Produção Nacional
Revisado por pares
Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells
2005; Elsevier BV; Volume: 280; Issue: 26 Linguagem: Inglês
10.1074/jbc.m500625200
ISSN1083-351X
AutoresMaria Sueli Soares Felipe, Rosângela Vieira de Andrade, Fabrício Barbosa Monteiro Arraes, André Moraes Nicola, Andréa Queiróz Maranhão, Fernando Araripe Gonçalves Torres, Ildinete Silva-Pereira, Márcio José Poças-Fonseca, Élida G. Campos, Lídia Maria Pepe de Moraes, Patrícia A. Andrade, Aldo Henrique Tavares, Simoneide Souza Titze-de-Almeida, Cynthia Maria Kyaw, Diorge P. Souza, PbGenome Network, Maristela Pereira, Rosália Santos Amorim Jesuíno, Edmar Vaz de Andrade, Juliana Alves Parente, Gisele S. Oliveira, Mônica Santiago Barbosa, N. F. Martins, Ana Lúcia Fachin, Renato S. Cardoso, Geraldo Aleixo Silva Passos, Nalvo F. Almeida, Maria Emília M. T. Walter, Célia M.A. Soares, Maria José A. Carvalho, Marcelo M. Brígido,
Tópico(s)Plant Pathogens and Fungal Diseases
ResumoParacoccidioides 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. 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. The dimorphic human pathogenic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM) 1The 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. 1The 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. (1Franco M. J. Med. Vet. Mycol. 1987; 25: 5-18Crossref PubMed Scopus (213) Google Scholar), 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 (2Restrepo A. McEwen J.G. Castaneda E. Med. Mycol. 2001; 39: 233-241Crossref PubMed Scopus (202) Google Scholar). 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 (1Franco M. J. Med. Vet. Mycol. 1987; 25: 5-18Crossref PubMed Scopus (213) Google Scholar). Chronic severe multifocal PCM may also cause granulomatous lesions in the central nervous system (3de Almeida S.M. Queiroz-Telles F. Teive H.A. Ribeiro C.E. Werneck L.C. J. Infect. 2004; 48: 193-198Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). 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 (4San-Blas G. Nino-Vega G. Iturriaga T. Med. Mycol. 2002; 40: 225-242Crossref PubMed Scopus (166) Google Scholar). Pathogenicity has been intimately associated with this process, since P. brasiliensis strains unable to differentiate into the yeast form are avirulent (5San-Blas G. Nino-Vega G. Fungal Pathogenesis: Principles and Clinical Applications. Marcel Dekker, New York2001: 205-226Google Scholar). Mammalian estrogens inhibit dimorphism, explaining the lower incidence of disease in females (6Salazar M.E. Restrepo A. Stevens D.A. Infect. Immun. 1988; 56: 711-713Crossref PubMed Google Scholar). 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 (7Cano M.I. Cisalpino P.S. Galindo I. Ramirez J.L. Mortara R.A. da Silveira J.F. J. Clin. Microbiol. 1998; 36: 742-747Crossref PubMed Google Scholar). A study of P. brasiliensis gene density suggests that this fungus contains between 7,500 and 9,000 genes, 2C. Reinoso, G. Niño-Vega, G. San-Blas, and A. Dominguez (2003) IV Congreso Virtual de Micologia, personal communication. 2C. Reinoso, G. Niño-Vega, G. San-Blas, and A. Dominguez (2003) IV Congreso Virtual de Micologia, personal communication. 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. 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 λZAPII 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 (8Felipe M.S. Andrade R.V. Petrofeza S.S. Maranhão 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. Leitao 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. Simoes 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. Yeast. 2003; 20: 263-271Crossref PubMed Scopus (70) Google Scholar). EST assembly was performed using the software package CAP3 (9Huang X. Madan A. Genome Res. 1999; 9: 868-877Crossref PubMed Scopus (4026) Google Scholar) 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 (10Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar) was used for annotation along with GenBank™ 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 (11Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9328) Google Scholar) to compare assemblies with Saccharomyces cerevisiae and Schizosaccharomyces pombe predicted polypeptides. The INTERPROSCAN program (12Apweiler R. Biswas M. Fleischmann W. Kanapin A. Karavidopoulou Y. Kersey P. Kriventseva E.V. Mittard V. Mulder N. Phan I. Zdobnov E. Nucleic Acids Res. 2001; 29: 44-48Crossref PubMed Scopus (77) Google Scholar) was used to obtain domain and family classification of the assemblies. Metabolic pathways were analyzed using maps obtained in the KEGG Web site (13Kanehisa M. Goto S. Nucleic Acids Res. 2000; 28: 27-30Crossref PubMed Scopus (16238) Google Scholar) 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 (14Audic S. Claverie J.M. Genome Res. 1997; 7: 986-995Crossref PubMed Scopus (2337) Google Scholar) 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 × 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 [α-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 α-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 [α-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 (15Quackenbush J. Nat. Genet. 