Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing

Abstract

Coccidioides immitis and C. posadasii are dimorphic fungi that transform from mycelia with internal arthroconidia in the soil to a tissue form known as a spherule in mammals. This process can be recapitulated in vitro by increasing the temperature, CO₂ and changing other culture conditions. In this study, we have analyzed changes in gene expression in mycelia and young and mature spherules. Genes that were highly upregulated in young spherules include a spherule surface protein and iron and copper membrane transporters. Genes that are unique to Coccidioides spp. are also overrepresented in this group, suggesting that they may be important for spherule differentiation. Enriched GO terms in young spherule upregulated genes include oxidation-reduction, response to stress and membrane proteins. Downregulated genes are enriched for transcription factors, especially helix-loop-helix and C2H2 type zinc finger domain-containing proteins, which is consistent with the dramatic change in transcriptional profile. Almost all genes that are upregulated in young spherules remain upregulated in mature spherules, but a small number of genes are differentially expressed in those two stages of spherule development. Mature spherules express more Hsp31 and amylase and less tyrosinase than young spherules. Some expression of transposons was detected and most of the differentially expressed transposons were upregulated in spherules.

Keywords: Coccidioides immitis; RNA-Seq; coccidioidomycosis; differential gene expression; differentiation; dimorphic fungus; fungus; mycelium; spherule.

Figure 1

Differential expression of genes in mycelia, young and mature spherules. Legend Figure 1: mRNA was obtained from mycelia, young spherules and mature spherules for RNA-Seq. The morphology of the organism and the number of differentially expressed genes is shown.

Figure 2

Length of genus-specific and conserved genes. Legend Figure 2: Lengths (Y-axis) of conserved and differentially expressed genus-specific proteins.

Figure 3

Phylogenetic tree of C2H2 transcription factors and FC. Legend: Phylogenetic tree was built on protein sequence. FC (young spherule/mycelium) values of C. immitis C2H2 transcription factors. Gene IDs within tan boxes are downregulated more than 4-fold; those that are within yellow boxes are downregulated more than 2-fold but less than 4-fold; those not within boxes are not significantly differentially expressed and those within blue boxes are upregulated more than 2-fold. None of these genes were upregulated more than 4-fold. All values are arithmetic.

Figure 4

Comparison of FC values in this study to a previous study by Whiston et al. Legend: Genes with log₂ FC values (young spherules/mycelia) > 2 or < –2 in the current study were selected. FC values of genes in the current study are shown on the X axis and the FC values of matched genes from Whiston study [7] are shown on the Y axis. The regression line was derived from the linear model and the shaded area indicates the 95% confidence limits. The plot was made with ggplot2 R package and the regression line was drawn with the geom smooth option.

Figure 5

Overlap of up- and downregulated genes in young and mature spherules. Legend: Comparison of up- and downregulated genes in young spherules and mature spherules. Blue represents young spherules, yellow represents mature spherules.