ABSTRACT
BACKGROUND: Mycelium-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. RESULTS: In 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-beta-glucosidase) in mycelium cells; and ags (an alpha-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. CONCLUSION: Taken 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.
Subject(s)
Gene Expression Regulation, Fungal/genetics , Mycelium/genetics , Paracoccidioides/genetics , Yeasts/genetics , Biological Transport/genetics , Blotting, Northern/methods , Cation Transport Proteins/genetics , Cell Wall/genetics , Cell Wall/metabolism , Cysteine/biosynthesis , Cytoskeleton/genetics , Cytoskeleton/metabolism , Expressed Sequence Tags , Fungal Proteins/genetics , Gene Expression Profiling/methods , Ions/metabolism , Oligonucleotide Array Sequence Analysis/methods , Yeasts/cytology , beta-Glucosidase/geneticsABSTRACT
Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.
