Yoshiko Umeda

Agrobacterium tumefaciens-mediated transformation of the dermatophyte, Trichophyton mentagrophytes: an efficient tool for gene transfer.

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to facilitate gene transfer into the clinically important dermatophyte, Trichophyton mentagrophytes (teleomorph: Arthroderma vanbreuseghemii). A binary vector containing a hygromycin B resistance cassette was introduced into A. tumefaciens, and the resultant strain was co-cultivated with fungal small conidia. Transformation yielded a large number of hygromycin B-resistant transformants. Hybridization analysis showed that most of the transformants harboured a single copy of T-DNA randomly integrated into the genome. Transformation frequency was increased to more than 200 per 10(7) conidia by optimizing the co-cultivation time and temperature. ATMT was then used for targeted gene disruption mediated by homologous recombination. Using a PCR-based strategy, we isolated the areA/nit-2-like nitrogen regulatory gene (tnr:Trichophytonnitrogen regulator) from T. mentagrophytes. A binary vector containing two regions of the tnr locus flanking the hygromycin B resistance cassette was constructed and introduced into T. mentagrophytesvia ATMT. Transformants with disruption of the areA/nit-2-like gene (tnr) were obtained in three of four independent disruption experiments, most of which showed homologous recombination via double crossover without additional ectopic integration of the disruption construct.

Translation elongation factor 1-α gene as a potential taxonomic and identification marker in dermatophytes

Intra- and interspecies variations of the translation elongation factor 1-α (Tef-1α) gene were evaluated as a new identification marker in a wide range of dermatophytes, which included 167 strains of 30 species. An optimized pan-dermatophyte primer pair was designed, and the target was sequenced. Consensus sequences were used for multiple alignment and phylogenetic tree analysis and the levels of intra- and interspecific nucleotide polymorphism were assessed. Between species, the analyzed part of the Tef-1α gene varied in length from 709 to 769 nucleotides. Significant numbers of species including Trichophyton rubrum, T. tonsurans, T. schoenleinii, T. concentricum, T. violaceum, Epidermophyton floccosum, Microsporum ferrugineum, M. canis, M. audouinii, T. equinum, T. eriotrephon, and T. erinacei were invariant in Tef-1α and had sufficient barcoding distance with neighboring species. Although overall consistency was found between ITS phylogeny as the current molecular marker of dermatophytes and Tef-1α, a higher discriminatory power of Tef-1α appeared particularly useful in some clades of closely related species such as the A. vanbreuseghemii, T. rubrum, A. benhamiae, and A. otae complexes. Nevertheless, we stress that a single gene can not specify species borderlines among dermatophytes and multiple lines of evidence based on a multilocus inquiry may ascertain an incontrovertible evaluation of kinship.

Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes

The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes. The Ku genes, Ku70 and Ku80, encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes, the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80. The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens-mediated genetic transformation. The average homologous recombination frequency was 73.3 +/- 25.2% for the areA/nit-2-like nitrogen regulatory gene (tnr) in Ku80(-) mutants, about 33-fold higher than that in wild-type controls. A high frequency (c. 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80(-) mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes, providing valuable information on the basic mechanisms of host invasion.

Detection and identification of probable endemic fungal pathogen, Cryptococcus gattii, and worldwide pathogen, Cryptococcus neoformans, by real‐time PCR

A real‐time PCR method for detection and identification of Cryptococcus neoformans and Cryptococcus gattii was developed and evaluated using DNA from single‐colony or koala nasal smears. Two TaqMan minor groove binder probes that distinguished between these species were designed corresponding to the internal sequences of the CAP59 gene for both species. The real‐time PCR assay had 100% specificity, as assessed using 13 reference strains and 300 environmental strains. Twelve smear samples from healthy koalas were analyzed by direct real‐time PCR. This method successfully detected C. gattii and C. neoformans in one and three koalas, respectively.

