The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.

The Antidepressant Sertraline Affects Cell Signaling and Metabolism in Trichophyton rubrum.

The dermatophyte Trichophyton rubrum is responsible for most human cutaneous infections. Its treatment is complex, mainly because there are only a few structural classes of fungal inhibitors. Therefore, new strategies addressing these problems are essential. The development of new drugs is time-consuming and expensive. The repositioning of drugs already used in medical practice has emerged as an alternative to discovering new drugs. The antidepressant sertraline (SRT) kills several important fungal pathogens. Accordingly, we investigated the inhibitory mechanism of SRT in T. rubrum to broaden the knowledge of its impact on eukaryotic microorganisms and to assess its potential for future use in dermatophytosis treatments. We performed next-generation sequencing (RNA-seq) to identify the genes responding to SRT at the transcript level. We identified that a major effect of SRT was to alter expression for genes involved in maintaining fungal cell wall and plasma membrane stability, including ergosterol biosynthetic genes. SRT also altered the expression of genes encoding enzymes related to fungal energy metabolism, cellular detoxification, and defense against oxidative stress. Our findings provide insights into a specific molecular network interaction that maintains metabolic stability and is perturbed by SRT, showing potential targets for its strategic use in dermatophytosis.

The Transcription Factor StuA Regulates the Glyoxylate Cycle in the Dermatophyte Trichophyton rubrum under Carbon Starvation.

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.

Synergism between the Antidepressant Sertraline and Caspofungin as an Approach to Minimise the Virulence and Resistance in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is responsible for several superficial human mycoses. Novel strategies aimed at controlling this pathogen are being investigated. The objective of this study was to evaluate the antifungal activity of the antidepressant sertraline (SRT), either alone or in combination with caspofungin (CASP). We calculated the minimum inhibitory concentrations of SRT and CASP against T. rubrum. Interactions between SRT and CASP were evaluated using a broth microdilution chequerboard. We assessed the differential expression of T. rubrum cultivated in the presence of SRT or combinations of SRT and CASP. We used MTT and violet crystal assays to compare the effect of SRT alone on T. rubrum biofilms with that of the synergistic combination of SRT and CASP. A human nail infection assay was performed. SRT alone, or in combination with CASP, exhibited antifungal activity against T. rubrum. SRT targets genes involved in the biosyntheses of cell wall and ergosterol. Furthermore, the metabolic activity of the T. rubrum biofilm and its biomass were affected by SRT and the combination of SRT and CASP. SRT alone, or in combination, shows potential as an approach to minimise resistance and reduce virulence.

Alternative Splicing in Trichophyton rubrum Occurs in Efflux Pump Transcripts in Response to Antifungal Drugs.

Dermatophytes are challenging to treat because they have developed many strategies to neutralize the stress triggered by antifungals. Drug tolerance is achieved by mechanisms such as drug efflux and biofilm formation, and cellular efflux is a consequence of the synergistic and compensatory regulation of efflux pumps. Alternative splicing (AS) has also been considered as a mechanism that enhances fungal adaptive responses. We used RNA-seq data from the dermatophyte Trichophyton rubrum exposed to undecanoic acid (UDA) to search for and validate AS in genes encoding efflux pumps. The magnitude of this phenomenon was evaluated using UDA and other antifungals (caspofungin, itraconazole, and terbinafine) in planktonic and biofilm cultures. In addition to the conventional isoforms, the efflux pump encoded by TERG_04309 presented two intron-retained isoforms. Biofilms trigger the simultaneous production of at least two isoforms. The intron-retained isoforms showed short lengths and topologically different organization. Furthermore, we identified the putative interaction of efflux pumps (TERG_04309 and TERG_04224). Co-expression of these genes suggests a synergistic action in antifungal resistance. Our data provide new insights into drug tolerance related to differential isoform usage and the co-expression of stress-responsive genes, which may lead to higher antifungal resistance, mainly in biofilms.

