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The aim of the study was to optimize culture conditions and medium composition to accelerate the biodegradation of chicken feather waste by keratinolytic soil strains of Trichophyton ajelloi , which are poorly known in this respect, as well as to propose hitherto unconsidered culture conditions for these fungi in order to obtain a biopreparation with a high fertilization value. Different pH of the medium, incubation temperatures, amounts of chicken feathers, additional carbon sources, and culture methods were tested. The process of optimizing keratin biodegradation was evaluated in terms of measuring the activity of keratinase, protease, disulfide reductase, concentration of released soluble proteins and peptides, total pool of amino acids, ammonium and sulfate ions, changes in medium pH, and feather weight loss. It was found that the studied fungal strains were capable of decomposing and mineralizing keratin from feather waste. Regarding the fertilizer value of the obtained hydrolysates, it was shown that the release of sulfate and ammonium ions was highest in a stationary culture containing 2% feathers with an initial pH of 4.5 and a temperature of 28 °C. Days 14-21 of the culture were indicated as the optimal culture time for these fungi to obtain biopreparations of high fertilizing value.

Isoflavaspidic acid PB (PB), a phloroglucinol derivative extracted from aerial parts of Dryopteris fragrans (L.) Schott, had antifungal activity against several dermatophytes. This study was aimed at exploring the antifungal mechanism of PB against Trichophyton rubrum ( T. rubrum ). The effectiveness of PB in inhibiting T. rubrum growth was detected by time-kill kinetics study and fungal biomass determination. Studies on the mechanism of action were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), sorbitol and ergosterol assay, nucleotide leakage measurement, and UPLC-based test and enzyme-linked immunosorbent assay. Fungicidal activity of PB was concentration- and time-dependent at 2 × MIC (MIC: 20  μ g/mL) after 36 h. The total biomass of T. rubrum was reduced by 64.17%, 77.65%, and 84.71% in the presence of PB at 0.5 × MIC, 1 × MIC, and 2 × MIC, respectively. SEM analysis showed that PB changed mycelial morphology, such as shrinking, twisting, collapsing, and even flattening. TEM images of treated cells exhibited abnormal distributions of polysaccharide particles, plasmolysis, and cytoplasmic content degradation accompanied by plasmalemma disruption. There were no changes in the MIC of PB in the presence of sorbitol. However, the MIC values of PB were increased by 4-fold with exogenous ergosterol. At 4 h and 8 h, PB increased nucleotide leakage. Besides, ergosterol content in T. rubrum membrane treated with PB at 0.5 × MIC, 1 × MIC, and 2 × MIC was decreased by 9.58%, 15.31%, and 76.24%, respectively. There was a dose-dependent decrease in the squalene epoxidase (SE) activity. And the reduction in the sterol 14 α -demethylase P450 (CYP51) activity was achieved after PB treatments at 1 × MIC and 2 × MIC. These results suggest that PB displays nonspecific action on the cell wall. The membrane damaging effects of PB were attributed to binding with ergosterol to increase membrane permeability and interfering ergosterol biosynthesis involved with the reduction of SE and CYP51 activities. Further study is needed to develop PB as a natural antifungal candidate for clinical use., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Zhisi Zhang et al.)

Background: Trichophyton schoenleinii is an anthropophilic dermatophyte that causes tinea favosa. Nowadays, it remains an important pathogen in some regions of the world, mainly epidemic in Africa and West Asia. Despite the medical importance of T. schoenleinii infections, a high-quality reference genome for T. schoenleinii is still unavailable, neither its transcriptomic profile., Objectives: The aim of the current study was to improve understanding of the underlying pathogenic mechanism of T. schoenleinii, and to define the candidate pathogenic genes of T. schoenleinii., Methods: Comprehensive genomic analysis of T. schoenleinii was carried out by Illumina and PacBio sequencing platforms. Transcriptome profiles of T. schoenleinii cultured in vitro in two media containing either keratin or soy protein were determined using RNA sequencing (RNA-seq) technology., Results: Here, we present the first draft genome sequence of T. schoenleinii strain T2s, which consists of 11 scaffolds containing 7474 predicted genes. Transcriptome analysis showed that genes involved in keratin hydrolysis have higher expression in T. schoenleinii grown in keratin medium, including genes encoding proteases, cysteine dioxygenase and acetamidase. Other genes with higher expression include genes encoding the components of the pH-responsive signal transduction pathways and transcription factors, many of which may play a role in pathogenicity., Conclusion: In summary, this study provides new insights into the pathogenic mechanism of T. schoenleinii and highlights candidate genes for further development of novel targets in disease diagnosis and treatment of tinea favosa., (© 2021 Wiley-VCH GmbH.)

The environmental challenges imposed onto fungal pathogens require a dynamic metabolic modulation, which relies on activation or repression of critical factors and is essential for the establishment and perpetuation of host infection. Wherefore, to overcome the different host microenvironments, pathogens not only depend on virulence factors but also on metabolic flexibility, which ensures their dynamic response to stress conditions in the host. Here, we evaluate Trichophyton rubrum interaction with keratin from a metabolic perspective. We present information about gene modulation of the dermatophyte during early infection stage after shifting from glucose- to keratin-containing culture media, in relation to its use of glucose as the carbon source. Analyzing T. rubrum transcriptome using high-throughput RNA-sequencing technology, we identified the modulation of essential genes related to nitrogen, fatty acid, ergosterol, and carbohydrate metabolisms, among a myriad of other genes necessary for the growth of T. rubrum in keratinized tissues. Our results provide reliable and critical strategies for adaptation to keratin and confirm that the urea-degrading activity associated with the reduction in disulfide bonds and proteolytic activity facilitated keratin degradation. The global modulation orchestrates the responses that support virulence and the proper adaptation to keratin compared with glucose as the carbon source. The gene expression profiling of the host-pathogen interaction highlights candidate genes involved in fungal adaptation and survival and elucidates the machinery required for the establishment of the initial stages of infection., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Trichophyton rubrum ( T. rubrum ) is one of the most important agents of dermatophyte infection in humans. The aim of this experiment was to evaluate the effect of HaCaT cells on T. rubrum , investigate the responsible mechanism of action, and explore the role of reactive oxygen species (ROS) and nitric oxide (NO) in the inhibition of T. rubrum growth by HaCaT cells. The viability of fungi treated with HaCaT cells alone and with HaCaT cells combined with pretreatment with the NADPH oxidase inhibitor (DPI) or the nitric oxide synthase (NOS) inhibitor L-NMMA was determined by enumerating the colony-forming units. NOS, ROS, and NO levels were quantified using fluorescent probes. The levels of the NOS inhibitor asymmetric dimethylarginine (ADMA) were determined by enzyme-linked immunosorbent assay (ELISA). Micromorphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, fungal keratinase activity was assessed by measuring dye release from keratin azure. In vitro fungal viability, keratinase activity, and ADMA content decreased after HaCaT cell intervention, whereas the levels of ROS, NO, and NOS increased. The micromorphology was abnormal. Fungi pretreated with DPI and L-NMMA exhibited opposite effects. HaCaT cells inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism is that ROS and NO play an important role in the inhibition of T. rubrum growth by HaCaT cells., Competing Interests: The authors declare that they have no conflicts of interest to disclose., (Copyright © 2020 Meiling Huang et al.)