Utilization of matrix‐assisted laser desorption and ionization time‐of‐flight mass spectrometry for identification of infantile seborrheic dermatitis‐causing Malassezia and incidence of culture‐based cutaneous Malassezia microbiota of 1‐month‐old infants

Matrix‐assisted laser desorption and ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) has been utilized for identification of various microorganisms. Malassezia species, including Malassezia restricta, which is associated with seborrheic dermatitis, has been difficult to identify by traditional means. This study was performed to develop a system for identification of Malassezia species with MALDI‐TOF‐MS and to investigate the incidence and variety of cutaneous Malassezia microbiota of 1‐month‐old infants using this technique. A Malassezia species‐specific MALDI‐TOF‐MS database was developed from eight standard strains, and the availability of this system was assessed using 54 clinical strains isolated from the skin of 1‐month‐old infants. Clinical isolates were cultured initially on CHROMagar Malassezia growth medium, and the 28S ribosomal DNA (D1/D2) sequence was analyzed for confirmatory identification. Using this database, we detected and analyzed Malassezia species in 68% and 44% of infants with and without infantile seborrheic dermatitis, respectively. The results of MALDI‐TOF‐MS analysis were consistent with those of rDNA sequencing identification (100% accuracy rate). To our knowledge, this is the first report of a MALDI‐TOF‐MS database for major skin pathogenic Malassezia species. This system is an easy, rapid and reliable method for identification of Malassezia.

Human external ear canal as the specific reservoir of Malassezia slooffiae.

The incidence of Malassezia species recovered from the external ear canal was characterized using culture medium optimized for Malassezia spp., CHROMagar Malassezia. The results of this study indicated that in healthy individuals M. slooffiae was the dominant Malassezia species followed by M. restricta.

Characterization of fungi isolated from the equipment used in the International Space Station or Space Shuttle.

As a part of a series of studies regarding the microbial biota in manned space environments, fungi were isolated from six pieces of equipment recovered from the Japanese Experimental Module “KIBO” of the International Space Station and from a space shuttle. Thirty‐seven strains of fungi were isolated, identified and investigated with regard to morphological phenotypes and antifungal susceptibilities. The variety of fungi isolated in this study was similar to that of several previous reports. The dominant species belonged to the genera Penicillium, Aspergillus and Cladosporium, which are potential causative agents of allergy and opportunistic infections. The morphological phenotypes and antifungal susceptibilities of the strains isolated from space environments were not significantly different from those of reference strains on Earth.

Characterization of the translation elongation factor 1-α gene in a wide range of pathogenic Aspergillus species

Purpose. We aimed to evaluate the resolving power of the translation elongation factor (TEF)‐1&agr; gene for phylogenetic analysis of Aspergillus species. Methodology. Sequences of 526 bp representing the coding region of the TEF‐1&agr; gene were used for the assessment of levels of intra‐ and inter‐specific nucleotide polymorphism in 33 species of Aspergillus, including 57 reference, clinical and environmental strains. Results. Analysis of TEF‐1&agr; sequences indicated a mean similarity of 92.6 % between the species, with inter‐species diversity ranging from 0 to 70 nucleotides. The species with the closest resemblance were A. candidus/A. carneus, and A. flavus/A. oryzae/A. ochraceus, with 100 and 99.8 % identification, respectively. These species are phylogenetically very close and the TEF‐1&agr; gene appears not to have sufficient discriminatory power to differentiate them. Meanwhile, intra‐species differences were found within strains of A. clavatus, A. clavatonanicus, A. candidus, A. fumigatus, A. terreus, A. alliaceus, A. flavus, Eurotium amstelodami and E. chevalieri. The tree topology with strongly supported clades (≥70 % bootstrap values) was almost compatible with the phylogeny inferred from analysis of the DNA sequences of the beta tubulin gene (BT2). However, the backbone of the tree exhibited low bootstrap values, and inter‐species correlations were not obvious in some clades; for example, tree topologies based on BT2 and TEF‐1&agr; genes were incompatible for some species, such as A. deflectus, A. janus and A. penicillioides. Conclusion. The gene was not phylogenetically more informative than other known molecular markers. It will be necessary to test other genes or larger genomic regions to better understand the taxonomy of this important group of fungi.

Cross-reactivity in Cryptococcus antigen latex agglutination test in two commercial kits

This article presents an examination of the cross-reactivity of pathogenic fungi with Cryptococcus neoformans in two commercial Cryptococcus antigen latex agglutination tests performed across 39 fungal strains. Some fungi were newly indicated as Cryptococcus cross-reactive, and the two kits showed differences in cross-reactive fungi.