Publications by authors named "Tohru Gonoi"

90 Publications

Clarithromycin As an Alternative and Prophylactic Agent in a Hematopoietic Stem Cell Transplantation Patient.

Am J Case Rep 2021 Jun 15;22:e931731. Epub 2021 Jun 15.

Medical Mycology Research Center, Chiba University, Chiba, Japan.

BACKGROUND Nocardia infections have rarely been reported in hematopoietic stem cell transplantation (HSCT) patients, who usually receive the prophylactic use of sulfamethoxazole/trimethoprim (ST) against Pneumocystis jiroveci. However, the ST prophylaxis, sensitive to Nocardia species, sometimes induces renal toxicities. Therefore, alternative prophylactic or therapeutic drugs are required for nocardiosis in HSCT patients. CASE REPORT A 34-year-old Japanese man with acute mixed phenotypic leukemia with t(9; 22) received allogenic peripheral blood HSCT from a haplo-identical sibling donor. He developed graft versus host disease (GVHD) with grade II, and was treated with prednisolone and cyclosporine A with concurrent ciprofloxacin, fluconazole, valacyclovir, and ST. However, the prophylactic ST was ceased because of its renal toxicity. He developed a pulmonary nodular lesion with elevated ß-D-glucan and Aspergillus galactomannan antigen. Repeated blood and sputum culture isolated no pathogens. Voriconazole treatment administered once improved these lesions and laboratory findings. One month later, he presented with right pleuritic chest pain and multiple ring-enhancing cavitation lesions along the ribs. A needle biopsy demonstrated Nocardia elegans, which is an extremely rare infection induced by Nocardia species, in the cavitation lesions, shown by 16S rRNA gene sequencing. He was started on doripenem and liposomal amphotericin B, and a subsequent treatment kept him free from Nocardia elegans infection, without any adverse effects, while continuing the cyclosporine A and prednisolone treatment for chronic GVHD. CONCLUSIONS Clarithromycin has fewer adverse effects than ST. This case suggests that clarithromycin is an appropriate alternative and prophylactic therapy for patients with nocardiosis and ST toxicities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12659/AJCR.931731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216568PMC
June 2021

Disseminated nocardiosis due to Nocardia terpenica.

J Infect Chemother 2021 Sep 25;27(9):1365-1368. Epub 2021 Apr 25.

Department of Pulmonary Medicine, St. Luke's International Hospital, Tokyo, Japan.

We report the first case of disseminated nocardiosis due to trimethoprim/sulfamethoxazole-resistant Nocardia terpenica successfully treated with meropenem and clarithromycin. The patient travelled to Japan from Australia via Southeast Asia, which led to differential diagnoses of multiple lung nodules including miliary tuberculosis and melioidosis as well as nocardiosis. Because of variety of susceptibility depending on the Nocardia species, clinicians need to confirm the species and investigate its susceptibility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jiac.2021.04.014DOI Listing
September 2021

Phenotypic and Molecular Biological Analysis of Polymycovirus AfuPmV-1M From : Reduced Fungal Virulence in a Mouse Infection Model.

Front Microbiol 2020 11;11:607795. Epub 2020 Dec 11.

Medical Mycology Research Center, Chiba University, Chiba, Japan.

The filamentous fungal pathogen is one of the most common causal agents of invasive fungal infection in humans; the infection is associated with an alarmingly high mortality rate. In this study, we investigated whether a mycovirus, named AfuPmV-1M, can reduce the virulence of in a mouse infection model. AfuPmV-1M has high sequence similarity to AfuPmV-1, one of the polymycovirus that is a capsidless four-segment double-stranded RNA (dsRNA) virus, previously isolated from the genome reference strain of , Af293. However, we found the isolate had an additional fifth dsRNA segment, referred to as open reading frame 5 (ORF5), which has not been reported in AfuPmV-1. We then established isogenic lines of virus-infected and virus-free strains. Mycovirus infection had apparent influences on fungal phenotypes, with the virus-infected strain producing a reduced mycelial mass and reduced conidial number in comparison with these features of the virus-free strain. Also, resting conidia of the infected strain showed reduced adherence to pulmonary epithelial cells and reduced tolerance to macrophage phagocytosis. In an immunosuppressed mouse infection model, the virus-infected strain showed reduced mortality in comparison with mortality due to the virus-free strain. RNA sequencing and high-performance liquid chromatography (HPLC) analysis showed that the virus suppressed the expression of genes for gliotoxin synthesis and its production at the mycelial stage. Conversely, the virus enhanced gene expression and biosynthesis of fumagillin. Viral RNA expression was enhanced during conidial maturation, conidial germination, and the mycelial stage. We presume that the RNA or translation products of the virus affected fungal phenotypes, including spore formation and toxin synthesis. To identify the mycovirus genes responsible for attenuation of fungal virulence, each viral ORF was ectopically expressed in the virus-free KU strain. We found that the expression of ORF2 and ORF5 reduced fungal virulence in the mouse model. In addition, ORF3 affected the stress tolerance of host in culture. We hypothesize that the respective viral genes work cooperatively to suppress the pathogenicity of the fungal host.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.607795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794001PMC
December 2020

sp. nov. and sp. nov. isolated from sludge of a wastewater treatment plant.

