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Originally published In Press as doi:10.1074/jbc.M500625200 on May 3, 2005 Originally published In Press as doi:10.1074/jbc.M500625200 on April 27, 2005 Originally published In Press as doi:10.1074/jbc.M500625200 on April 22, 2005

J. Biol. Chem., Vol. 280, Issue 26, 24706-24714, July 1, 2005
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Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells*{boxs}

Maria Sueli S. Felipe,ab Rosângela V. Andrade,ac Fabrício B. M. Arraes,ac André M. Nicola,ac Andréa Q. Maranhão,a Fernando A. G. Torres,a Ildinete Silva-Pereira,a Márcio J. Poças-Fonseca,a Élida G. Campos,a Lídia M. P. Moraes,a Patrícia A. Andrade,a Aldo H. F. P. Tavares,a Simoneide S. Silva,a Cynthia M. Kyaw,a Diorge P. Souza,a PbGenome Network,d Maristela Pereira,a Rosália S. A. Jesuíno,e Edmar V. Andrade,e Juliana A. Parente,e Gisele S. Oliveira,e Mônica S. Barbosa,e Natália F. Martins,f Ana L. Fachin,g Renato S. Cardoso,g Geraldo A. S. Passos,gh Nalvo F. Almeida,i Maria Emília M. T. Walter,j Célia M. A. Soares,e Maria José A. Carvalho,ac and Marcelo M. Brígidoac

From the aDepartamento de Biologia Celular, Universidade de Brasília, 70910-900, Brasília, DF, Brazil, jDepartamento de Ciência da Computação, Universidade de Brasília, 70910-900, Brasília, DF, Brazil, fEmbrapa-Recursos Genéticos e Biotecnologia, W5 Norte, 70770-900, Brasília, DF, Brazil, eDepartamento de Bioquímica, Universidade Federal de Goiás, 74001-970, Goiânia, GO, Brazil, iDepartamento de Computação e Estatística, Universidade Federal de Mato Grosso do Sul, 79070-900, Campo Grande, Mississippi, Brazil, gDepartamento de Genética, Universidade de São Paulo, 14040-900, Ribeirão Preto, SP, Brazil, and hFaculdade de Odontologia, Universidade de São Paulo, 14040-900, Ribeirão Preto, SP, Brazil

Received for publication, January 18, 2005 , and in revised form, March 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The dimorphic human pathogenic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM)1 (1), a major health problem in Latin America. High positive skin tests (75%) in the adult population reinforce the importance of the mycosis in endemic rural areas, where it has been estimated to affect around 10 million individuals, 2% of whom will develop the fatal acute or chronic disease (2). The acute form of PCM chiefly compromises the reticuloendothelial system; the chronic form mainly affects adult males with a high frequency of pulmonary and/or mucocutaneous involvement (1). Chronic severe multifocal PCM may also cause granulomatous lesions in the central nervous system (3). Regardless of the affected organ, PCM usually evolves to the formation of fibrotic sequelae, permanently hindering the patient's health.

P. brasiliensis Undergoes a Dimorphic Process in Vivo—It is assumed that the fungus exists as a soil saprophyte, producing propagules that can infect humans and produce disease after transition to the pathogenic yeast form (4). Pathogenicity has been intimately associated with this process, since P. brasiliensis strains unable to differentiate into the yeast form are avirulent (5). Mammalian estrogens inhibit dimorphism, explaining the lower incidence of disease in females (6). The mycelium-to-yeast transition in P. brasiliensis is governed by the rise in temperature that occurs upon contact of mycelia or conidia with the human host. In vitro, it can be reversibly reproduced by shifting the growth temperature between 22 and 36 °C. Molecular events related to genes that control signal transduction, cell wall synthesis, and integrity are likely to be involved in this dimorphic transition.

P. brasiliensis genome size was estimated to be ~30 Mb (7). A study of P. brasiliensis gene density suggests that this fungus contains between 7,500 and 9,000 genes,2 which is in agreement with the estimated gene number for ascomycete fungi genomes.

Here are presented the results of an effort to achieve a comprehensive metabolic view of the P. brasiliensis dimorphic life cycle based on analysis of 6,022 groups generated from both mycelium and yeast phases. This view arises from both a general metabolism perspective and the identification of the precise metabolic points that distinguish both morphological phases. Overexpressed genes and those that are up- or down-regulated in both stages were identified. Expression levels were assessed by cDNA microarrays and some were confirmed by Northern blot. Drug targets and genes related to virulence were also detected in several metabolic pathways. Finally, the majority of genes involved in signal transduction pathways (cAMP/protein kinase A, Ca2+/calmodulin, and MAPKs) possibly participating in cell differentiation and infection were annotated, and now we are able to describe the corresponding signaling systems in P. brasiliensis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fungus—P. brasiliensis isolate Pb01 (ATCC MYA-826) was grown at either 22 °C in the mycelium form (14 days) or 36 °C as yeast (7 days) in semisolid Fava Neto's medium. Following incubation, cells were collected for immediate RNA extraction with Trizol reagent (Invitrogen).