Dermatophytes are among the most successful fungal pathogens in humans, but their virulence mechanisms have not yet been fully characterized. Dermatophytic fungi secrete proteases in vivo, which are responsible for fungal colonization and degradation of the keratinized tissue during infection. In the present study, we used PCR to investigate the presence of genes encoding fungalysins (MEP) and subtilisins (SUB) in three dermatophyte species whose incidence is increasing in Europe: the anthropophilic Trichophyton rubrum (n = 58), zoophilic Microsporum canis (n = 33), and Trichophyton benhamiae (n = 6). MEP2 and SUB4 genes were significantly correlated with T. rubrum; MEP3 and SUB1 were mostly frequently harbored by M. canis; and MEP1, 2, and 4 and SUB3-7 were most frequently harbored by T. benhamiae isolates (p < 0.05). Furthermore, MEP1-5 and SUB1-3 genes were significantly more prevalent among human clinical isolates of M. canis (n = 17) than among asymptomatic cat isolates of M. canis (n = 16; p < 0.05). Unidentified MEP and/or SUB genes in some isolates in the current study may suggest that other gene repertoires may be involved in the degradation of keratin. The presented analysis of the incidence of MEP and SUB virulence genes in three dermatophyte species of diverse origins provides an insight into the host-fungus interaction and dermatophyte pathogenesis.

Background: Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80-90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum., Results: The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing., Conclusions: We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

Fungal biofilms are invariably recalcitrant to antifungal drugs and thus can cause recurrent serious infections. The aim of this work was to prepare highly effective form of the antifungal drug griseofulvin using the chloroform solvate embedded into different polymeric matrices. Based on their solid solubility, solvated (chloroform) and non-solvated (methanol and acetone) solid dispersions were prepared using different materials: silica, microcrystalline cellulose, polyvinylpyrrolidone and hydroxypropyl methylcellulose acetate succinate (HPMCAS) by which HPMCAS dispersions showed the highest solubility of about 200 μg/mL compared with ∼30 μg/mL for pure griseofulvin. The anti fungal potential of griseofulvin was assessed against the dermatophytes T. rubrum. Metabolic and protease activity of T. rubrum NCPF 935 with and without the presence of GF:HPMCAS chloroform solvates showed significant reduction compared to the untreated control after 24 h period. Confocal laser scanning microscopy showed thin hyphae compared to Control and GF:HPMCAS (non solvated). Dynamic vapour sorption data showed that HPMCAS formed most stable solvate structure preventing recrystallization and solvate expulsion, which could explain the disruptive effect of the biofilms. This could be explained by the formed hydrogen bonds as revealed by the solid and liquid state NMR data, which was further confirmed via thermal and FTIR analyses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus , Candida albicans , and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum , which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance., (Copyright © 2019 Monod et al.)

Trichophyton rubrum is a human pathogenic fungus. As a dermatophyte it causes athlete's foot, fungal infection of nails, jock itch and ringworm. The pigmentation of T. rubrum is variable and can range from white or yellow to wine-red. We demonstrate that the pigmentation is strongly influenced by pH. Under alkaline conditions, T. rubrum has a red pigmentation, whereas at acid conditions, T. rubrum has a yellow pigmentation. Moreover, the color change immediately from yellow to red by adding NaOH and reverse immediately from red to yellow by adding HCl. We suggest that the chemical compound Xanthomegnin is responsible for red as well for yellow pigmentation in T. rubrum. To figure out, why T. rubrum has red pigmentation on Trichophyton medium, adjust to alkaline, but not on Synthetic-Complete medium, also adjusted to alkaline, we measure the pH of liquid media, adjusted to pH 3.5, 6 and 8, over a period of four weeks. The pH of both cultivation media changes significantly, with a maximum of five pH levels. Whereas the Trichophyton medium, initially adjusted to pH 8, stays alkaline, the pH of the Synthetic-Complete medium drops to acid conditions. The acidification of the SC medium and the alkalization of the Trichophyton medium explains the different pigment color of the T. rubrum colonies., Competing Interests: The authors have declared that no competing interests exist.

Author: Recently, increasing research in siderophores has been dedicated to their possible medical applications in diagnostics and therapeutics for human pathogenic infections. Fusarinine C (FsC) is a natural hydroxamate siderophore that harbors three amino groups, which allow the easy chemical modification of FsC for the design of novel multifunctional conjugates. However, low production of FsC has hampered its extensive exploitation.Herein, we rewired the FsC biosynthetic pathway in the Aureobasidium melanogenum HN6.2 strain to achieve a self-supplying l-ornithine with component-simplified and enhanced production of extracellular siderophores, for which the FsC accounted for 94%, its final titer being approximately 1.7 g L -1 . The convenient acquisition of FsC effectuated our exploitation for its application. We employed in vitro and in vivo assays to show that FsC is an active targeting molecule that acts on the human pathogenic fungi Trichophyton rubrum and Candida albicans ; this demonstrates the potential to use FsC for the development of novel antifungal targeting reagents in the future.

