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 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.

Cellular and Molecular Response of Macrophages THP-1 during Co-Culture with Inactive Trichophyton rubrum Conidia.

Trichophyton rubrum is causing an increasing number of invasive infections, especially in immunocompromised and diabetic patients. The fungal invasive infectious process is complex and has not yet been fully elucidated. Therefore, this study aimed to understand the cellular and molecular mechanisms during the interaction of macrophages and T. rubrum. For this purpose, we used a co-culture of previously germinated and heat-inactivated T. rubrum conidia placed in contact with human macrophages cell line THP-1 for 24 h. This interaction led to a higher level of release of interleukins IL-6, IL-2, nuclear factor kappa beta (NF-κB) and an increase in reactive oxygen species (ROS) production, demonstrating the cellular defense by macrophages against dead fungal elements. Cell viability assays showed that 70% of macrophages remained viable during co-culture. Human microRNA expression is involved in fungal infection and may modulate the immune response. Thus, the macrophage expression profile of microRNAs during co-culture revealed the modulation of 83 microRNAs, with repression of 33 microRNAs and induction of 50 microRNAs. These data were analyzed using bioinformatics analysis programs and the modulation of the expression of some microRNAs was validated by qRT-PCR. In silico analysis showed that the target genes of these microRNAs are related to the inflammatory response, oxidative stress, apoptosis, drug resistance, and cell proliferation.

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.

Expression of genes containing tandem repeat patterns involved in the fungal-host interaction and in the response to antifungals in Trichophyton rubrum.

BACKGROUND: Trichophyton rubrum is the most common aetiological agent of human dermatophytoses. These infections mainly occur in keratinised layers such as skin, hair and nails because the fungus uses keratin as a nutrient source. Fluconazole and amphotericin are antifungal agents most commonly used to treat dermatophytoses and acts on cell membrane ergosterol. Despite the clinical importance of T rubrum, the mechanisms underlying the fungal-host relationship have not yet been clarified. Tandem repeats (TRs) are short DNA sequences that are involved in a variety of adaptive functions, including the process of fungal infection. It is known that the larger the number of TRs in the genome, the greater the capacity of cell-cell junction and surface adhesion, especially when these repeats are present in regions encoding cell surface proteins. OBJECTIVES: To identify in silico T rubrum genes containing TR patterns and to analyse the modulation of these genes in culture medium containing keratin (a model simulating skin infection) and antifungal drugs. METHODS: The Dermatophyte Tandem Repeats Database (DTRDB) and the FaaPred tool were used to identify four T rubrum genes containing TR patterns. Quantitative real-time (RT) PCR was used to evaluate the gene expression during the growth of T rubrum on keratin and in the presence of fluconazole, amphotericin B and Congo red (acts in the cell wall). RESULTS: The expression of these genes was found to be induced in culture medium containing keratin. In addition, these genes were induced in the presence of antifungal agents, especially fluconazole, indicating an adaptive response to the stress caused by this drug. CONCLUSION: The results suggest an important role of genes containing TRs in the fungal-host interaction and in the susceptibility to inhibitory compounds, indicating these sequences as new potential targets for the development of antifungal agents.

Trans-chalcone activity against Trichophyton rubrum relies on an interplay between signaling pathways related to cell wall integrity and fatty acid metabolism.

BACKGROUND: Trichophyton rubrum is the main etiological agent of skin and nail infections worldwide. Because of its keratinolytic activity and anthropophilic nature, infection models based on the addition of protein substrates have been employed to assess transcriptional profiles and to elucidate aspects related to host-pathogen interactions. Chalcones are widespread compounds with pronounced activity against dermatophytes. The toxicity of trans-chalcone towards T. rubrum is not fully understood but seems to rely on diverse cellular targets. Within this context, a better understanding of the mode of action of trans-chalcone may help identify new strategies of antifungal therapy and reveal new chemotherapeutic targets. This work aimed to assess the transcriptional profile of T. rubrum grown on different protein sources (keratin or elastin) to mimic natural infection sites and exposed to trans-chalcone in order to elucidate the mechanisms underlying the antifungal activity of trans-chalcone. RESULTS: Overall, the use of different protein sources caused only slight differences in the transcriptional profile of T. rubrum. The main differences were the modulation of proteases and lipases in gene categories when T. rubrum was grown on keratin and elastin, respectively. In addition, some genes encoding heat shock proteins were up-regulated during the growth of T. rubrum on keratin. The transcriptional profile of T. rubrum exposed to trans-chalcone included four main categories: fatty acid and lipid metabolism, overall stress response, cell wall integrity pathway, and alternative energy metabolism. Consistently, T. rubrum Mapk was strongly activated during the first hours of trans-chalcone exposure. Noteworthy, trans-chalcone inhibited genes involved in keratin degradation. The results also showed effects of trans-chalcone on fatty acid synthesis and metabolic pathways involved in acetyl-CoA supply. CONCLUSION: Our results suggest that the mode of action of trans-chalcone is related to pronounced changes in fungal metabolism, including an imbalance between fatty acid synthesis and degradation that interferes with cell membrane and cell wall integrity. In addition, this compound exerts activity against important virulence factors. Taken together, trans-chalcone acts on targets related to dermatophyte physiology and the infection process.

Caffeic acid and licochalcone A interfere with the glyoxylate cycle of Trichophyton rubrum.

Trichophyton rubrum is the most common causative agent of dermatomycoses worldwide. Despite the increasing incidence of fungal infections, the number of commercially available antifungal drugs is limited, mainly because of the biochemical similarities between fungal and mammalian cells. Biomolecules of different origins might lead to the discovery of new pharmacological targets that are more specific to the fungal cell. In this respect, caffeic acid (CA) and licochalcone A (LicoA) exhibit activity against some human pathogenic fungi by acting on important fungal molecular targets. The glyoxylate cycle is involved in the adaptation of fungal cells inside the human cell and is well established for some fungi of clinical interest. Activation of this cycle is related to the survival of fungi in nutrient-limited environments. However, little is known about the involvement of the glyoxylate cycle in this process in dermatophytes. The objective of this study was to evaluate the antifungal activity of CA and LicoA against T. rubrum, investigating specifically the effect of these compounds on important antifungal targets such as ergosterol synthesis, cell wall and glyoxylate cycle. The minimum inhibitory concentration was 86.59 µM for CA and 11.52 µM for LicoA. Plasma membrane damage and a reduction in ergosterol levels were observed after the exposure of T. rubrum to CA, but not to LicoA. Evaluation of gene expression in T. rubrum co-cultured with human keratinocytes (HaCat) in the absence of the antifungal compounds showed induction of genes related to the ergosterol biosynthesis pathway and genes encoding enzymes involved in cell wall synthesis and in the glyoxylate cycle. The same genes were significantly repressed after exposure of the co-culture to subinhibitory concentrations of CA and LicoA. The enzymatic activity of isocitrate lyase was reduced in the presence of LicoA and a moderate reduction was observed in the presence of CA. These results indicate that CA and LicoA act on targets that play important roles in pathogen-host interactions, in antifungal activity and, especially, in the glyoxylate cycle.