Construction of cDNA Libraries and Sequencing—Poly(A)+ mRNA was isolated from total mycelium and yeast RNA through oligo(dT)-cellulose columns (Stratagene). Unidirectional cDNA libraries were constructed in {lambda}ZAPII following supplier's instructions (Stratagene). Phagemids containing fungal cDNA were then mass-excised and replicated in XL-1 Blue MRF' cells. In order to generate ESTs, single pass 5'-end sequencing of cDNAs was performed by standard fluorescence labeling dye terminator protocols with T7 flanking vector primer. Samples were loaded onto a MegaBACE 1000 DNA sequencer (Amersham Biosciences) for automated sequence analysis.

EST Processing Pipeline and Annotation—PHRED quality assessment and computational analysis were carried out as previously described (8). EST assembly was performed using the software package CAP3 (9) plus a homemade scaffolding program. Sequences of at least 100 nucleotides, with PHRED ≥20, were considered for clustering. A total of 20,271 ESTs were selected by these exclusion criteria. Contaminant and rRNA sequences were then removed to generate a set of 19,718 ESTs, which was submitted to CAP3 clustering, generating 2,655 contigs and leaving 3,367 ESTs as singlets. Contigs plus singlets comprise the base set of 6,022 P. brasiliensis assembled EST sequences (PbAESTs) that underwent further analysis. Annotation was carried out using a system that essentially compared these assemblies with sequences available in public databases. The BLASTX program (10) was used for annotation along with GenBankTM nonredundant (nr), cluster of orthologous groups (COG), and gene ontology (GO) data bases. The GO data base was also used to assign EC numbers to assemblies. Additionally, we used the FASTA program (11) to compare assemblies with Saccharomyces cerevisiae and Schizosaccharomyces pombe predicted polypeptides. The INTERPROSCAN program (12) was used to obtain domain and family classification of the assemblies. Metabolic pathways were analyzed using maps obtained in the KEGG Web site (13) with annotated EC numbers, and this information was used to help in assigning function to PbAESTs.

Differential Expression Analysis in Silico by Electronic Subtraction—To assign a differential expression character, the contigs formed with mycelium and yeast ESTs were statistically evaluated using a test previously described (14) with a confidence of 95%.

cDNA Microarrays and Data Analysis—A set of two microarrays containing a total of 1,152 clones in the form of PCR products was spotted in duplicate on 2.5 x 7.5-cm Hybond N+ nylon membranes (Amersham Biosciences). Arrays were prepared using a Generation III Array Spotter (Amersham Biosciences). Complementary DNA inserts of both P. brasiliensis libraries were amplified in 96-well plates using vector-PCR amplification with T3 forward and T7 reverse universal primers. Membranes were first hybridized against the T3 [{alpha}-33P]dCTP-labeled oligonucleotide. The amount of DNA deposited in each spot was estimated by the quantification of the obtained signals. After stripping, membranes were used for hybridization against {alpha}-33P-labeled cDNA complex probes. The latter were prepared by reverse transcription of 10 µg of filamentous or yeast P. brasiliensis total RNA using oligo(dT)12–18 primer. One hundred microliters of [{alpha}-33P]cDNA complex probe (30–50 million cpm) was hybridized against nylon microarrays. Imaging plates were scanned by a phosphor imager (Cyclone; Packard Instruments) to capture the hybridization signals. BZScan software was employed to quantify the signals with background subtraction. Spots were matched with a template grid. The ratio between vector and cDNA complex probe hybridization values for each spot was used as the reference normalization value. Total intensity normalization using the median expression value was adopted as previously described (15). Gene expression data analyzed here were obtained from three independent determinations for each phase (filamentous or yeast). We used the significance analysis of microarrays method (16) to assess the significant variations in gene expression between both mycelium and yeast. Briefly, this method is based on t test statistics, specially modified to high through-put analysis. A global error chance, the false discovery rate, and a gene error chance (q value) are calculated by the software.

