Trichophyton rubrum conidia modulate the expression and transport of Toll-like receptor 2 in HaCaT cell

Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that T. rubrum can modulate the innate immune responses of host cells, which result in the failure of host cells to recognize T. rubrum and initiate effective immune responses. In this study we show how T. rubrum conidia modulate the expression and transport of Toll-like receptor 2 in HaCaT cell. Flow cytometric analysis showed that the surface and total expression of Toll-like receptor 2 were upregulated at the very early stage when keratinocytes were exposed to T. rubrum conidia regardless of the dose, and the upregulation of surface TLR2 was much more significant than that of total TLR2. Moreover, TLR2 expression was suppressed after upregulation in the initial stage of T. rubrum exposure, and the decrease of total TLR2 was earlier than that of surface TLR2. Our results suggest that in the early stage, TLR2 of keratinocytes were upregulated and transported to the cell surface. After then, the expression of TLR2 was suppressed by T. rubrum conidia.     

Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation

In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei.     

Transformation of Acremonium coenophialum,a protective fungal symbiont of the grass Festuca arundinacea

Summary Acremonium coenophialum is a mutualistic mycosymbiont and natural agent of biological protection of the widely distributed grass Festuca arundinacea (tall fescue). An electroporative transformation system was developed for A. coenophialum. Segments of DNA 5 to the -tubulin gene (tub2) of the closely related ascomycete Epichloë typhina, fused to the Escherichia coli hph gene encoding hygromycin B phosphotransferase, conferred hygromycin resistance when introduced into A. coenophialum by electroporation. The incorporation of the Emericella nidulans trpC terminator greatly increased protoplast germination on selective medium and improved transformation efficiencies 30–200% depending on the plasmid construct. Plasmid pCSN43, which incorporates the trpC controlling elements for hph expression, was also used to transform A. coenophialum. Southern blot analysis of ten pCSN43 transformants indicated the possibility of random integration of this vector into the genome.     

Transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia

A highly efficient and reproducible procedure for the transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia is reported. Development of the procedure involved a detailed study of the germination process followed by extensive calibration of the electrical-pulse and selection conditions. Optimization of the transformation protocol was facilitated by the use of the green fluorescent protein that helped in the identification of stable transformants and in a fast assessment of transgene expression levels, colony homogeneity and genetic stability. Under optimal conditions, over 80 stable transformants/cuvette were obtained. Plasmid integration was predominantly homologous, but high transformation rates were obtained both with homologous and non-homologous vectors. The method described not only opens up opportunities for the genetic manipulation of C. gloeosporioides f. sp. aeschynomene, but also provides a framework for the development and optimization of transformation in other fungi. Received: 27 November 1998 / 6 March 1999     

Identification and differentiation of Trichophyton rubrum clinical isolates using PCR-RFLP and RAPD methods

Trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections. Several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels. The purpose of this study was to apply selected DNA fingerprinting methods to the identification and strain discrimination of T. rubrum clinical isolates. Fifty-seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and strain differentiation using a randomly amplified polymorphic DNA (RAPD) method, with two primers designated 1 and 6. Using PCR-RFLP, 55 of the isolates studied were confirmed to be T. rubrum. Among those, a total of 40 and five distinct profiles were obtained by RAPD with primers 1 and 6, respectively. The combination of profiles from both RAPD assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4%. A dendrogram analysis performed on the profiles generated by RAPD with primer 1 showed most of the isolates (87.3%) to be genetically related. PCR-RFLP serves as a rapid and reliable method for the identification of T. rubrum species, while the RAPD analysis is rather a disadvantageous tool for T. rubrum strain typing.     

Transformation of Trichoderma species with dominant selectable markers

Summary We have developed a transformation system for Trichoderma hamatum and Trichoderma harzianum Rifai, using dominant markers for selection based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the -tubulin gene (bml) from Neurospora crassa, respectively. Transformation frequencies and protoplast regeneration were low in both species. All the T. hamatum hygromycin-resistant transformants analysed were mitotically stable, in contrast to those of T. harzianum derived by benomyl resistance, in which only 50% of the transformants analysed were stable. Molecular analysis of transformants showed the integration of the transforming DNA into the genome and indicated that the number and sites of integration varied among the transformants.     

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. marcospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the antitumor clavaric acid-producing basidiomycete <Emphasis Type="Italic">Hypholoma sublateritium</Emphasis>

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.     

