RNA-seq and transcriptome-wide analysis of Sporothrix schenckii yeast and mycelial forms

Objectives: Sporothrix genus contains four phylogenetically closely-related species (S. schenckii, S. brasiliensis, S. globosa and S. luriei) that form the so-called "pathogenic clade" and which are responsible for almost all cases of sporotrichosis occurring in the world each year. These fungi exhibit a temperature-dependent dimorphic switch and are therefore able to convert between two different morphologies (mold and yeast) in response to thermal stimuli. In the environment (at 25◦C), they grow as septate hyphae that produce conidia that, when transmitted to humans (at 37◦C) or other susceptible mammalian hosts, are capable to convert into pathogenic yeasts causing serious infections. The transition from hyphae or conidia to yeast at 37◦C is essential for virulence but the regulation of this morphologic shift is complex and still not well-understood. In this study, using whole RNA-sequencing and bioinformatics analysis, we investigate the transcriptomic modifications that occur between the mold and yeast phases of S. schenckii. Moreover, using RNA-seq data, we characterize and annotate the assembled S. schenckii reference genome, and launch the “Sporothrix genome database”, an online resource that will provide gene, protein and sequence information for multiple Sporothrix species along with web-based tools for accessing, analyzing and exploring genomics data. Methods: In this study we sequenced the whole transcriptome of the reference strain S. schenckii CBS 132984 (formerly 1099-18 strain) which genome was previously sequenced, assembled and deposited in the Genbank database under the accession number GCA 000961545.1. To obtain the mold and yeast forms, and to induce the expression of the genes associated with these two different morphologies, the strain was inoculated in yeast extract peptone dextrose (YEPD) broth and incubated at 25 and 37◦C respectively. Extracted RNA was used to prepare TruSeq Stranded mRNA-paired-end sequencing libraries (2 × 150 bp) that were sequenced using the Illumina platform NextSeq500. A total of three biological replicates were sequenced for each condition. Illumina raw reads were first processed to remove adapters and sequences with low Phred-scores (cutoff: ≥30) and subsequently used for downstream bioinformatics analysis including differential gene expression analysis using the AIR program (https://transcriptomics.sequentiabiotech.com). Results: Illumina whole transcriptome sequencing produced about 33 million reads for each paired-end library. After quality filtering and trimming, approximately 90% of high-quality reads were used for mapping against the S. schenckii reference assembly GCA_000961545.1 and over 99% of these mapped uniquely to the fungal genome. Differential expression analysis performed with AIR showed that over 5,000 genes (FDR <0.05) were differentially expressed (logFC values between 9.6 and -12.3) between the two different examined conditions (mold at 25◦C and yeast at 37◦C). However, because about 30% of the reads mapped to unannotated regions (intergenic) of the existing reference genome assembly, we carried out a comprehensive genome re-annotation that will be available online at the “Sporothrix genome database”. Conclusion: The release of transcriptome-wide data of Sporothrix schenckii yeast and mycelial forms represents an important milestone for Sporothrix research because it sets the framework for future genetic studies to determine the transcriptional network underlying the dimorphic switching and also provides a platform for future comparative studies between closely related Sporothrix specie