Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction

Abstract

Paracoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http:// www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53 %) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75 % of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47 % of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.Abbreviations: EST, expressed sequence tag; KEGG, Kyoto Encyclopedia of Gene and Genomes; sqRT-PCR, semiquantitative RT-PCR.3These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession numbers for the ESTs of Paracoccidioides brasiliensis identified in this study are EST1487-EST6420.Two supplementary tables listing the overexpressed and novel genes identified during this study and supplementary material describing the EST dataset analysed are available with the online version of this paper. Microbiology

Preferential transcription of Paracoccidioides brasiliensis genes: host niche and time-dependent expression

“…Our in silico subtractive analysis indicated that the cycle is up-regulated in yeast cells during liver infection, relative to in vitro cultured yeast cells, as demonstrated by the over-expression of the regulatory enzyme isocitrate lyase (Costa et al 2007). This gene is up-regulated in C. albicans during growth inside macrophages (Lorenz & Fink 2001).…”

“…Of special note, and corroborating our data, the predominance of glycolytic metabolism in Candida albicans infecting mouse kidney (Barelle et al 2006) has been described. The over-expression of carbohydrate metabolism enzymes and overproduction of ethanol suggest that this metabolic pathway could be particularly relevant during liver infection because of the abundant glucose in the host milieu, as described by Costa et al (2007). The abundance of carbohydrate and energy in this infection condition could also pertain to the over-expression of transcripts encoding lipid-biosynthesis enzymes.…”

“…By analyzing the most redundant ESTs obtained from cDNAs of yeast cells recovered from the livers of infected mice, it was possible to obtain insight into the features of fungal metabolism during the infective process in tissues (Bailão et al 2006, Costa et al 2007. Fig.…”

“…2. A total of 1,602 unique sequences were obtained from the non-normalized cDNA libraries of yeast cells recovered from infected mouse liver and 639 were obtained from mycelium in transition to yeast cells (Costa et al 2007, Bastos et al 2007. From the 1,602 unique sequences in the cDNA library of yeast cells recovered from infected mice, 649 (40.51%) represented novel transcripts and 344 (21.47%) represented overexpressed genes.…”

“…Additionally, Costa et al (2007) analyzed 4,934 expressed sequence tags (ESTs) from a non-normalized cDNA library derived from P. brasiliensis yeast cells recovered from infected mouse liver. Comparative analysis of the unigene sequences at (http://www.lbm.icb.…”