Dual RNA-Seq Analysis of Trichophyton rubrum and HaCat Keratinocyte Co-Culture Highlights Important Genes for Fungal-Host Interaction

Abstract

The dermatophyte Trichophyton rubrum is the major fungal pathogen of skin, hair, and nails that uses keratinized substrates as the primary nutrients during infection. Few strategies are available that permit a better understanding of the molecular mechanisms involved in the interaction of T. rubrum with the host because of the limitations of models mimicking this interaction. Dual RNA-seq is a powerful tool to unravel this complex interaction since it enables simultaneous evaluation of the transcriptome of two organisms. Using this technology in an in vitro model of co-culture, this study evaluated the transcriptional profile of genes involved in fungus-host interactions in 24 h. Our data demonstrated the induction of glyoxylate cycle genes, ERG6 and TERG_00916, which encodes a carboxylic acid transporter that may improve the assimilation of nutrients and fungal survival in the host. Furthermore, genes encoding keratinolytic proteases were also induced. In human keratinocytes (HaCat) cells, the SLC11A1, RNASE7, and CSF2 genes were induced and the products of these genes are known to have antimicrobial activity. In addition, the FLG and KRT1 genes involved in the epithelial barrier integrity were inhibited. This analysis showed the modulation of important genes involved in T. rubrum–host interaction, which could represent potential antifungal targets for the treatment of dermatophytoses.

The Transcriptional Profile of Trichophyton rubrum Co-Cultured with Human Keratinocytes Shows New Insights about Gene Modulation by Terbinafine

“…RNA was isolated and its integrity was analyzed as described by Petrucelli et al [26]. Only RNA with an RNA integrity number (RIN) > 7 was accepted for construction of the cDNA libraries.…”

“…The sequence data were analyzed as described by Petrucelli et al [26]. According to the distribution of the genes, those exhibiting statistical significance <0.05 and a log 2 fold change ≥1.5 or ≤−1.5 were classified as differentially expressed genes (Supplementary Figure S1).…”

“…In order to present a more in-depth view of the effect of terbinafine on T. rubrum genes modulation, the present study is the first to evaluate the effect of this drug using large-scale analysis of the expression profile of T. rubrum co-cultured with human keratinocytes in an attempt to mimic fungus-host interactions. In addition to this model, we applied the dual RNA-seq technique that is widely used for the study of these interactions [21] in bacteria [22], viruses [23], and fungi [24][25][26], and enable the entire transcriptome to be surveyed in a high-throughput and quantitative manner [20].…”

Attenuation of cGAS/STING Activity During Mitosis

“…We chose to investigate activity of the cGAS/STING pathway in HaCaTs, a spontaneously immortalized human keratinocyte cell line [57]. This cell line is a good model for primary keratinocytes that serve a barrier function and as host cells for a number of viral infections including papillomaviruses, herpesviruses, mosquito-transmitted alphaviruses and flaviviruses [58][59][60][61][62][63][64], and numerous bacterial and fungal pathogens [65,66]. As not all cell types express every PRR, we sought to ensure the cGAS/STING pathway was functional in HaCaTs and that this pathway was the primary mode of IRF3 phosphorylation in response to foreign DNA.…”

The Role of Reactive Oxygen Species and Nitric Oxide in the Inhibition of Trichophyton rubrum Growth by HaCaT Cells

“…Trichophyton rubrum (T. rubrum) is a dermatophyte responsible for the majority of fungal infections worldwide [1] and accounts for as many as 69.5% of dermatophytoses in humans [2]. Although dermatomycoses are frequently limited to superficial keratinized tissues such as nails, skin, and hair, fungi may invade the dermis and subcutaneous tissue, especially when precipitated by trauma, immunosuppression, or the use of topical corticosteroids [3].…”