Northern Blot Analysis—Total RNA (15 µg) was separated in a 1.5% denaturing formaldehyde agarose gel and transferred to a Hybond-N nylon membrane (GE Healthcare). Probes were radiolabeled with the random primers DNA labeling system (Invitrogen) using [{alpha}-32P]dATP. Membranes were incubated with the probes in hybridization buffer (50% formamide, 4x SSPE, 5x Denhardt's solution, 0,1% SDS, 100 µg/ml herring sperm DNA) at 42 °C overnight and then washed twice (2x SSC, 1% SDS) at 65 °C for 1 h. Signal bands were visualized using a Typhoon 9210 phosphor imager (GE Healthcare).

URLs—Details of the results and raw data are available for download from the World Wide Web: Pbgenome project Web site (www.biomol.unb.br/Pb); Gene Ontology Consortium (www.geneontology.org); Cluster of Ortologous Genes (www.ncbi.nlm.nih.gov/COG); INTER-PROSCAN (www.ebi.ac.uk/interpro/); National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/); Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp/kegg); BZScan Software (tagc.univ-mrs.fr); Audic and Claverie statistical test (telethon.bio.unipd.it/bioinfo/IDEG6_form/); Significance Analysis of Microarrays method (www-stat.stanford.edu/~tibs/SAM/); Candida albicans data base (genolist.pasteur.fr/CandidaDB/); genomes from Aspergillus nidulans and Neurospora crassa (www.broad.mit.edu/annotation/fungi/aspergillus/).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transcriptome Features—In sequencing the P. brasiliensis transcriptome, EST data were generated from nonnormalized cDNA libraries of mycelium and yeast cells. The size range of the cDNA inserts ranged from 0.5 to 2.5 kb. Single pass 5' sequencing was performed on 25,598 cDNA clones, randomly selected from both libraries. Upon removal of bacterial and rRNA contaminant sequences, a total of 19,718 high quality ESTs underwent CAP3 assembly, yielding 2,655 contigs and 3,367 singlets, which constitute the so-called 6,022 P. brasiliensis Assembled EST (PbAEST) data base. Contigs presented an average size of 901 bp, and the number of ESTs assembled into contigs varied from 2 to 657 in the largest one (PbAEST 1068), which corresponds to M51, a previously reported P. brasiliensis mycelium-specific transcript (17). Of the 6,022 PbAESTs, 4,198 (69.4%) showed a probable homologue in GenBankTM, and 4,130 (68.3%) showed a fungus homologue (Fig. 1A and Supplemental Table I). We had used MIPS functional categories to classify 2,931 PbAESTs into 12 major groups. P. brasiliensis showed a slightly higher percentage of PbAESTs (4%) related to cellular communication and signal transduction (Fig. 1B) compared with S. cerevisiae functional categorization (3.4%).

Highly and Differentially Expressed Genes—The 27 highly transcribed genes found in the P. brasiliensis transcriptome, using a cut-off of 50 reads, are shown in Supplemental Table II. Some of them were previously reported (8). Also, up- and down-regulated genes in mycelium and yeast cells were detected by statistical comparison of the number of sequences in corresponding PbAESTs (Table I). In order to support the electronic subtraction data, cDNAs from each phase were used to probe cDNA microarrays membranes containing 1,152 clones, which were selected based on the following criteria: (i) ESTs exclusive for a particular morphotype; (ii) ESTs corresponding to genes more expressed in mycelium or yeast cells; and (iii) some ESTs equally expressed in both cell types. From the 1,152 clones, 328 genes were up-regulated during the dimorphic transition: 58 in mycelium and 270 in yeast (data not shown).


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  		TABLE I
Differentially expressed genes in mycelium and yeast cells detected by electronic subtraction and cDNA microarray analysis The PbAESTs were analyzed as to their differential expression by two methods: a statistical analysis of the number of mycelium and yeast ESTs clustered in each PbAEST (14) and a cDNA microarray analysis of 1,152 PbAESTs, chosen according to the electronic subtraction criteria. A differential pattern of genes encoding enzymes was used in the analysis of the differential metabolism.

 



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  		FIG. 1.
P. brasiliensis transcriptome characterization. A, distribution of blast best hit among organisms. Each PbAEST was tested against the GenBankTM nr data base, and the best hit organism was computed. A PbAEST was considered as not assigned when the best hit exceed an E value of 10-10. B, functional categorization of the PbAESTs using MIPS classification. We included 2931 curator-reviewed annotations in this analysis.

 
The cDNA microarray experiment confirmed most of the electronic subtraction data and also points out to new differentially expressed genes. Among them, a subclass of about 40 up-regulated genes in mycelium and yeast are described in Table I, which includes M51, M32, hydrophobin 1/2, the highly expressed yeast PbY20 protein, and some other genes that have previously been described as differentially expressed in P. brasiliensis by different approaches (17–20). Other key up-regulated genes related to the metabolism of P. brasiliensis (Table I) are described and discussed elsewhere in this work. Interestingly, we have found a yeast phase preferentially expressed gene that possibly encodes a previously characterized P. brasiliensis estradiol-binding protein (21), also described in C. albicans and in other fungi (22). It is speculated that the interaction of the 17-{beta}-estradiol hormone with a cytoplasmic protein inhibits the mycelium-to-yeast transition, explaining the lower incidence of PCM in females.

