XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:3-1

Oral / Poster

3-1	*Analysis of Paracoccidioides brasiliensis* nitrosative stress response by transcriptional and proteomic assays**
Authors:	Juliana Alves Parente (UFG - Universidade Federal de Goias) ; Priscila Elias Campos Naves (UFG - Universidade Federal de Goias) ; Clayton Luiz Borges (UFG - Universidade Federal de Goias) ; Ana Flavia Alves Parente (UFG - Universidade Federal de Goias) ; Alexandre Melo Bailao (UFG - Universidade Federal de Goias) ; Celia Maria de Almeida Soares (UFG - Universidade Federal de Goias)

Abstract

The nitrosative stress response in human pathogenic fungi, such as P. brasiliensis, is an important response to the successful infection establishment since phagocyte host cells response includes reactive nitrogen nitrogen species (RNS) that react with cellular components, including metals, lipids, and DNA bases. These perturbations result cellular damages, inhibition of replication/respiration, and inactivation of enzymes. Because the ability of P. brasiliensis to survive during RNS stress is important to pathogenesis, the goal of this work is to elucidate candidate genes that could contribute to the RNS response. P. brasiliensis yeast cells viability was evaluated with several sodium nitrite concentrations during 6 h. The sodium nitrite concentration that do not inhibit yeast cells growth was selected to perform transcriptional and proteomic assays. To perform transcriptional analysis, total RNA of P. brasiliensis yeast cells was extracted and used to obtain cDNA that were used to construct library. The cloned products were submitted to automated sequencer MegaBACE 1000 (GE Healthcare) to obtain ESTs. Preliminary results show that transcripts related to RNS response, such as molybdenum cofactor biosynthesis, required for nitrate reductase activity and peptide transporter PTR2, required for H+/nitrate transport were detected. Additionally, transcripts encoding proteins related to cell wall stress were identified, such as calcofluor white hypersensitive protein and glucan 1,3-beta-glucosidase. The protein profile was analyzed by 2D electrophoresis. Protein spots were selected based on staining intensity as determined by Image Master Platinum software (GE Healthcare). Preliminary analysis identified 15 spots detected in the RNS stress condition that were not detected by Comassie blue stain in the control condition. Volume ratios between matched spots were analyzed using statistical test Anova that was carried out using SPSS 13 for Windows. Proteins spots that showed a significant variation in their abundance were excised and digested with trypsin. Identification of the digested spots is under progress by using mass spectrometry MALDI-Q-TOF (Synapt, Waters). The results obtained by transcriptional and proteomic assays will be compared and could be important to identify gene and genetic pathways used by P. brasiliensis to survive during RNS stress conditions.

Keyword: Nitrosative stress, pathogenesis, transcriptome, proteome

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