In the dimorphic fungus Paracoccidioides brasiliensis, α -1,3-glucan is the major neutral cell wall polysaccharide of the pathogenic yeastlike phase, organized as a sort of outer capsule . It replaces almost entirely the β -1,3-glucan which comprises the neutral polysaccharide of the vegetative mycelial phase, and has been pointed out as virulence factor in P. brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum . In order to clone the α -1,3-glucan synthase gene from P. brasiliensis (PbrAGS1), DNA was extracted as before . From the alignment of the amino acid sequences from several fungal α -1,3 glucan synthases, high homology areas were determined on which primers were designed. For the isolation of the PbrAGS1 gene, the 900 bp PCR product was used as a probe. From Southern analysis, a HindIII partial genomic library was constructed. Screening of this library by colony hybridization resulted in 4 positives clones containing inserts of 4.2 kb. One of the clones, plasmid pLBM1, was selected for further analysis. Nucleotide sequence analysis of the insert revealed the presence of an approximately 4 Kb open reading frame (ORF) for the 5’ region of the gene, with high homology to fungal a-glucan synthase genes. However, it missed the 3’ region of the gene, which represents about 40% of the gene, as compared with the Aspergillus fumigatus AGS1 gene, the one with the highest homology to PbrAGS1. In order to identify and clone the missing region of the gene, a second Southern analysis of genomic DNA of P. brasiliensis was performed, using the 300 bp downstream BamHI-HindIII fragment obtained from the pLBM1 insert. A P. brasiliensis partial genomic DNA library was constructed and the PCR amplified product used as probe to search for recombinant colonies carrying the fragment under study. A 4 Kb fragment was obtained, which is under sequencing for further studies. The gene is expressed in the yeast phase but not in the mycelial phase of P. brasiliensis, as deduced from RT-PCR, an information that matches the reported presence of α -1,3- glucan in the yeast cell wall of P. brasiliensis and its absence from the mycelial cell wall . 1.- San-Blas G. et al. 2002. Med. Mycol. 40: 225-242. 2.- Nií±o-Vega G.A. et al. 2000. Med. Mycol. 38: 31-39.
Full conference title:
14th Annual Focus on Fungal Infections
- FFI 14th (2004)