Metabolic Overview—P. brasiliensis seems to be capable of producing ATP from the classical pathways of glycolysis, alcohol fermentation, and oxidative phosphorylation, since alcohol dehydrogenase, cytochrome genes, ATP synthase subunits, and pyrophosphatase genes were annotated. All genes encoding glycolytic enzymes were identified in both mycelium and yeast. Genes corresponding to the citrate cycle enzymes and to the components of complexes I, II, III, and IV were found, reflecting the ability of the fungus to perform complete aerobic pyruvate degradation and oxidative phosphorylation. Its putative capacity to also grow in anaerobiosis was evidenced by the alternative conversion of pyruvate to ethanol. Last, it may be able to utilize two-carbon sources in the form of acetate and ethanol through the glyoxylate cycle and obtain sulfite and nitrite from the environment.

In order to validate the carbon source utilization profile predicted by the transcriptome data, two P. brasiliensis isolates (Pb01 and Pb18) were grown in McVeigh-Morton minimum medium supplemented with different carbon sources and growth patterns were qualitatively evaluated (Supplemental Table III). We observed that, in accordance to the transcriptome analysis prediction, several mono- and disaccharides, such as D-glucose, D-fructose, D-galactose, D-mannose, D-sorbitol, {alpha}-trehalose, maltose, and sucrose were indeed utilized. On the other hand, the predicted assimilation of D-inositol was not confirmed. Transcripts related to the consumption of L-sorbose and L-lactose were not detected; in fact, P. brasiliensis was unable to grown in L-sorbose as the sole carbon source. We consider that the unpredicted fungal growth in L-lactose can be explained by the fact that the P. brasiliensis cDNA libraries were not constructed under induction conditions. The observation that fructose, galactose, and glycerol were only utilized by Pb01 and not by Pb18 isolate may simply reflect strain biological variability as previously observed (7). A detailed description of P. brasiliensis metabolism, including a list of PbAESTs, is shown in Supplemental Table IV.

Differential Metabolism between Mycelium and Yeast—The up-regulated genes encoding enzymes in mycelium and yeast cells listed in Table I are highlighted in Fig. 2. The differential expression pattern of these genes (with the exception of glucokinase from mycelium cells) was confirmed by Northern blot analysis (Fig. 3). In general, the gene overexpression pattern suggests that mycelium saprophytic cells possess an aerobic metabolism, in contrast with yeast cells. Actually, mycelium up-regulated genes correspond to the main regulatory points of the citrate cycle, such as the genes coding for isocitrate dehydrogenase and succinyl-CoA synthetase; this strongly suggests a metabolic shunt to oxidative phosphorylation. Also, glucokinase is induced, producing glucose 6-phosphate, which is possibly converted through the oxidative pentose phosphate pathway to ribose 5-phosphate, and then to salvage pathways of purine and pyrimidine biosynthesis. In fact, this correlates well with the overexpression of adenylate kinase and uridine kinase genes. The excess of ribose 5-phosphate is probably converted to fructose 6-phosphate and glyceraldehyde 3-phosphate by the nonoxidative pentose phosphate pathway catalyzed by the overexpressed transaldolase. Those sugars are converted to pyruvate and acetyl-CoA for the citrate cycle in aerobic conditions.

In contrast, P. brasiliensis yeast cells overexpress the genes encoding alcohol dehydrogenase I and pyruvate dehydrogenase E1 subunit (Table I and Fig. 3); the latter can be detected in high levels in cultures of S. cerevisiae grown both anaerobically and aerobically in the presence of ethanol (23). The carbohydrate metabolism is probably shifted toward ethanol production, reflecting the anaerobic behavior of the yeast form as previously reported (24). Several pathways that provide substrates for the glyoxylate cycle are up-regulated in the yeast cells (Table I and Fig. 3). First, isocitrate lyase redirects the metabolic flow using ethanol and acetate as two-carbon sources and generating oxaloacetate, which can be reconverted to glucose. In addition, the branched-chain aminotransferase gene is also overexpressed (as are other aminotransferase genes, such as those of aspartate and histidinol-P) and converts valine, leucine, and isoleucine to acetyl-CoA, which is then fed to the cycle. The yeast differential acetamidase also contributes to this pathway by deriving acetate from acetamide. Furthermore, the up-regulated acyl-CoA synthetase generates acetyl-CoA in the first step of {beta}-oxidation, which may also be taken up by the cycle. Finally, the generation of sulfite by phosphoadenylyl sulfate reductase provides acetate for the glyoxylate cycle as mentioned above. The overall analysis suggests that ATP production through alcohol fermentation and the respiratory chain occurs in a biased pattern, the former being preferential in the yeast form and the latter in mycelium.