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.

The Trichophyton rubrum genome contains six proteins containing two or more lysin M (LysM) domains. We have characterized two of these proteins, LysM1 and LysM2, and demonstrated that these proteins have the capacity to bind two substrates, chitin and N-linked oligosaccharides associated with human skin glycoproteins. We have characterized the individual LysM domains in LysM1, and shown that the protein contains two functional LysM domains. Each of these domains can bind to chitin, to N-linked oligosaccharides in human skin glycoproteins, and to N-linked oligosaccharides on fungal glycoproteins. We hypothesize that LysM proteins could provide the pathogen with three important functions. First, the T. rubrum LysM proteins could shield host cell wall chitin from the human immune system. Second, the LysM proteins could shield the pathogen's glycoproteins from host degradation and immune surveillance. Third, the LysM proteins could help facilitate pathogen adhesion to human skin., Competing Interests: The authors have declared that no competing interests exist.

Dermatophytes are fungi that have an ability to invade keratinised structures. Enzymes secreted by dermatophytes can underlie fungal survival on the host and development of infection. It is possible that the range of activity of keratinases from various dermatophytes is limited to specific species of animals and groups of people. The aim of this study was to carry out phenotypic analysis of the degree of keratinolytic activity of Trichophyton verrucosum strains using hairs of humans and various animal species as substrates. Our results indicated that the activity of keratinases is substrate-induced. The host range of T. verrucosum can be defined as wide. The highest activity of keratinases was recorded in media containing keratin from cow (Bos taurus) and sheep (Ovis aries) hairs in comparison with that from other tested species. The production of keratin-degrading enzymes is a function of time, with the peak of their activity occurring on day 15 of incubation. The role of keratin-degrading enzymes in the pathogenesis of dermatophytosis is becoming increasingly clearer. Given the conceptual understanding that keratin breakdown may require more than just one enzyme, the use of phenotypic methods is an optimal approach to in vitro study of the decomposition of species-specific keratin., (© 2018 Blackwell Verlag GmbH.)

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.

Introduction: Dermatophytosis is a superficial fungal infection limited to the stratum corneum of the epidermis, or to the hair and nails, and constitutes an important public health problem because of its high prevalence and associated morbidity. Dermatophyte fungi, especially 2 species, Trichophyton rubrum and Trichophyton mentagrophytes , are the predominant pathogens. Topical antifungal drugs, mainly azoles or allyamines, are currently used for the treatment of dermatophytoses, although in some cases, such as in nail and hair involvement, systemic treatment is required. However, therapeutic efficacy of current antifungal agents can be limited by their side effects, costs, and the emergence of drug resistance among fungi. Plant extracts represent a potential source of active antimicrobial agents, due to the presence of a variety of chemical bioactive compounds. In the present work, we evaluated in silico and in vitro the antifungal activity of an extract of the medicinal plant Cardiospermum halicacabum against T. rubrum suggesting a potential interaction with Hsp90 as playing an important role in both pathogenicity and drug susceptibility of T. rubrum ., Methods: We investigated in vitro the effect of different concentrations of C. halicacabum (from 500 to 31.25 µg) against a clinical isolate of T. rubrum . Furthermore, using a computational assessment, the interaction between different C. halicacabum active compounds and the fungal Hsp90 was also investigated., Results: Our results indicate a clear-cut antifungal activity of the total plant extract at the highest concentrations (500 and 250 µg). Among all tested C. halicacabum compounds, the luteolin and rutin molecules have been identified in silico as the most important potential inhibitors of Hsp90. Based on these data, luteolin and rutin were also individually assessed for their antifungal activity. Results demonstrate that both substances display an antifungal effect, even if lower than that of the total plant extract., Conclusion: Our data indicate a strong fungistatic effect of C. halicacabum against T. rubrum , suggesting its potential therapeutic efficacy in the treatment of dermatophytoses. Additionally, C. halicacabum compounds, and particularly luteolin and rutin, are all possible Hsp90 interactors, explaining their fungistatic activity., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

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 (C 11:0 ), 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.

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., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: Trichophyton mentagrophytes is an important zoonotic dermatophytic (ringworm) pathogen; causing severe skin infection in humans and other animals worldwide. Fortunately, commonly used fungal skin disease prevention and treatment measures are relatively simple. However, T. mentagrophytes is primarily studied at the epidemiology and drug efficacy research levels, yet current study has been unable to meet the needs of clinical medicine. Zinc is a crucial trace element for the growth and reproduction of fungi and other microorganisms. The metal ions coordinate within a variety of proteins to form zinc finger proteins, which perform many vital biological functions. Zinc transport regulatory networks have not been resolved in T. mentagrophytes. The T. mentagrophytes transcriptome will allow us to discover new genes, particularly those genes involved in zinc uptake., Result: We found T. mentagrophytes growth to be restricted by zinc deficiency; natural T. mentagrophytes growth requires zinc ions. T. Mentagrophytes must acquire zinc ions for growth and development. The transcriptome of T. mentagrophytes was sequenced by using Illumina HiSeq™ 2000 technology and the de novo assembly of the transcriptome was performed by using the Trinity method, and functional annotation was analyzed. We got 10,751 unigenes. The growth of T. mentagrophytes is severely inhibited and there were many genes showing significant up regulation and down regulation respectively in T. mentagrophytes when zinc deficiency. Zinc deficiency can affect the expression of multiple genes of T. mentagrophytes. The effect of the zinc deficiency could be recovered in the normal medium. And we finally found the zinc-responsive activating factor (ZafA) and speculated that 4 unigenes are zinc transporters. We knocked ZafA gene by ATMT transformation in T. mentagrophytes, the result showed that ZafA gene is very important for the growth and the generation of conidia in T. mentagrophytes. The expression of 4 zinc transporter genes is potentially regulated by the zinc-responsive activating factor. The data of this study is also sufficient to be used as a support to study T. mentagrophytes., Conclusion: We reported the first large transcriptome study carried out in T. mentagrophytes where we have compared physiological and transcriptional responses to zinc deficiency, and analyzed the expression of genes involved in zinc uptake. The study also produced high-resolution digital profiles of global genes expression relating to T. mentagrophytes growth.

