Influence of 17β-Estradiol on Gene Expression of Paracoccidioides during Mycelia-to-Yeast Transition

BACKGROUND: Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17β-estradiol, E(2)) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E(2) inhibits M-to-Y transition. METHODOLOGY: We assessed temporal gene expression in strain Pb01 in the presence or absence of E(2) at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E(2)-regulated clones were sequenced to identify genes and biological function. PRINCIPAL FINDINGS: E(2)-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p≤0.001). Genes with low expression after 4 or 12 h exposure to E(2) belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, α-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E(2)-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E(2), whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E(2). CONCLUSION: This study characterizes the effect of E(2) at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E(2) may be working through signaling genes that regulate dimorphism

Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers

Diversity in 26 microsatellite loci from section Caulorrhizae germplasm was evaluated by using 33 accessions of A. pintoi Krapov. & W.C. Gregory and ten accessions of Arachis repens Handro. Twenty loci proved to be polymorphic and a total of 196 alleles were detected with an average of 9.8 alleles per locus. The variability found in those loci was greater than the variability found using morphological characters, seed storage proteins and RAPD markers previously used in this germplasm. The high potential of these markers to detect species-specific alleles and discriminate among accessions was demonstrated. The set of microsatellite primer pairs developed by our group for A. pintoi are useful molecular tools for evaluating Section Caulorrhizae germplasm, as well as that of species belonging to other Arachis sections

Analysis of the sequenced clones showing sequence similarity with retrotransposon elements.

<p>Sequenced clones showing sequence similarities with retrotransposon elements are subjected to Sequencher software that resulted in 4 contigs and 4 singlets. GI: GenBank accession number from <a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>. BLASTX expect value ≤1e-04 was considered significant.</p

E₂ regulated Pb01 genes during M-to-Y transition.

<p>In comparison to controls, E₂ regulated Pb01 genes assigned to a functional category that have significant change of expression values at selected time points during M-to-Y transition. 1; Energy production, 2; Yeast genes, 3; Mycelial gene, 4; Transporters, 5; Heat-shock response, 6; Oxidative stress, 7; Ribosomal proteins, 8; Ubiquitin-mediated protein degradation, 9; Signal transduction, 10; RNA processing, 11; Chromatin structure, 12; Others (2A). E₂ regulated retrotransposable elements; 31 sequenced clones with sequence similarity to retrotransposon elements (2B). Red represents significantly higher expression and green represents a significantly low level of expression, from microarray data. Genes with a p≥0.001 were set to black at all time points. The heat-map was prepared using online software available at <a href="http://www.bioinformatics.ubc.ca" target="_blank">http://www.bioinformatics.ubc.ca</a>.</p

Temperature induced mycelia-to-yeast transition in Pb01.

<p>Temperature induced mycelia-to-yeast transition in Pb01. at various time-points in control and E₂-treated samples, ×400 magnification (1A). Number of array elements showing significant (p≤0.001) differential expression in E₂-treated samples in comparison to control at various times during morphologic transition (1B).</p

Sequence analysis of E₂ regulated gene and expression values in comparison to controls at various time-points during M-to-Y transition.

<p>Geometric mean of linear values from microarray data; E₂ (17β-estradiol) vs Controls (untreated control plus ethanol-treated control), M: Mycelia. 6: Oxidative stress, 7: Ribosomal proteins, 8: Ubiquitin-mediated protein degradation, 9: Signal transduction, 10: RNA processing, 11: Chromatin structure, 12: Others. GI: GenBank accession number from <a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>. BLASTX expect value ≤1e-04 was considered significant.</p

Alignment of 19 retrotransposable sequences found in contig 1 that are differentially regulated in E₂-treated samples in comparison to control.

<p>Sequence domains were identified from conserved hypothetical protein (ORF1: Retrotransposon gag protein) of <i>Penicillium marneffei</i> (GI: 212525972), ORF2: Retrovirus polyprotein, putative, of <i>P. marneffei</i> (GI: 212525970) and hypothetical protein of <i>A. nidulans</i> (GI: 67524427).</p

Sequence analysis of E₂ regulated gene and expression values in comparison to controls at various time-points during M-to-Y transition.

<p>Geometric mean of linear values from microarray data; E₂ (17β-estradiol) vs Controls (untreated control plus ethanol-treated control), M: Mycelia. 1: Energy production, 2: Yeast genes 3: Mycelial genes, 4: Transporters, 5: Heat-shock response.</p><p>*Mitochondrial encoded gene. GI: GenBank accession number from <a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>. BLASTX expect value ≤1e-04 was considered significant.</p

Quantification of expression of genes by controls and E₂-treated Pb01 cells during M-to-Y transition.

<p>Panel A illustrates <i>hsp90</i>, B; <i>hsp70</i>, C; <i>hsp40</i>, D; hydrophobin, E; <i>Y20</i>, F; chitin synthase, G; <i>gfa1</i>, H; <i>gls1</i>, I; <i>mok11</i>, J; GTPase RhoA, K; phosphatidylinositol 4-kinase, L; <i>cdr4</i>, M; <i>ubr11</i>, and N; retropepsin. The expression value of each gene was obtained from β-tubulin-normalized quantitative real-time PCR (qRT-PCR), plotting controls (C; untreated plus ethanol-treated control) vs E₂-treated at different time points. The measured quantity of each Pb01 gene from each sample was normalized by using C_T values obtained for the β-tubulin qRT-PCR on the same plate. The relative quantification of each gene and β-tubulin gene expression was determined by a standard curve (i.e. C_T values plotted against logarithm of the DNA copy number). The values represent the number of copies of the cDNA of each gene divided by the number of copies of the cDNA of the β-tubulin gene. The data represent the mean (±SEM) of triplicate of qRT-PCR runs from RNA samples of two independent biological replicates used in the microarray hybridization. Statistical comparison between C (controls) and E₂ (E₂-treated) samples was done at each time point and significant differences denoted on the figure above the bars by one or more asterisks, which indicate the p value (* p<0.05, ** p<0.01, *** p<0.001). The box on the right side of each figure shows the result of statistical comparison between C (controls) or E₂ (E₂-treated) samples at various time points and the 0 h baseline time point.</p