Int J Syst Evol Microbiol 2020 Jun;70(6):3718-3723

Medical Mycology Research Center (MMRC), Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba, Chiba 260-8673, Japan.

Two novel actinobacteria, designated NBRC 107696 and NBRC 107697, were isolated from sludge samples from a wastewater treatment plant and their taxonomic positions were investigated by a polyphasic approach. The cells of the strains were aerobic, rod-shaped, non-motile and non-endospore-forming. The strains contained glutamic acid, alanine and -diaminopimelic acid in the peptidoglycan. Galactose and arabinose were detected as cell-wall sugars. The predominant menaquinone was identified as MK-9(H) and the major fatty acids were C, Cω9c and Cω7c. The DNA G+C contents of NBRC 107696 and NBRC 107697 were 68.07 and 68.99 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequence comparisons revealed that NBRC 107696 and NBRC 107697 were a clade with members of the genus . The highest 16S rRNA gene sequence similarity values were obtained with IFM 10211 (98.9 %) for NBRC 107697, and IMMIB WWCC-22, AD-6 and CC-12301 (98.1 %) for NBRC 107696, respectively. The digital DNA-DNA relatedness data coupled with the combination of genotypic and phenotypic data indicated that the two strains are representatives of two novel separate species. The names proposed to accommodate these two strains are sp. nov. and sp. nov., and the type strains are NBRC 107696 (=IFM 10067=TBRC 11239) and NBRC 107697 (=IFM 10881=TBRC 11240), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/ijsem.0.004225DOI Listing
June 2020

Analysis of an Intrinsic Mycovirus Associated With Reduced Virulence of the Human Pathogenic Fungus .

Front Microbiol 2019 17;10:3045. Epub 2020 Jan 17.

Medical Mycology Research Center, Chiba University, Chiba, Japan.

is an airborne fungal pathogen that causes severe infections with invasive growth in immunocompromised patients. Several mycoviruses have recently been isolated from strains, but there are presently no reports of mycoviral-mediated reduction or elimination of fungal pathogenicity in vertebrate models. Here, we report the biological features of a novel mycovirus, chrysovirus 41362 (AfuCV41362), isolated from the hypovirulent strain IFM 41362. The AfuCV41362 genome is comprised of four dsRNAs, each of which contains a single ORF (ORF1-4). ORF1 encodes a protein with sequence similarity to RNA-dependent RNA polymerases of viruses in the family Chrysoviridae, while ORF3 encodes a putative capsid protein. Viral RNAs are expressed primarily during the germination stage, and RNA-seq analysis of virus-infected at the germination stage suggested that the virus suppressed expression of several pathogenicity-associated host genes, including hypoxia adaptation and nitric oxide detoxification genes. functional analysis revealed that the virus-infected strain had reduced tolerance to environmental stressors. Virus-infected strain IFM 41362 had reduced virulence compared to the virus-free strain in a mouse infection model. Furthermore, introduction of the mycovirus to a natively virus-free KU strain induced virus-infected phenotypes. To identify mycovirus genes responsible for the reduced virulence of , each viral ORF was ectopically expressed in the virus-free KU strain. Ectopic expression of the individual ORFs only nominally reduced virulence of the host fungus in a mouse infection model. However, we found that ORF3 and ORF4 reduced tolerance to environmental stresses in analysis. Based on these results, we suggest that the AfuCV41362 mycovirus ORF3 and ORF4 reduce fungal virulence by suppressing stress tolerance together with other viral genes, rather than alone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.03045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978690PMC
January 2020

Case of localized cutaneous nocardiosis caused by Nocardia cyriacigeorgica.

J Dermatol 2019 Dec 25;46(12):e452-e454. Epub 2019 Sep 25.

Division of Dermatological Science, Department of Dermatology, Nihon University School of Medicine, Tokyo, Japan.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1346-8138.15094DOI Listing
December 2019

The neutral N-linked glycans of the Basidiomycetous yeasts Pseudozyma antarctica and Malassezia furfur (Subphylum Ustilaginomycotina).

J Gen Appl Microbiol 2019 May 10;65(2):53-63. Epub 2018 Oct 10.

International Center for Biotechnology, Osaka University.

