ISSN 1347−6920

MMRC
ANNUAL REPORT OF MEDICAL MYCOLOGYRESEARCH CENTER, CHIBA UNIVERSITY 2014
千葉大学 真菌医学研究センター

18

平成26年

報告

目

次

Content
はじめに Preface
病原機能分野 川本 PI（分子細胞シグナリング解析）プロジェクト

Project for Molecular Signaling Analysis

病原機能分野 知花 PI（カンジダ・グラブラータ

3

フェノーム）プロジェクト

Candida glabrata Phenome Project

感染免疫分野 米山 PI（感染応答）プロジェクト

Project for Immune Response in Infections Diseases

感染免疫分野 西城 PI（サイトカイン）プロジェクト

Project for Cytokine Research

臨床感染症分野 亀井 PI（臨床感染症）プロジェクト

11
13
16

Project to Link Basic Sciences and Clinical Medicine

20

微生物資源分野 五ノ井 PI（真菌・放線菌と宿主の分子相互作用研究）
プロジェクト
Project for Host Pathogen（fungi/actinomycetes）Molecular Interaction

33

微生物資源分野 高橋 PI（微生物創生）プロジェクト

Project for Systems Biology of Microorganisms

47

微生物資源分野 バイオリソース管理室

Management of Unit of Microbiological Resources

49

文部科学省 ナショナルバイオリソースプロジェクト「病原微生物」

Ministry of Education, Culture, Sports, Science and Technology
National BioResource Project“Pathogenic Microorganisms”

53

長崎大学熱帯医学研究拠点特定領域共同研究

Cooperative Research of Priority Areas with NEKKEN, Nagasaki University

54

アスペルギルス症を中心とした新興真菌症制圧プロジェクト

The Project on Controlling Aspergillosis and the Related Emerging Mycoses55

平成 25 年度 共同利用・共同研究報告2013 Fiscal Year Cooperative Research Program Report56

平成 25 年度 共同利用・共同研究研究会報告2013 Fiscal Year Cooperative Research Meetings Report642014 年講演会
2014 Scientific Meetings & Seminars

66

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に

千葉大学真菌医学研究センターは , 真菌感染症の基盤・臨床研究 , 共同利用・共同研究拠点事業 , 真菌バイオ

リソース事業を活動の３本柱とする , 我が国で唯一の真菌感染症の研究・教育に特化した公立の大学附置研究センターです .

平成26年７月 , 千葉大学亥鼻キャンパスでは , 次世代対応型医療人の育成と「治療学」拠点形成を目的として ,徳久剛史学長の主導のもとに , 医学研究院の中山俊憲教授を機構長とする「未来医療教育研究機構」が設立されました . 真菌医学研究センターは , 医学部 , 薬学部 , 看護学部とともに機構のメンバーとして研究・教育活動に参加しています . これに伴い , 本センターには , 准教授 , 助教ポジションと事業費が配分されました . また学長裁量による特別経費で , 新たな研究チームを立ち上げるために , センター A 棟の諸設備の更新 , 実験室新設 , 客員教授

居室新設等 , 一連の工事を行っています . また平成27年３月までには , A 棟の耐震補強工事が完了するとともに , B
棟１階には BSL-3レベル実験施設が設置され , より安全性の高い病原真菌の研究環境が整備される予定です .

本センターでは , 超高齢社会における真菌感染症の増加 , 及び経済のグローバリゼーションを通じてもたらされる輸入真菌症など , 新たな課題の解決に貢献するため , 真菌感染症の診断・治療への取り組みを強化すると共に , 深在性真菌症を中心とする難治性感染症研究拠点の形成を目指して , 医学部附属病院との連携を強化しました . 具体的なアクションとして , 平成26年11月に附属病院において , 亀井克彦教授 , 渡邉哲准教授が「真菌症専門外来」を開設しました . また11月には , 医学部附属病院の感染症管理治療部講師石和田稔彦先生を准教授として迎えました . その結果 , 本センターと附属病院との連携が従来にもまして深まり , 本センターにおける感染症臨
床研究のさらなる発展が期待されます . 一方で , 病原真菌の基礎研究の強化及び感染免疫研究の裾野を広げる目

的で , 共同利用・共同研究拠点「真菌感染症研究拠点」事業を活用して , 異分野との連携を積極的に行い , 平成
26年度は全国の研究機関と24件の共同研究課題を実施しました . また , 平成25年度の研究成果は本年報に掲載してありますが , 一部の採択課題の成果について , 平成26年３月７日に東大医科学研究所と合同で開催した成果報
告会にて報告を行いました .