Background: Dermatophytes, the most common cause of fungal infections, affect millions of individuals worldwide. They pose a major threat to public health because of the severity and longevity of infections caused by dermatophytes and their refractivity to therapy. Trichophyton rubrum (T. rubrum), the most common dermatophyte species, is a promising model organism for dermatophyte research. Post-translational modifications (PTMs) have been shown to be essential for many biological processes, particularly in the regulation of key cellular processes that contribute to pathogenicity. Although PTMs have important roles, little is known about their roles in T. rubrum and other dermatophytes. Succinylation is a new PTM that has recently been identified. In this study, we assessed the proteome-wide succinylation profile of T. rubrum. This study sought to systematically identify the succinylated sites and proteins in T. rubrum and to reveal the roles of succinylated proteins in various cellular processes as well as the differences in the succinylation profiles in different growth stages of the T. rubrum life cycle., Results: A total of 569 succinylated lysine sites were identified in 284 proteins. These succinylated proteins are involved in various cellular processes, such as metabolism, translation and epigenetic regulation. Additionally, 24 proteins related to pathogenicity were found to be succinylated. Comparison of the succinylome at the conidia and mycelia stages revealed that most of the succinylated proteins and sites were growth-stage specific. In addition, the succinylation modifications on histone and ribosomal proteins were significantly different between these two growth stages. Moreover, the sequence features surrounding the succinylated sites were different in the two stages, thus indicating the specific recognition of succinyltransferases in each growth phase., Conclusions: In this study, we explored the first T. rubrum succinylome, which is also the first PTM analysis of dermatophytes reported to date. These results revealed the major roles of the succinylated proteins involved in T. rubrum and the differences in the succinylomes between the two major growth stages. These findings should improve understanding of the physiological and pathogenic properties of dermatophytes and facilitate future development of novel drugs and therapeutics for treating superficial fungal infections.

We report the successful implementation of a novel melt co-extrusion process to fabricate ca. 1 μm diameter fibers of poly(caprolactone) (PCL) containing the antifungal compound clotrimazole in concentrations between 4 and 8 wt%. The process involves co-extrusion of a clotrimazole-loaded PCL along with poly(ethylene oxide) (PEO) as a co-feed, with subsequent removal of PEO to isolate PCL-clotrimazole fibers. In vitro tests of the clotrimazole-containing fibers against the fungus Aspergillus fumigatus, Candida albicans, and Trichophyton mentagrophytes strains demonstrated good antifungal activity which was maintained for more than 3 weeks. An in vivo study using a mouse model showed the lowest tissue fungal burden for PCL-clotrimazole when compared to a PCL-only patch and untreated controls. Comparative studies were conducted with clotrimazole-containing PCL fibers fabricated by electrospinning. Our data showed that the co-extruded, clotrimazole-containing fibers maintain activity for longer times vs. electrospun samples. This, coupled with the much higher throughput of the co-extrusion process vs. electrospinning, renders this new approach very attractive for the fabrication of drug-releasing polymer fibers.

Purpose: The aim of this study was to evaluate the in vitro and ex vivo biofilm-forming ability of dermatophytes on a nail fragment., Methodology: Initially, four isolates of Trichophyton rubrum, six of Trichophyton tonsurans, three of Trichophyton mentagrophytes, ten of Microsporum canis and three of Microsporum gypseum were tested for production biomass by crystal violet assay. Then, one strain per species presenting the best biofilm production was chosen for further studies by optical microscopy (Congo red staining), confocal laser scanning (LIVE/DEAD staining) and scanning electron (secondary electron) microscopy., Results: Biomass quantification by crystal violet assay, optical microscope images of Congo red staining, confocal microscope and scanning electron microscope images revealed that all species studied are able to form biofilms both in vitro and ex vivo, with variable density and architecture. M. gypseum, T. rubrum and T. tonsurans produced robust biofilms, with abundant matrix and biomass, while M. canis produced the weakest biofilms compared to other species., Conclusion: This study sheds light on biofilms of different dermatophyte species, which will contribute to a better understanding of the pathophysiology of dermatophytosis. Further studies of this type are necessary to investigate the processes involved in the formation and composition of dermatophyte biofilms.

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.

Most of the dermatophyte species have the ability to perforate hair structure and consume its contents of keratin through secretion of keratinase enzyme. Two clinical isolated species of dermatophytes were diagnosed as Trichophyton mentagrophytes var. granulosum and Trichophyton verrucosum. To determine the perforation ability, the isolated fungi were incubated with the hair of four different animals (rabbit, rat, cow and cat) and human hairs with three different colours (black, albino and bleached yellow) for different periods. Variable perforating ability of T. mentagrophytes was shown in rat hair and all types of human hair after 15 days, while other mammalian hairs exhibited resistance to this fungal activity. On the other hand, T. verrucosum was revealed a slow perforating ability, which needed a long time (60 days) for most of tested hairs. Perforation of human black hair could be considered a diagnostic test for differentiation between T. mentagrophytes and T. verrucosum. In conclusion, the treated or untreated human hair was considered more suitable to use as a source of keratin for the growth of dermatophytes than animal hairs. The human black hair was a best type of hair to use in the perforating test for differentiation between T. mentagrophytes and T. verrucosum., (© 2016 Blackwell Verlag GmbH.)

