Alternative splicing is a driving force that tunes metabolic adaptations to virulence traits in the dermatophyte Trichophyton rubrum

Abstract

Introduction: Alternative splicing (AS), a common process in pathogenic fungal species, is not fully understood. We hypothesized that AS is a critical regulatory mechanism that enables species to undergo continuous adaptations during interactions with challenging host environments.

Methods: We utilized the model species Trichophyton rubrum to contextualize the role of AS in fungal physiology and virulence. We performed transcriptome-wide splicing analysis to search for AS events in RNA-sequencing data of T. rubrum grown in keratin. This scenario mimicked infection in vitro and allowed us to map biologically relevant splicing events.

Results and discussion: Overall, the results showed that AS is recruited to regulate approximately 12.6% of the T. rubrum genome under an infection-like scenario. We extended this analysis to ex vivo infection models of T. rubrum grown on human nails and cocultured them with human HaCaT keratinocytes. We found that AS affects a wide range of cellular processes, including amino acid and carbohydrate metabolism, cell signaling, protein folding and transport, transcription, and translation. We showed that transcription factors such as PacC and Ap1 govern the major features of fungal virulence and metabolism and are controlled by the spliceosome machinery under different infection-like conditions.

Conclusions: Our data indicate that mRNA isoforms originating from AS contribute to the adaptation of T. rubrum, demonstrating that AS of transcription factor genes plays a central role in fungal pathogenesis. The transcription and splicing machinery tune fungal physiology to achieve an optimal metabolic balance in virulence traits during infection.

Keywords: Ap1; Con7; PacC; alternative splicing; fungal pathogen; intron retention; metabolism; transcription factor.

Figure 1

Distribution of genes modulated in response to keratin compared with the glucose (control). (A) Venn diagram of the number of alternative splicing genes (ASGs) and differentially expressed genes (DEGs) in response to keratin. (B) Venn diagram of alternative splicing (AS) events at 24, 48, and 96 h. (C) Total number of up- and down-regulated AS events at each time point. (D) Heatmap of functional enrichment analysis of ASGs based on Gene Ontology (GO) molecular function categories. The yellow-to-red color gradient indicates the fold-enrichment indices, which range from 0 to 16. Only the 30 most representative GO categories are displayed.

Figure 2

Functional characterization of genes with alternative splicing (AS) events in response to keratin. Functional annotation is categorized into biological processes (BP), molecular functions (MF), and cellular components (CC). (A) Functional characterization of 132 genes (from Figure 1B ) that exhibited AS across all tested time points. (B) Functional categorization of genes with repressed AS in keratin. Sphere size represents the number of genes, whereas the color gradient, from red to green, indicates the p-value, ranging from smallest to largest.

Figure 3

Expression network between differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) in Trichophyton rubrum in response to keratin. Network representation of DEGs and ASGs across all experimental conditions. Small blue nodes represent individual genes, whereas gray lines indicate connections between regulatory mechanisms and conditions.

Figure 4

Venn diagrams showing the number of transcription factor (TF) genes differentially expressed (DEG) and alternatively spliced (ASG) in response to keratin. (A) Number of TF-coding genes showing alternative splicing (AS) events and were differentially expressed when Trichophyton rubrum was exposed to keratin. (B) Number of TF genes showing AS at different times of exposure to keratin.

Figure 5

Schematization of transcription factor isoforms after conventional or alternative splicing. The transcripts and putative proteins resulting from each isoform are shown. Blue boxes represent exons, and introns are depicted as lines. Domains are indicated in the protein structure. IR shows the number of retained introns.

Figure 6

Expression of transcription factor isoforms of Trichophyton rubrum under in vitro and ex vivo conditions. (A) Expression analysis of pacC, con7, ap1, and c6 isoforms in T. rubrum upon exposure to several challenges. The control conditions were as follows: keratin (glucose 24 and 96 h), keratinocytes (RPMI 24 h), and nails (glucose 96 h). Paired controls, indicated by gray bars in each graph, were used as modulation references. Statistical analyses were performed using t-tests. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Intron retention (IR) percentage of pacC, con7, ap1, and c6 transcripts of T. rubrum exposed to different conditions. The proportions of alternative isoform IR are represented in blue, and the conventional isoform is in gray. The numbers above each bar represent the percentage of alternate splicing isoforms.

Figure 7

Expression of prps (splicing factors) of Trichophyton rubrum exposed to keratin. Statistical analysis was performed using t-test. *p < 0.05, **p < 0.01. Paired controls, indicated by the gray bar on each graph, were used as a modulation reference.

Figure 8

Comparison of putative PacC protein isoforms of Trichophyton rubrum. Three-dimensional structures of PacC protein enzymatically processed, in blue (A), from IR-2, in green (B), and their superposition (C). Additionally, alignment of the amino acid sequences of these two protein variants is shown (D).