本センターでは７名の Principal investigator（PI）による研究グループが研究・教育活動を行い , 世界レベルの研究成果が発信できる研究センターを目指しています . 本年度は , 西城忍特任准教授が九大の山崎晶教授（本センター客員教授）との共同研究を行い , その成果が Immunity 誌に発表され , 新聞等でも紹介され注目を集めました .
また米山光俊教授が , トムソンロイターが本年発表した ,「Highly Cited Researchers」に , 免疫学の分野で世界87名の最も影響力のある研究者の一人に選ばれました（センターホームページ参照）. 一方 , 病原真菌の基礎研究では ,11月に川本進教授が , 「Cryptococcus neoformans の細胞周期制御と低酸素ストレス応答の分子細胞シグナリング解析」
で , 日本医真菌学会より学会賞を受賞されました . これらは , いずれも日頃の研究活動の成果が評価されたものでありセンターとしても大変喜ばしいことであります .

センターでは , 平成26年６月より国内外から第一線で活躍する研究者をセンターへ招き , ６回の「Monthly
seminar」を開催しました . また11月15日には共同利用・共同研究拠点事業の一環として , 医学研究院の横須賀収先生及び教室の方々の御支援をいただき ,「感染症研究グローバルネットワークフォーラム2014」を開催しました .
今回で３回目となり , 学内外から著名な先生をお招きして講演会を行い , 成功裏に終了することができました .
本年度は , これら一連の活動を通じて , 本センターに対する , 社会 , 大学 , 研究コミュニティーからの期待が極めて大きいことを改めて認識させられました .
我が国はすでに超高齢社会に突入し , 本センターには真菌をはじめとする難治性感染症の基盤研究を推進し , それらの研究成果を臨床や創薬開発へ繋げてゆくことが強く求められています . 一方 , 世界規模では , 高度病原性病
原体による感染症の脅威に加えて , 多剤耐性病原体の拡散 , あるいは高度医療の普及に伴う難治性感染症の増加が大きな問題となっています . 本センターでは , これらの現状と今後の社会情勢を踏まえ , 学内外からの御支援を賜り , 次世代の研究者の育成と同時に , 医療へ貢献できる研究センターを目指す所存でおります .

平 成 27 年 1 月
千葉大学真菌医学研究センター長

笹

川

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Preface
It is my great pleasure to present our Annual Report 2014 for the Medical Mycology Research Center（MMRC）,

Chiba University. MMRC was originally founded in 1946 as the Institute for Food-Microbiology Chiba Medical College.

The Institute was renovated to the Research Institute for Chemobiodynsmics in 1973, and after 4 years the Institute wasrebuild to focus largely on pathogenic fungi and its infectious diseases, for which the Institute was renamed as MMRC

in 1997. In 2004, MMRC was certified to act as National University Cooperation. In 2010, MMRC was reorganized
into 1 department composing of 4 research divisions to further strengthen the research activity, those include divisions ofmolecular biology of pathogenic fungi, infection and immunology, clinical research, and bioresources. In 2010, MMRC

was certified for Joint/Research Center for promoting the central role in leading medical mycology research in Japan.
We are aiming at achievement of high level of research and clinical activities, for which we employ multi-disciplinary

approaches to infection biology, comprising concepts and methodologies of molecular genetics, bioinformatics,
immunology, cell biology, protein chemistry, and clinical research. MMRC also encourages to promote the applicationsof their researches toward prevention, diagnosis, and control of fugal and bacterial infectious diseases by cooping with theFaculty of Medicine, Medical Hospital, and Faculty of Pharmacy at Chiba University.