We investigated the antifungal properties and anti-candidal mechanism of antimicrobial peptide APP. The minimum inhibitory concentration of APP was 8 μM against Candida albicans and Aspeogillus flavus, the concentration against Saccharomyces cerevisiae and Cryptococcus neoformans was 16 μM, while 32 μM inhibited Aspergilla niger and Trichopyton rubrum. APP caused slight depolarization (12.32 ± 0.87%) of the membrane potential of intact C. albicans cells when it exerted its anti-candidal activity and only caused 21.52 ± 0.48% C. albicans cell membrane damage. APP interacted with cell wall membrane, caused potassium efflux and nucleotide leakage. However, confocal fluorescence microscopy experiment and flow cytometry confirmed that FITC-labeled APP penetrated C. albicans cell membrane with 52.31 ± 1.88% cell-penetrating efficiency and accumulated in the cytoplasm. Then, APP interact with C. albicans genomic DNA and completely suppressed DNA migration above weight ratio (peptide/DNA) of 2, and significantly arrested cell cycles during the S-phase (S-phase cell population was 27.09 ± 0.73%, p < 0.05) after penetrating the cell membrane. Results indicated that APP kills C. albicans for efficient cell-penetrating efficiency, strong DNA-binding affinity and significant physiological changes inducing S-phase arrest in intracellular environment.

We evaluated luliconazole nail solution, originally generated formulation, for the topical treatment of onychomycosis by two infection models. First, a suspension of Trichophyton mentagrophytes was dropped onto the ventral layer of human nail plate and these nails were set in Franz diffusion cells. After 9-day culture, luliconazole nail solutions (1, 3, and 5%) were applied to the dorsal surface of the nails once a day for 7 days. After application, fungal viability was assessed by measuring the ATP contents of the samples. The dose-dependent efficacy was confirmed, with 3% and 5% luliconazole nail solutions producing significantly lower ATP levels at 7-day treatment. When 3% and 5% luliconazole nail solutions were evaluated in a rabbit model of onychomycosis, both concentrations completely inhibited the recovery of fungi on culture after 4-week treatment. We therefore think these results indicate that 5% luliconazole nail solution is sufficiently potent for treatment of onychomycosis.

Background: Trichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively., Methods: The culture supernatants of two strains (T1a, T XHB ) were compared for the β-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The β-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test., Results: The T. rubrum strains (T1a and T XHB ) released β-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than T XHB . The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than T XHB . The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than T XHB . After a long-time contact, all the elevated defense genes decreased after 24 h., Conclusion: The culture supernatant of T. rubrum could directly and transiently activate the innate immune response of keratinocytes.

Here, we aimed to discriminate the Trichophyton rubrum and Trichophyton mentagrophytes complexes, via detection of xanthomegnin using HPLC. We concluded that it is not a reliable technique for discriminating the two complexes, because strains belonging to both T. rubrum and T. mentagrophytes complexes displayed xanthomegnin activity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Antimicrobial photodynamic inhibition (aPI) utilizes radical stress generated from the excitation of a photosensitizer (PS) with light to destroy pathogens. Its use against Trichophyton rubrum, a dermatophytic fungus with increasing incidence and resistance, has not been well characterized. Our aim was to evaluate the mechanism of action of aPI against T. rubrum using curcumin as the PS in both free and nanoparticle (curc-np) form. Nanocarriers stabilize curcumin and allow for enhanced solubility and PS delivery. Curcumin aPI, at optimal conditions of 10 μg/mL of PS with 10 J/cm² of blue light (417 ± 5 nm), completely inhibited fungal growth (p<0.0001) via induction of reactive oxygen (ROS) and nitrogen species (RNS), which was associated with fungal death by apoptosis. Interestingly, only scavengers of RNS impeded aPI efficacy, suggesting that curcumin acts potently via a nitrosative pathway. The curc-np induced greater NO˙ expression and enhanced apoptosis of fungal cells, highlighting curc-np aPI as a potential treatment for T. rubrum skin infections.

Context: Trichophyton rubrum is the most common fungus causing chronic dermatophytosis in humans. Antifungal activity of promising agents is of great interest. Geraniol and citronellol are monoterpenes with antimicrobial properties., Objective: This study aimed to investigate the inhibitory effects and possible mechanism of antifungal activity of geraniol and citronellol against strains of T. rubrum., Materials and Methods: The minimum inhibitory concentration (MIC) of each drug against 14 strains was determined by broth microdilution. The effects of the drugs on dry mycelial weight, conidial germination, infectivity on human nail fragments, and morphogenesis of T. rubrum were analyzed. The effects on the cell wall (test with sorbitol) and cell membrane (release of intracellular material and ergosterol biosynthesis) were investigated., Results: MIC values of geraniol ranged between 16 and 256 µg/mL while citronellol showed MIC values from 8 to 1024 µg/mL. The drugs (MIC and 2 × MIC) inhibited the mycelial growth, conidia germination, and fungal growth on nail fragments. The drugs (half of MIC) induced the formation of wide, short, and crooked hyphae in T. rubrum morphology. With sorbitol, geraniol MIC was increased by 64-fold and citronellol by 32-fold. The drugs caused leakage of intracellular material and inhibited ergosterol biosynthesis., Discussion: The results suggest that the drugs damage cell wall and cell membrane of T. rubrum through a mechanism that seems to involve the inhibition of the ergosterol biosynthesis., Conclusion: This study confirms that geraniol and citronellol can be regarded as potential drugs for controlling T. rubrum growth, with great potential against agents of dermatophytosis.

Dermatophytes are some of the most common fungal pathogens in both humans and animals. These fungi release enzymes (e.g., keratinases) that play roles in their pathogenesis. Little is known about their haemolytic and co-haemolytic (CAMP-like) activities; however, in bacteria, these components play significant roles in pathogenesis. This study characterised these two factors in 45 dermatophyte strains (representing the genera Arthroderma, Epidermophyton, Microsporum and Trichophyton) using Columbia agar (CA) supplemented with 5% bovine, ovine and equine erythrocytes. Haemolysis was best observed on CA supplemented with ovine erythrocytes followed by equine and bovine erythrocytes, while CAMP-like reactions occurred using bovine and ovine but not equine erythrocytes. Haemolytic and CAMP-like activities were best observed using ovine and bovine erythrocytes in CA in 44 and 38 strains at 7 and 3 days respectively. Most dermatophytes recovered from both symptomatic and asymptomatic lesions had haemolytic and CAMP-like activities. We suggest that the haemolytic and CAMP-like activities are not correlated with ecological characteristics, isolation sites or clinical manifestations of dermatophytic fungi. We also believe that this study has the potential to contribute to the existing literature on dermatophytes and dermatophyte pathogenesis., (© 2014 Blackwell Verlag GmbH.)

