxes) binding sites.
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Alterations of the external osmolarity by KCl supplementation of the culture medium led to growth halt when KCl concentration was 0.6 M or above; instead, a 0.3 M concentration allowed growth at a lower rate (0.16 h1) than control cultures grown in KCl-deprived YPD (0.87 h1); so, 0.3 M KCl was used for transcription analysis. As deduced from quantitative RT-PCR, transcription levels of both CHS4 and CHS5 were higher in the absence of KCl than at higher osmotic conditions, e.g., in the presence of 0.3 M KCl (Fig 2). On the other hand, low glucose concentrations (0.05 % instead of 3 %) provoked a derepression of transcription in both genes within the first 2 h, only to drop afterwards (Fig 3), as reported for Adr1 (alcohol dehydrogenase gene regulator 1)-dependent genes in Saccharomyces cerevisiae (Agricola et al. 2004). Oxidative stress due to mycelial growth in presence of 5 mM H2O2, induced higher transcription levels of PbrCHS4 and PbCHS5 when compared with cultures growing in absence of H2O2 (Fig 4).
Discussion Previous results on partial sequences (Nin˜o-Vega et al. 2000) suggested a 6.7 kb transcript size for CHS5, and an amino acid sequence highly similar to other class V fungal chitin synthases, results that were confirmed herein. Highest identities were found with Coccidioides posadasii Chs5 and Coccidioides immitis Chs5 (80 %), Aspergillus oryzae ChsY, Emericella nidulans CsmA, and Aspergillus fumigatus ChsE (75 %), Wangiella dermatitidis Chs5 (72 %), and Blumeria graminis Chs2 (69 %). Two domains were identified; the first one, characteristic of class V chitin synthases, towards the N-terminal end of the protein (aa 16–786), presents partial identity to myosin motor-like domains, e.g., P-loop ‘‘GESGSGKT’’ (aa 102–109), switch I ‘‘ASKAG’’ (aa 152–156) and switch II ‘‘DFPGFA’’ (aa 410–415)
Fig 2 – Transcriptional levels of CHS4 and CHS5 of Paracoccidioides brasiliensis, mycelial phase, in response to alteration of the external osmolarity. Quantitative RT-PCR analysis was performed on total RNA of P. brasiliensis ATCC 32071 grown on YPD and YPD D 0.3 M KCl liquid medium. As normalizer gene control, the 18S rRNA gene was used under each osmotic condition.
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Fig 3 – Transcriptional levels of CHS4 and CHS5 of Paracoccidioides brasiliensis, mycelial phase, in response to changes in glucose content of YPD liquid medium. Quantitative RT-PCR analysis was performed on total RNA extracted from P. brasiliensis ATCC 32071 grown on YPD with 3 % glucose for 48 h (R, repressing conditions), after which the culture was washed and resuspended in YPD with a lower glucose content (0.05 %, derepressing conditions), and aliquots of resuspended cells taken at 2, 4, and 8 h post-resuspension in the lower glucose condition medium. As normalizer gene control, the 18S rRNA gene was used.
sequences. This domain plays important roles in polarized cell wall synthesis and maintenance of normal hyphal growth (Takeshita et al. 2006). The second domain towards the C-terminal end (aa 1221–1752) has homology to fungal chitin synthases. Contrariwise, PbrChs4, while being a protein as large as PbrChs5, lacked such sequences, a feature that classifies it in class VII, apart from PbrChs5 (Nin˜o-Vega et al. 2004a).
Fig 4 – Transcriptional levels of CHS4 and CHS5 of Paracoccidioides brasiliensis, mycelial phase, in response to oxidative stress. Quantitative RT-PCR analysis was performed on total RNA of P. brasiliensis ATCC 32071 grown on YPD and YPD D 5 mM H2O2 liquid medium. As normalizer gene control, the 18S rRNA gene was used.
Up-regulation of P. brasiliensis CHS4 and CHS5
The head-to-head arrangement in Paracoccidioides brasiliensis CHS4 and CHS5 has also been reported in genes coding for other classes V and VII chitin synthases in Aspergillus nidulans, C. posadasii and Fusarium oxysporum (Mandel et al. 2006; Martı´n-Urdı´roz et al. 2008; Takeshita et al. 2006). A common transcriptional regulation for csmA (coding a class V chitin synthase) and csmB (coding a class VII chitin synthase) in A. nidulans has been suggested (Takeshita et al. 2002; Takeshita et al. 2006). This feature may also be suggested for P. brasiliensis CHS4 and CHS5, in view of the fact that analysis in silico of its shared promoter region yielded sequences that matched several binding sites for transcription factors and response elements. Among them, there are three potential sites for the transcription activator Rlm1p, known to be stimulated by hypo-osmotic stress in Saccharomyces cerevisiae MAP kinases (Dodou & Treisman 1997; Jung & Levin 1999), playing important roles in cell wall integrity (Lagorce et al. 2003); also RlmAp in A. niger, required for wall reinforcement in response to cell wall stress (Damveld et al. 2005). Adr1p functions in S. cerevisiae as repressor of genes under high glucose concentrations, although it must be present for derepression to occur under low glucose conditions (Agricola et al. 2004; Geng & Laurent 2004). The presence of clusters of Adr1p binding sites in the common 50 UTR region of P. brasiliensis CHS4 and CHS5 (Fig 1) seems to have the same regulatory effect, inasmuch growth under derepressing conditions led to an increase in the levels of transcription of both genes during the first 2 h, followed by a sharp drop in mRNA levels afterwards (Fig 3). Additionally, some stress response elements (STREs) were identified; they might be regulating both chitin synthase genes, as observed in the increase of transcription levels in both PbrCHS4 and PbCHS5 when the fungus is grown under conditions of oxidative stress, namely 5 mM H2O2 (Fig 4). Similar effects have been reported in response to stress in S. cerevisiae (Estruch 2000; Nevitt et al. 2004). Till now, in P. brasiliensis only one single case of shared intergenic promoter region had been reported between two genes, MDJ1 and LON (Batista et al. 2007), whose products Mdj1 and Lon are, respectively, a mitochondrial protein of the heat-shock protein family Hsp40 and a conserved ATPbinding, heat-inducible serine proteinase. Similarly to our findings in the common promoter region between CHS4 and CHS5, MDJ1 and LON have regulatory elements in their shared intergenic region that control transcription in both genes and stimulate their response to heat shock and oxidative stress. Our data represent the second report of such occurrence in P. brasiliensis. Further investigation into gene regulation in P. brasiliensis will help us to understand the relationships among genes involved in its cell wall synthesis, and may provide knowledge on eventual genes for future development of new specific antifungals.
Acknowledgements This work was supported by research project 112 from Instituto Venezolano de Investigaciones Cientı´ficas.
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