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  		FIG. 2.
Comparison of the expression pattern of genes encoding for enzymes in mycelium-to-yeast cell differentiation of P. brasiliensis. For the detailed metabolic comparison between mycelium and yeast metabolism, see Supplemental Table IV, since we have presented in this figure only the central pathways for carbohydrate metabolism and citrate cycle. Genes that are overexpressed are boxed, either in mycelium or yeast cells, according to the criteria described in Table I.

 



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  		FIG. 3.
Northern blot analysis of mycelium and yeast up-regulated genes of P. brasiliensis. Total RNA samples from both mycelium (M) and yeast (Y) were blotted onto nylon membranes and hybridized against gene-specific radiolabeled probes. ICDH, isocitrate dehydrogenase; SCS, {beta}-succinyl-CoA-synthetase, TAL, transaldolase; ADK, adenylate kinase; UDK, uridylate kinase; ICL, isocitrate lyase; acyl-CS, acyl-CoA synthetase; PDH, pyruvate dehydrogenase; ADH, alcohol dehydrogenase; PAPsR, phosphoadenylyl sulfate reductase; ACA, acetamidase; AMT, aminotransferase. The constitutive 60 S ribosomal protein L34 was used as a loading control.

 
Yeast cells are rich in chitin; the high expression of chitin deacetylase reveals its possible involvement in cell wall loosening, reorganization, and synthesis of newly components during cell growth and budding of yeast cells. This enzyme is not present in humans and thus represents a possible drug target. In mycelium, overexpression of cystathionine {beta}-synthase and nitroalkane oxidase strongly suggests that mycelium cells take up sulfite and nitrogen, respectively, from the environment for metabolic processing. Finally, the probable role of the remaining overexpressed gene encoding palmitoylthioesterase remains unclear. In contrast, the enzymes isochorismatase and ubiquinone-reductase are greatly up-regulated in the yeast form, strongly suggesting a high production of ubiquinone by P. brasiliensis, which could be involved in cellular oxidative stress under anaerobic conditions. The high yeast expression of dihydropteroate synthase produces, as a consequence, high levels of tetrahydrofolate, which probably will increase the metabolic flow toward purine biosynthesis. The meaning of the high expression in yeast of choline sulfatase, gluconate dehydrogenase, glutamyl-tRNA amidotransferase, and phenylacetate hydroxylase also remains unclear.

Cell Cycle and Genetic Information—The main genes involved in cell cycle and in the basic genetic information flow machinery (DNA replication, repair, recombination, transcription, RNA processing, translation, and post-translational modifications) are well conserved in comparison with their counterparts from S. cerevisiae. Also, sequences related to mitochondrial replication, budding, sporulation, and mating were also annotated (Supplemental Table V).

From the cell cycle-related orthologues identified in P. brasiliensis, those related to the structure and assembly of the cytoskeleton, chromatin structure, chromosome segregation, cyclins, and cell cycle control genes were highlighted. Genes related to the major DNA repair mechanisms found in yeast (mismatch, base excision, and recombination systems) were identified in P. brasiliensis, although not every component was represented, since cells were not subjected to DNA-damaging conditions. The RAD52 gene, which plays an essential role in S. cerevisiae recombination, is also present in the P. brasiliensis transcriptome.

Among the identified transcription factors, the orthologues for MAT, MCM1, and NsdD are of relevance, since they are implicated in ascomycete sexual reproduction. These genes represent a strong evidence for mating in P. brasiliensis, so far not yet described, which is reinforced by the detection of six transcripts involved in meiotic recombination.

Stress Responses—Cell differentiation in P. brasiliensis requires a temperature shift, which might be associated with a stress response. We have found 48 sequences encoding molecular chaperones and their associated co-chaperones in P. brasiliensis transcriptome (Supplemental Table VI). These sequences were divided into nine groups: small chaperones (four genes), HSP40 (9), HSP60 (10), HSP70 (7), HSP90 (4), HSP100 (4), 14-3-3 (2), calnexin (1), and immunophilins (7). Eight of these are differentially expressed: calnexin, cct7 (cytoplasmic hsp60) and sba1 (HSP90/70 co-chaperone) for the mycelium form and cpr1 (HSP90/70 co-chaperone), hsp42, hsp60, ssc1 (HSP70), and hsp90 for the yeast form. From these, hsp60 and hsp70 had been previously characterized as differentially expressed in yeast (25, 26). cDNA microarray analysis confirmed the differential expression pattern of sba1. Furthermore, the number of chaperone and co-chaperone ESTs is 38% larger in the yeast cDNA library than in the mycelium library. These data represent an evidence of an altogether higher expression of HSPs in yeast cells, compatible with growth at 37 °C.