Despite that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool in the clinical microbiology setting, few studies have till now focused on MALDI-TOF MS-based identification of dermatophytes. In this study, we analyze dermatophytes strains isolated from clinical samples by MALDI-TOF MS to supplement the reference database available in our laboratory. Twenty four dermatophytes (13 reference strains and 11 field isolated strains), identified by both conventional and molecular standard procedures, were analyzed by MALDI-TOF MS, and the spectra obtained were used to supplement the available database, limited to a few species. To verify the robustness of the implemented database, 64 clinical isolates other than those used for the implementation were identified by MALDI-TOF MS. The implementation allowed the identification of the species not included in the original database, reinforced the identification of the species already present and correctly identified those within the Trichophyton mentagrophytes complex previously classified as Trichophyton. tonsurans by MALDI-TOF MS. The dendrogram obtained by analyzing the proteic profiles of the different species of dermatophytes reflected their taxonomy, showing moreover, in some cases, a different clusterization between the spectra already present in the database and those newly added. In this study, MALDI-TOF MS proved to be a useful tool suitable for the identification of dermatophytes for diagnostic purpose.

Bakuchiol was an active antifungal compound isolated from Psoraleae Fructus by means of bioassay-guided fractionation in our previous study. The present work aimed to investigate the underlying mechanisms and the therapeutic effect of bakuchiol in Trichophyton mentagrophytes-induced tinea pedis. After exposure to bakuchiol at 0.25-fold, 0.5-fold and 1-fold of minimum inhibitory concentration (MIC) (3.91 μg/ml) for 24h, the fungal conidia of T. mentagrophytes demonstrated a significant dose-dependent increase in membrane permeability. Moreover, bakuchiol at 1-fold MIC elicited a 187% elevation in reactive oxygen species (ROS) level in fungal cells after a 3-h incubation. However, bakuchiol did not induce DNA fragmentation. In a guinea pig model of tinea pedis, bakuchiol at 1%, 5% or 10% (w/w) concentration in aqueous cream could significantly reduce the fungal burden of infected feet (p<0.01-0.05). In conclusion, this is the first report to demonstrate that bakuchiol is effective in relieving tinea pedis and in inhibiting the growth of the dermatophyte T. mentagrophytes by increasing fungal membrane permeability and ROS generation, but not via induction of DNA fragmentation., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including age-related macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 μM Pc 4 followed by 670 to 675 nm light at 2.0 J/cm(2) reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

This study is to investigate the effect of Euphorbia humifusa effective fraction (EHEF) on the CYP51 enzyme activity, the lanosterol content and the MEP, SUB gene expression of Trichophyton rubrum. Trichophyton rubrum was treated by EHEF for 7 days at 26 degrees C. The activity of CYP51 enzyme of Trichophyton rubrum in the cell membrane was determined by using ELISA kit, and the lanosterol content was investigated by using high performance liquid chromatography (HPLC), and the MEP, SUB gene expression of Trichophyton rubrum was detected with the reverse transcription polymerase chain reaction (RT-PCR) method. Results showed that EHEF can decrease the membrane CYP51 enzyme activity, and it also can accumulate the fungal lanosterol in a dose-dependent manner, and it also can decrease the gene expression of MEP and SUB. The antifungal mechanism of EHEF may be related to the inhibition on CYP51 enzyme activity, and to the effects on fungal cell membrane ergosterol biosynthesis. It may also play an antifungal effect by inhibiting the MEP, SUB gene expression of fungal proteases.

Dermatophytes are fungi responsible for a disease known as dermatophytosis. Biofilms are sessile microbial communities surrounded by extracellular polymeric substances (EPS) with increased resistance to antimicrobial agents and host defenses. This paper describes, for the first time, the characteristics of Trichophyton rubrum and T. mentagrophytes biofilms. Biofilm formation was analyzed by light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) as well as by staining with crystal violet and safranin. Metabolic activity was determined using the XTT reduction assay. Both species were able to form mature biofilms in 72 h. T. rubrum biofilm produced more biomass and EPS and was denser than T. mentagrophytes biofilm. The SEM results demonstrated a coordinated network of hyphae in all directions, embedded within EPS in some areas. Research and characterization of biofilms formed by dermatophytes may contribute to the search of new drugs for the treatment of these mycoses and might inform future revisions with respect to the dose and duration of treatment of currently available antifungals.

Twelve thiazoles and their fused derivatives were tested for their antimicrobial activity against Trichophyton rubrum, T. terrestre, Epidermophyton floccosum, and Microsporum gypseum. Most of the synthesized compounds were inhibitory to the tested fungi. The most effective compound was 5-(4-ethoxybenzylidene-4,5-dihydro-4-oxothiazol-2-yl)-N,3-diphenylbut-2-namide (3c) followed by 2-(4-oxo-4,5-dihydrothiazol-2-yl)-3-phenyl-but-2-enoic acid-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-amide (2b). These compounds were more efficacious than terbinafine, the reference drug. The tested compounds caused variable reduction in the activity of keratinase of the dermatophytes, depending on the azole derivative and the test fungus. Thiazole derivatives (2b) and (3c) exhibited the highest efficacy in decreasing ergosterol biosynthesis of the tested dermatophytes. The treatment of guinea pigs with compound (3c) induced complete curing in the case of all the test dermatophytes 30days post-treatment. The percent curing for compounds (3c) and (2b) was better than the reference drug., (Copyright © 2013. Published by Elsevier SAS.)

