Tetsuhiro Matsuzawa

C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRγ-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRγ, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to α-mannose but not mannan. Thus, Mincle may recognize specific geometry of α-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle−/− mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle−/− mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus.

Identification of Distinct Ligands for the C-type Lectin Receptors Mincle and Dectin-2 in the Pathogenic Fungus Malassezia

Various C-type lectin receptors (CLRs), including Mincle and Dectin-2, function as pattern recognition receptors and play a central role in immunity to fungal pathogens. However, the precise structures of the CLR ligands in various pathogenic fungi have yet to be completely defined. Here we report that Malassezia, an opportunistic skin fungal pathogen, is cooperatively recognized by Mincle and Dectin-2 through distinct ligands. Solvent-based fractionation revealed that Mincle and Dectin-2 recognize lipophilic and hydrophilic components of Malassezia, respectively. Mass spectrometry and nuclear magnetic resonance (NMR) revealed glyceroglycolipid and unique mannosyl fatty acids linked to mannitol as two Mincle ligands. An O-linked mannobiose-rich glycoprotein was identified as a Malassezia ligand for Dectin-2. Cytokine production in response to the Mincle ligands and the Dectin-2 ligand was abrogated in Mincle(-/-) and Dectin-2(-/-) dendritic cells, respectively. These results demonstrate that Mincle and Dectin-2 recognize distinct ligands in Malassezia to induce host immune responses.

New species in Aspergillus section Fumigati from reclamation sites in Wyoming (U.S.A.) and revision of A. viridinutans complex

The Aspergillus viridinutans complex includes morphologically similar, soil-inhabiting species. Although its species boundaries have not been fully defined, many isolates from the complex have been isolated as opportunistic human and animal pathogens. In the present study, these species were dominant in spoil sites subjected to various types of reclamation management after coal mining. These species were characterised using two different PCR-fingerprinting methods, sequence data from the β-tubulin (benA) and calmodulin (caM) genes, macro- and micromorphology (optical and scanning electron microscopy), maximum growth temperatures and mating experiments. In addition, RNA polymerase II gene (RPB2), actin (act1) and ITS sequences were deposited for the ex-type isolates of newly described species. The mating experiment results, phylogenetic analyses and ascospore morphology suggested the presence of five species in the A. viridinutans complex. Aspergillus aureolus (syn. Neosartorya aureola) was the only homothallic species. Three species, A. felis, A. udagawae (syn. N. udagawae) and A. wyomingensis sp. nov., were heterothallic and their morphologically distinguishable teleomorph was induced by systematic mating experiments. Aspergillus viridinutans s. str. seems to be a very rare species and was represented only by the ex-type isolate in which the MAT1-1 locus was amplified. Aspegillus viridinutans and A. aureolus were typified in accordance with the rules of the new botanical code. Other species outside the A. viridinutans complex isolated from the reclamation sites were A. fumigatiaffinis and A. lentulus as well as two new sister species, A. brevistipitatus sp. nov. and A. conversis sp. nov. which were closely related each to other and to N. papuensis. Both new species are phylogenetically distant from all anamorphic species and resemble A. brevipes, A. duricaulis and A. unilateralis in micromorphology and are distinguishable from each other by the slower growth of A. conversis on all tested media. Interestingly, no isolate from the reclamation sites represented A. fumigatus s. str. which is usually reported as the dominant species from the section Fumigati in soil.

A genome sequence-based approach to taxonomy of the genus Nocardia.

The genus Nocardia includes both pathogens and producers of useful secondary metabolites. Although 16S rRNA analysis is required to accurately discriminate among phylogenetic relationships of the Nocardia species, most branches of 16S rRNA-based phylogenetic trees are not reliable. In this study, we performed in silico analyses of the genome sequences of Nocardia species in order to understand their diversity and classification for their identification and applications. Draft genome sequences of 26 Nocardia strains were determined. Phylogenetic trees were prepared on the basis of multilocus sequence analysis of the concatenated sequences of 12 genes (atpD-dnaJ-groL1-groL2-gyrB-recA-rpoA-secA-secY-sodA-trpB-ychF) and a bidirectional best hit. To elucidate the evolutionary relationships of these genes, the genome-to-genome distance was investigated on the basis of the average nucleotide identity, DNA maximal unique matches index, and genome-to-genome distance calculator. The topologies of all phylogenetic trees were found to be essentially similar to each other. Furthermore, whole genome-derived and multiple gene-derived relationships were found to be suitable for extensive intra-genus assessment of the genus Nocardia.

Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome.

Birt‐Hogg‐Dubé syndrome (BHD) is an autosomal dominant disorder characterized by fibrofolliculomas, renal tumors and pulmonary cysts with repeated pneumothorax. This disorder is caused by mutations in the gene that encodes folliculin (FLCN). FLCN is known to be involved in the signaling of mammalian target of rapamycin (mTOR). We investigated the lung of a BHD patient who presented with a unique mutation. A 33‐year‐old woman visited our hospital due to repeated pneumothorax. Histopathologic study of the resected lung demonstrated multiple epithelial cysts. An increase of blood vessels was observed in the vicinity of subpleural cysts. Genomic DNA analysis revealed heterozygous mutation at the 3′ end of intron 5 of the FLCN gene. Total mRNA and protein were extracted from the resected lung tissue. RT‐PCR and sequence analysis demonstrated the production of exon 6‐skipped FLCN mRNA. In Western blotting, the band intensities of phospho‐mTOR, phospho‐S6, phospho‐Akt, hypoxia‐inducible factor (HIF)‐1α and vascular endothelial growth factor (VEGF) were increased in the BHD lung compared with normal lungs. Histopathologic analysis demonstrated strong immunostainings of mTOR signaling molecules in cyst‐lining cells. Collective data indicates that dysregulation of mTOR signaling facilitates S6‐mediated protein synthesis and HIF‐1α‐mediated angiogenesis, which may contribute to the development of pulmonary cysts in this disorder.