Oxidative agents may cause stress and damage to P. brasiliensis cells. They may originate from the activity of host macrophages or from intracellular oxidative species. P. brasiliensis contains several genes encoding enzymes with known or putative antioxidant properties, such as superoxide dismutases, catalases (two isoenzymes), peroxiredoxins, and a novel cytochrome c peroxidase (Supplemental Table VII). Homologues to genes encoding secondary antioxidant enzymes belonging to the glutathione S-transferase family were also found. Several transcription factors may be involved in the induction of antioxidant defenses in P. brasiliensis. Homologues to YAP1, HAP3, and SKN7 from S. cerevisiae (27) were discovered in the transcriptome, showing that the oxidative stress regulators from P. brasiliensis and baker's yeast might be conserved.

Signal Transduction Pathways—Transcriptome analysis and reverse annotation revealed several putative components of the biosignaling pathways in P. brasiliensis (Supplemental Table VIII), such as (i) MAPK signaling for cell integrity, cell wall construction, pheromone/mating, and osmotic regulation; (ii) cAMP/protein kinase A, regulating fungal development and virulence, and (iii) calcium-calmodulin-calcineurin, controlling growth at high temperature. Furthermore, a ras homologue sequence was detected raising the possibility of cross-talk among the distinct signal transduction pathways (Fig. 4).

In budding yeast, the MAPK cascade responsible for cell integrity mediates cell cycle regulation and cell wall synthesis, responding to different signals including temperature, changes in external osmolarity, and mating pheromone. Components of this pathway identified in P. brasiliensis encompass the most classical steps, with the exception of a cell surface tyrosine kinase-like receptor that was not found in the transcriptome so far analyzed. Rho1p is a small GTP-binding protein of the Rho subfamily required for cell growth and coordinated regulation of cell wall construction (28) through the synthesis of {beta}-1,3-glucan. It also activates Pkc1p, which in turn regulates the MAPK pathway.

Transcripts related to the pathway for activation by mating pheromone were identified in the P. brasiliensis transcriptome. The intermediary components appear to be constitutively expressed in both mycelium and yeast forms. Intriguingly, mating has not yet been described in P. brasiliensis. Conversely, the Hog1 MAPK cascade is activated when there is an increase in the environment osmolarity. One of its targets, Glo1p, which controls genes required for cell adaptation and survival upon osmotic stress in S. cerevisiae (29), was also detected in P. brasiliensis.

The cAMP/protein kinase A is a cascade known to regulate fungal differentiation and virulence. From the genes identified in P. brasiliensis, we highlight a homologue to several fungal adenylate cyclases; the low affinity cAMP phosphodiesterase, encoded by the gene Pde1; homologues to both the regulatory and the catalytic subunits of protein kinase A, which is involved in the regulation of the cell surface flocculin Flo11p/Muc1p (30). In P. brasiliensis exogenous cAMP is known to inhibit the process of filamentation (31). Both the catalytic (CnaA) and the Ca+2-binding regulatory B (CnaB) subunits of calcineurin were found in P. brasiliensis. In dimorphic fungi, cAMP- and calcineurin-dependent pathways seem to be involved in differentiation. As in the pathogenic fungus Cryptococcus neoformans (32), calcineurin might also play a role in mating of P. brasiliensis. In several pathogenic and nonpathogenic fungi, RAS is involved in filamentation, pseudohyphal/hyphal growth, and mating (33). A RAS-related transcript was identified in P. brasiliensis, but further studies are required to elucidate its function in mycelium-to-yeast transition and in the mechanism of pathogenicity.

Virulence Genes, Drug Targets and Resistance—In order to identify genes that could be related to P. brasiliensis virulence, its transcriptome has been searched for orthologues assigned as virulence factors in human pathogenic fungi, as defined by Falkow's postulate (34). Table II lists 28 P. brasiliensis sequences, which were previously experimentally established as virulence or essential genes in C. albicans, C. neoformans, and Aspergillus fumigatus. They were subdivided into four classes: metabolism-, cell wall-, and signal transduction-related and others. Some of these genes has been considered for antifungal therapy and are also listed in Table III as potential drug targets.


