Rosângela V. Andrade

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature‐dependent cell morphology change from mycelium (22° C) to yeast (36° C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3938 (Y = 1654 and M = 2274) ESTs were sequenced and clustered into 597 contigs and 1563 singlets, making up a total of 2160 genes, which possibly represent one‐quarter of the complete gene repertoire in P. brasiliensis. From this total, 1040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes—cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage‐specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis. All EST sequences have been deposited in GenBank under Accession Nos CA580326–CA584263. Copyright

The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability.

BackgroundParacoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target.ResultsIn this work, we decided to further study the Pbhsp90 gene, its expression and role in cell viability because it plays important roles in fungal physiology and pathogenesis. Thus, we have sequenced a Pbhsp90 cDNA and shown that this gene is present on the genome as a single copy. We have also confirmed its preferential expression in the yeast phase and its overexpression during dimorphic transition and oxidative stress. Treatment of the yeast with the specific HSP90 inhibitors geldanamycin and radicicol inhibited growth at 2 and 10 μM, respectively.ConclusionThe data confirm that the Pbhsp90 gene encodes a morphologically regulated and stress-responsive protein whose function is essential to cell viability of this pathogen. This work also enforces the potential of HSP90 as a target for antifungal therapies, since the use of HSP90 inhibitors is lethal to the P. brasiliensis yeast cells in a dose-responsive manner.

Oxidative stress response in Paracoccidioides brasiliensis: assessing catalase and cytochrome c peroxidase.

Paracoccidioides brasiliensis is a dimorphic fungus that infects humans and establishes infection in the yeast form. We are interested in the mechanisms this fungus uses to evade the human immune system, and in its survival strategies within infected host cells. Reactive oxygen species play an important role in host defence, but are detoxified by pathogen-derived antioxidant enzymes to prevent oxidative damage. The transcriptional and post-transcriptional regulation of P. brasiliensis catalase and cytochrome-c peroxidase (CCP) antioxidant enzymes upon culture treatment with hydrogen peroxide (H(2)O(2)) is described. High H(2)O(2) concentrations (up to 100 mm) still permitted 70-100% survival of exponential and stationary phase yeast cells, though stationary phase cells were consistently more resistant. P. brasiliensis has both cytosolic and peroxisomal catalase isoenzymes and a single cytochrome-c peroxidase. High-dose treatments with H(2)O(2) led to an early increase in total catalase and CCP enzymatic activities, indicative of post-transcriptional regulation. The expression levels of the catalase genes increased three to fourfold when the cells were treated with 50 mm H(2)O(2) for 40 or 50 min. Lipid peroxidation, as assessed by the thiobarbituric acid method, was relatively low upon treatment with H(2)O(2), which was consistent with our results demonstrating that P. brasiliensis has a powerful antioxidant defence system enabling it to survive H(2)O(2)-mediated stress.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

BackgroundMycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above.ResultsIn this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-β-glucosidase) in mycelium cells; and ags (an α-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase.ConclusionTaken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

The kex2 gene from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis

Kexin‐like protein is a component of the subtilase family of proteinases involved in the processing of proproteins to their active forms. Kexin‐like proteins are also synthesized as a propeptide and this is involved in (auto)inhibition, correct folding and subcellular sorting of proteins. The kexin‐like protein was described as the product of the kex2 gene for Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Yarrowia lipolytica and other fungi. Disruption of the kex2 gene in C. albicans and Y. lipolytica affects hyphae production and induces morphological cell defects, strongly suggesting a possible role of kexin‐like proteins in dimorphism of human pathogenic fungi. In this work, we report the nucleotide sequence of the kex2 gene cloned from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis (Pbkex2). An open reading frame (ORF) of 2622 bp was identified in the complete sequence, interrupted by only one intron of 93 bp. The 5′ non‐coding region contains consensus sequences such as canonical TATA, CAAT boxes and putative motifs for transcriptional factors binding sites, such as HSE‐like regulating genes involved in thermo‐dependent processes; Xbp1, reported as a transcriptional factor that may control genes involved in cell morphology; and StuAp, which may regulate spore differentiation and pseudohyphal growth in fungi. In the 3′ non‐coding region were observed the canonical motifs necessary for correct mRNA processing and polyadenylation. The deduced protein sequence consists of 842 amino acid residues, showing identity to kexin‐like proteinases from A. niger (55%), Emericella nidulans (53%) and C. albicans (48%). Comparative sequence analysis of P. brasiliensis kexin‐like protein reveals the presence of homologous regions related to a signal peptide, a propeptide, a subtilisin‐like catalytic domain, a P domain, a S/T rich region and a transmembrane domain. A putative Golgi retrieval signal (YEFEMI) has also been found in the cytoplasmic tail. The complete nucleotide sequence of Pbkex2 and its flanking regions have been submitted to GenBank database under Accession No. AF486805. Copyright

Overview and perspectives on the transcriptome of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.