Pseudozyma antarctica and Malassezia furfur are basidiomycetous yeasts under the subphylum Ustilaginomycotina. P. antarctica is a commensal organism found in certain plant species, while M. furfur is associated with several skin diseases of animals including humans. N-linked glycans of P. antarctica and M. furfur were prepared, digested with glycosidases, and structurally analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). Analyses revealed the presence of neutral N-linked glycans ranging in length from ManGlcNAc-PA to ManGlcNAc-PA. The two species shared the most abundant neutral N-linked glycan: Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M8A). The second and third most abundant neutral N-linked glycans for P. antarctica were Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M9A) and Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M5A), respectively. In the case of M. furfur, Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M7A) was the second most abundant, while both M8A and M9A were tied for the third most abundant. The presence of putative galactose residues in the hypermannosylated neutral N-linked glycans is also discussed. This report is the first to analyze the neutral N-linked glycans of P. antarctica and M. furfur.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2323/jgam.2018.05.003DOI Listing
May 2019

[Investigation of Virulence Factors by "Omics" Approaches].

Med Mycol J 2018 ;59(2):J35-J40

Medical Mycology Research Center, Chiba University.

 Aspergillus fumigatus is the predominant fungal pathogen responsible for life-threatening systemic infections in humans. Recently developed high-throughput whole genome sequencing (WGS) and RNA-Seq technologies have proven to be powerful tools for systematically investigating pathogenic organisms. In this review, we present new virulence factors obtained through our "omics" researches on A. fumigatus. We first sequenced genomes of A. fumigatus stains isolated from one infected patient at different time points, and made an important finding that although the genome (microsatellites) type of the infected strain remained unchanged, the strain exhibited several genetic changes, including acquiring therapeutic drug resistance, during patient treatment for 1.5 years. Of the various presentations of aspergillosis, pulmonary aspergilloma (PA) is one of the most common forms of A. fumigatus infection, where fungus balls are composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. We compared genomes of strains individually isolated from eight PA and eight CNPA patients in Japan, and found that the PA and CNPA strains show indiscernible genetic and ancestral backgrounds as far as genomic SNPs of the strains are concerned. The main route of infections caused by A. fumigatus is via inhalation of conidia. Inhaled conidia rapidly adhere to pulmonary epithelial cells. Nevertheless, little is known of the molecular mechanism of adherence in A. fumigatus resting conidia. We assumed corresponding adhesion molecules were highly expressed in high-adhesion conidia during conidia maturation, and exhaustively searched adhesion molecules by comparing gene expression levels in high- and low-adherence strains using the RNA-Seq technique. We found several factors involved in conidial adhesion and suggest that composite actions of these molecules have roles in conidial adhesion to human pulmonary epithelial cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3314/mmj.18.005DOI Listing
August 2018

Airborne transmission of invasive fusariosis in patients with hematologic malignancies.

PLoS One 2018 26;13(4):e0196426. Epub 2018 Apr 26.

Medical Mycology Research Center, Chiba University, Chiba, Japan.

From 2006 to 2013, an increasing incidence of fusariosis was observed in the hematologic patients of our University Hospital. We suspected of an environmental source, and the indoor hospital air was investigated as a potential source of the fungemia. Air samplings were performed in the hematology and bone marrow transplant (BMT) wards using an air sampler with pre-defined air volumes. To study the molecular relationship among environmental and clinical isolates, 18 Fusarium spp. recovered from blood cultures were included in the study. DNA sequencing of a partial portion of TEF1α gene was performed for molecular identification. Molecular typing was carried out by multi-locus sequence typing (MLST) using a four-gene scheme: TEF1α, rDNA, RPB1 and RPB2. One hundred four isolates were recovered from the air of the hematology (n = 76) and the BMT (n = 28) wards. Fusarium isolates from the air were from five species complexes: Fusarium fujikuroi (FFSC, n = 56), Fusarium incarnatum-equiseti (FIESC, n = 24), Fusarium solani (FSSC, n = 13), Fusarium chlamydosporum (FCSC, n = 10), and Fusarium oxysporum (FOSC, n = 1). Fifteen Fusarium isolates recovered from blood belonged to FSSC, and three to FFSC. MLST identified the same sequence type (ST) in clinical and environmental isolates. ST1 was found in 5 isolates from blood and in 7 from the air, both identified as FSSC (Fusarium petroliphilum). STn1 was found in one isolate from blood and in one from the air, both identified as FFSC (Fusarium napiforme). F. napiforme was isolated from the air of the hospital room of the patient with fungemia due to F. napiforme. These findings suggested a possible clonal origin of the Fusarium spp. recovered from air and bloodcultures. In conclusion, our study found a diversity of Fusarium species in the air of our hospital, and a possible role of the air as source of systemic fusariosis in our immunocompromised patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196426PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919535PMC
August 2018

Reclassification of Nocardia species based on whole genome sequence and associated phenotypic data.

