Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis

Decreased expression of 14-3-3 inParacoccidioides brasiliensisconfirms its involvement in fungal pathogenesis

“…coli DH5a grown at 37 C on LB medium supplemented with 0 mg mL ¡1 kanamycin was used as host for plasmid propagation and cloning. 69 Obtaining P. brasiliensis with 14-3-3 silencing We followed the anti-sense strategy and A. tumefaciens-mediated transformation protocol described previously by Almeida et al 70 and Ruiz et al 71 to obtain P. brasiliensis mutants with silencing of Pb14-3-3. DNA from Pb18 was extracted from yeast cultures during exponential growth, and a high-fidelity proofreading DNA polymerase (Taq DNA Polymerase High-Fidelity, Invitrogen, USA) was employed to amplify an aRNA oligonucleotide sequence targeting the exon coding sequence of Pb14-3-3 (GenBank accession number AY462124); the oligonucleotide primers used are Pb14-3-3 Forward (5 0 -CCGCTCGAGCG-GAAGGGGGACTACCACCGCT-3 0 ) and Pb14-3-3 Reverse (5 0 -GGCGCGCCCTGAGCAACCTCAGTTGCAT-3 0 ).…”

A 32-Kilodalton Hydrolase Plays an Important Role in Paracoccidioides brasiliensis Adherence to Host Cells and Influences Pathogenicity

“…Plasmid construction for aRNA and ATMT of P. brasiliensis were performed as described previously (1,2). Briefly, the amplified PbHAD32 aRNA oligonucleotides were inserted into the pCR35 plasmid under the control of the calciumbinding protein 1 (CBP-1) promoter region from H. capsulatum (28).…”

“…Protein sequence analysis revealed a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, P. brasiliensis Had32p (PbHad32p). Using antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation (ATMT), we constructed a mitotically stable P. brasiliensis PbHAD32 aRNA strain with consistently reduced gene expression (1,2). Yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to epithelial cells.…”

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

“…However, up to now, none of these genes has been confirmed as important in fungal virulence. More efficient molecular genetic systems for transformation and gene expression have been described only recently, and these will provide new opportunities to study the role of P. brasiliensis genes in pathogenesis [17]. It is likely that this fungus will produce a plethora of virulence factors.…”