This annual report summarizes our scientific achievements in 2014, which I hope will be useful to promote domestic

as well as worldwide collaboration with our scientists, and ultimately contribute to medial fungal research and medicine.
January 26, 2015

Chihiro Sasakawa
Director of MMRC

川本 PI（分子細胞シグナリング解析）
プロジェクト
Project for Molecular Signaling Analysis
研究概要（Summary）
生化学・分子生物学・細胞生物学等の手法を用い , 病原酵母・糸状菌の分子細胞研究を行い , 病原機能などに関連するシグナリング解析を進め , 抗真菌薬シーズ創出など真菌症の分子制御に向けた分子細胞医真菌学への貢献を目指す .

We are conducting basic research on the molecular and cellular biology of pathogenic fungi using
biochemistry and molecular biology methods based on gene and protein science as well as ultrastructuralmorphology and cell biology methods such as electron microscopy.
教

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グランドフェロー
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技 術 補 佐 員

1.

川本
進
横山 耕治
清水 公徳
大楠美佐子
山口 正視
萩原 大祐
中野百実子

Professor

Associate Professor
Assistant Professor

Research Technician
Grandfellow

Research Assistant Professor

Research Promotion Technician

Structure based Functional Distinction between Cln1 andCln2 Depends on the Ubiquitin-Proteasome Pathway.
1

1

2

Akiko Suganami , Norio Takase , Hajime Sugiyama , Eric V
3

3

1

Virtudazo , Susumu Kawamoto , Yutaka Tamura

1

2

3

Department of Bioinformatics, Graduate School of
Medicine, Chiba University, Chiba 260-8670, Japan

Bio IT Development, Fujitsu Limited, Chiba 261-8588,
Japan

Division of Molecular Biology, Medical Mycology ResearchCenter, Chiba University, Chiba 260-8673, Japan

Susumu Kawamoto
Koji Yokoyama
Kiminori Shimizu
Misako Ohkusu
Masashi Yamaguchi
Daisuke Hagiwara
Yumiko Nakano

Cdc28 complexes. Our in silico simulations suggested thatthe interaction of Cln1 and Cln2 with Cdc28 were in the two

distinct situations, designated as flip and flop conformation,at the extra amino acid region in the cyclin box of Cln1 andCln2. We speculated the trigger of this flip-flop conversion of

the extra amino acid region in the cyclin box of Cln1-Cdc28
and Cln2-Cdc28 might be regulated by the ubiquitination of

the sequences rich in Pro（P）, Glu（E）, Ser（S）and Thr（T）,
so-called PEST motifs, in Cln1 and Cln2. Furthermore, we
presumed that the functional superiority between Cln1 andCln2 in the G1/S phase of S. cerevisiae might be controlled by
flip-flop conversion and ubiquitin-proteasome pathway.

Cln1 and Cln2, G1/S cyclins of the ascomycetous budding

yeast Saccharomyces cerevisiae（S. cerevisiae）, oscillate during

the cell cycle, rising in late G1 and falling in early S phase.
We have been tried to elucidate the structure basis of thefunctional distinction between Cln 1 and Cln 2 . Here we
performed in silico simulations: construction and evaluationof three dimensional structures of Cln 1-Cdc28 and Cln2-

千葉大学

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3

Functional characterization of PMT2 , encoding a2.

protein-O-mannosyltransferase, in the human pathogen

Cryptococcus neoformans.

Kiminori Shimizu1, Yumi Imanishi1,2, Akio Toh-e1, Jun Uno1,
Hiroji Chibana1, Christina M. Hull 3,4, Susumu Kawamoto1
1

2

4

Susumu Kawamoto1, Haruo Takahashi1,5, Koji Yokoyama1

Department of Applied Material and Life Science, College

21-50-1, Kanazawa-ku, Yokohama 236-8501, Japan

3

and Public Health, University of Wisconsin–Madison, WI

4

Department of Medical Microbiology & Immunology, School

5

Department of Biomolecular Chemistry, School of Medicine
53706, USA

of Medicine and Public Health, University of Wisconsin–

Madison, WI 53706, USA

fumonisin B2 biosynthesis in Aspergillus niger.

