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

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.

Figure 1

Northern blot analysis of mycelium and yeastup-regulated genes of P. brasiliensis. Total RNA samples from both mycelium (M) and yeast (Y) were blotted onto nylon membranes and hybridised against gene-specific radiolabelled probes: (a) Control of cell organisation: hex – Hexagonal peroxisome protein, bgl –1,3 beta-glucosidase, ags – alpha 1,3-glucan synthase, cda – Chitin deacetylase, vrp – Verprolin; (b) Ion transporters: isc –Iron-sulphur cluster-like protein, ktp – Potassium transporter, pct – Putative P-type Cu(2+) transporting ATPase; (c) Sulphur metabolism: chs – Choline sulphatase, ats – ATP sulphurylase. The constitutive 60S ribosomal protein L34 was used as a loading control.

Figure 2

Up-regulated genes encoding enzymes from the cysteine de novo biosynthesis pathway. Arrows indicate enzymes identified as up-regulated both by in silico subtraction, cDNA microarray and confirmed by northern blotting experiments. (*) enzyme identified as up-regulated by both in silico subtraction and cDNAs microarray but not assayed by northern blotting. (**) indicates an enzyme not found in the transcriptome of P. brasiliensis.

Figure 3

Cell differentiation of P. brasiliensis in modified MVM medium without inorganic sulphate. The fungus was grown in four different concentrations of sulphate salts (0, 8, 12 and 17 mM; the latter is the original concentration of MVM medium). (A) The appearance of yeast cells was verified daily in the transition from mycelium to yeast after temperature shift to 37°C, (B) The disappearance of yeast cells was verified daily in the transition from yeast to mycelium after temperature shift to 22°C. Triple samples were counted for each time point. The coloured boxes indicate the average of the three samples and bars represent the standard deviation of the mean. As observed, the presence or absence of inorganic sulphate did not affect transition in either direction.

Figure 4

Genes involved in cell organisation (cell wall, membrane and cytoskeleton), sulphur metabolism and ion transport. Genes that were identified as up-regulated in mycelium (22°C) or yeast (36°C) cells of P. brasiliensis are represented by black arrowheads. Electron microscopy was performed by Silva et al. [78].