Peptidase Regulation in Trichophyton rubrum Is Mediated by the Synergism Between Alternative Splicing and StuA-Dependent Transcriptional Mechanisms.

Trichophyton rubrum is the most common causative agent of dermatophytosis worldwide and uses keratinized substrates such as skin and nails as its main source of nutrition during infection. Its pathogenic character relies on colonization and viability maintenance at the target host sites. Since fungal physiology must adapt and respond to host conditions for the successful establishment of infection, biological mechanisms are constantly being triggered by T. rubrum to guarantee its survival in the host environment. The ability of this fungus to sense and modulate the secretion of specific proteases according to environmental pH signaling is considered as a pivotal virulence factor for effective invasion and persistence of infection in the host. Transcriptional regulation of genes encoding specific proteases, such as peptidases, is a key biological process that drives physiological modulation to meet fungal requirements. It accomplishes a robust balance among transcript isoforms that can be directed to perform distinct cellular functions. Thus, alternative splicing mechanisms are suitable for fungal cells to establish a balance toward reprogramming protein translation to impair or boost physiological conditions. In this study, we investigated the role of alternative splicing, especially intron retention events, in generating isoforms of virulence factors in T. rubrum mediated by transcriptional coordination of the protein StuA, a recently described transcription factor in this fungus. By analyzing the previous gene expression data provided by RNA-sequencing and after validation by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), we observed that two peptidase-coding genes (TERG_00734 and TERG_04614) could be direct targets of alternative splicing in the presence of keratin. Furthermore, protease isoforms generated by alternative splicing in T. rubrum were also detected in a co-culture with human keratinocytes, highlighting the role of these proteins in keratin deconstruction. Our results strongly suggest the influence of StuA on the regulation of virulence factors in T. rubrum and dermatophyte infections by triggering the transcription of the peptidase genes mentioned above in an alternative splicing-independent balance. The results elucidate how fungal cells drive alternate splicing to promote physiological adaptations and show that transcriptional regulation and virulence traits are robust elements required for dermatophyte infection.

StuA-Regulated Processes in the Dermatophyte Trichophyton rubrum: Transcription Profile, Cell-Cell Adhesion, and Immunomodulation.

Fungal infections represent a significant concern worldwide, contributing to human morbidity and mortality. Dermatophyte infections are among the most significant mycoses, and Trichophyton rubrum appears to be the principal causative agent. Thus, an understanding of its pathophysiology is urgently required. Several lines of evidence have demonstrated that the APSES family of transcription factors (Asm1p, Phd1p, Sok2p, Efg1p, and StuA) is an important point of vulnerability in fungal pathogens and a potential therapeutic target. These transcription factors are unique to fungi, contributing to cell differentiation and adaptation to environmental cues and virulence. It has recently been demonstrated that StuA plays a pleiotropic role in dermatophyte pathophysiology. It was suggested that it functions as a mediator of crosstalk between different pathways that ultimately contribute to adaptive responses and fungal-host interactions. The complex regulation of StuA and its interaction pathways are yet to be unveiled. Thus, this study aimed to gain a deeper understanding of StuA-regulated processes in T. rubrum by assessing global gene expression following growth on keratin or glucose sources. The data showed the involvement of StuA in biological processes related to central carbon metabolism and glycerol catabolism, reactive oxygen species metabolism, and cell wall construction. Changes in carbohydrate metabolism may be responsible for the significant alteration in cell wall pattern and consequently in cell-cell interaction and adhesion. Loss of StuA led to impaired biofilm production and promoted proinflammatory cytokine secretion in a human keratinocyte cell line. We also observed the StuA-dependent regulation of catalase genes. Altogether, these data demonstrate the multitude of regulatory targets of StuA with a critical role in central metabolism that may ultimately trigger a cascade of secondary effects with substantial impact on fungal physiology and virulence traits.

Reassessing the Use of Undecanoic Acid as a Therapeutic Strategy for Treating Fungal Infections.

