The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium altered during cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Pothomorphe umbellata: Antifungal activity against strains of Trichophyton rubrum

Trichophyton rubrum is a dermatophyte, which can cause infections in human skin, hair and nail. Pothomorphe umbellata (L.) Miq. (Piperaceae) is a native Brazilian plant, in which phytochemical studies have demonstrated the presence of steroids, 4-nerolidylcatechol, sesquiterpenes and essential oils. The objective of this study was to analyze the in vitro activity of extracts and fractions of P. umbellata on resistant strains of T. rubrum. The microdilution plate method was utilized to test Tr1, H6 and Delta TruMDR2 strains of T rubrum; Delta TruMDR2 strain was obtained from H6 by TruMDR2 gene rupture, which is involved in multiple drugs resistance. The highest antifungal activity to all strains was observed for dichloromethane and hexane fractions of the 70% ethanolic extract which showed minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC) of 78.13 mu g/mL. This antifungal activity was also obtained by 70% ethanolic extract, which presented MIC and MFC of 78.13 mu g/mL to Delta TruMDR2, whereas the MIC values for Tr1 and H6 were 78.13 and 156.25 mu g/mL, respectively. Our results suggest the potential for future development of new antifungal drugs from P umbellata, especially to strains presenting multiple resistance. (C) 2012 Elsevier Masson SAS. All rights reserved.

Análise de genes diferencialmente expressos por Trichophyton rubrum na presença de queratina e tipagem molecular

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

cDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin

Dermatophytes are adapted to infect skin, hair and nails by their ability to utilize keratin as a nutrient source. Trichophyton rubrum is an anthropophilic fungus, causing up to 90% of chronic cases of dermatophytosis. The understanding of the complex interactions between the fungus and its host should include the identification of genes expressed during infection. To identify the genes involved in the infection process, representational difference analysis (RDA) was applied to two cDNA populations from T. rubrum, one transcribed from the RNA of fungus cultured in the presence of keratin and the other from RNA generated during fungal growth in minimal medium. The analysis identified differentially expressed transcripts. Genes related to signal transduction, membrane protein, oxidative stress response, and some putative virulence factors were up-regulated during the contact of the fungus with keratin. The expression patterns of these genes were also verified by real-time PCR, in conidia of T. rubrum infecting primarily cultured human keratinocytes in vitro, revealing their potential role in the infective process. A better understanding of this interaction will contribute significantly to our knowledge of the process of dermatophyte infection.

Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. IMPORTANCE Athlete's foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete's foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host's immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.

Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum é um importante agente causal de dermatomicose. Os métodos de tipagem molecular têm sido recentemente desenvolvidos para responder questões sobre epidemiologia e auxiliar no esclarecimento de recidivas, após o tratamento. As seqüências aleatórias 1- (5'-d[GGTGCGGGAA]-3') e 6- (5'-d[CCCGTCAGCA]-3') foram usadas para tipagem molecular deste fungo por RAPD produzindo variabilidade intraespecífica. Cinco padrões foram observados entre os 10 isolados de T. rubrum, com ambas as seqüências. Foi concluído que a análise por RAPD pode ser utilizada para estudos epidemiológicos.

Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5'-d[GGTGCGGGAA]-3' and 5'-d[CCCGTCAGCA]-3', as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPID products showed a high degree of similarity (> 90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60% similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPID analysis is especially suitable for molecular typing in T. rubrum.

In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The use of high frequency waves to treat onychomycosis-preliminary communication of three cases

The research evaluated the efficacy of high frequency waves in the treatment of onychomycosis in three patients during twelve months through the clinical examination of nails and also through mycological examination. The causative agent of the mycosis, in the three patients, was the dermatophyte Trichophyton rubrum and after application of high frequency, it was possible to notice a great improvement in the appearance of nails and also growth inhibition in culture despite the fact that the mycological examination remained positive. The preliminary study of the three cases demonstrated that the fungistatic activity of high frequency waves is a promising method to be used in combination with conventional drugs.

