Dermatophytes are opportunistic pathogens that specifically colonize keratinized tissues. These pathogens engage in complex coevolutionary dynamics with their hosts, shaping their ability to colonize and infect keratinized tissues, such as the skin and nails, while influencing host immune responses (Deng et al., 2023; Gupta et al., 2023; Peres et al., 2010). Anthropophilic dermatophytes are an increasingly relevant medical concern affecting approximately 25% of the world’s population and cause chronic infections that can progress to invasive diseases in patients who are immunocompromised (Martinez-Rossi et al., 2021). Trichophyton rubrum is the most prevalent and clinically significant species worldwide, accounting for over 60% of all dermatophytosis cases (Zheng et al., 2020; Wang et al., 2021). Moreover, the emergence of resistant strains poses a challenge to the effective management of fungal infections (Bristow and Joshi, 2023; Martinez-Rossi et al., 2018; Kruithoff et al., 2023). Continuous adaptation of these organisms and their ability to resist conventional antifungal treatments emphasizes the need for ongoing research to determine the mechanisms underlying their infection persistence and develop novel therapeutic approaches.

Carbohydrate substrates are scarce in host tissues; therefore, dermatophytes degrade more complex molecules during infection. The main sources of available nitrogen and carbon are proteins, notably keratin. T. rubrum metabolic plasticity occurs via nitrogen catabolite repression, which is coordinated by the GATA zinc-finger transcription factors (TFs) (Martins et al., 2020). This process allows the use of complex nitrogen sources when preferential nitrogen sources such as glutamine are unavailable. However, keratin has a rigid structure formed by disulfide bonds, which prevent the usual proteolytic digestion of this molecule. Dermatophytes can produce urea and sulfites secreted by the Ssu1 efflux pump, which reduces covalent bonding (Martins et al., 2020; Grumbt et al., 2013). Keratin degradation and the metabolism of specific amino acids such as glycine to generate acetyl-CoA promote ammonia release and consequent alkalinization of the medium, which can increase pH values to as high as 9.0 (Ferreira-Nozawa et al., 2003; Maranhão et al., 2007). As the infection progresses, repression/activation of pH-regulated pathways and enzymes occurs in response to cell demands and environmental changes.

In this scenario, effective adaptation during infection requires fungi to coordinate diverse metabolic responses. Fungal cells activate transcriptional programs according to the surrounding conditions, such as cellular stress and host infection sites (Martinez-Rossi et al., 2021; Park et al., 2013). Arguably, the most important components governing these response programs are TFs, which regulate gene expression through hierarchical interactions between signaling and response networks (Song et al., 2016). In dermatophytes, many attributes related to virulence and susceptibility to stressors, as well as tolerance to different antifungals, are regulated by TFs such as PacC, Dnr1, Ap1, HacA, SteA, and StuA (Kröber et al., 2017; Yamada et al., 2006; Ferreira-Nozawa et al., 2006; Peres et al., 2022; Bitencourt et al., 2020; Lang et al., 2020; Martins-Santana et al., 2025).

The post-transcriptional regulation of gene expression by alternative splicing (AS) is a common process in fungi (Grützmann et al., 2014). AS may influence the activity of many genes in response to the various conditions imposed on these organisms (Grützmann et al., 2014; Mendes et al., 2018; Neves-da-Rocha et al., 2019; Lopes et al., 2022). Intron retention (IR) events are the most common type of AS in fungi (Grützmann et al., 2014; Kempken, 2013; Jeon et al., 2022; Li et al., 2021). In addition, important correlations exist between AS and distinct fungal species, with multicellular complexity, pathogen lifestyle, and a younger evolutionary age being associated with higher AS rates (Grützmann et al., 2014). Nevertheless, the extent of AS and the biological relevance of this post-transcriptional mechanism in fungal pathogenesis remain unclear. To investigate and correlate AS with fungal biology, we assessed global AS patterns in the transcriptome of T. rubrum exposed to keratin, which enabled mapping of biologically relevant AS events in an infection-like manner. We further performed functional enrichment analysis to investigate the physiological consequences of AS regulation. IR events in major fungal TFs were validated using in vitro and ex-vivo infection models to demonstrate the practical relevance of our findings. Because dermatophytes are representative filamentous pathogenic fungi in which AS is prevalent, this study improved the understanding of the strategies employed by pathogens to adapt to and colonize host tissues. Furthermore, our data provide critical fundamental information for guiding future studies on AS and medical mycology.