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  		TABLE II
Putative virulence or essential genes found in P. brasiliensis transcriptome related to the experimentally confirmed orthologues of C. albicans, C. neoformans, and/or A. fumigatus

 


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  		TABLE III
Potential drug targets genes found in P. brasiliensis transcriptome with no homologues in the human genome

 
MAPK-related sequences, whose orthologues in C. albicans were experimentally correlated to hyphal formation and virulence, were also detected. The extrapolation to the P. brasiliensis model is not direct, since yeast, not hyphae, is the pathogenic cell type, but several MAPK homologues are found in species exhibiting diverse morphology and infection habits (35). A cavps34 orthologue, identified in P. brasiliensis transcriptome (vps34), is implicated in the protein/lipid transport from the Golgi apparatus/endosome to the vacuole and has been proved to be important to C. albicans virulence (36).

Noteworthy is the finding of glyoxylate cycle genes in P. brasiliensis, since its activity has been reported as a fungal virulence requirement (37). The activity of the key enzymes malate synthase and isocitrate lyase was reported to be up-regulated in C. albicans upon phagocytosis (38). Both enzymes were detected in the P. brasiliensis transcriptome, with isocitrate lyase being overexpressed in the yeast phase, as confirmed by Northern blot analysis (Fig. 3).



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  		FIG. 4.
Signaling pathways in P. brasiliensis. Shown are cAMP/protein kinase A regulating fungal development and virulence; MAPK signaling for cell integrity, cell wall construction, pheromone/mating, and osmoregulation; calcium-calmodulin-calcineurin controlling cell survival under stress conditions; and Ras allowing cross-talk of extracellular signals. For abbreviations of gene names see Supplemental Table VIII.

 
The cell wall, as the most obvious difference between human and fungal cells, represents a prime target for antifungals. Genes involved in its biogenesis and assembly can act as virulence factors and therefore are putative drug targets. We have identified orthologues to chitin synthase 3 (chs3), glucosamine-6-phosphate acetyltransferase (gna1), mannosyltransferase (pmt1), and {alpha}1,2-mannosyltransferase (mnt1) genes and glycosidases Phr1p and Phr2p. The expression of the two last genes in C. albicans is responsive to the product of prr2 (39), a pH-related transcription factor also present in the P. brasiliensis transcriptome. The detection of chitin deacetylase, as an overexpressed yeast gene confirmed by cDNA microarray and Northern blot (data not shown), points out to a novel target for drug research in P. brasiliensis.

Microbe resistance to reactive oxygen and nitrogen intermediates plays an important role in virulence (40). We were able to identify sequences that are oxidative stress response orthologues, including an alternative oxidase (aox1), a copper/zinc superoxide dismutase (sod1), and two different catalase orthologues, one of them a peroxisomal cat1, as recently described (41).

The urate oxidase gene detected in the P. brasiliensis transcriptome, but not in S. cerevisiae, C. albicans, and Homo sapiens genomes, suggests that uric acid could be degraded to allantoin. In addition, the presence of a C. neoformans urease orthologue also probably reflects the degradation of urea to ammonia and carbamate. A role in virulence and sporulation has been assigned for both genes (42). The production of urea has been involved in an improved in vitro survival for those microorganisms exposed to an acidic environment. In this view, it could be related to the survival of the fungus in the host cells.

The development of new drugs is crucial, considering the problem of emerging drug resistance and toxicity (37). Novel drug targets have been found through the analysis of genome sequences. The genes listed in Table III have no homologues in the human genome and therefore could be considered for the development of new antifungal drugs. Most therapies designed to treat fungal infections target the ergosterol biosynthetic pathway (43). The orthologue of C-24 sterol methyltransferase (ERG6) is present in P. brasiliensis. In addition, modulation of sphingolipid metabolism exerts a deep impact on cell viability. The synthesis of inositol-phosphoryl-ceramide from phytoceramide catalyzed by the product of the aur1 gene, present in P. brasiliensis, corresponds to the first specific step of this pathway (44). Translation elongation factors have also been pointed out as drug targets (37). In the P. brasiliensis transcriptome, we have found an elongation factor-3 sequence that is absent in human genome (45) and thus can be addressed for pharmaceutical purposes.