The mechanism of action of efinaconazole, a new triazole antifungal, was investigated with Trichophyton mentagrophytes and Candida albicans. Efinaconazole dose-dependently decreased ergosterol production and accumulated 4,4-dimethylsterols and 4α-methylsterols at concentrations below its MICs. Efinaconazole induced morphological and ultrastructural changes in T. mentagrophytes hyphae that became more prominent with increasing drug concentrations. In conclusion, the primary mechanism of action of efinaconazole is blockage of ergosterol biosynthesis, presumably through sterol 14α-demethylase inhibition, leading to secondary degenerative changes.

Trichophyton rubrum var. raubitschekii is a rare anthropophilic dermatophyte isolated around the world from tinea corporis, tinea cruris, tinea pedis and tinea unguium. In this study, the isolation rate of T. rubrum var. raubitschekii was studied in 200 cases of tinea pedis and tinea unguium in Japan. The 200 clinical isolates were shown to be of downy type as their colonies on Sabouraud's dextrose agar were white to cream, suede-like to downy, with a yellow-brown to wine-red reverse, and they produced few macroconidia. The type strain of T. rubrum var. raubitschekii (CBS 100084) and one clinical isolate (KMU 8337; isolated at Kanazawa) of downy type tested positive for urease, but the reference strain of T. rubrum (CBS 392.58) and the remaining 199 clinical isolates tested negative. Further epidemiological investigations are required to study human cases of infection with the granular type of T. rubrum and T. rubrum var. raubitschekii in Japan., (© 2012 Japanese Dermatological Association.)

Background: Trichophyton tonsurans is the foremost fungal pathogen of minority children in the U.S. Despite overwhelming infection rates, it does not appear that this fungus infects children in a non-specific manner., Objective: This study was designed to identify genes that may predispose or protect a child from T. tonsurans infection., Methods: Children participating in an earlier longitudinal study wherein infection rates could be reliably determined were eligible for inclusion. DNA from a subset (n=40) of these children at the population extremes underwent whole genome genotyping (WGG). Allele frequencies between cases and controls were examined and significant SNPs were used to develop a candidate gene list for which the remainder of the cohort (n=115) were genotyped. Cumulative infection rate was examined by genotype and the ability of selected genotypes to predict the likelihood of infection explored by multivariable analysis., Results: 23 genes with a putative mechanistic role in cutaneous infection were selected for evaluation. Of these, 21 demonstrated significant differences in infection rate between genotypes. A risk index assigned to genotypes in the 21 genes accounted for over 60% of the variability observed in infection rate (adjusted r(2)=0.665, p<0.001). Among these, 8 appeared to account for the majority of variability that was observed (r(2)=0.603, p<0.001). These included genes involved in: leukocyte activation and migration, extracellular matrix integrity and remodeling, epidermal maintenance and wound repair, and cutaneous permeability., Conclusions: Applying WGG to individuals at the extremes of phenotype can help to guide the selection of candidate genes in populations of small cohorts where disease etiology is likely polygenic in nature., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Entomopathogens are significant natural enemies for mosquitoes. We have investigated the adulticidal efficacies of metabolites of Trichophyton ajelloi and Lagenidium giganteum against Culex quinquefasciatus and Aedes aegypti simultaneously. The T. ajelloi was grown on Sabouraud's dextrose broth medium at 25 ± 2°C and relative humidity at 75 ± 5% for 15 days. L. giganteum was grown in peptone yeast extract glucose broth at 25 ± 2°C and relative humidity 75 ± 5% for 15 days. The filtrations of metabolites have been made by using Whatman-1 filter paper then with the flash chromatograph. The bioassays were conducted as per the World Health Organization's methods and protocols (2006). In this significant investigations, the metabolites of T. ajelloi have been found highly susceptible against A. aegypti with LC(99)-7.24 ml after an exposure time of 24 h with a comparison, the LC(99)-66 ml was observed against C. quinquefasciatus after exposure of 24 h. Moreover, the L. giganteum metabolites have shown higher pathogenicity against C. quinquefasciatus with LC(99)-11.3 ml and A. aegypti with LC(99)-15.49 ml. Although, the efficacy in adults could be achieved with higher concentration can be significant also. Their adulticidal activities in different climatic zones are plausible with metabolites which have better LT values of T. ajelloi.

Background: To develop a new green approach for biosynthesis of silver nanoparticles, myconanotechnology has been represented as a novel field of study in nanotechnology. In this study, we have reported the extracellular synthesis of highly stable silver nanoparticles using three species of dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis., Methods: Clinical strains of these species were grown in a liquid medium containing mineral salt and incubated at 25°C for 5-7 days. The cell-free filtrate of each culture was obtained and subjected to synthesize silver nanoparticles in the presence of 1 mM AgNO3., Results: The reduction of Ag+ ions in metal nanoparticles was investigated virtually by tracing the solution color which was switched into reddish-light brown after 72 h. For T. mentagrophytes, a UV-visible spectra demonstrating a strong, quite narrow peak located between 422 and 425 nm was obtained. For M. canis, a fairly wide peak centering at 441 nm and for T. rubrum, a weak spectrum to decipher were observed. According to transmission electron microscopy (TEM) results, fairly uniform, spherical, and small in size with almost less than 50 nm particles were forms in case of T. mentagrophytes. For the other two species, TEM images showed existence of small spherical nanosilvers but not as small as nanoparticles synthesized by T. mentagrophytes., Conclusion: We observed that species belong to a single genus of the fungi have variable ability to synthesize silver nanoparticles extracellulary with different efficiency. Furthermore, the extracellular synthesis may make the process simpler and easier for following processes.