Treating fungal infections is challenging and frequently requires long-term courses of antifungal drugs. Considering the limited number of existing antifungal drugs, it is crucial to evaluate the possibility of repositioning drugs with antifungal properties and to revisit older antifungals for applications in combined therapy, which could widen the range of therapeutic possibilities. Undecanoic acid is a saturated medium-chain fatty acid with known antifungal effects; however, its antifungal properties have not been extensively explored. Recent advances indicate that the toxic effect of undecanoic acid involves modulation of fungal metabolism through its effects on the expression of fungal genes that are critical for virulence. Additionally, undecanoic acid is suitable for chemical modification and might be useful in synergic therapies. This review highlights the use of undecanoic acid in antifungal treatments, reinforcing its known activity against dermatophytes. Specifically, in Trichophyton rubrum, against which the activity of undecanoic acid has been most widely studied, undecanoic acid elicits profound effects on pivotal processes in the cell wall, membrane assembly, lipid metabolism, pathogenesis, and even mRNA processing. Considering the known antifungal activities and associated mechanisms of undecanoic acid, its potential use in combination therapy, and the ability to modify the parent compound structure, undecanoic acid shows promise as a novel therapeutic against fungal infections.

HacA Governs Virulence Traits and Adaptive Stress Responses in Trichophyton rubrum.

The ability of fungi to sense environmental stressors and appropriately respond is linked to secretory system functions. The dermatophyte infection process depends on an orchestrated signaling regulation that triggers the transcription of genes responsible for adherence and penetration of the pathogen into host-tissue. A high secretion system is activated to support the host-pathogen interaction and assures maintenance of the dermatophyte infection. The gateway of secretion machinery is the endoplasmic reticulum (ER), which is the primary site for protein folding and transport. Current studies have shown that ER stress that affects adaptive responses is primarily regulated by UPR and supports fungal pathogenicity; this has been assessed for yeasts and Aspergillus fumigatus, in regard to how these fungi cope with host environmental stressors. Fungal UPR consists of a transmembrane kinase sensor (Ire1/IreA) and a downstream target Hac1/HacA. The active form of Hac is achieved via non-spliceosomal intron removal promoted by endonuclease activity of Ire1/IreA. Here, we assessed features of HacA and its involvement in virulence and susceptibility in Trichophyton rubrum. Our results showed that exposure to antifungals and ER-stressing agents initiated the activation of HacA from T. rubrum. Interestingly, the activation occurs when a 20 nt fragment is removed from part of the exon-2 and part of intron-2, which in turn promotes the arisen of the DNA binding site motif and a dimer interface domain. Further, we found changes in the cell wall and cellular membrane composition in the ΔhacA mutant as well as an increase in susceptibility toward azole and cell wall disturbing agents. Moreover, the ΔhacA mutant presented significant defects in important virulence traits like thermotolerance and growth on keratin substrates. For instance, the development of the ΔhacA mutant was impaired in co-culture with keratinocytes or human nail fragments. Changes in the pro-inflammatory cytokine release were verified for the ΔhacA mutant during the co-culture assay, which might be related to differences in pathogen-associated molecular patterns (PAMPs) in the cell wall. Together, these results suggested that HacA is an integral part of T. rubrum physiology and virulence, implying that it is an important molecular target for antidermatophytic therapy.

Genes coding for LysM domains in the dermatophyte Trichophyton rubrum: A transcription analysis.

The filamentous fungus Trichophyton rubrum is a pathogen that causes superficial mycoses in humans, predominantly in keratinized tissues. The occurrence of dermatophytoses has increased in the last decades, mainly in immunocompromised patients, warranting research on the mechanisms involved in dermatophyte virulence. The genomes of dermatophytes are known to be enriched in genes coding for proteins containing the LysM domain, a carbohydrate-binding module, indicating the possible involvement of these genes in virulence. Although the LysM domains have already been described in other fungi, their biological functions in dermatophytes are unknown. Here we assessed the transcription of genes encoding proteins containing the LysM domains in T. rubrum grown on different substrates using quantitative real-time polymerase chain reaction. Some of these genes showed changes in transcription levels when T. rubrum was grown on keratin. In silico analyses suggest that some of these proteins share features, namely, they are anchored in the plasma membrane and contain the catalytic domain chitinase II and signal peptide domains. Here we show a detailed study of genes encoding the proteins with LysM-containing domains in T. rubrum, aiming to contribute to the understanding of their functions in dermatophytes.