Twenty PbAESTs annotated as related to multiple drug resistance genes were identified (Supplemental Table IX). They include 12 S. cerevisiae orthologues, 10 of which are related to the ABC transporter and two to major facilitator superfamilies (46). One of them corresponds to Pfr1, a gene recently described in P. brasiliensis (47), and another is related to the CDR1 gene from C. albicans, which is up-regulated in the presence of human steroid hormones (48). It has been speculated that steroid hormones are involved in morphological changes as well as in pathogenicity in P. brasiliensis and also in drug resistance in C. albicans. Interestingly, the process of infection of P. brasiliensis is strongly biased toward males, albeit the role of steroid hormones in the expression of ABC transporters in this organism remains to be investigated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The P. brasiliensis transcriptome described here is represented by 6,022 EST clusters that may cover about 80% of the fungal total genome, whose gene number has been estimated to be ~8,000 genes.3 This number greatly exceeds the previous EST studies in this fungus (8, 49). The analysis compares the two fungal cell types as well as their metabolic behavior. The results obtained probably reflect the adaptations associated with the mycelium (soil) and yeast (human host) environments. Most importantly, they provide new insights with respect to signal transduction pathways, virulence genes, and drug targets for this pathogen.

The transcription profile of the mycelium infective phase suggests the shunting of pyruvate into aerobic metabolism, since the expression of the ESTs encoding enzymes of the trichloroacetic acid cycle are up-regulated in this fungal phase. In contrast, the yeast transcription profile evidenced the deviation of pyruvate from the glycolytic pathway into anaerobic metabolism; this observation is consistent with a lower oxygen level in infected tissues. Its putative ability to produce ethanol suggests a potential anaerobic pathway for P. brasiliensis, which is dependent on the metabolic state of the cell. It seems that the main regulatory effector on the shunting of the end product of glycolysis into aerobic or anaerobic metabolism is temperature; therefore, it can be hypothesized that this physical factor is the central trigger of all of these molecular events, since it was the only parameter changed in the in vitro cultivation of yeast and mycelium of P. brasiliensis. Experiments are currently being carried out in order to confirm the in vivo expression profile of the differentially expressed genes in macrophages and human pulmonary epithelial cells infected by P. brasiliensis.

Since P. brasiliensis is a medical problem in Latin America, the prediction of new drug targets from sequence information is of great importance. Chitin deacetylase, which is absent in humans and highly expressed in the parasitic yeast, could be a specific drug target for PCM therapy if it is shown to play a key role in the fungal metabolism during human infection. Functional analysis of the P. brasiliensis genes described in this work will lead to important information on cellular differentiation, pathogenicity, and/or virulence. These issues can only be addressed when molecular tools are developed for this organism. In conclusion, the knowledge of the transcribed sequences of P. brasiliensis will most likely facilitate the development of new therapeutics to PCM and other medically relevant mycosis.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) CA580326 [GenBank] -CA584263, CN238087 [GenBank] -CN253933, and CN373644 [GenBank] -CN373755.

Minimal information about cDNA microarray experiments was deposited in the MIAMExpress databank (EMBL) under the accession numbers E-MEXP-103 and A-MEXP-71. The sequences are also available at https://www.biomol.unb.br/Pb.

* This work was supported by MCT, CNPq, CAPES, FUB, UFG, and FUNDECT-MS. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains nine additional tables. Back

c These authors contributed equally to this work. Back

d PbGenome Network: Alda Maria T. Ferreira, Alessandra Dantas, Alessandra J. Baptista, Alexandre M. Bailão, Ana Lídia Bonato, André C. Amaral, Bruno S. Daher, Camila M. Silva, Christiane S. Costa, Clayton L. Borges, Cléber O. Soares, Cristina M. Junta, Daniel A. S. Anjos, Edans F. O. Sandes, Eduardo A. Donadi, Elza T. Sakamoto-Hojo, Flábio R. Araújo, Flávia C. Albuquerque, Gina C. Oliveira, João Ricardo M. Almeida, Juliana C. Oliveira, Kláudia G. Jorge, Larissa Fernandes, Lorena S. Derengowski, Luís Artur M. Bataus, Marcus A. M. Araújo, Marcus K. Inoue, Marlene T. De-Souza, Mauro F. Almeida, Nádia S. Parachin, Nadya S. Castro, Odair P. Martins, Patrícia L. N. Costa, Paula Sandrin-Garcia, Renata B. A. Soares, Stephano S. Mello, and Viviane C. B. Reis. Back

b To whom correspondence should be addressed. Tel.: 55-307-2423; Fax: 55-61-3498411; E-mail: msueli{at}unb.br.

1 The abbreviations used are: PCM, paracoccidioidomycosis; contig, group of overlapping clones; EST, expressed sequence tag; PbAEST, P. brasiliensis assembled EST sequence; MAPK, mitogen-activated protein kinase. Back

2 C. Reinoso, G. Niño-Vega, G. San-Blas, and A. Dominguez (2003) IV Congreso Virtual de Micologia, personal communication. Back

3 G. San-Blas, personal communication. Back


   ACKNOWLEDGMENTS
 
We are grateful to Hugo Costa Paes and Robert Miller for English text revision.



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