P421 Whole-transcriptome analysis of Sporothrix brasiliensis grown in mold- and yeast-inducing conditions

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Sporothrix brasiliensis is an emerging Sporothrix species limited to Brazil capable of causing sporotrichosis in humans and animals, especially in cats. Like other pathogenic Sporothrix species, S. brasiliensis exhibits a temperature-dependent dimorphic switch and is therefore, able to undergo a reversible morphological transition (mold and yeast), in response to environmental thermal stimuli.While dimorphism appears to be essential for virulence in Sporothrix spp, the molecular mechanisms involved in this phenomenon have not yet been fully elucidated.In this study, we used the strand-specific RNA-Seq technique and bioinformatics analysis to investigate the transcriptomic signatures associated with mold and yeast phases of S. brasiliensis. Furthermore, we generated an accurate version of the S. brasiliensis genome annotation in order to perform high-quality gene expression analysis and other functional or structural genomic studies.MethodsThe whole transcriptome of S. brasiliensis ATCC-MYA-4823, grown in both yeast-inducing (YPD medium at 37°C) and mold-inducing (YPD medium at 25°C) conditions, was sequenced in this study. High-quality RNA was used to prepare Illumina TruSeq Stranded mRNA-paired-end sequencing libraries (2 × 150 bp) that were sequenced using the HiSeq-2500 platform. A total of three biological replicates were sequenced for each condition.Before transcriptome assembly, adapters and low-quality reads (Phred-score <25) were removed. The StringTie software was used to assemble the transcriptomes imported into the Apollo webtool to manually curate the genome annotation. Transcripts were investigated using TransDecoder and CPC2 programs to determine whether a gene was potentially protein-coding or non-coding. Finally, differential gene expression analysis between yeast and mold forms of S. brasiliensis was conducted using the edgeR package.ResultsIllumina sequencing resulted in a total of ∼217 million raw reads. After quality filtering and trimming, ∼99.5% of reads were used for downstream bioinformatics analysis. The updated S. brasiliensis genome annotation consisted of a total of 14 664 genes of which 10 243 protein-coding genes, 4259 lncRNAs, 140 tRNAs, and 22 rRNAs.Gene expression analysis revealed a total of 13 838 and 13 938 transcripts expressed in mold- and yeast-form, respectively. Of these, 192 and 292 were expressed exclusively in the mold and yeast-phase, respectively. Moreover, a total of 6802 genes (FDR <0.05) were differentially expressed between the two examined conditions. In particular, 3420 of these genes were up-regulated in the yeast-form (2450 coding, 970 non-coding), and 3382 genes in the filamentous form (2507 coding, 875 non-coding). The raw reads have been deposited into the SRA database and are available under BioProjectID: PRJNA646214.ConclusionsThe characterization of the whole-transcriptome of S. brasiliensis mycelial and yeast-like forms represents an essential starting point for investigating the molecular pathways and regulatory frameworks associated with these two morphological stages. Our results provide new insight into global gene expression profiles of S. brasiliensis, emphasizing the role of non-coding RNAs in its complex transcriptional network.All transcriptomic data have also been integrated into the ‘Sporothrix Genome DataBase’ (www. sporothrixgenomedatabase.unime.it) in order to expand the current knowledge of Sporothrix genomics and to allow a more in-depth structural exploration of S. brasiliensis gene models, including gene expression patterns related to its saprophytic and pathogenic lifestyle.