Genes diferencialmente expressos em Paracoccidioides brasiliensis

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Paracoccidioides brasiliensis is a thermally dimorphic fungus causing paracoccidioidomycosis, an endemic disease widespread in Latin America. The dimorphic transition from the mycelia (22 C) to the yeast (36 C) form is induced by a shift from the environmental temperature to that of the mammalian host. Genes that are differentially expressed in phases of P. brasiliensis can be relevant to the dimorphism, and to the establishment of infection. Here we describe the analysis of differentially expressed genes of P. brasiliensis. The formamidase gene of P. brasiliensis was described as highly expressed in mycelia of P. brasiliensis, isolate Pb01. Formamide aminohydrolase (formamidase, EC 3.5.1.49) catalyzes the specific hydrolysis of formamide to produce ammonia and formate. We identified the formamidase of P. brasiliensis, which reacts with antibodies present in sera of P. brasiliensis infected patients. The cDNA of the gene was cloned and the heterologous protein was produced and purified. Also, the purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active and formamidase activity was also detected in P. brasiliensis yeast and mycelium protein extracts. The recombinant formamidase was used to produce polyclonal antibody in mice, which showed high specificity in western blot assay. We also performed purification of the native protein in two steps of chromatography. Fractions with formamidase activity were selected and analysed by SDS-PAGE, which revealed a protein with molecular mass of 180-kDa. The purified native protein was submitted to mass spectrometry and was identified as formamidase. Additionally, SDS-PAGE assay with heat-denatured protein extracts revealed a protein with molecular mass of 45-kDa. Those results suggest that P. brasiliensis formamidase is a tetrameric protein. The cellular localization of the native protein in fungal yeast cells was performed by confocal and transmission electron microscopy. The P. brasiliensis formamidase was found in cytoplasm and cell wall. In order to elucidate the role of this molecule, the cDNA encoding formamidase was used to screen a library constructed with P. brasiliensis yeast cells cDNAs using a Saccharomyces cerevisiae two-hybrid system. Proteins related with protein folding, processing and destination were found, which can be related to the cell wall localization of formamidase, as well as with the protein involvement in nitrogen metabolism of P. brasiliensis. A model for formamidase interactions is provided. We evaluated genes putatively involved in the establishment of yeast phase of P. brasiliensis. Changes in gene expression in the yeast phase were analyzed by comparison of the isolate Pb01 to a non-differentiating mycelia-like isolate using subtractive the cDNA-RDA methodological strategy. In an effort to help identify gene products associated with the yeast parasitic phase, cDNAs profiles were generated from yeast cells of isolate Pb01 and from isolate Pb4940, the last growing as mycelia at the host temperature. The isolate Pb01 induced the expression of a variety of transcripts encoding some proteins related to stress response, cell wall/membrane composition, compared to isolate Pb4940, probably reflecting the establishment/maintenance of the P. brasiliensis yeast parasitic phase.

ASSUNTO(S)

parcoccidioides brasiliensis biologia molecular análise da diferença representacional formamidasa

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