Abstract
Coccidioides immitis and C. posadasii, the causative agents of coccidioidomycosis, are dimorphic fungal pathogens, which grow as hyphae in the saprobic phase in the environment and as spherules in the parasitic phase in the mammalian host. In this study, we use comparative transcriptomics to identify gene expression differences between the saprobic and parasitic growth phases. We prepared Illumina mRNA sequencing libraries for saprobic-phase hyphae and parasitic-phase spherules in vitro for C. immitis isolate RS and C. posadasii isolate C735 in biological triplicate. Of 9,910 total predicted genes in Coccidioides, we observed 1,298 genes up-regulated in the saprobic phase of both C. immitis and C. posadasii and 1,880 genes up-regulated in the parasitic phase of both species. Comparing the saprobic and parasitic growth phases, we observed considerable differential expression of cell surface-associated genes, particularly chitin-related genes. We also observed differential expression of several virulence factors previously identified in Coccidioides and other dimorphic fungal pathogens. These included alpha (1,3) glucan synthase, SOWgp, and several genes in the urease pathway. Furthermore, we observed differential expression in many genes predicted to be under positive selection in two recent Coccidioides comparative genomics studies. These results highlight a number of genes that may be crucial to dimorphic phase-switching and virulence in Coccidioides. These observations will impact priorities for future genetics-based studies in Coccidioides and provide context for studies in other fungal pathogens.
Highlights
The methods for transcriptional profiling have changed dramatically in recent years from microarray-based techniques to full transcriptome sequencing using next-generation sequencing (NGS) technologies
Experimental Design To compare gene expression in the saprobic and parasitic growth phases between the sibling Coccidioides species, C. immitis and C. posadasii, we focused our analyses on actively-growing hyphae and pre-endosporulation spherules cultured for 96 hours in vitro in their respective growth conditions as described in the methods
The library preparation protocol includes an mRNA pull-down, not all of the rRNA was removed from the samples because assigned reads mapping to ribosomal RNA sequence accounted for a mean of 11.8% in saprobic-phase libraries and 1.3% in parasitic-phase libraries (p,0.0001)
Summary
The methods for transcriptional profiling have changed dramatically in recent years from microarray-based techniques to full transcriptome sequencing using next-generation sequencing (NGS) technologies. NGS offers many advantages over traditional microarrays, but the underlying principle of comparative transcriptomics remains the same: analysis of changes in gene expression between conditions can identify genes critical to cellular responses to environmental cues, morphological change and growth. Transcriptional profiling has been used in many fungal pathogens to identify genes critical to growth in a host environment [1,2,3,4]. Considered a single species, we know that there are two species of Coccidioides: C. immitis and C. posadasii [6]. There are no discernable phenotypic differences in pathogenicity between the two species, differences in salt tolerance and thermal tolerance have been observed [6,8,9]
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