2002; 32: 496-501Crossref PubMed Scopus (1466) Google Scholar). 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 (16Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9648) Google Scholar) 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 [α-32P]dATP. Membranes were incubated with the probes in hybridization buffer (50% formamide, 4× SSPE, 5× Denhardt's solution, 0,1% SDS, 100 μg/ml herring sperm DNA) at 42 °C overnight and then washed twice (2× 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/). 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 (17Venancio E.J. Kyaw C.M. Mello C.V. Silva S.P. Soares C.M. Felipe M.S. Silva-Pereira I. Med. Mycol. 2002; 40: 45-51Crossref PubMed Scopus (18) Google Scholar). Of the 6,022 PbAESTs, 4,198 (69.4%) showed a probable homologue in GenBank™, 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 (8Felipe M.S. Andrade R.V. Petrofeza S.S. Maranhão 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. Leitao 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. Simoes 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. Yeast. 2003; 20: 263-271Crossref PubMed Scopus (70) Google Scholar). 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).Table IDifferentially 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 (14Audic S. Claverie J.M. Genome Res. 1997; 7: 986-995Crossref PubMed Scopus (2337) Google Scholar) 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.PbAESTEC numberAnnotated functionNumber of readsaNumber of mycelium (M)- and yeast (Y)-derived ESTs in the PbAESTp valuebp value for the Audic and Claverie test-Fold changec-Fold change found for the microarray experimentsAccession number/Best hit organism/E valueMYMycelium up-regulated genes1068M51dPreviously shown to be differential by Northern blot or proteome analysis,eElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses65340.00000041666.0BE758605/P. brasiliensis /0.022744.4.1.5Lactoylglutathione lyaseeElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses7500.0000007.0NP_105614.1/Mesorhizobium loti/1e-112521Hydrophobin 1dPreviously shown to be differential by Northern blot or proteome analysis,fElectronic subtraction differential pattern and not assayed in cDNA microarray analysis5600.000000AAM88289.1/P. brasiliensis/2e-511789HSP90 co-chaperonefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis19100.018169CAD21185.1/N. crassa/4e-4825091.15.1.1Copper-zinc superoxide dismutasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis1450.010801Q9Y8D9/A. fumigatus/1e-682458UnknownfElectronic subtraction differential pattern and not assayed in cDNA microarray analysis1360.0253362478Hydrophobin 2dPreviously shown to be differential by Northern blot or proteome analysis,fElectronic subtraction differential pattern and not assayed in cDNA microarray analysis900.000951AAR11449.1/P. brasiliensis/2e-7012871.13.11.322-nitropropane dioxygenasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis810.008606CAB91335.2/N. crassa/e-1331318Amino acid permeaseeElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses800.00190750.4CAD21063.1/N. crassa/0.01470UnknowneElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses820.02157220.122692.7.4.3Adenylate kinasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis510.046263NP_011097.1/S. cerevisiae/1e-422364UnknowneElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses510.0462633.6379UnknowneElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses510.0462634.910924.2.1.22Cystathionine β-synthasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis400.030842AAL09565.1/Pichia pastoris/4e-9623562.2.1.2TransaldolasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis400.030842NP_013458.1/S. cerevisiae/e-10824763.1.2.22Palmitoyl-protein thioesterasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis400.030842I58097/H. sapiens/8e-4241351.1.1.41Isocitrate dehydrogenasegSinglets that are differential in cDNA microarray analysis100.2486903.1O13302/Acetobacter capsulatum/6e-3155306.2.1.5β-Succinyl CoA synthetasegSinglets that are differential in cDNA microarray analysis100.2486902.7T49777/N. crassa/9e-7347492.7.1.2GlucokinasegSinglets that are differential in cDNA microarray analysis100.2486901.7Q92407/Aspergillus niger/2e-5042462.7.1.48Uridine-kinasegSinglets that are differential in cDNA microarray analysis100.2486902.7T41020/S. pombe/3e-28Yeast up-regulated genes2536Y20 proteineElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses,dPreviously shown to be differential by Northern blot or proteome analysis27880.0000008.7AAL50803.1/P. brasiliensis/e-10624311.1.1.1Alcohol dehydrogenase IfElectronic subtraction differential pattern and not assayed in cDNA microarray analysis2450.000000P41747/Aspergillus flavus/e-1297373.5.1.41Xylanase/chitin deacetylaseeElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses8330.0000232.8NP_223015.1/Helicobacter pylori/e-113201Putative membrane protein