Project description:Paracoccidioides brasiliensis is a thermally dimorphic fungus, which causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has been rarely addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the organism’s genome, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared gDNA and known control genes were printed onto glass slides to generate a microarray of over 12,000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from mycelia, especially at earlier time-points, and that mycelial gene expression changed less than it did on yeasts over time. Genes, upregulated in yeasts, were found to be involved in methionine/cysteine metabolism, respiratory processes, metabolic processes (sugars, amino acids, proteins and lipids), transporters (small peptides, sugar, ions and toxin), regulatory proteins and transcription factors. Mycelia genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transporters showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analyzed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes coding for ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand knowledge about the different morphologic forms of P. brasiliensis during growth in culture.

Project description:Paracoccidioides brasiliensis is a thermally dimorphic fungus, which causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has been rarely addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the organismM-bM-^@M-^Ys genome, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared gDNA and known control genes were printed onto glass slides to generate a microarray of over 12,000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from mycelia, especially at earlier time-points, and that mycelial gene expression changed less than it did on yeasts over time. Genes, upregulated in yeasts, were found to be involved in methionine/cysteine metabolism, respiratory processes, metabolic processes (sugars, amino acids, proteins and lipids), transporters (small peptides, sugar, ions and toxin), regulatory proteins and transcription factors. Mycelia genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transporters showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analyzed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes coding for ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand knowledge about the different morphologic forms of P. brasiliensis during growth in culture. Briefly we aimed at generating and comparing gene expression patterns of P. brasiliensis mycelia and yeasts during growth in liquid modified McVeigh/Morton media for 5, 8 and 14 days. We extracted total RNA from two cultures per time point for each morphologic form. Hybridizations were done twice for each total RNA sample. We used sheared genomic DNA as reference sample (Cy3) on every slide. Normalization was done as follows: Raw data underwent global normalization using the R statistical package to perform quantile normalization of the global reference channel by computing the centroid of all global reference signatures, and performing a loess fit of each individual global reference signature to the centroid. The loess curve for a given sample was then used to scale both channels for each gene in that sample. After that data was loaded in GeneSpring GX 7.3.1 and underwent the following normalization steps: -per spot to the gDNA channel -per slide using 102 positive controls (listed below) -per gene to each geneM-bM-^@M-^Ys respective median Positive controls: JM_2d11 JM_5a12 JM_22h5 JM_120a12 JM_61c4 JM_15g2 JM_13g9 JM_27c1 JM_55a6 JM_42a8 JM_14e1 JM_66c4 JM_13a9 JM_109e5 JM_20c1 JM_100a11 JM_62d8 JM_105c11 JM_22e8 JM_6a7 JM_66a5 JM_1e7 JM_42f5 JM_99h9 JM_9c7 JM_123g1 JM_33e5 JM_112a2 JM_25g6 JM_114h6 JM_98b1 JM_38h9 JM_30e11 JM_10e10 JM_22g11 JM_120c9 JM_120g4 JM_87g2 JM_70e12 JM_119e12 JM_79b12 JM_99a8 JM_67b5 JM_41b11 JM_76a2 JM_131e11 JM_65c2 JM_104e12 JM_26h3 JM_18c11 JM_56a7 JM_111c8 JM_21c9 JM_5e1 JM_3g12 JM_64e7 JM_115e5 JM_74c5 JM_117a5 JM_17b12 JM_10a5 JM_118e2 JM_15e5 JM_73e8 JM_89c6 JM_1c5 JM_5c8 JM_2e1 JM_83f10 JM_81e10 JM_83f11 JM_116a3 JM_5b11 JM_4c4 JM_86g6 JM_66e10 JM_44a6 JM_22a1 JM_22b4 JM_16a2 JM_22b5 JM_79g2 JM_26a11 JM_94b5 JM_25c2 JM_17e3 JM_66g1 JM_54a9 JM_84a11 JM_74b11 JM_115c6 JM_14a6 JM_122c9 JM_133e9 JM_11b9 JM_51b4 JM_24c7 JM_52b2 JM_66f3 JM_10c8 JM_57c1 JM_8b2

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Paracoccidioides brasiliensis

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