Aspergillus fumigatus Extracellular Vesicles Display Increased Galleria mellonella Survival but Partial Pro-Inflammatory Response by Macrophages.

Fungal extracellular vesicles (EVs) mediate intra- and interspecies communication and are critical in host-fungus interaction, modulating inflammation and immune responses. In this study, we evaluated the in vitro pro- and anti-inflammatory properties of Aspergillus fumigatus EVs over innate leukocytes. A. fumigatus EVs induced a partial proinflammatory response by macrophages, characterized by increased tumor necrosis factor-alpha production, and increased gene expression of induced nitric oxide synthase and adhesion molecules. EVs induce neither NETosis in human neutrophils nor cytokine secretion by peripheral mononuclear cells. However, prior inoculation of A. fumigatus EVs in Galleria mellonella larvae resulted in increased survival after the fungal challenge. Taken together, these findings show that A. fumigatus EVs play a role in protection against fungal infection, although they induce a partial pro-inflammatory response.

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.

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.

Extracellular Vesicles From the Dermatophyte Trichophyton interdigitale Modulate Macrophage and Keratinocyte Functions.

The release of biomolecules critically affects all pathogens and their establishment of diseases. For the export of several biomolecules in diverse species, the use of extracellular vesicles (EVs) is considered to represent an alternative transport mechanism, but no study to date has investigated EVs from dermatophytes. Here, we describe biologically active EVs from the dermatophyte Trichophyton interdigitale, a causative agent of mycoses worldwide. EV preparations from T. interdigitale were examined using nanoparticle-tracking analysis, which revealed vesicular structures 20-380 nm in diameter. These vesicles induced the production of proinflammatory mediators by bone marrow-derived macrophages (BMDMs) and keratinocytes in a dose-dependent manner, and an addition of the EVs to BMDMs also stimulated the transcription of the M1-polarization marker iNOS (inducible nitric oxide synthase) and diminished the expression of the M2 markers arginase-1 and Ym-1. The observed M1 macrophages' polarization triggered by EVs was abolished in cells obtained from knockout Toll-like receptor-2 mice. Also, the EVs-induced productions of pro-inflammatory mediators were blocked too. Furthermore, the EVs from T. interdigitale enhanced the fungicidal activity of BMDMs. These results suggest that EVs from T. interdigitale can modulate the innate immune response of the host and influence the interaction between T. interdigitale and host immune cells. Our findings thus open new areas of investigation into the host-parasite relationship in dermatophytosis.

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.

In silico characterization of tandem repeats in Trichophyton rubrum and related dermatophytes provides new insights into their role in pathogenesis.

Database URL: http://comp.mch.ifsuldeminas.edu.br/dtrdb/.

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.

Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest.

Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA1) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis.

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

BACKGROUND: Trichophyton 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. RESULTS: A 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. CONCLUSION: These 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.

Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum.

BACKGROUND: Fatty acid synthase (FAS) is a promising antifungal target due to its marked structural differences between fungal and mammalian cells. The aim of this study was to evaluate the antifungal activity of flavonoids described in the scientific literature as FAS inhibitors (quercetin, trans-chalcone, ellagic acid, luteolin, galangin, and genistein) against the dermatophyte Trichophyton rubrum and their effects on fatty acid and ergosterol synthesis. METHODS: The antifungal activity of the natural products was tested by the microdilution assay for determination of the minimum inhibitory concentration (MIC). The effect of the compounds on the cell membrane was evaluated using a protoplast regeneration assay. Ergosterol content was quantified by spectrophotometry. Inhibition of FAS by flavonoids was evaluated by an enzymatic assay to determine IC50 values. Quantitative RT-PCR was used to measure transcription levels of the FAS1 and ERG6 genes involved in fatty acid and ergosterol biosynthesis, respectively, during exposure of T. rubrum to the flavonoids tested. RESULTS: The flavonoids quercetin and trans-chalcone were effective against T. rubrum, with MICs of 125 and 7.5 µg/mL for the wild-type strain (MYA3108) and of 63 and 1.9 µg/mL for the ABC transporter mutant strain (ΔTruMDR2), respectively. The MICs of the fluconazole and cerulenin controls were 63 and 125 µg/mL for the wild-type strain and 30 and 15 µg/mL for the mutant strain, respectively. Quercetin and trans-chalcone also reduced ergosterol content in the two strains, indicating that interference with fatty acid and ergosterol synthesis caused cell membrane disruption. The MIC of quercetin reduced the number of regenerated protoplasts by 30.26% (wild-type strain) and by 91.66% (mutant strain). Half the MIC (0.5 MIC) of quercetin did not reduce the number of regenerated wild-type fungal colonies, but caused a 36.19% reduction in the number of mutant strain protoplasts. In contrast, the MIC and 0.5 MIC of trans-chalcone and cerulenin drastically reduced protoplast regeneration in the two strains. The FAS1 gene was repressed in the presence of MICs of quercetin, trans-chalcone, fluconazole and cerulenin. The ERG6 gene was induced in the presence of MICs of fluconazole and cerulenin and was repressed in the presence of MICs of trans-chalcone and quercetin. Trans-chalcone and quercetin inhibited the enzymatic activity of FAS, with IC50 values of 68.23 and 17.1 µg/mL, respectively. CONCLUSION: Trans-chalcone and quercetin showed antifungal activity against T. rubrum, reducing ergosterol levels and modulating the expression of FAS1 and ERG6.

Antidermatophytic and antileishmanial activities of essential oils from Lippia gracilis Schauer genotypes.

Lippia gracilis, popularly known in Brazil as 'alecrim-de-tabuleiro', is used for many purposes, especially antimicrobial and antiseptic activities. The leaves of three L. gracilis genotypes, including LGRA-106, LGRA-109 and LGRA-110 were collected from the Active Germplasm Bank located in the "Campus Rural da UFS" research farm at the São Cristóvão country, Sergipe State, Brazil. The essential oils were obtained from leaves of L. gracilis plants by hydrodistillation. Chemical analysis of the essential oils was performed by gas chromatography-mass spectrometry (GC-MS). The susceptibility of Trichophyton rubrum strains, MYA3108 and TruMDR2, to the two L. gracilis genotypes (LGRA-106 and LGRA-109) essential oils was determined by the serial microdilution method. Leishmanicidal activity of essential oil from LGRA-106 and LGRA-110 was assayed by tetrazolium-dye (MTT) colorimetric method. The oxygenated monoterpene thymol was the main component of the essential oil from genotype LGRA-106, while Carvacrol was more abundant in LGRA-109 and LGRA-110. The concentrations of LGRA-106 and LGRA-109 essential oils that completely eliminate the fungi were determined and these concentrations were similar to those observed for fluconazole, a common antifungal drug. Among the genotype tested, LGRA-106 essential oil exhibited the best fungicidal activity at 46.87µgmL(-1). Regarding to leishmanicidal activity, the IC50, for LGRA-106 and LGRA-110, was 86.32 and 77.26µgmL(-1), respectively. The results showed that L. gracilis essential oil, rich in thymol and thymol itself presented best antidermatophytic activity, while the best leishmanicidal activity was obtained with essential oil from genotype rich in Carvacrol and Carvacrol itself.