J Antibiot (Tokyo) 2018 07 4;71(7):633-641. Epub 2018 Apr 4.

Biological Resource Center, National Institute of Technology and Evaluation (NBRC), 2-49-10 Nishihara, Shibuya-ku, Tokyo, 151-0066, Japan.

Type strains of 72 validated Nocardia species were phylogenetically analyzed based on the multilocus sequence analysis (MLSA) concatenated atpD-groL1-groL2-recA-rpoA-secY-sodA-ychF. Furthermore, their similarity based on digital DNA-DNA hybridization (dDDH) was calculated. Nocardia soli, Nocardia cummidelens and Nocardia salmonicida, Nocardia nova and Nocardia elegans, Nocardia exalbida and Nocardia gamkensis, and Nocardia coubleae and Nocardia ignorata formed coherent clades, respectively. Moreover, each set showed over 70% relatedness by dDDH and shared common phenotypic characteristics. Therefore, we propose a reclassification of Nocardia soli and Nocardia cummidelens as a later heterotypic synonym of Nocardia salmonicida, Nocardia elegans as a later heterotypic synonym of Nocardia nova, Nocardia gamkensis as a later heterotypic synonym of Nocardia exalbida, and Nocardia coubleae as a later heterotypic synonym of Nocardia ignorata.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41429-018-0043-1DOI Listing
July 2018

Visible DNA Microarray System as an Adjunctive Molecular Test in Identification of Pathogenic Fungi Directly from a Blood Culture Bottle.

J Clin Microbiol 2018 05 25;56(5). Epub 2018 Apr 25.

Department of Internal Medicine, Faculty of Medical Sciences, University of Campinas, Campinas, Sao Paulo, Brazil

A DNA microarray platform, based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene, was developed to identify 32 fungal pathogens at the species level. The probe sequences were spotted onto polycarbonate slides with a mini-microarray printer, and after the hybridization, the results were visible with the naked eye. The performance of the microarray platform was evaluated against the commercial automated systems (Vitek 2 and BD Phoenix systems) and DNA sequencing (gold standard). A total of 461 blood culture bottles were tested: 127 positive for fungi, 302 positive for bacteria, and 32 that were negative. Once the microorganisms were identified by automated systems, fungal DNA was extracted directly from the blood culture bottles. The DNA products were tested using the microarray platform, and DNA sequencing was performed. The results of the microarray and DNA sequencing were concordant in 96.7% of cases, and the results from the automated systems and DNA sequencing were concordant in 98.4%. Of all the nucleotide sequences contained in the microarray platform, the microarray failed to identify four fungal isolates (one , two , and one ). Of note, the microarray detected DNA in two blood cultures from the same patient, whereas the automated system was only positive for Our microarray system provided reliable and fast fungal identification compared to that from DNA sequencing and the automated systems. The simplicity of reading the results by the naked eye made this DNA platform a suitable method for fungal molecular diagnosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.01908-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925724PMC
May 2018

Fucose-specific lectin of Aspergillus fumigatus: binding properties and effects on immune response stimulation.

Med Mycol 2019 Jan;57(1):71-83

Medical Mycology Research Center, Chiba University, Japan.

Aspergillus fumigatus is the major causative fungus of aspergillosis, and many studies have explored the relationship between A. fumigatus and pathogenicity. In the current study, we focused on a fucose-specific lectin, FleA, as a novel molecule which related to the pathogenicity of A. fumigatus. The disruption of the fleA gene did not lead to clear morphological changes compared to parental strain under several stress conditions in culture, but germination become earlier. In comparison with parental strain, the pathogenicity of disruptant was enhanced in a mouse infection model. The pattern of conidial phagocytosis and adhesion to cultured cells did not explain this enhanced pathogenicity. FleA was reported to contain six conserved fucose-binding sites; the analysis of constructed FleA point mutants revealed nonequivalent contribution of the fucose-binding sites to fucose binding. Based on the immune response induced in the cultured cells upon exposure to wild-type and mutant FleA, we propose a model of the FleA molecule in A. fumigatus infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/mmy/myx163DOI Listing
January 2019

meningitis in a diffuse large B-cell lymphoma patient with CD4-positive lymphocytopenia and persistent oligoclonal CD8-positive lymphocytes in the peripheral blood.

Int J Clin Exp Pathol 2018 1;11(1):455-461. Epub 2018 Jan 1.

Medical Mycology Research Center, Chiba University Chuo-Ku, Chiba, Japan.