Global Analysis of Cell Wall Genes Revealed Putative Virulence Factors in the Dermatophyte Trichophyton rubrum.

The fungal cell wall is a structure in constant contact with the external environment. It confers shape to the cell and protects it from external threats. During host adaptation, the cell wall structure of fungal pathogens is continuously reshaped by the orchestrated action of numerous genes. These genes respond to environmental stresses and challenging growth conditions, influencing the infective potential of the fungus. Here, we aimed to identify cell wall biosynthesis-related genes that putatively encode virulence factors in Trichophyton rubrum. We used RNA-seq to examine the impact of two drugs, namely undecanoic acid, and acriflavine as well as the effects of the carbon source switching from glucose to keratin on T. rubrum cell wall metabolism. By using functional annotation based on Gene Ontology terms, we identified significantly differentially expressed cell wall-related genes in all stress conditions. We also exposed T. rubrum to osmotic and other cell wall stressors and evaluated the susceptibility and gene modulation in response to stress. The changes in the ambient environment caused continuous cell wall remodeling, forcing the fungus to undergo modulatory restructuring. The influence of the external challenges indicated a highly complex response pattern. The genes that were modulated simultaneously in the three stress conditions highlight potential targets for antifungal development.

Alternative Splicing in Heat Shock Protein Transcripts as a Mechanism of Cell Adaptation in Trichophyton rubrum.

Heat shock proteins (HSPs) are involved in critical processes like host tissue invasion, resistance, and pathogenicity in dermatophytes. RNA-Seq analysis of Trichophyton rubrum exposed to undecanoic acid (UDA) revealed intron retention events in HSP transcripts. Because HSPs are modulated in response to various stimuli and as alternative splicing (AS) can result in a broad diversity in the proteome of eukaryotic cells, our objective was to confirm the aforementioned retention events, investigating their consequences and extent. Furthermore, we aimed to determine: (1) the expression profile of HSP genes in an infection-like scenario and (2) the importance of Hsp90 for the keratinolytic potential of T. rubrum. RT and qPCR analyses comparing the exposure to UDA and terbinafine (TRB) confirmed the presence of two mRNA isoforms of the hsp7-like gene, with distinct expression patterns in response to UDA and TRB. The HSP expression profile revealed two upregulated, three downregulated, and four unmodulated transcripts; Hsp90 inhibition by 17-AAG resulted in a significant decrease in keratinolytic potential at 37 °C. Altogether, these results broaden the current knowledge on the importance of HSP-mediated pathways for cell adaptation and other aspects of dermatophyte biology, indicating that HSP network proteins can be potential targets for antifungal therapy.

The pH Signaling Transcription Factor PAC-3 Regulates Metabolic and Developmental Processes in Pathogenic Fungi.

The zinc finger transcription factor PAC-3/RIM101/PacC has a defined role in the secretion of enzymes and proteins in response to ambient pH, and also contributes to the virulence of species. Herein we evaluated the role of PAC-3 in the regulation of Neurospora crassa genes, in a model that examined the plant-fungi interactions. N. crassa is a model fungal species capable of exhibiting dynamic responses to its environment by employing endophytic or phytopathogenic behavior according to a given circumstance. Since plant growth and productivity are highly affected by pH and phosphorus (P) acquisition, we sought to verify the impact that induction of a Δpac-3 mutation would have under limited and sufficient Pi availability, while ensuring that the targeted physiological adjustments mimicked ambient pH and nutritional conditions required for efficient fungal growth and development. Our results suggest direct regulatory functions for PAC-3 in cell wall biosynthesis, homeostasis, oxidation-reduction processes, hydrolase activity, transmembrane transport, and modulation of genes associated with fungal virulence. Pi-dependent modulation was observed mainly in genes encoding for transporter proteins or related to cell wall development, thereby advancing the current understanding regarding colonization and adaptation processes in response to challenging environments. We have also provided comprehensive evidence that suggests a role for PAC-3 as a global regulator in plant pathogenic fungi, thus presenting results that have the potential to be applied to various types of microbes, with diverse survival mechanisms.