Transformation of Botrytis cinerea with the hygromycin B resistance gene,hph

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), confering hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2–10 transformants per g of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.     

Expression of the Escherichia coli β-glucuronidase gene in industrial and phytopathogenic filamentous fungi

Summary The plant pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed using two positive selection systems, one based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the other on the Neurospora crassa -tubulin gene bml which encodes resistance to the methyl benzimidazole carbamate fungicides. Both selection systems gave a transformation frequency of 1–20 transformants g^–1 DNA. The vector DNA was integrated into the genome and the number and sites of integration varied among the transformants. The hph transformants were mitotically stable and the transformed gene was transmitted through spores. In contrast the bml transformants were less stable.     

Gene-disruption of the entomopathogenic fungus Beauveria bassiana incubated with dsRNA

The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp-double-stranded RNA (dsRNA) to a previously generated egfp-expressing B. bassiana transformant (Bb-egfp#3) using a protoplast integration method. The Bb-egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl₂ conditions and liquid-cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb-egfp#3/egfp-dsRNA cultures showed very low fungal growth (OD₆₃₀ = 0.2) compared to the control culture, Bb-egfp#3 only (OD₆₃₀ = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA-seq of Bb-egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.     

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.     

Substrate adaptation of Trichophyton rubrum secreted endoproteases

Trichophyton rubrum is the most common pathogen caused the dermatophytosis of nail and skin in human. The secreted proteases were considered to be the most important virulence factors. However, the substrates adaptation of T. rubrum secreted proteases is largely unknown. For the first time, we use the keratins from human nail and skin stratum corneum as the growth medium to investigate the different expression patterns of T. rubrum secreted endoproteases genes. During grow in both keratin-containing media SUB7 and MEP2 were the highest expressed gene in each family. These results indicated that SUB7 and MEP2 may be the dominant endoproteases secreted by T. rubrum during host infection and the other proteases may play a supplementary role. The direct comparison of T. rubrum grown on skin and nail medium showed different substrate favorite of secreted endoproteases. The genes MEP2, SUB5, SUB2 and SUB3 were more active during growth in skin medium, possibly these proteases have a higher affinity for skin original keratins. While the structures of SUB1, SUB4, and MEP4 maybe more suitable for the degradation of nail original keratins. This work presents useful molecular details for further understanding the pathogenesis of secreted proteases and the wide adaptation of T. rubrum.     

Genetic differentiation of cercariae infrapopulations of the avian schistosome <Emphasis Type="Italic">Trichobilharzia szidati</Emphasis> based on RAPD markers and mitochondrial <Emphasis Type="Italic">cox1</Emphasis> gene

Avian schistosome Trichobilharzia szidati is a member of the largest genus within the family Schistosomatidae (Trematoda). Population genetic structure of Trichobilharzia spp. schistosomes, causative agents of cercarial dermatitis in humans, has not been studied yet. The knowledge of the genetic structure of trichobilharzian populations is essential for understanding the host–parasite coevolutionary dynamics and epidemiology strategies. Here we examined genetic diversity in three geographically isolated local populations of T. szidati cercariae inhabiting Russia based on nuclear (randomly amplified polymorphic DNA, RAPD) and mt (cox1) markers. We analyzed T. szidati cercariae shed from seven naturally infected snails of Lymnaea stagnalis. Using three random primers, we demonstrated genetic variation among populations, thus posing genetic structure across geographic sites. Moreover, T. szidati cercariae have been genetically structured among hosts (infrapopulations). Molecular variance analysis was performed to test the significance of genetic differentiation within and between local populations. Of total parasitic diversity, 18.8% was partitioned between populations, whereas the higher contribution (48.9%) corresponds to the differences among individual cercariae within infrapopulations. In contrast to RAPD markers, a 1,125-bp fragment of cox1 mt gene failed to provide any significant within-species structure. The lack of geographic structuring was detected using unique haplotypes which were determined in the current work for Moscow and Western Siberian local populations as well as obtained previously for European isolates (Czech Republic and Germany). All T. szidati/Trichobilharzia ocellata haplotypes were found to be mixed across their geographical origin.     

