Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro

Abstract

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and 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 rarely been 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 genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (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 those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport 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 analysed 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 encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.

Fig. 1.

Pie chart representing 481 sequenced clones showing the general distribution of categories after sequence analysis. Y, yeast, M, mycelia.

Fig. 2.

Real-time RT-PCR results for eight specific genes obtained from the same RNA samples used in the microarray hybridizations; each group of bars represents days 5, 8 and 14 from left to right. mannosyl transf, mannosyltranferase gene.

Fig. 3.

Correlation plot of mycelial and yeast datasets with eigenarrays as determined by PCA. Correlations with eigenarray 1 (x axis), which captured 60.84 % of the variance present in the data, show opposing behaviours in mycelia and yeast, which could be related to morphology and/or temperature. Correlations with eigenarray 2 (y axis), which captured 20.91 % of the variance present in the data, show that mycelial datasets are more closely related to each other than to yeast datasets, suggesting more changes in yeast over the sample times examined.

Fig. 4.

Hierarchical clustering of all genes present in the microarray using Pearson correlation as the similarity measure. Relative gene expression is centred around the median expression value per gene, where bright red is twofold expression or greater with respect to the median, black is expression equal to the median value and bright green is twofold expression or less with respect to the median. M, mycelia; Y, yeast; Time, days of growth in culture. Cluster 1, genes overexpressed in yeast at all time points; cluster 2, genes overexpressed in yeast with increased levels over time; cluster 3, genes overexpressed in mycelia with decreased levels over time; cluster 4, genes that show stable expression in mycelia and increased expression in yeast over time.

Fig. 5.

Clustering of 521 known genes (on the left) present in the microarray using Pearson correlation as the similarity measure. Relative gene expression is centred around the median expression value per gene, where bright red is twofold expression or greater with respect to the median, black is expression equal to the median value and bright green is twofold expression or less with respect to the median. All genes have more than 50 % of pixels in each spot above the Cy5 background by one standard deviation in at least one time point. M, mycelia; Y, yeast; Time, days of growth in culture. The Y Genes centre panel is a close-up view of the corresponding cluster on the tree to the left. The M Genes panel on the right is a close-up view of the corresponding cluster on the tree to the left.