The prp4 kinase gene and related spliceosome factor genes in Trichophyton rubrum respond to nutrients and antifungals.

PURPOSE: Trichophyton rubrum is a dermatophyte that causes most human superficial mycoses worldwide. The spliceosome, a large ribonucleoprotein complex responsible for pre-mRNA processing, may confer adaptive advantages to deal with different stresses. Here, we assessed the structural aspects of the Prp4 kinase protein and other pre-mRNA-splicing factors (Prps) in T. rubrum grown in different protein sources and exposed to antifungal drugs. METHODOLOGY: Quantitative Reverse Transcription PCR (RT-PCR) assessed the modulation of prp1, prp31, prp8 and prp4 kinase genes after exposure of T. rubrum to sub-lethal doses of amphotericin B, caspofungin and acriflavine, or after T. rubrum growth on keratin sources for 48 and 72 h. We also performed the in silico analysis of the domain organization of Prps orthologues from filamentous fungi and yeasts. RESULTS: The prp4 gene was modulated in a time-dependent manner. Transcription levels were mostly up-regulated when T. rubrum was grown on keratin for 72 h, while exposure to amphotericin B promoted prp4 gene down-regulation at the same time point. We also observed co-expression of prp1 and prp31, and their down-regulation after amphotericin B exposure. In silico analysis revealed a conserved domain organization for most Prps orthologues with slight differences, which were mostly related to structural elements such as repetition domains in Prp1 and complexity in motif assembly for the Prp4 kinase. These differences were mainly observed in dermatophyte species and may alter protein interactions and substrate affinity. CONCLUSION: Our results improve the understanding of spliceosome proteins in fungi as well as their roles in adaptation to different environmental situations.

STE20/PAKA Protein Kinase Gene Releases an Autoinhibitory Domain through Pre-mRNA Alternative Splicing in the Dermatophyte Trichophyton rubrum.

Signaling pathways are highly diverse in filamentous fungi, allowing the cells to receive and process ambient information. Interaction of components from different pathways results in signaling networks. The mitogen-activated protein kinase (MAPK) pathway is dependent on phosphorylation that is accomplished by kinase proteins. Thus, the STE/PAK protein kinase family plays essential roles in MAPK signal transduction, regulating several cellular functions. The STE/PAK protein displays an autoinhibitory (Cdc42/Rac interactive binding-CRIB) domain on its N-terminal portion, which interacts with the C-terminal catalytic kinase domain. Based on current knowledge, for the STE/PAK kinase to be activated, molecular signals (e.g., interaction with the activated form of Rac1 and Cdc42 proteins) or proteolytic cleavage by caspase 3 is necessary. Both mechanisms release the kinase domain from the CRIB interaction. Here, we hypothesize a novel molecular mechanism for the activation of STE20/PAKA kinase in Trichophyton rubrum based on an alternative pre-mRNA splicing process. Our data suggest that, because of the retention of intron 1 of this gene, it is theoretically possible that the translation of STE20/PAKA kinase will be free of its autoinhibitory CRIB domain. These findings indicate a rapid response system to environmental changes. Furthermore, STE20/PAKA may be a potential T. rubrum virulence factor and an interesting target for new drugs against dermatophytes.

mus-52 disruption and metabolic regulation in Neurospora crassa: Transcriptional responses to extracellular phosphate availability.