Nocardiosis, sometimes presenting with multiple granulomatous lesions, is a rare opportunistic infection occurring in immunocompromised patients. However, its immunological features remain largely unaddressed. We investigated the immunological characteristics of human nocardiosis and examined the component cells of the granulomatous lesions. A 66-year-old man with diffuse large B-cell lymphoma presented with fever and multiple nodules in the lung during chemotherapy. The blood culture formed white colonies, but their characterization was difficult by routine microbiological laboratory methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the colonies as . Meanwhile, the patient suddenly experienced an epileptic seizure without a brain abscess. His cerebrospinal fluid (CSF) showed neutrophilic pleocytosis (108/mm). The conventional agar culturing failed to isolate colonies, but culturing with brain-heart infusion agar generated colonies. These colonies were completely concordant with those from the blood, as confirmed by 16S rRNA gene sequencing. Therefore, the patient had developed meningitis through sepsis induced by . His CD4-positive T-lymphocyte counts were low, and oligoclonal CD8-positive αβ T-lymphocytes were present in the blood prior to the first and after three cycles of chemotherapy. He had bone marrow granulomatous lesions comprising lymphoma and CD8-positive αβ T-cells. Treatment with sulfamethoxazole/trimethoprim relieved all of his symptoms. The combined analysis by microbiological and molecular methods determined the cause of his epileptic seizure. His immunological characteristics, including low CD4-positive or CD8-positive αβ T-lymphocytes, may have contributed to the unusual clinical presentations by , which rarely involves the central nervous system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957946PMC
January 2018

A case of empyema and septic arthritis due to .

Clin Case Rep 2017 12 24;5(12):1976-1979. Epub 2017 Oct 24.

Department of Respiratory Medicine Saitama Cardiovascular and Respiratory Center Saitama Japan.

Septic arthritis due to sp. should be suspected when a patient with risk factors such as pneumoconiosis or diabetes mellitus develops joint symptoms, especially if the patient has had nocardiosis in other sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ccr3.1228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715427PMC
December 2017

Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554 in the presence of animal cells.

J Nat Med 2018 Jan 5;72(1):280-289. Epub 2017 Dec 5.

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan.

A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554 in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from D-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a "co-culture" in which the genus Nocardia was cultured in the presence of an animal cell line.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11418-017-1161-yDOI Listing
January 2018

Global gene expression reveals stress-responsive genes in Aspergillus fumigatus mycelia.

BMC Genomics 2017 Dec 4;18(1):942. Epub 2017 Dec 4.

Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Japan.

Background: Aspergillus fumigatus is a human fungal pathogen that causes aspergillosis in immunocompromised hosts. A. fumigatus is believed to be exposed to diverse environmental stresses in the host cells. The adaptation mechanisms are critical for infections in human bodies. Transcriptional networks in response to diverse environmental challenges remain to be elucidated. To gain insights into the adaptation to environmental stresses in A. fumigatus mycelia, we conducted time series transcriptome analyses.

Results: With the aid of RNA-seq, we explored the global gene expression profiles of mycelia in A. fumigatus upon exposure to diverse environmental changes, including heat, superoxide, and osmotic stresses. From the perspective of global transcriptomes, transient responses to superoxide and osmotic stresses were observed while responses to heat stresses were gradual. We identified the stress-responsive genes for particular stresses, and the 266 genes whose expression levels drastically fluctuated upon exposure to all tested stresses. Among these, the 77 environmental stress response genes are conserved in S. cerevisiae, suggesting that these genes might be more general prerequisites for adaptation to environmental stresses. Finally, we revealed the strong correlations among expression profiles of genes related to 'rRNA processing'.

Conclusions: The time series transcriptome analysis revealed the stress-responsive genes underlying the adaptation mechanisms in A. fumigatus mycelia. These results will shed light on the regulatory networks underpinning the adaptation of the filamentous fungi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-017-4316-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715996PMC
December 2017

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses.

Cell Microbiol 2018 03 22;20(3). Epub 2017 Dec 22.

Medical Mycology Research Center, Chiba University, Chiba, Japan.

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K-means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high-adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin-like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cmi.12802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838799PMC
March 2018

Temperature during conidiation affects stress tolerance, pigmentation, and trypacidin accumulation in the conidia of the airborne pathogen Aspergillus fumigatus.

PLoS One 2017 9;12(5):e0177050. Epub 2017 May 9.

Medical Mycology Research Center (MMRC), Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Japan.

