The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.

Molecular Signaling and Metabolic Responses during the Interaction between Human Keratinocytes (HaCaT) and the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is the leading causative agent of dermatophytosis worldwide. Keratinocytes are the first line of defense that drives an immune response against fungal invasion. Host-specific pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) to trigger immunological pathways. Fungal cell wall components are the primary sources of fungal PAMPs, and some pathogens increase cell wall rearrangement to evade the immune system. Glycolysis and enhanced lactate levels are critical for improving host immune responses to fungal infections. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), we evaluated the transcriptional responses of human genes involved in fungal recognition and glycolytic metabolism and fungal cell-wall-related genes in a co-culture model of human keratinocytes with T. rubrum. We observed the upregulation of several Toll-like receptors (TLRs), NOD-like receptors (NLRs), and glycolytic genes. Complementarily, we measured intra- and extracellular glucose levels and the increase in lactate production in the co-culture supernatant. We noted a distinct transcriptional regulation pattern of fungal cell-wall-related genes from fungal growth on keratin as the primary carbon source compared to co-culture with human keratinocytes. Our results showed new insights into the transcriptional adaptation of keratinocytes, particularly in regulating genes involved in sensing and metabolic processes, during the interaction with T. rubrum.

Transcriptome Analysis of Co-Cultures of THP-1 Human Macrophages with Inactivated Germinated Trichophyton rubrum Conidia.

Although most mycoses are superficial, the dermatophyte Trichophyton rubrum can cause systemic infections in patients with a weakened immune system, resulting in serious and deep lesions. The aim of this study was to analyze the transcriptome of a human monocyte/macrophage cell line (THP-1) co-cultured with inactivated germinated T. rubrum conidia (IGC) in order to characterize deep infection. Analysis of macrophage viability by lactate dehydrogenase quantification showed the activation of the immune system after 24 h of contact with live germinated T. rubrum conidia (LGC). After standardization of the co-culture conditions, the release of the interleukins TNF-α, IL-8, and IL-12 was quantified. The greater release of IL-12 was observed during co-culturing of THP-1 with IGC, while there was no change in the other cytokines. Next-generation sequencing of the response to T. rubrum IGC identified the modulation of 83 genes; of these, 65 were induced and 18 were repressed. The categorization of the modulated genes showed their involvement in signal transduction, cell communication, and immune response pathways. In total, 16 genes were selected for validation and Pearson's correlation coefficient was 0.98, indicating a high correlation between RNA-seq and qPCR. Modulation of the expression of all genes was similar for LGC and IGC co-culture; however, the fold-change values were higher for LGC. Due to the high expression of the IL-32 gene in RNA-seq, we quantified this interleukin and observed an increased release in co-culture with T. rubrum. In conclusion, the macrophages-T. rubrum co-culture model revealed the ability of these cells to modulate the immune response, as demonstrated by the release of proinflammatory cytokines and the RNA-seq gene expression profile. The results obtained permit to identify possible molecular targets that are modulated in macrophages and that could be explored in antifungal therapies involving the activation of the immune system.

The Transcription Factor StuA Regulates the Glyoxylate Cycle in the Dermatophyte Trichophyton rubrum under Carbon Starvation.

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.

The Transcriptional Regulation of Genes Involved in the Immune Innate Response of Keratinocytes Co-Cultured with Trichophyton rubrum Reveals Important Roles of Cytokine GM-CSF.

Trichophyton rubrum is the most causative agent of dermatophytosis worldwide. The keratinocytes are the first line of defense during infection, triggering immunomodulatory responses. Previous dual RNA-seq data showed the upregulation of several human genes involved in immune response and epithelial barrier integrity during the co-culture of HaCat cells with T. rubrum. This work evaluates the transcriptional response of this set of genes during the co-culture of HaCat with different stages of T. rubrum conidia development and viability. Our results show that the developmental stage of fungal conidia and their viability interfere with the transcriptional regulation of innate immunity genes. The CSF2 gene encoding the cytokine GM-CSF is the most overexpressed, and we report for the first time that CSF2 expression is contact and conidial-viability-dependent during infection. In contrast, CSF2 transcripts and GM-CSF secretion levels were observed when HaCat cells were challenged with bacterial LPS. Furthermore, the secretion of proinflammatory cytokines was dependent on the conidia developmental stage. Thus, we suggest that the viability and developmental stage of fungal conidia interfere with the transcriptional patterns of genes encoding immunomodulatory proteins in human keratinocytes with regard to important roles of GM-CSF during infection.

