Maia, Z.A. ¹*; Castro, N.S. ¹; Felipe, M.S.S. ²; Pereira, M. ¹; Soares, C.M.A. ¹

1 Universidade Federal de Goiás, Goiânia, GO, Brazil. ² Universidade de Brasília, Brasília, DF, Brazil. *E-mail: zilmabio@bol.com.br

Paracoccidioides brasiliensis causes paracoccidioidomycosis the most prevalent systemic mycosis in Latin America. It is a thermally dimorphic fungus that exists in a mycelial form at environmental temperatures (e.g. 26°C) and in the yeast form at the mammalian host temperature (37°C). The morphological change in P. brasiliensis is accompanied by extensive changes in the cell wall and cell membrane structure and composition. It has been shown that both yeast and mycelial forms have chitin as a common structural polysaccharide in different concentrations at each stage of the parasite. In addition a (1-3) glucan was found in the yeast phase, while b (1-3) glucan was encountered in the mycelial form. The lipid composition of the membrane also changes during the morphological transition. It is possible that these changes in the composition of the cell membrane could be required to the regulation of the activity of synthases and hydrolases that modify the composition of the cell wall since most of those proteins are integral to the membrane. DFG5 is a probable GPI-anchored membrane protein involved in the cell wall biosynthesis and putatively required for filamentous growth, cell polarity, cell elongation and also for expression of hypha-specific genes and filamentation under alkaline conditions.

DNA of P. brasiliensis will be extracted and digested with specific restriction enzymes and separated on 0,8% agarose gel. DNA will be transferred to plus membranes and hybridized with the radiolabeled DFG5 probe.

The RNA of P. brasiliensis, mycelial and yeast phase, will be isolated and separated on 1% formaldehyde-agarose gels. RNA will be transferred to plus membranes and hybridized with the radiolabeled DFG5 probe.

P. brasiliensis organisms will be grown in medium at range of ambient pHs prior to isolation of RNA and Northern analysis.

The cDNA of Pbdfg5 will be isolated for PCR and fractionated by electrophoresis in a 0,8% agarose gel, excised from the gel and purification ( Amersham Biosciences ® ) The isolated PCR product will be then cloned into the vector pET100/D – TOPO ( Amersham Biosciences ® ) to expression in E. coli (BL21) according to the manufacturer’ s instructions.

• We isolated the sequence of a cDNA encoding a DFG5 homologue of Paracoccidioides brasiliensis (Pbdfg5) during the screening of cDNA library of the mycelial phase using the probe PbYmnt (GenBank: AF374353) encoding a mannosyltransferase;
• The translated amino acid sequence predicted a protein of 448 residues with a molecular mass of 49.9 kDa and isoelectric point (pI) of 5.4;
• The cDNA Pbdfg5 manifest a domain characteristic of the glycosyl hydrolase family 76 and also presents homology to bacterial alpha 1,6-mannanase;
• N-glycosylation sites and a cell attachment sequence (RGD) were predicted by protein sequence analysis using the PROSITE algorithm. The RGD tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion;
• Sequence analysis of the P. brasiliensis DFG5, performed by PSORT, reveled that 22 amino acid at the N-terminus have the characteristic of a signal peptide, with a predicted cleavage site between A²² and L²³.
• The highest identity was found between the DFG5 proteins of P. brasiliensis, Neurospora crassa, Schizosaccharomyces pombe and DCW1 of Saccharomyces cerevisiae ;
• Studies in Saccharomyces cerevisiae suggest that Dfg5p act as key molecules in the cell wall biosynthesis pathway. This may lead to the development to new antifungal drugs, as the cell wall biosynthesis pathway is specific to fungi and not found in higher eukaryotes such as animals and plants.

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KITAGAKI H, WU H, SHIMOI H, ITO K. Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae. Mol Microbiol. 2002 Nov;46(4):1011-22.

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NINO-VEGA GA, MUNRO CA, SAN-BLAS G, GOODAY GW, GOW NA. Differential expression of chitin synthase genes during temperature-induced dimorphic transitions in Paracoccidioides brasiliensis. Med Mycol. 2000 Feb;38(1):31-9.

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