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusarium solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusarium genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1α gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n=4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis.

Method for identifying heat-resistant fungi of the genus Neosartorya.

Species of the genus Neosartorya are heat-resistant fungi that cause the spoilage of heat-processed acidic foods due to the formation of heat-resistant ascospores, and they produce mycotoxins, such as fumitremorgins and gliotoxin. Their anamorphs are phylogenetically and morphologically very close to Aspergillus fumigatus, which has never been reported as a spoilage agent in heat-processed food products. Therefore it is important to discriminate between the species of Neosartorya and A. fumigatus in the food industry. In the present study, we examined β-tubulin and calmodulin genes to identify Neosartorya and A. fumigatus at the species level and found a region for specifically detecting these species. We succeeded in developing the PCR method of differentiating and identifying Neosartorya and A. fumigatus using specific primer sets. Moreover, we developed specific primer sets to identify Neosartorya species, N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri, and N. spinosa-complex, which are important in food spoilage; these fungi vary in heat resistance and productivity of mycotoxins, depending on the species. PCR using these primer sets did not detect other fungi involved in food spoilage and environmental contamination. These identification methods are rapid and simple with extremely high specificity.

In Vitro Antifungal Activity of Epigallocatechin 3-O-Gallate against Clinical Isolates of Dermatophytes

Previously, we reported that epigallocatechin 3-O-gallate (EGCg) has growth-inhibitory effect on clinical isolates of Candida species. In this study, we investigated the antifungal activity of EGCg and antifungal agents against thirty-five of dermatophytes clinically isolated by the international guidelines (M38-A2). All isolates exhibited good susceptibility to EGCg (MIC50, 2-4 µg/mL, MIC90, 4-8 µg/mL, and geometric mean (GM) MICs, 3.36-4 µg/mL) than those of fluconazole (MIC50, 2-16 µg/mL, MIC90, 4-32 µg/mL, and GM MICs, 3.45-25.8 µg/mL) and flucytosin (MIC50, MIC90, and GM MICs, >64 µg/mL), although they were less susceptible to other antifungal agents, such as amphotericin B, itraconazole, and miconazole. These activities of EGCg were approximately 4-fold higher than those of fluconazole, and were 4 to 16-fold higher than flucytosin. This result indicates that EGCg can inhibit pathogenic dermatophyte species. Therefore, we suggest that EGCg may be effectively used solely as a possible agent or combined with other antifungal agents for antifungal therapy in dermatophytosis.

A reappraisal of Aspergillus section Nidulantes with descriptions of two new sterigmatocystin-producing species

Aspergillus section Nidulantes is a speciose group of microscopic fungi whose species are important in indoor air quality, food spoilage, mycotoxin production and human pathogenicity. We assembled as many species from the section as possible with either type specimens or protologues for analysis. DNA sequences were obtained from up to four loci to conduct phylogenetic analysis of sect. Nidulantes and other species in subgenus Nidulantes. Comparisons of phenetic characters were made. Our goal was to recognize monophyletic taxa for section Nidulantes as well as the other taxa in the subgenus. Phylogenetic analysis indicated that sections Versicolores and Aenei formed subclades in the larger clade containing the type species of section Nidulantes. In order to maintain monophyly in section Nidulantes, we synonymized sects. Versicolores and Aenei with sect. Nidulantes. In place of sections, we have identified six subclades in sect. Nidulantes that also have shared phenetic character sets that allow for easier morphological identification of species. Earlier reports showed that standard β-tubulin primers can occasionally amplify the tubC gene rather than the benA gene they were designed for. We show that codon usage analysis can easily distinguish between these paralogues, and we provide primer pair options to allow benA amplifications. Two new sect. Nidulantes species are described from European caves, A. croceus (Spain) and A. askiburgiensis (Czech Republic). The analysis of exometabolites showed that both species are able to produce the mycotoxin sterigmatocystin and some additional compounds.

A rapid method for identifying Byssochlamys and Hamigera.

Heat-resistant fungi, genera Byssochlamys, Talaromyces, Neosartorya, and Hamigera, contribute significantly to the spoilage of heat-processed acidic foods, due to the formation of heat-resistant ascospores. Here, we first evaluated the differences in the beta-tubulin gene between Byssochlamys and Hamigera and developed specific primers to identify the Byssochlamys species fulva, nivea, and spectabilis, and Hamigera. Using primers designed for B. fulva and B. nivea (B1F/1R), specific PCR products were detected for B. fulva and B. nivea, as well as B. langunculariae and B. zollerniae, two closely related species. Similarly, the Pae4F/4R-1 and H2F/2R primers produced specific PCR products for B. spectabilis and Hamigera, respectively. Using these three primer sets, strains involved in acidic food spoilage and environmental contamination were not detected. The detection limits of all primer sets were 1 ng of DNA by PCR and 10 pg of DNA by nested PCR. Each PCR assay was specific, even if the sample was contaminated 1,000-fold by other fungal DNA. Thus, this method has proved to possess an extremely high degree of specificity.