Asexual spores (conidia) are reproductive structures that play a crucial role in fungal distribution and survival. As fungal conidia are, in most cases, etiological agents of plant diseases and fungal lung disease, their stress resistance and interaction with their hosts have drawn increasing attention. In the present study, we investigated whether environmental temperature during conidiation affects the stress tolerance of the conidia of the human pathogenic fungus Aspergillus fumigatus. Conidia from a 25°C culture showed a lower tolerance to heat (60°C) and oxidative (H2O2) stresses and a marked resistance to ultraviolet radiation exposure, compared with those produced at 37 and 45°C. The accumulation of trehalose was lower in the conidia from the 25°C culture. Furthermore, the conidia from the 25°C culture showed darker pigmentation and increased transcripts of dihydroxynaphthalene (DHN)-melanin biosynthesis-related genes (i.e., pksP, arp1, and arp2). An RNA-sequencing analysis revealed that the transcription level of the trypacidin (tpc) gene cluster, which contains 13 genes, was sharply and coordinately activated in the conidia from the 25°C culture. Accordingly, trypacidin was abundant in the conidia from the 25°C culture, whereas there was little trypacidin in the conidia from the 37°C culture. Taken together, these data show that the environmental temperature during conidiation affects conidial properties such as stress tolerance, pigmentation, and mycotoxin accumulation. To enhance our knowledge, we further explored the temperature-dependent production of DHN-melanin and trypacidin in clinical A. fumigatus isolates. Some of the isolates showed temperature-independent production of DHN-melanin and/or trypacidin, indicating that the conidia-associated secondary metabolisms differed among the isolates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177050PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423626PMC
September 2017

First case report of pulmonary nocardiosis caused by .

JMM Case Rep 2016 Aug 30;3(4):e005054. Epub 2016 Aug 30.

Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuoku, Chiba City, Chiba, 260-8673, Japan.

Introduction: species usually cause opportunistic infections, and the frequency of these infections is increasing owing to the growing population of immunocompromised hosts. However, species may sometimes cause an infection disease in immunocompetent hosts. infections are the least common and are very rare.

Case Presentation: Herein, we report the first case of a pulmonary infection with in a 61-year-old Japanese woman with a history of hyperlipidaemia and bronchiectasis and a 6-month history of non-productive hacking cough. A sample of bronchial lavage fluid obtained by bronchofiberscopy showed filamentous branching gram-positive rods and acid-fast filamentous branching rods, and a colony of suspected was cultured. Based on 16S rRNA, , and gene sequence analyses and biochemical and physiological properties, the strain was identified as The strain was resistant to the antimicrobial agents amoxicillin-clavulanic acid, clarithromycin, minocycline, gentamycin, tobramycin, ciprofloxacin and trimethoprim-sulfamethoxazole. The patient was treated with biapenem followed by intravenous amikacin and oral linezolid.

Conclusion: Despite its rarity, the species require attention owing to the existence of multidrug-resistant strains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jmmcr.0.005054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330244PMC
August 2016

Transcription factor Afmac1 controls copper import machinery in Aspergillus fumigatus.

Curr Genet 2017 Aug 18;63(4):777-789. Epub 2017 Feb 18.

Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Chiba, Japan.

Copper (Cu) is an essential metal for all living organisms, although it is toxic in excess. Filamentous fungus must acquire copper from its environment for growth. Despite its essentiality for growth, the mechanisms that maintain copper homeostasis are not fully understood in filamentous fungus. To gain insights into copper homeostasis, we investigated the roles of a copper transcription factor Afmac1 in the life-threatening fungus Aspergillus fumigatus, a homolog of the yeast MAC1. We observed that the Afmac1 deletion mutant exhibited not only significantly slower growth, but also incomplete conidiation including a short chain of conidia and defective melanin. Moreover, the expressions of the copper transporters, ctrA1, ctrA2, and ctrC, and metalloreductases, Afu8g01310 and fre7, were repressed in ∆Afmac1 cells, while those expressions were induced under copper depletion conditions in wild-type. The expressions of pksP and wetA, which are, respectively, involved in biosynthesis of conidia-specific melanin and the late stage of conidiogenesis, were decreased in the ∆Afmac1 strain under minimal media condition. Taken together, these results indicate that copper acquisition through AfMac1 functions in growth as well as conidiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00294-017-0681-zDOI Listing
August 2017

Zamamidine D, a Manzamine Alkaloid from an Okinawan Amphimedon sp. Marine Sponge.

J Nat Prod 2017 04 16;80(4):1196-1199. Epub 2017 Feb 16.

Graduate School of Pharmaceutical Sciences, Hokkaido University , Sapporo 060-0812, Japan.

A new manzamine alkaloid, zamamidine D (1), was isolated from an Okinawan Amphimedon sp. marine sponge. The structure of zamamidine D (1) including the relative configuration was elucidated on the basis of spectroscopic data. Zamamidine D (1) is the first manzamine alkaloid possessing a 2,2'-methylenebistryptamine unit as the aromatic moiety instead of a β-carboline unit. Zamamidine D (1) showed antimicrobial activity against several bacteria and fungi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jnatprod.6b01110DOI Listing
April 2017

Nabscessins A and B, Aminocyclitol Derivatives from Nocardia abscessus IFM 10029.

J Nat Prod 2017 02 23;80(2):565-568. Epub 2017 Jan 23.

Graduate School of Pharmaceutical Sciences, Chiba University , 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.