We comprehensively evaluated the functional categories of genes that undergo AS in the human pathogen T. rubrum using a model that mimics infection. Our findings revealed that a large proportion of genes, approximately 12.6% of the genome, underwent AS, and approximately 39% of these genes are differentially expressed. This percentage of 12.6% is high considering the rate of AS observed in other pathogenic fungi, such as Sclerotinia sclerotiorum (5.6%) (Cheng et al., 2022), Aspergillus oryzae (8.55%) (Wang et al., 2010), and Ustilago maydis 3.6% (Ho et al., 2007), assayed under different conditions. Indeed, identification of TF genes that undergo many AS events in other pathogenic fungi suggests that AS regulates a broad spectrum of processes, highlighting the complex interplay of mechanisms regulating these genes in T. rubrum. Notably, at least 61% of ASGs did not exhibit differential expression, suggesting that AS plays a crucial role in regulating T. rubrum infection beyond the level of gene expression. These findings have significant implications for the understanding of pathogen–host interactions.

As a major step towards characterizing the role of the spliceosome machinery in dermatophytes, we assessed the global impacts of AS regulation on T. rubrum physiology. Functional enrichment analysis revealed that AS might contribute extensively to fungal metabolic adaptation during changes in nutrition sources, with direct impacts on the infection dynamics. During keratin degradation compared to glucose growth, AS influenced sensing and regulatory processes that are required for efficient physiological responses, such as kinase and phosphatase activities, transcription factors and splicing factors, transporters, enzyme activators, chaperones, and histone modifications ( Figure 1D ). Interestingly, considering the genes modulated in common in all time points in response to keratin, T. rubrum showed increased spliceosome influence over central carbon and nitrogen metabolism, affecting glycolysis, glycogen biosynthesis, amino acid metabolism, and proteasome function ( Figure 2A ). Those pathways indicate that dermatophyte physiology requires specific tuning of function to degrade complex substrates such as keratin, which is potentially influenced by nutrient limitation during keratin-based host colonization. Those findings are in accordance with previous data, which demonstrate keratin influence over dermatophyte central metabolism, including the control of nitrogen catabolite repression for regulating the TCA and urea cycles (Martins et al., 2020).

The number of ASGs in T. rubrum is particularly important during the initial phase of infection, when the pathogen must rapidly adapt to the new environment. Based on these findings, splicing factor genes such as prp1, prp22, prp28, and prp43, which are crucial in this process, were induced upon exposure to keratin, mainly at 24 h. This activation underscores the pivotal role of these four splicing factors in adaptation. Furthermore, AS events in TFs were present at all three tested time points, indicating that this phenomenon is an indispensable regulatory pathway for the adaptation, establishment, and maintenance of T. rubrum infection. The extensive range of categories that perform vital functions in cells under control of the splicing machinery in this infection-like context enhances the understanding of the crucial role of AS in fungal adaptation and pathogenicity. These findings may lead to the development of new strategies for combating fungal infections. The four selected TF genes revealed different expression profiles and IR in keratin medium, keratinocytes, and human nails. These results suggest that the fungus uses an intricate mechanism in its interaction with the host, depending on the tissue type and culture time.

The PacC signal transduction pathway, a crucial element in fungal infection, regulates the synthesis of a diverse range of enzymes and proteins with optimal activity at specific pH values dictated by fungal metabolism. This complex process enables the fungus to adhere to and penetrate host tissue, obtain nutrients, and subvert host defense mechanisms. Because human skin has an acidic pH, the first contact of dermatophytes during infection occurs in an acidic environment. We previously showed that the growth of pacC mutant strains of various fungi, including T. rubrum, is severely impaired under alkaline conditions (Ferreira-Nozawa et al., 2006; Trushina et al., 2013; Wiemann et al., 2009; Barda et al., 2020). The strong expression of pacC observed in keratin reinforces the involvement of this gene in keratin metabolism (Martins et al., 2020). The presence of IR-2 in pacC transcripts leads to the formation of protein isoforms that maintain three zinc-finger domains. In addition, in silico analyses indicated that the putative PacC protein generated by splicing (274 amino acids (aa)) resembled the activated form of the homolog protein of Aspergillus nidulans after proteolytic cleavage (254 aa) in terms of size, sequence, and domain number (three zinc finger domains) ( Supplementary Figure S4 ). Functional activation of PacC in A. nidulans is a two-step proteolytic process, one of which is mediated by PalB, a calpain-family protease (Tilburn et al., 1995; Martinez-Rossi et al., 2012; Li et al., 2022), generating a minor protein containing three zinc fingers domains (Tilburn et al., 1995). Similarly, in silico data suggested that the putative version of PacC enzymatically activated in T. rubrum exhibited a conformation, size, and domain number similar to the putative isoform generated by IR-2 ( Figure 8 ). Our RNA-seq data revealed high pacC expression, whereas differential expression of the T. rubrum palB homolog was not detected. This observation suggests the potential for alternative pathways to activate PacC protein at an acidic pH when culturing T. rubrum in glucose. Previous studies suggested that PacC protein in A. nidulans can be activated through an alternative pathway to PalB proteolysis due to the induction of an alternative palB isoform that is potentially nonfunctional, whereas PacC is functional (Trevisan et al., 2011). Our results suggest that IR-2 of the pacC pre-mRNA is an alternative mechanism to enzymatic activation of PacC at acidic pH in T. rubrum.

