Tatiana Takahasi Komoto

Transcription profile of Trichophyton rubrum conidia grown on keratin reveals the induction of an adhesin-like protein gene with a tandem repeat pattern

BackgroundTrichophyton rubrum is a cosmopolitan filamentous fungus that can infect human keratinized tissue (skin, nails and, rarely, hair) and is the major agent of all chronic and recurrent dermatophytoses. The dermatophyte infection process is initiated through the release of arthroconidial adhesin, which binds to the host stratum corneum. The conidia then germinate, and fungal hyphae invade keratinized skin structures through the secretion of proteases. Although arthroconidia play a central role in pathogenesis, little is known about the dormancy and germination of T. rubrum conidia and the initiation of infection. The objective of this study was to evaluate the transcriptional gene expression profile of T. rubrum conidia during growth on keratin- or elastin-containing medium, mimicking superficial and deep dermatophytosis, respectively.ResultsA transcriptional profiling analysis was conducted using a custom oligonucleotide-based microarray by comparing T. rubrum conidia grown on elastin and keratin substrates. This comparison shows differences according to protein source used, but consisted of a very small set of genes, which could be attributed to the quiescent status of conidia. The modulated genes were related to the dormancy, survival and germination of conidia, including genes involved in the respiratory chain, signal transduction and lipid metabolism. However, an induction of a great number of proteases occurred when T. rubrum was grown in the presence of keratin such as the subtilisin family of proteases (Sub 1 and Sub 3) and leucine aminopeptidase (Lap 1 and Lap 2). Interestingly, keratin also promoted the up-regulation of a gene encoding an adhesin-like protein with a tandem repeat sequence. In silico analysis showed that the protein contains a domain related to adhesin that may play a role in host-pathogen interactions. The expression of this adhesin-like gene was also induced during the co-culture of T. rubrum with a human keratinocyte cell line, confirming its role in fungal-host interactions.ConclusionThese results contribute to the discovery of new targets involved in the adhesion of conidia and the maintenance of conidial dormancy, which are essential for triggering the process of infection and the chronicity of dermatophytosis.

Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents

Trichophyton rubrum is the most common causative agent of dermatomycoses worldwide, causing infection in the stratum corneum, nails, and hair. Despite the high prevalence of these infections, little is known about the molecular mechanisms involved in the fungal-host interaction, particularly during antifungal treatment. The aim of this work was to evaluate the gene expression of T. rubrum cocultured with keratinocytes and treated with the flavonoid trans-chalcone and the glycoalkaloid α-solanine. Both substances showed a marked antifungal activity against T. rubrum strain CBS (MIC = 1.15 and 17.8 µg/mL, resp.). Cytotoxicity assay against HaCaT cells produced IC50 values of 44.18 to trans-chalcone and 61.60 µM to α-solanine. The interaction of keratinocytes with T. rubrum conidia upregulated the expression of genes involved in the glyoxylate cycle, ergosterol synthesis, and genes encoding proteases but downregulated the ABC transporter TruMDR2 gene. However, both antifungals downregulated the ERG1 and ERG11, metalloprotease 4, serine proteinase, and TruMDR2 genes. Furthermore, the trans-chalcone downregulated the genes involved in the glyoxylate pathway, isocitrate lyase, and citrate synthase. Considering the urgent need for more efficient and safer antifungals, these results contribute to a better understanding of fungal-host interactions and to the discovery of new antifungal targets.

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.

Chalcones Repressed the AURKA and MDR Proteins Involved in Metastasis and Multiple Drug Resistance in Breast Cancer Cell Lines

In the present investigation, trans-chalcone and licochalcone A were tested against MCF-7 and BT-20 breast cancer cell lines for anti-tumor activity. We found that both chalcones down regulated important genes associated to cancer development and inhibited cell migration of metastatic cells (BT-20). Finally, we observed that licochalcone A reduces the MDR-1 protein, while both chalcones suppress the AURKA protein in a dose-dependent manner. In conclusion, we observed the trans-chalcone and licochalcone A affected the cell viability of breast cancer cell lines MCF-7 and BT-20 and presents anti-metastatic and anti-resistance potential, by the repression of AUKA and MDR-1 proteins.

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.

Effect of chalcones in the modulation of Trichophyton rubrum cell wall synthesis genes

Background Trichophyton rubrum is a dermatophyte that causes mostly superficial mycoses in skin, hair and nails, but an invasive course has been described in immunocompromised patients [1]. The resistance to usual antifungal drugs has been observed in T.rubrum what drives an increasing demand for new antifungal drugs. Chalcones are flavonoids found in plants that exhibit pronounced antifungal activity, most likely acting on the cell wall [2,3]. The aim of this study was to evaluate the modulation of expression of genes involved in cell wall synthesis of T.rubrum in the presence of chalcones.

Evaluation of antifungal and cytotoxic activity of trans -Chalcone and α-Solanine

Background Dermatophytes are adapted to grow in keratinized tissues such as skin, nail and hair. Trichophyton rubrum is the most frequent cause of dermatophytosis in Brazil and in the world [1]. Despite its incidence there are only a limited number of antifungal drugs available for clinical use and some drugs are highly toxic to humans. In this regard, chalcones and alkaloids are phytochemical products which provide a rich source of chemical diversity for the development of new antifungals. Chalcones inhibit the biosynthesis of the cell wall and activity of fatty acid synthase in yeast [2,3]. The glycoalkaloid a-Solanine purified from potate sprout presents antifungal activity by altering cell membrane integrity and inhibition of sporulation [4]. The aim of the present study was to evaluate the minimum inhibitory concentration (MIC) and cytotoxicity (by MTT) of trans-Chalcone and a-Solanine toward strain MYA3108 of T.rubrum and the keratinocyte cell line HaCat, in order to evaluate the potential use of these phytochemicals against fungal skin infection.

Evaluation RNAi silencing in the DH82 canine histiocytic sarcoma cell line

Background Cancer is a leading cause of death among dogs worldwide [1]. The disease results from alterations in expression of genes that control the cell cycle, including proliferation, differentiation and programmed cell death [2]. The interest in understanding the molecular aspects of cancer in humans and dogs is driven by the possibilities of identification of novel targets for the development of new anticancer compounds [3]. A molecular tool that has been used for this purpose is the RNA interference (RNAi) technique which exploits the mechanism of posttranscriptional silencing, mediated by molecules of double-stranded RNA that trigger degradation of complementary mRNAs [4]. However, for the application of this technique, important aspects must be investigated, since not all cell strains are susceptible to silencing by RNAi, and often the intracellular release of the interfering RNAs has low efficiency [5]. In this work we evaluated the efficiency of RNAi in silencing of topoisomerase IIA, a target for many anticancer compounds, in the canine histiocytic sarcoma DH82 cell line.