Two new aminocyclitol amide derivatives, nabscessins A (1) and B (2), were isolated from the culture broth of a pathogenic actinomycete species, Nocardia abscessus IFM 10029. The structures of nabscessins A and B were elucidated by spectral studies, and the compounds showed antifungal activity against Cryptococcus neoformans, with IC values of 32 and 16 μg/mL, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jnatprod.6b00935DOI Listing
February 2017

Discrepancy between Hepatitis C Virus Genotypes and NS4-Based Serotypes: Association with Their Subgenomic Sequences.

Int J Mol Sci 2017 Jan 17;18(1). Epub 2017 Jan 17.

Department of Molecular Virology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.

Determination of hepatitis C virus (HCV) genotypes plays an important role in the direct-acting agent era. Discrepancies between HCV genotyping and serotyping assays are occasionally observed. Eighteen samples with discrepant results between genotyping and serotyping methods were analyzed. HCV serotyping and genotyping were based on the HCV nonstructural 4 (NS4) region and 5'-untranslated region (5'-UTR), respectively. HCV core and NS4 regions were chosen to be sequenced and were compared with the genotyping and serotyping results. Deep sequencing was also performed for the corresponding HCV NS4 regions. Seventeen out of 18 discrepant samples could be sequenced by the Sanger method. Both HCV core and NS4 sequences were concordant with that of genotyping in the 5'-UTR in all 17 samples. In cloning analysis of the HCV NS4 region, there were several amino acid variations, but each sequence was much closer to the peptide with the same genotype. Deep sequencing revealed that minor clones with different subgenotypes existed in two of the 17 samples. Genotyping by genome amplification showed high consistency, while several false reactions were detected by serotyping. The deep sequencing method also provides accurate genotyping results and may be useful for analyzing discrepant cases. HCV genotyping should be correctly determined before antiviral treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms18010172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297804PMC
January 2017

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions.

PLoS Pathog 2017 01 4;13(1):e1006096. Epub 2017 Jan 4.

Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215518PMC
January 2017

Nocardia elegans infection: a case report and literature review.

Int J Infect Dis 2017 Jan 5;54:15-17. Epub 2016 Nov 5.

Department of Microbiology, Tokyo Medical University, Tokyo, Japan.

A case of disseminated nocardiosis caused by Nocardia elegans in a 72-year-old man with rheumatoid arthritis, treated with tacrolimus and prednisolone, is reported herein. The patient had impaired vision and was diagnosed with endophthalmitis and an abdominal skin abscess. He was started on trimethoprim-sulfamethoxazole treatment, followed by cefepime. The patient was then switched to a combination of imipenem-cilastatin and minocycline. Although the patient survived as a result of surgery and prolonged antibiotic treatment, he eventually lost vision after the infection became resistant to antibiotic treatment. Molecular analysis of samples from the abscess and vitreous fluid confirmed the extremely rare pathogen N. elegans, which accounts for only 0.3-0.6% of infections caused by Nocardia species. This organism is almost always associated with pulmonary infection, and disseminated infections are rare. As with previously reported norcardial infections, the current case was treated successfully with trimethoprim-sulfamethoxazole, carbapenems, and aminoglycosides. However, the clinical characteristics of this organism remain unclear. Further studies are therefore required to develop more effective treatment protocols for disseminated nocardiosis caused by this problematic pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijid.2016.10.031DOI Listing
January 2017

Hyrtinadines C and D, New Azepinoindole-Type Alkaloids from a Marine Sponge Hyrtios sp.

Chem Pharm Bull (Tokyo) 2016 ;64(7):975-8

Showa Pharmaceutical University.

New bisindole alkaloids, hyrtinadines C (1) and D (2), have been isolated from an Okinawan marine sponge Hyrtios sp. The structures of hyrtinadines C (1) and D (2) were elucidated based on analyses of the spectral data. Hyrtinadines C (1) and D (2) were the relatively rare alkaloids possessing a 3,4-fused azepinoindole skeleton. Hyrtinadines C (1) and D (2) showed antimicrobial activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1248/cpb.c16-00201DOI Listing
January 2017

Nocardia shinanonensis sp. nov., isolated from a patient with endophthalmitis.

Int J Syst Evol Microbiol 2016 Sep 2;66(9):3324-3328. Epub 2016 Jun 2.

Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan.