Although IR-3 in the con7 transcripts resulted in a minor protein (197 aa) compared to the conventional isoform (263 aa), the zinc-finger domain was present in both isoforms. The prediction of phosphorylation sites in Con7 protein isoforms also revealed interesting differences between the two isoforms. These isoforms share 20 phosphorylation sites on the serine, threonine, and tyrosine residues. However, the alternative isoform had seven distinct phosphorylation sites resulting from the alternatively generated amino acid sequence, with five serine and two threonine residues ( Supplementary Figure S2 ). These findings have important functional implications. Changes in the phosphorylation profile of TFs represent a mechanism employed by cells to mediate the activity of these proteins in response to environmental changes by regulating their transcriptional activity, cellular localization, stability, protein–protein interactions, DNA binding, and coregulators (Riedl and Egly, 2000; Whitmarsh and Davis, 2000; Zhou et al., 2020). Moreover, the IR-3 of con7 transcripts of T. rubrum was more prevalent when the fungus was cultured with keratinocytes than in the control ( Figure 6B ), suggesting a role of this isoform in tissue-specific interaction with the host. The role of this gene in fungal virulence and host interactions has been revealed in Fusarium oxysporum (Ruiz-Roldán et al., 2015). con7 in Magnaporthe grisea and F. oxysporum also have splicing variants, including isoforms generated through IR during vegetative growth and infection (Odenbach et al., 2007; Ruiz-Roldán et al., 2015).

IR-1 in ap1 transcripts generated a small protein of 153 amino acids lacking the essential leucine zipper domain, indicating loss of its function as a transcriptional regulator. However, notably, IR-1 isoform showed a high percentage among ap1 transcripts when cultured with keratinocytes (63%), suggesting that this isoform is essential for interactions with HaCaT cells ( Figure 6B ). This alternative isoform may lead to the formation a putative mini-protein without a typical Ap1 domain. However, we cannot rule out the possibility that the RNA produced participates in fungal interactions with keratinocytes. As previously reported, the Δap1 mutant of T. rubrum exhibited increased growth in cocultures with keratinocytes and nail fragments compared to the wild type, suggesting that ap1 participates in the negative control of virulence-related attributes and contributes to the chronicity of infection caused by this dermatophyte (Peres et al., 2022). The regulation of fungal virulence traits affects the chronicity of the disease and evasion of the host immune system (Martinez-Rossi et al., 2021; Peres et al., 2022). We hypothesized that the correct balance in the production of these ap1 isoforms contributes to the control of the chronic infection.

The presence of IR-2 in the c6 isoforms resulted in a minor protein, with a partial loss of the fungal-specific TF domain. However, the Zn(2) Cys(6) DNA-binding domain was maintained, suggesting that the function of this putative protein differed from that of the conventional isoform. In keratin, human nails, and keratinocytes, the IR-2 isoform of c6 was more prevalent than in its respective controls, suggesting that it functions under infectious conditions ( Figure 6B ). These data revealed the post-transcriptional regulation of the mechanisms involved in fungal pathogenicity. In Magnaporthe oryzae, TFs are a vital group of genes regulated by AS during fungal infections, with the C6 TF group being the most common (Jeon et al., 2022). The C6 TF of Aspergillus fumigatus (AFUB_043270) exhibits IR, which is a potential mechanism of self-protein activation control (Sieber et al., 2018). These data highlight the importance of C6-type TF AS events in several fungal infections.

Finally, the presence of alternative isoforms, even under control conditions (glucose or RPMI), indicates that infection conditions do not always dictate the presence of AS but instead influence the proportion of these events. These data underscore the importance of investigating the proportion of alternative isoforms compared to total isoforms, providing precise quantification of this phenomenon.

In conclusion, our data for these TF genes validate AS events and corroborate the idea that pathogenic fungi show different AS patterns in their infection environments (Jeon et al., 2022; Sieber et al., 2018; Cheng et al., 2022; Ibrahim et al., 2021). Our results also improve the understanding of the involvement of TF isoforms in this process, as pacC, con7, ap1, and c6 are crucial TFs for virulence and fungal adaptation to the host environment (van der Does and Rep, 2017; Wang et al., 2024; John et al., 2021). Exposure of the fungus to keratinolytic substrates induces the modulation of transcriptional and post-transcriptional responses, particularly of virulence factors and metabolic regulation. The regulatory amplitude exerted by AS under infection-like conditions suggests the impact of this mechanism on providing adequate responses to T. rubrum pathogenesis. The consequences observed in the TF genes from this regulation clarify how this mechanism contributes to the strategies employed by the fungus to adapt and colonize the host. This study provides a foundation for future fungal infection management by improving the understanding of the molecular mechanisms employed by pathogens to infect their hosts.