Expressed Sequence Tag Analysis of the Human Pathogen Paracoccidioides brasiliensis Yeast Phase: Identification of Putative Homologues of Candida albicans Virulence and Pathogenicity Genes

Abstract

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5 and 3 ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.

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

“…Pb14-3-3 expression was evaluated in PbWT, PbEV and Pb14-3-3 aRNA yeast cells; b-tubulin was selected as the endogenous control. 74 The sequences of the oligonucleotide primers used for the amplification of the target gene were Pb14-3-3RT-forward (5 0 -AATCTGCTTTCCGTTGCCTA-3 0 ) and Pb14-3-3RT-reverse (5 0 GTTTTGGCGGTACTCCTTGA-3 0 ), the qRT-PCR product span any of the region targeted by antisense. For the b-tubulin gene, the primer sequences were b-tubulin RT-forward (5 0 -GTGGACCAGGTGATCGATGT-3 0 ) and b-tubulin RT-reverse (5 0 -ACCCTGGAGGCAGTCACA-3 0 ).…”

C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit a serine-thiol proteinase activity from Paracoccidioides brasiliensis

“…Although a couple of peptides would match those of subtilisins, the number of identical amino acids was not enough to guarantee reliable protein identification. One difficulty to analyze generated peptides is the lack of complete genome information for P. brasiliensis, although there are two GenBank databases of partial translated sequences [19,20]. It is important to point out that there is a 481-amino acid P. brasiliensis subtilase of the S8 family deposited in the GeneBank (Accession number AAP83193).…”

The putative flavin carrier family FlcA-C is important forAspergillus fumigatusvirulence

“…The pyrG used in the A. fumigatus cassette for generating the mutant strains were used as marker for prototrophy and was amplified from pCDA21 plasmid. 56 The DNA fragments together with plasmid linearized pRS426 BamHI/ EcoRI were transformed into S. cerevisiae strain SC9721 (FGSC) by the lithium acetate method 57 and DNA of the transformants extracted as previously described 58 TaKaRa Ex Taq TM DNA Polymerase (Clontech Takara Bio) was used for DNA amplification and Southern blot analyses demonstrated that the transformation cassettes had integrated homologously at the targeted A. fumigatus loci. A. fumigatus transformation was performed as described by de Castro et al 48 The complementing strains were constructed by first isolating from the corresponding deletion strain, a pyrG ¡ auxotroph sector resistant to 0.75 mg/ml of 5-FOA (5-Fluoroorotic acid, Sigma-Aldrich), a fluorinated derivative of the pyrimidine precursor orotic acid.…”