A nocardioform strain IFM 11456T was isolated from the aqueous humor from a patient with endophthalmitis and was characterized to its taxonomic position. IFM 11456T contained arabinose, galactose and meso-diaminopimelic acid in whole-cell hydrolysates and mycolic acids that co-migrated with those from the type strain of Nocardiaasteroides. The acyl type of muramic acid was N-glycolyl. The diagnostic polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and two unidentified glycolipids and the predominant menaquinone was MK-8(H4, ω-cycl.). These characteristics are typical of members of the genus Nocardia. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate represented a novel species of the genus Nocardia and was most closely related to the type strains of Nocardia mikamii JCM 15508T (98.1 %) and Nocardiaaobensis IFM 0372T (98.1 %). However, analysis of partial gyrB sequences showed that strain IFM 11456T had 90.2 % similarity to Nocardia concava IFM 0354T and 90 % to Nocardianiigatensis IFM 0330T. The DNA-DNA relatedness values for strain IFM 11456T compared with N. mikamii JCM 15508T, N. aobensisIFM 0372T and N. concava IFM 0354T ranged from 24.4 to 39.9 %. Phenotypic characteristics that differentiated IFM 11456T from phylogenetically related species were growth at 45 °C, utilization of citrate and growth with inositol as a sole carbon source. On the basis of this polyphasic study, the isolate represents a novel species within the genus Nocardia, for which the name Nocardia shinanonensis sp. nov. is proposed. The type strain is IFM 11456T (=NBRC 109590T=TBRC 5149T).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/ijsem.0.001197DOI Listing
September 2016

Comparative transcriptome analysis revealing dormant conidia and germination associated genes in Aspergillus species: an essential role for AtfA in conidial dormancy.

BMC Genomics 2016 05 17;17:358. Epub 2016 May 17.

Medical Mycology Research Center (MMRC), Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Japan.

Background: Fungal conidia are usually dormant unless the extracellular conditions are right for germination. Despite the importance of dormancy, little is known about the molecular mechanism underlying entry to, maintenance of, and exit from dormancy. To gain comprehensive and inter-species insights, transcriptome analyses were conducted across Aspergillus fumigatus, Aspergillus niger, and Aspergillus oryzae.

Results: We found transcripts of 687, 694, and 812 genes were enriched in the resting conidia compared with hyphae in A. fumigatus, A. niger, and A. oryzae, respectively (conidia-associated genes). Similarly, transcripts of 766, 1,241, and 749 genes were increased in the 1 h-cultured conidia compared with the resting conidia (germination-associated genes). Among the three Aspergillus species, we identified orthologous 6,172 genes, 91 and 391 of which are common conidia- and germination-associated genes, respectively. A variety of stress-related genes, including the catalase genes, were found in the common conidia-associated gene set, and ribosome-related genes were significantly enriched among the germination-associated genes. Among the germination-associated genes, we found that calA-family genes encoding a thaumatin-like protein were extraordinary expressed in early germination stage in all Aspergillus species tested here. In A. fumigatus 63 % of the common conidia-associated genes were expressed in a bZIP-type transcriptional regulator AtfA-dependent manner, indicating that AtfA plays a pivotal role in the maintenance of resting conidial physiology. Unexpectedly, the precocious expression of the germination-associated calA and an abnormal metabolic activity were detected in the resting conidia of the atfA mutant, suggesting that AtfA was involved in the retention of conidial dormancy.

Conclusions: A comparison among transcriptomes of hyphae, resting conidia, and 1 h-grown conidia in the three Aspergillus species revealed likely common factors involved in conidial dormancy. AtfA positively regulates conidial stress-related genes and negatively mediates the gene expressions related to germination, suggesting a major role for AtfA in Aspergillus conidial dormancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-016-2689-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869263PMC
May 2016

Multi-azole resistant Aspergillus fumigatus harboring Cyp51A TR46/Y121F/T289A isolated in Japan.

J Infect Chemother 2016 Aug 16;22(8):577-9. Epub 2016 Feb 16.

Division of Clinical Research, Medical Mycology Research Center, Chiba University, Japan.

Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same clade of the ones which were clinically isolated in France and Germany. A. fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jiac.2016.01.015DOI Listing
August 2016

Identification and Functional Analysis of the Nocardithiocin Gene Cluster in Nocardia pseudobrasiliensis.

PLoS One 2015 20;10(11):e0143264. Epub 2015 Nov 20.

Medical Mycology Research Center, Chiba University, Chiba, Japan.

Nocardithiocin is a thiopeptide compound isolated from the opportunistic pathogen Nocardia pseudobrasiliensis. It shows a strong activity against acid-fast bacteria and is also active against rifampicin-resistant Mycobacterium tuberculosis. Here, we report the identification of the nocardithiocin gene cluster in N. pseudobrasiliensis IFM 0761 based on conserved thiopeptide biosynthesis gene sequence and the whole genome sequence. The predicted gene cluster was confirmed by gene disruption and complementation. As expected, strains containing the disrupted gene did not produce nocardithiocin while gene complementation restored nocardithiocin production in these strains. The predicted cluster was further analyzed using RNA-seq which showed that the nocardithiocin gene cluster contains 12 genes within a 15.2-kb region. This finding will promote the improvement of nocardithiocin productivity and its derivatives production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143264PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654471PMC
June 2016
-->