Advances in the understanding of molecular systems depend on specific tools like the disruption of genes to produce strains with the desired characteristics. The disruption of any mutagen sensitive (mus) genes in the model fungus Neurospora crassa, i.e. mus-51, mus-52, or mus-53, orthologous to the human genes KU70, KU80, and LIG4, respectively, provides efficient tools for gene targeting. Accordingly, we used RNA-sequencing and reverse transcription-quantitative polymerase chain reaction amplification techniques to evaluate the effects of mus-52 deletion in N. crassa gene transcriptional modulation, and thus, infer its influence regarding metabolic response to extracellular availability of inorganic phosphate (Pi). Notably, the absence of MUS-52 affected the transcription of a vast number of genes, highlighting the expression of those coding for transcription factors, kinases, circadian clocks, oxi-reduction balance, and membrane- and nucleolus-related proteins. These findings may provide insights toward the KU molecular mechanisms, which have been related to telomere maintenance, apoptosis, DNA replication, and gene transcription regulation, as well as associated human conditions including immune system disorders, cancer, and aging.

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.

Heat Shock Proteins in Dermatophytes: Current Advances and Perspectives.

Heat shock proteins (HSPs) are proteins whose transcription responds rapidly to temperature shifts. They constitute a family of molecular chaperones, involved in the proper folding and stabilisation of proteins under physiological and adverse conditions. HSPs also assist in the protection and recovery of cells exposed to a variety of stressful conditions, including heat. The role of HSPs extends beyond chaperoning proteins, as they also participate in diverse cellular functions, such as the assembly of macromolecular complexes, protein transport and sorting, dissociation of denatured protein aggregates, cell cycle control, and programmed cell death. They are also important antigens from a variety of pathogens, are able to stimulate innate immune cells, and are implicated in acquired immunity. In fungi, HSPs have been implicated in virulence, dimorphic transition, and drug resistance. Some HSPs are potential targets for therapeutic strategies. In this review, we discuss the current understanding of HSPs in dermatophytes, which are a group of keratinophilic fungi responsible for superficial mycoses in humans and animals. Computational analyses were performed to characterise the group of proteins in these dermatophytes, as well as to assess their conservation and to identify DNA-binding domains (5'-nGAAn-3') in the promoter regions of the hsp genes. In addition, the quantification of the transcript levels of few genes in a pacC background helped in the development of an extended model for the regulation of the expression of the hsp genes, which supports the participation of the pH-responsive transcriptional regulator PacC in this process.

Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum.

Treatment of fungal infections is difficult due to several reasons, such as side effects of drugs, emergence of resistant strains, and limited number of molecular targets for the drug compounds. In fungi, heat shock proteins (Hsps) have been implicated in several processes with the conserved molecular chaperone Hsp90 emerging as a potential target for antifungal therapy. It plays key cellular roles by eliciting molecular response to environmental changes, morphogenesis, antifungal resistance, and fungal pathogenicity. Here, we evaluated the transcription profiles of hsp genes of the most prevalent dermatophyte Trichophyton rubrum in response to different environmental challenges including nutrient availability, interaction with cells and molecules of the host tissue, and drug exposure. The results suggest that each Hsp responds to a specific stress condition and that the cohort of Hsps facilitates fungal survival under various environmental challenges. Chemical inhibition of Hsp90 resulted in increased susceptibility of the fungus to itraconazole and micafungin, and decreased its growth in human nails in vitro. Moreover, some hsp and related genes were modulated by Hsp90 at the transcriptional level. We are suggesting a role of Hsp90 in the pathogenicity and drug susceptibility of T. rubrum as well as the regulation of other Hsps. The synergism observed between the inhibition of Hsp90 and the effect of itraconazole or micafungin in reducing the fungal growth is of great interest as a novel and potential strategy to treat dermatophytoses.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum.

BACKGROUND: Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. RESULTS: The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. CONCLUSION: In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.