RESUMEN
A gene encoding the entire highly expressed protein previously identified in the proteome of Paracoccidioides brasiliensis yeast cells as PbY20 has been isolated. The pby20 sequence reveals an open reading frame of 1364bp and a deduced amino acid sequence of 203 residues, which shows high identity to benzoquinone reductase from Phanerochaete chrysosporium (72.0%), Saccharomyces cerevisiae Ycp4 (65%), and Schizosaccharomyces pombe p25 (59%), and to allergens from Alternaria alternata Alt a7 (70%) and from Cladosporium herbarum, Cla h5 (68%). Low levels of the pby20 transcript in the mycelium and highly induced ones in infective yeast cells during the transition of this dimorphic fungus indicate transcriptional control of its expression. PbY20 was immunologically detected only in yeast cell extract, suggesting an important role in cell differentiation or even in the maintenance of the yeast form. Immunoelectron microscopy showed that PbY20 is found inside large granules and vacuoles, in the nucleus, and also in the cytoplasm. Through sequence comparisons analysis and fluorescence emission assay, PbY20 was recognized as a member of the flavin mononucleotide flavodoxin-like WrbA family, which are involved in heat shock and oxidative stress in biological systems. Assuming that PbY20 belongs to this family, a similar role could be attributed to this protein.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Paracoccidioides/genética , Alérgenos/genética , Alternaria/genética , Secuencia de Aminoácidos , Secuencia de Bases , Núcleo Celular/química , Cladosporium/genética , Gránulos Citoplasmáticos , Vesículas Citoplasmáticas/química , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , Flavodoxina/química , Flavodoxina/genética , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oxidorreductasas/genética , Phanerochaete/genética , ARN de Hongos/análisis , ARN Mensajero/análisis , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
A proteomic approach was used to identify a 39 kDa antigen of Paracoccidioides brasiliensis. Amino acid sequences of the N-terminal and of endoproteinase Lys-C digested peptides revealed the protein to be a fructose 1,6-biphosphate aldolase (FBA) Class II of P. brasiliensis. Two cDNA homologues, Pbfba1 and Pbfba2, were cloned and characterized. Pbfba1 encoded a predicted polypeptide of 360 amino acids that was highly homologous in the primary structure to the same enzyme from fungi and bacteria. The other DNA, Pbfba2, encoded a polypeptide predicted to be 363 amino acids. The sequence of Pbfba2 differed significantly from Pbfba1. Phylogenetic and molecular analysis supports the concept of gene duplication for FBAs in P. brasiliensis, constituting a two-member family. Expression analysis demonstrated differential expression for both fbas genes in P. brasiliensis cells.
Asunto(s)
ADN de Hongos , Fructosa-Bifosfato Aldolasa/genética , Paracoccidioides/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Fructosa-Bifosfato Aldolasa/aislamiento & purificación , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Duplicación de Gen , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Paracoccidioides/genética , Filogenia , Proteoma/análisis , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.
Asunto(s)
ADN Complementario/genética , Paracoccidioides/genética , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón Iniciador/genética , Codón de Terminación/genética , Secuencia Conservada/genética , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Punto Isoeléctrico , Datos de Secuencia Molecular , Peso Molecular , Señales de Localización Nuclear/genética , Sistemas de Lectura Abierta/genética , Señales de Poliadenilación de ARN 3'/genética , ARN de Hongos/análisis , ARN de Hongos/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia , Transcripción Genética/fisiologíaRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. Here we report the sequence and analysis of a novel developmentally regulated gene and cDNA (Pbgadph), encoding a GAPDH homologue (PbGAPDH), of the pathogenic dimorphic fungus Paracoccidioides brasiliensis. We have analyzed the protein, the cDNA and genomic sequences to provide insights into the structure, function, and potential regulation of PbGAPDH. That Pbgapdh encodes PbGAPDH was demonstrated by micro-sequencing of the native protein homologue isolated from the fungus proteome. The deduced amino acid sequence of Pbgapdh showed identity to those of from other species (88-76%). Phylogenetic analysis indicated that GAPDH could be useful for the determination of evolutionary relationships. Expression of the Pbgapdh gene and the cognate protein were developmentally regulated in phases of P. brasiliensis, with a higher expression in the yeast parasitic phase and was induced during the transition from mycelium to yeast and decreased during the reverse process, transition from yeast to mycelium.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Paracoccidioides/enzimología , Paracoccidioides/crecimiento & desarrollo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Clonación Molecular , ADN Complementario , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Intrones/genética , Datos de Secuencia Molecular , Micelio/genética , Paracoccidioides/genética , Filogenia , ARN de Hongos/análisis , ARN de Hongos/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia , Transcripción Genética , Levaduras/genéticaRESUMEN
Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.
Asunto(s)
Calmodulina/genética , Calmodulina/fisiología , Paracoccidioides/citología , Paracoccidioides/genética , Secuencia de Aminoácidos , Secuencia de Bases , Calmodulina/química , Quelantes/farmacología , ADN de Hongos/química , Ácido Egtácico/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Dosificación de Gen , Genes Fúngicos/fisiología , Genoma Fúngico , Imidazoles/farmacología , Intrones , Datos de Secuencia Molecular , Micelio , Paracoccidioides/efectos de los fármacos , Paracoccidioides/metabolismo , Alineación de Secuencia , Homología de Secuencia , Trifluoperazina/farmacología , LevadurasRESUMEN
We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.
Asunto(s)
Manosiltransferasas/genética , Paracoccidioides/enzimología , Paracoccidioides/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/aislamiento & purificación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Manosiltransferasas/química , Manosiltransferasas/inmunología , Manosiltransferasas/aislamiento & purificación , Datos de Secuencia Molecular , Paracoccidioides/inmunología , Paracoccidioidomicosis/inmunología , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We report the cloning and sequence analysis of a genomic clone encoding a Paracoccidioides brasiliensis ClpB chaperone homologue (PbClpB). The clpb gene was identified in a lambda Dash II library. Sequencing of Pbclpb revealed a long open reading frame capable of encoding a 792 amino acid, 87.9 kDa protein, pI of 5.34. The predicted polypeptide contains several consensus motifs of the ClpB proteins. Canonical sequences such as two putative nucleotide-binding sites, chaperonins ClpA/B signatures and highly conserved casein kinase phosphorylation domains are present. ClpB is 69% to 49% identical to members of the ClpB family from several organisms from prokaryotes to eukaryotes. The transcript of PbclpB was detected as a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phase of the fungus. A 89 kDa protein was also detected in yeast cells of P. brasiliensis.