Investigating dominant selection markers for <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis>: a carboxin resistance system and re-evaluation of hygromycin and phleomycin resistance vectors

Dominant selectable markers are beneficial for transformation of many fungi, particularly those model species where repeated transformations may be required. A carboxin resistance allele of the Coprinopsis cinerea sdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the histidine block responsible for binding of the associated iron ion. This modified gene was used successfully to confer carboxin resistance upon transformation of C. cinerea protoplasts. Plasmids previously used to establish hygromycin transformation systems of several basidiomycete species, such as pAN7-1 and phph004, failed to give rise to hygromycin-resistant transformants of C. cinerea, whilst pPHT1 was successful. Sequencing of these constructs showed that the hygromycin resistance gene in pAN7-1 and phph004 had been modified removing the codons encoding two lysine residues following the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two codons for expression in C. cinerea. Phleomycin-resistant (ble) transformants were also obtained, but only with the intron-containing construct pblei004, showing that an intron is necessary to obtain phleomycin-resistant C. cinerea. This contrasts with hygromycin-resistance, where introns are not required for expression, emphasising the variability in importance of these elements.     

A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes

A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15–20 g of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pANsCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.Dedicated to Professor Dr. K. Esser on the occasion of his 70th birthday     

Increasing Prevalence of Trichophyton rubrum Identified through an Analysis of 115,846 Cases over the Last 37 Years

Trichophyton rubrum is the most common dermatophyte in the world with the highest prevalence in Korea. There are few reports about epidemiological and mycological characteristics of T. rubrum based on long-term, large-scale studies. The purpose of this study was to investigate the clinical and epidemiological characteristics of T. rubrum infections in Korea. We retrospectively investigated with patients'' records about the epidemiological and mycological status of 115,846 cases with T. rubrum infection that was mycologically diagnosed at Catholic Skin Disease Clinic from 1979 to 2013. Direct microscopy in 15% KOH solution and culture was done in each case. The annual incidence of patients with T. rubrum infection had been increasing during the period; and of 131,122 patients with dermatophytosis, 115,846 patients (88.35%) had T. rubrum infection. Disease was most prevalent among patients in their twenties in the 1970s and 1980s; in their thirties in the 1990s; in their forties in the 2000s; and in their fifties in the 2010s. The sex ratio was 1.5:1. T. rubrum infection was most commonly seen in summer and was found predominantly in patients living in urban areas. Toe webs were most frequently involved, followed by toenails and groin. This epidemiologic findings provide useful information for prevention of T. rubrum infection and future dermatophytosis prospects.

Graphical Abstract

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Genetic transformation of the fungal plant wilt pathogen,Fusarium oxysporum

Summary A system for transformation of the fungal plant pathogen Fusarium oxysporum has been developed. The system employs plasmids which contain a bacterial hygromycin B phosphotransferase gene (hph) linked to Aspergillus regulatory sequences and which confer hygromycin B resistance in Fusarium. Transformation resulted from integration of the vectors into heterologous regions of the Fusarium genome and occurred at a frequency of approximately one transformant per µg DNA. No evidence was found for autonomous replication of the vector in the fungus. The transformed, drug resistant phenotype was mitotically stable with or without selection. However, modification of integrated DNA could occur during vegetative growth.     

First Isolation of Trichophyton fischeri in the United States

A 49-year-old male with Pneumocystis carinii pneumonia was seen at Bellevue Hospital in New York, N.Y. Sputum samples yielded cultures of Candida lusitaniae, Mycobacterium avium, and a filamentous fungus, Trichophyton fischeri. T. fischeri is a nonpathogenic fungus which resembles the dermatophyte Trichophyton rubrum. This is the first record of the species from U.S. sources. This case exemplifies the ecological differences between T. fischeri and T. rubrum and illustrates how correct identification of the former species can minimize diagnostic confusion. The two species are distinguished from each other by the type of growth on Casamino Acids-erythritol-albumin agar and by micromorphological differences.     

A transformation procedure for Botryotinia squamosa

Summary The onion leaf blight fungus, Botrytis squamosa, was transformed to hygromycin B resistance using a plasmid (pDH25) containing a bacterial gene for hygromycin phosphotransferase (hph) fused to promoter elements from Aspergillus. Southern hybridization of transformants indicated that single or multiple copies of the vector were integrated into heterologous regions of the B. squamosa genome. Free plasmid was found in undigested preparations of transformant DNA, but was not detected after 3–5 passages of selective transfer. Most transformants were mitotically stable in both selective and non-selective growth; however, both genetic rearrangements and loss of integrated DNA occurred during vegetative growth.