Nce2eElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses0270.00000025.2NP_015475.1/S. cerevisiae/5e-087973.1.6.6Choline sulfataseeElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses3150.0016024.8NP_248721.1/P. aeruginosa/e-104814Glyoxylate pathway regulatoreElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses0150.00001617.7NP_009936.1/S. cerevisiae/4e-37170460S ribosomal protein L19fElectronic subtraction differential pattern and not assayed in cDNA microarray analysis0140.000032NP_596715.1/S. pombe/6e-4915851.8.4.8PAPS reductaseeElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses1120.0008155.1AAG24520.1/Penicillium chrysogenum/e-12163Putative methyltransferaseeElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses3110.0113142.5CAD21381.1/N. crassa/2e-46778Putative estradiol-binding proteineElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses3110.01131429.5NP_012049.1/S. cerevisiae/1e-31136UnknowndPreviously shown to be differential by Northern blot or proteome analysis,fElectronic subtraction differential pattern and not assayed in cDNA microarray analysis4100.0309503.9767UnknowneElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses3100.0177322.27011.2.4.1Pyruvate dehydrogenasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis190.004973Q10489/ S. pombe/1e-721724Putative sterol transportereElectronic subtraction and cDNA microarray analysis; differential pattern in both analyses060.00791529.3NP_013748.1/S. cerevisiae/4e-121712.6.1.42Branched-chain aminotransferasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis050.015790NP_012078.1/S. cerevisiae/7e-8719831.6.5.3NADH dehydrogenase (ubiquinone reductase)fElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496S47150/N. crassa/1e-192441.1.1.69Gluconate dehydrogenasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496NP_471610.1/Listeria innocua/1e-092583.3.2.1IsochorismatasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496NP_436193.1/Sinorhizobium meliloti/1e-202792.5.1.15Dihydropteroate synthasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496T49535/N. crassa/1e-383142.6.1.1Aspartate aminotransferasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496NP_509047.1/Caenorhabditis elegans/4e-965556.2.1.3Acyl-CoA synthetasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496NP_275799.1/Methanothermobacter thermautotrophicus/9e-897566.3.5.7Glutamyl-tRNA amidotransferasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496Q33446/A. nidulans/1e-158654.1.3.1Isocitrate lyasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496AAK72548.2/Coccidioides immitis/e-1199632.6.1.9Histidinol-phosphate aminotransferasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496P36605/S. pombe/4e-879803.5.1.4AcetamidasefElectronic subtraction differential pattern and not assayed in cDNA microarray analysis040.031496AAK31195.1/Aspergillus terreus/2e-0930731.14.13.3Phenylacetate hydroxylasegSinglets that are differential in cDNA microarray analysis010.2499982.3AAF21760.1/P. chrysogenum/2e-48a Number of mycelium (M)- and yeast (Y)-derived ESTs in the PbAESTb p value for the Audic and Claverie testc -Fold change found for the microarray experimentsd Previously shown to be differential by Northern blot or proteome analysise Electronic subtraction and cDNA microarray analysis; differential pattern in both analysesf Electronic subtraction differential pattern and not assayed in cDNA microarray analysisg Singlets that are differential in cDNA microarray analysis Open table in a new tab 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 (17Venancio E.J. Kyaw C.M. Mello C.V. Silva S.P. Soares C.M. Felipe M.S. Silva-Pereira I. Med. Mycol. 2002; 40: 45-51Crossref PubMed Scopus (18) Google Scholar, 18Albuquerque P. Kyaw C.M. Saldanha R.R. Brigido M.M. Felipe M.S. Silva-Pereira I. Fungal Genet. Biol. 2004; 41: 510-520Crossref PubMed Scopus (19) Google Scholar, 19Cunha A.F. Sousa M.V. Silva S.P. Jesuino R.S. Soares C.M. Felipe M.S. Med. Mycol. 1999; 37: 115-121Crossref PubMed Google Scholar, 20Marques E.R. Ferreira M.E. Drummond R.D. Felix J.M. Menossi M. Savoldi M. Travassos L.R. Puccia R. Batista W.L. Carvalho K.C. Goldman M.H. Goldman G.H. Mol. Genet. Genomics. 2004; 271: 667-677Crossref PubMed Scopus (50) Google Scholar). 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 (21Loose D.S. Stover E.P. Restrepo A. Stevens D.A. Feldman D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7659-7663Crossref PubMed Scopus (83) Google Scholar), also described in C. albicans and in other fungi (22Madani N.D. Malloy P.J. Rodriguez-Pombo P. Krishnan A.V. Feldman D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 922-926Crossref PubMed Scopus (102) Google Scholar). It is speculated that the interaction of the 17-β-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, α-trehalose, maltose, and sucrose were indeed utilized. On the other hand, the predicted assimilation of d-inositol was not confirmed. Transcripts related to
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