Peptidase Regulation in Trichophyton rubrum Is Mediated by the Synergism Between Alternative Splicing and StuA-Dependent Transcriptional Mechanisms.

Trichophyton rubrum is the most common causative agent of dermatophytosis worldwide and uses keratinized substrates such as skin and nails as its main source of nutrition during infection. Its pathogenic character relies on colonization and viability maintenance at the target host sites. Since fungal physiology must adapt and respond to host conditions for the successful establishment of infection, biological mechanisms are constantly being triggered by T. rubrum to guarantee its survival in the host environment. The ability of this fungus to sense and modulate the secretion of specific proteases according to environmental pH signaling is considered as a pivotal virulence factor for effective invasion and persistence of infection in the host. Transcriptional regulation of genes encoding specific proteases, such as peptidases, is a key biological process that drives physiological modulation to meet fungal requirements. It accomplishes a robust balance among transcript isoforms that can be directed to perform distinct cellular functions. Thus, alternative splicing mechanisms are suitable for fungal cells to establish a balance toward reprogramming protein translation to impair or boost physiological conditions. In this study, we investigated the role of alternative splicing, especially intron retention events, in generating isoforms of virulence factors in T. rubrum mediated by transcriptional coordination of the protein StuA, a recently described transcription factor in this fungus. By analyzing the previous gene expression data provided by RNA-sequencing and after validation by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), we observed that two peptidase-coding genes (TERG_00734 and TERG_04614) could be direct targets of alternative splicing in the presence of keratin. Furthermore, protease isoforms generated by alternative splicing in T. rubrum were also detected in a co-culture with human keratinocytes, highlighting the role of these proteins in keratin deconstruction. Our results strongly suggest the influence of StuA on the regulation of virulence factors in T. rubrum and dermatophyte infections by triggering the transcription of the peptidase genes mentioned above in an alternative splicing-independent balance. The results elucidate how fungal cells drive alternate splicing to promote physiological adaptations and show that transcriptional regulation and virulence traits are robust elements required for dermatophyte infection.

Epidemiology and Diagnostic Perspectives of Dermatophytoses.

Dermatophytoses affect about 25% of the world population, and the filamentous fungus Trichophyton rubrum is the main causative agent of this group of diseases. Dermatomycoses are caused by pathogenic fungi that generally trigger superficial infections and that feed on keratinized substrates such as skin, hair, and nails. However, there are an increasing number of reports describing dermatophytes that invade deep layers such as the dermis and hypodermis and that can cause deep infections in diabetic and immunocompromised patients, as well as in individuals with immunodeficiency. Despite the high incidence and importance of dermatophytes in clinical mycology, the diagnosis of this type of infection is not always accurate. The conventional methods most commonly used for mycological diagnosis are based on the identification of microbiological and biochemical features. However, in view of the limitations of these conventional methods, molecular diagnostic techniques are increasingly being used because of their higher sensitivity, specificity and rapidity and have become more accessible. The most widely used molecular techniques are conventional PCR, quantitative PCR, multiplex PCR, nested, PCR, PCR-RFLP, and PCR-ELISA. Another promising technique for the identification of microorganisms is the analysis of protein profiles by MALDI-TOF MS. Molecular techniques are promising but it is necessary to improve the quality and availability of the information in genomic and proteomic databases in order to streamline the use of bioinformatics in the identification of dermatophytes of clinical interest.

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

The dermatophyte Trichophyton rubrum is the main causative agent of dermatophytoses worldwide. Although a superficial mycosis, its incidence has been increasing especially among diabetic and immunocompromised patients. Terbinafine is commonly used for the treatment of infections caused by dermatophytes. However, cases of resistance of T. rubrum to this allylamine were reported even with the efficacy of this drug. The present study is the first to evaluate the effect of terbinafine using a co-culture model of T. rubrum and human keratinocytes, mimicking a fungus-host interaction, in conjunction with RNA-seq technique. Our data showed the repression of several genes involved in the ergosterol biosynthesis cascade and the induction of genes encoding major facilitator superfamily (MFS)- and ATP-binding cassette superfamily (ABC)-type membrane transporter which may be involved in T. rubrum mechanisms of resistance to this drug. We observed that some genes reported in the scientific literature as candidates of new antifungal targets were also modulated. In addition, we found the modulation of several genes that are hypothetical in T. rubrum but that possess known orthologs in other dermatophytes. Taken together, the results indicate that terbinafine can act on various targets related to the physiology of T. rubrum other than its main target of ergosterol biosynthetic pathway.

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

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.