Five water-soluble chitosan derivatives were carried out by quaternizing either iodomethane or N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat188) as a quaternizing agent under basic condition. The degree of quaternization (DQ) ranged between 28±2% and 90±2%. The antifungal activity was evaluated by using disc diffusion method, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) methods against Trichophyton rubrum (T. rubrum), Trichophyton mentagrophyte (T. mentagrophyte), and Microsporum gypseum (M. gypseum) at pH 7.2. All quaternized chitosans and its derivatives showed more effective against T. rubrum than M. gypseum and T. mentagrophyte. The MIC and MFC values were found to range between 125-1000 μg/mL and 500-4000 μg/mL, respectively against all fungi. Our results indicated that the quaternized N-(4-N,N-dimethylaminocinnamyl) chitosan chloride showed highest antifungal activity against T. rubrum and M. gypseum compared to other quaternized chitosan derivatives. The antifungal activity tended to increase with an increase in molecular weight, degree of quaternization and hydrophobic moiety against T. rubrum. However, the antifungal activity was depended on type of fungal as well as chemical structure of the quaternized chitosan derivatives., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Microsporum canis and Trichophyton mentagrophytes are zoophilic dermatophytes which can cause skin infections in animals and humans. The clinical expression of this infection strongly varies depending on host, fungal species as well as enzyme production. No comparative studies are available on the enzymatic activities of M. canis and T. mentagrophytes isolated from breeding rabbits. Thus, the aim of this work was to assess the capability of M. canis and T. mentagrophytes isolated from rabbits both with and without lesions in producing different enzymes. The relationship of dermatophyte enzymatic activities and presence/absence of skin lesions has also been investigated. A total of 260 isolates of T. mentagrophytes and 25 isolates of M. canis sampled both from healthy and lesioned skin of rabbits, as well as from air samples of positive farms were examined. The results showed that T. mentagrophytes and M. canis from rabbits produce different enzymes. However, only elastase and gelatinase were linked to the appearance of lesions in T. mentagrophytes infections, whereas lipase in those by M. canis., (© 2011 Blackwell Verlag GmbH.)

Dermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.

Trichophyton rubrum (T. rubrum) is a common superficial fungus. Molecular and genetic studies of T. rubrum are still limited. In this paper, we report the global analysis of gene expression profiles at different growth phases using cDNA microarray technology. A total of 2044 differentially expressed genes were obtained and clustered into three expression patterns. Our data confirmed previous results that many mRNAs were pre-stored in the conidia of T. rubrum. Transcriptional profiling and function analysis showed that some glycolytic enzymes share similar expression patterns and may be coregulated during the transition of growth phases. Some genes involved in small GTPase signaling pathways, and in cAMP-dependent and MAPK regulation pathways were induced in response to the growth dynamics of T. rubrum. Although the detailed biological roles of these T. rubrum genes are still unknown, our results suggest that these genes may be involved in regulation mechanisms in the life cycle of the fungus.

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts overexpressed in T. rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-β-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

The increasing resistance of pathogenic fungi to antifungal compounds and the reduced number of available drugs led to the search for therapeutic alternatives among natural products, including xanthones. The antifungal activity of 27 simple oxygenated xanthones was evaluated by determination of their minimal inhibitory concentration on clinical and type strains of Candida, Cryptococcus, Aspergillus and dermatophytes, and their preponderance on the dermatophytic filamentous fungi was observed. Furthermore, a simple and efficient HPLC method with UV detection to study the effect of the active xanthones on the biosynthesis of ergosterol was developed and validated. Using this methodology, the identification and quantification of fungal sterols in whole cells of Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Trichophyton mentagrophytes were accomplished. In summary, 1,2-dihydroxyxanthone was found to be the most active compound against all strains tested, showing its effect on sterol biosynthesis by reducing the amount of ergosterol detected., (© 2011 John Wiley & Sons A/S.)

Trichophyton spp. which are among the agents of dermatophytosis with high morbidity, produce many virulence factors including hemolysins that exhibit toxic activity on immune system cells. Since relapses and chronicity are common problems related to dermatophytosis, prompt and appropriate treatment is of crucial importance. However, treatment is getting difficult due to the choice of inappropriate antifungals and increasing rates of cross-resistance among antifungal agents. The aims of this study were to investigate the hemolytic activities of Trichophyton rubrum strains isolated from patients with dermatophytosis and to detect the in vitro susceptibilities of those strains to ketoconazole, itraconazole, sulconazole, econazole and terbinaphine. Hair, skin and nail samples of patients were examined with direct microscopy using potassium hydroxide and cultivated on mycobiotic agar and Sabouraud dextrose agar. To determine hemolytic activities of T.rubrum strains, they were subcultured in Columbia Agar with 5% sheep blood and incubated for 7-14 days at 25°C in aerobic conditions. Media which displayed hemolysis were further incubated for 1-5 days at 37°C to increase hemolytic activity. Antifungal susceptibility testing was done with broth microdilution method guided by Clinical and Laboratory Standards Institute (CLSI) M38-A document. A total of 79 T.rubrum strains which exhibited negative urease and hair perforation tests, yielded pigmentation in potato-dextrose agar, were evaluated in the study. Hemolytic activity was detected in 71 strains (89.9%). Fifty strains showed incomplete (alpha) hemolysis and 21 strains showed complete (beta) hemolysis, whereas hemolysis was absent in eight of the isolates. Larger colonies created a larger zone of hemolysis and the smaller ones created a smaller zone. However, alpha-hemolysis did not turn to beta-hemolysis following further enlargement of the colony. According to antifungal susceptibility testing, the minimum inhibitory concentration (MIC) ranges, MIC50 and MIC90 values were found 0.0125-4 µg/ml, 0.5 and 2 µg/ml for ketoconazole; 0.0625-2 µg/ml, 0.5 and 1 µg/ml for itraconazole; 0.0313-4 µg/ml, 0.25 and 1 µg/ml for sulconazole; 0.0313-0.125 µg/ml, 0.0313 and 0.0625 µg/ml for econazole; 0.0313-0.0313 µg/ml, 0.0313 and 0.0313 µg/ml for terbinaphine, respectively. When the MIC values of hemolytic and non-hemolytic T.rubrum strains were compared, it was detected that hemolytic activity had no effect on MIC values. Our data have indicated that terbinaphine was the most effective antifungal agent against T.rubrum, while MIC values for itraconazole which is in common clinical use, were higher than expected and MIC values for econazole were lower than expected.