Optimization of Conditions for Feather Waste Biodegradation by Geophilic Trichophyton ajelloi Fungal Strains towards Further Agricultural Use.

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 Extracted from Dryopteris fragrans (L.) Schott Inhibits Trichophyton rubrum Growth via Membrane Permeability Alternation and Ergosterol Biosynthesis Disruption.

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 µg/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 14α-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.

Comprehensive genome and transcriptome analysis of the dermatophyte Trichophyton schoenleinii reveals the candidate pathogenic genes.

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.

Comprehensive analysis of the dermatophyte Trichophyton rubrum transcriptional profile reveals dynamic metabolic modulation.

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.

The Role of Reactive Oxygen Species and Nitric Oxide in the Inhibition of Trichophyton rubrum Growth by HaCaT Cells.

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.

Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein.

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.

Genes Encoding Proteolytic Enzymes Fungalysin and Subtilisin in Dermatophytes of Human and Animal Origin: A Comparative Study.

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.

Griseofulvin solvate solid dispersions with synergistic effect against fungal biofilms.

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.

Influence of the cultivation medium and pH on the pigmentation of Trichophyton rubrum.

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.

Trichophyton rubrum Azole Resistance Mediated by a New ABC Transporter, TruMDR3.

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.

Metabolic Rewiring Improves the Production of the Fungal Active Targeting Molecule Fusarinine C.

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.

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.

Trichophyton rubrum LysM proteins bind to fungal cell wall chitin and to the N-linked oligosaccharides present on human skin glycoproteins.

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.

The host range of dermatophytes, it is at all possible? Phenotypic evaluation of the keratinolytic activity of Trichophyton verrucosum clinical isolates.

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.

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.

Antifungal activity of Cardiospermum halicacabum L. (Sapindaceae) against Trichophyton rubrum occurs through molecular interaction with fungal Hsp90.

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.

Transcriptome-wide survey of gene expression changes and alternative splicing in Trichophyton rubrum in response to undecanoic acid.

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.

Transcriptome sequencing and analysis of zinc-uptake-related genes in Trichophyton mentagrophytes.

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.

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.

The first succinylome profile of Trichophyton rubrum reveals lysine succinylation on proteins involved in various key cellular processes.

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.