Dermatophytes comprise pathogenic fungi that have a high affinity for the keratinized structures present in nails, skin, and hair, causing superficial infections known as dermatophytosis. A reasonable number of antifungal drugs currently exist on the pharmaceutical market to control mycoses; however, their cellular targets are restricted, and fungi may exhibit tolerance or resistance to these agents. For example, the stress caused by antifungal and cytotoxic drugs in sub-inhibitory concentrations promotes compensatory stress responses, with the over-expression of genes involved in cellular detoxification, drug efflux, and signaling pathways being among the various mechanisms that may contribute to drug tolerance. In addition, the ATP-binding cassette transporters in dermatophytes that are responsible for cellular efflux can act synergistically, allowing one to compensate for the absence of the other, revealing the complexity of drug tolerance phenomena. Moreover, mutations in genes coding for target enzymes could lead to substitutions in amino acids involved in the binding of antifungal agents, hindering their performance and leading to treatment failure. The relevance of each one of these mechanisms of resistance to fungal survival is hard to define, mainly because they can act simultaneously in the cell. However, an understanding of the molecular mechanisms involved in the resistance/tolerance processes, the identification of new antifungal targets, as well as the prospective of new antifungal compounds among natural or synthetic products, are expected to bring advances and new insights that facilitate the improvement or development of novel strategies for antifungal therapy.

BackgroundFatty 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.MethodsThe 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.ResultsThe 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.ConclusionTrans-chalcone and quercetin showed antifungal activity against T. rubrum, reducing ergosterol levels and modulating the expression of FAS1 and ERG6.

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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.

While fatty acids are known to be toxic to dermatophytes, key physiological aspects of the Trichophyton rubrum response to undecanoic acid (UDA), a medium chain saturated fatty acid (C11:0), are not well understood. Thus, we analysed RNA-seq data from T. rubrum exposed to sub-lethal doses of UDA for 3 and 12 h. Three putative pathways were primarily involved in UDA detoxification: lipid metabolism and cellular membrane composition, oxidative stress, and pathogenesis. Biochemical assays showed cell membrane impairment, reductions in ergosterol content, and an increase in keratinolytic activity following UDA exposure. Moreover, we assessed differential exon usage and intron retention following UDA exposure. A key enzyme supplying guanine nucleotides to cells, inosine monophosphate dehydrogenase (IMPDH), showed high levels of intron 2 retention. Additionally, phosphoglucomutase (PGM), which is involved in the glycogen synthesis and degradation as well as cell wall biosynthesis, exhibited a significant difference in exon 4 usage following UDA exposure. Owing to the roles of these enzymes in fungal cells, both have emerged as promising antifungal targets. We showed that intron 2 retention in impdh and exon 4 skipping in pgm might be related to an adaptive strategy to combat fatty acid toxicity. Thus, the general effect of UDA fungal toxicity involves changes to fungal metabolism and mechanisms for regulating pre-mRNA processing events.

Aspergillus flavus, a ubiquitous and saprophytic fungus, is the second most common cause of aspergillosis worldwide. Several mechanisms contribute to the establishment of the fungal infection. Extracellular vesicles (EVs) have been described as “virulence factor delivery bags” in several fungal species, demonstrating a crucial role during the infection. In this study, we evaluated production of A. flavus EVs and their immunomodulatory functions. We verified that A. flavus EVs induce macrophages to produce inflammatory mediators, such as nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β. Furthermore, the A. flavus EVs enhance phagocytosis and killing by macrophages and induce M1 macrophage polarization in vitro. In addition, a prior inoculation of A. flavus EVs in Galleria mellonella larvae resulted in a protective effect against the fungal infection. Our findings suggest that A. flavus EVs are biologically active and affect the interaction between A. flavus and host immune cells, priming the innate immune system to eliminate the fungal infection. Collectively, our results suggest that A. flavus EVs play a crucial role in aspergillosis. IMPORTANCE Immunocompromised patients are susceptible to several fungal infections. The genus Aspergillus can cause increased morbidity and mortality. Developing new therapies is essential to understand the fungal biology mechanisms. Fungal EVs carry important virulence factors, thus playing pivotal roles in fungal pathophysiology. No study to date has reported EV production by Aspergillus flavus, a fungus considered to be the second most common cause of aspergillosis and relevant food contaminator found worldwide. In this study, we produced A. flavus EVs and evaluated the in vitro immunomodulatory effects of EVs on bone marrow-derived macrophages (BMDMs) and in vivo effects in a Galleria mellonella model.

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 burden of fungal infections is not widely appreciated. Although these infections are responsible for over one million deaths annually, it is estimated that one billion people are affected by severe fungal diseases. Mycoses of nails and skin, primarily caused by fungi known as dermatophytes, are the most common fungal infections. Trichophyton rubrum appears to be the most common causative agent of dermatophytosis, followed by Trichophyton interdigitale. An estimated 25% of the world’s population suffers from dermatomycosis. Although these infections are not lethal, they compromise the quality of life of infected patients. The outcome of antidermatophytic treatments is impaired by various conditions, such as resistance and tolerance of certain dermatophyte strains. The adage “know your enemy” must be the focus of fungal research. There is an urgent need to increase awareness about the significance of these infections with precise epidemiological data and to improve knowledge regarding fungal biology and pathogenesis, with an emphasis on adaptive mechanisms to tackle adverse conditions from host counteractions. This review outlines the current knowledge about dermatophyte infections, with a focus on signaling pathways required for fungal infection establishment and a broad perspective on cellular and molecular factors involved in antifungal resistance and tolerance.