In Vitro Photodynamic Inactivation of Cryptococcus neoformans Melanized Cells with Chloroaluminum Phthalocyanine Nanoemulsion

The selection of fungi resistant to currently used fungicides and the emergence of new pathogenic species make the development of alternative fungus-control techniques highly desirable. Photodynamic antimicrobial chemotherapy (PACT) is a promising method which combines a nontoxic photosensitizer (PS) with visible light to cause selective killing of microbial cells. The development of PACT to treat mycoses or kill fungi in the environment depends on identifying effective PS for the different pathogenic species and delivery systems able to expand and optimize their use. In the present study, the in vitro susceptibility of Cryptococcus neoformans melanized cells to the photodynamic effects of the PS agent ClAlPc in nanoemulsion (ClAlPc/NE) was examined. Cells were killed in a PS concentration- and light dose-dependent manner. Treatment with ClAlPc/NE, using PS concentrations (e.g. 4.5 mu m) and light doses (e.g. 10 J cm-2) compatible with PACT, resulted in a reduction of up to 6 logs in survival. Washing the cells to remove unbound PS before light exposure did not inhibit fungal photodynamic inactivation. Internalization of ClAlPc by C.neoformans was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on PS concentration.

Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii.

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

Dihydroisocoumarin from Xyris pterygoblephara active against dermatophyte fungi

The ethanol extract from Xyris pteygoblephara aerial parts was evaluated against five microorganism strains, by the microdilution and agar diffusion methods. Extract fractionation led to the isolation of three compounds, whose structures were assigned by spectrometric data (113 and 2D NMR, IR, MS and UV) as (3R,4R)-(-)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one (1), moronic acid and quercetin. The absolute configuration of I was defined by circular dichroism spectroscopy and comparison with data reported for other dihydroisocoumarins. Assay of 1 (100 mu g/disc) by the agar diffusion method against clinical isolates of the dermatophytes Epidermophyton floccosum (inhibition zone, mm +/- s.d.: 4.5 +/- 0.8), Trichophyton mentagrophytes (4.8 +/- 0.4) and Trichophyton rubrum (10.2 +/- 0.8) revealed similar inhibition zones to the positive control amphotericin B (32 mu g/disc; 5.0 +/- 0.2; 5.0 +/- 0.6 and 8.8 +/- 1.2, respectively). The result corroborates the ethnomedical use of Xyris species to treat dermatitis. (C) 2007 Elsevier Ltd. All rights reserved.

Susceptibilities of the dermatophytes Trichophyton mentagrophytes and T. rubrum microconidia to photodynamic antimicrobial chemotherapy with novel phenothiazinium photosensitizers and red light

Photodynamic antimicrobial chemotherapy (PACT) is a promising alternative to conventional chemotherapy that can be used to treat localized mycosis. The development of PACT depends on identifying effective and selective PS for the different pathogenic species. The in vitro susceptibilities of Trichophyton mentagrophytes and Trichophyton rubrum microconidia to PACT with methylene blue (MB), toluidine blue o (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer S137 were investigated. The efficacy of each PS was determined based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of PACT with NMBN and S137 on the survival of the microconidia of both species. 5137 showed the lowest MIC. MIC for S137 was 2.5 mu M both for T. mentagrophytes and T. rubrum, when a light dose of 5J cm(-2) was used. PACT with NMBN (10 mu M and 20J cm(-2)) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. PACT with S137 at 1 mu M and 20J cm(-2) resulted in a reduction of approximately 3 logs in the survival of both species. When a S137 concentration of 10 mu M was used, no survivor was observed for both species at all light doses (5, 10 and 20J cm(-2)). (C) 2012 Elsevier B.V. All rights reserved.

Copper oxide nanoparticles: an antidermatophytic agent for Trichophyton spp.

Copper oxide (CuO) is one of the most important transition metal oxides due to its unique properties. It is used in various technological applications such as high critical temperature, superconductors, gas sensors, in photoconductive applications and so on. Recently, it has been used as an antimicrobial agent against various pathogenic bacteria. In the present investigation, we studied the structural and antidermatophytic properties of CuO nanoparticles (NPs) synthesized by a precipitation technique. Copper sulfate was used as a precursor and sodium hydroxide as a reducing agent. Scanning electron microscopy (SEM) showed flower-shaped CuO NPs and X-ray diffraction (XRD) pattern showed the crystalline nature of CuO NPs. These NPs were evaluated against two prevalent species of dermatophytes, i.e. Trichophyton rubrum and T. mentagrophytes by using the broth microdilution technique. Further, the NPs activity was also compared with synthetic sertaconazole. Although better antidermatophytic activity was exhibited with sertaconazole as compared to NPs, being synthetic, sertaconazole may not be preferred, as it shows different adverse effects. Trichophyton mentagrophytes is more susceptible to NPs than T. rubrum. A phylogenetic approach was applied for predicting differences in susceptibility of pathogens.