Daisuke Hagiwara 1, Yoshiki Onji 4, Katsuyoshi Asano 4,

1

Inohana 1-8-1, Chuo-ku, Chiba 260-8673, JapanThe a-oxoamine synthase gene fum8 is involved in

Kiminori Shimizu1, Hiroyuki Nakagawa2, Ruiko Hashimoto3,

Medical Mycology Research Center, Chiba University,of Engineering, Kanto Gakuin University, Mutsuura-higashi

3

3.

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

National Agriculture and Food Research Organization,
National Food Research Institute, Tsukuba, Ibaraki, Japan

Division of Bacteriology, Chiba Prefectural Institute of
Public Health, Chiba, JapanAir Environment Division, Nara Prefectural Institute for
Hygiene and Environment, Nara, Japan

Division of Microbiology, National Institute of Health
Sciences, Tokyo, JapanOne of the secondary metabolite gene clusters of Aspergillus

Diazobenzoic acid B（DBB）, also known as diazonium blue

niger possesses highly homologous genes with those within theyeasts from ascomycetes. This chemical has long been usedconsidered to be present within approximately 50 kb genomic

B or fast blue B, can be used to distinguishbasidiomycetous

fumonisin gene cluster of Fusarium spp. At least 14 genes arefor the taxonomic study of yeast species at the phylum level,

region. A number of strains of A. niger have been shown to

unknown. To identify molecular targets of DBB staining, we

direct evidence that the gene cluster is responsible for FB 2

of Cryptococcus neoformans, a basidiomycetous pathogenicof the cluster in FB2 biosynthesis. The disruption of fum8,

but the mechanism underlying the DBB staining remains
isolated Agrobacterium tumefaciens-mediated insertional mutants

produce fumonisin B2（FB2）, however, there has been no

production in this fungus. We investigated the involvement

yeast, which were negative to DBB staining. In one of thesecoding for an a-oxoamine synthase, resulted in loss of FB2Omannosyltransferase, was interrupted by a T-DNA insertion.

growth and sensitivity to high temperature or UV irradiation.

mutants, we found that the PMT 2 gene, encoding a protein-

A complete gene knockout of the PMT2 gene revealed thatthe gene was responsible for DBB staining in C. neoformans,suggesting that one of the targets of Pmt2-mediated glycosylationis responsible for interacting with DBB. We also determinedbiosynthesis in A. niger, but did not influence the vegetative

The gene expression patterns of the transcription factor genefum21 and fum8 were determined, but were different fromthose in F. verticillioides.

that Cryptococcus gattii, a close relative of C. neoformans, was

not stained by DBB when the PMT2 gene was deleted. Ourfinding suggests that the protein-O-mannosylation by the PMT2

gene product is required for DBB staining in Cryptococcus species

in general. We also showed that glycosylation in Cryptococcus by
Pmt2 plays important roles in controlling cell size, resistanceto high temperature and osmolarity, capsule formation, sexual

reproduction, and virulence.

4

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真菌医学研究センター報告

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4.

The role of AtfA and HOG MAPK pathway in stress
tolerance in conidia of Aspergillus fumigatus.

Daisuke Hagiwara1, Satoshi Suzuki2, Katsuhiko Kamei1,

Tohru Gonoi1, Susumu Kawamoto1
1

Medical Mycology Research Center（ MMRC）, Chiba

2

National Food Research Institute（NFRI）, Ibaraki, Japan

University, Chiba, Japan

Aspergillus fumigatus is a life-threatening pathogenic fungus,

whose conidium is the infectious agent of aspergillosis. To

better understand the mechanism underlying the long-term
viability of conidia, we characterized a bZip transcription

factor, AtfA, with special reference to stress-tolerance in

5.

Whole-Genome Comparison of Aspergillus fumigatus
Strains Serially Isolated from Patients with Aspergillosis.

Daisuke Hagiwara, Hiroki Takahashi, Akira Watanabe,Azusa Takahashi-Nakaguchi, Susumu Kawamoto, KatsuhikoKamei, Tohru Gonoi

Medical Mycology Research Center（ MMRC ）, Chiba
University, Chiba, JapanThe emergence of azole-resistant strains of Aspergillus

fumigatus during treatment for aspergillosis occurs by amutation selection process. Understanding how antifungal

resistance mechanisms evolve in the host environment during

infection is of great clinical importance and biological interest.

conidia. The atfA deletion mutant conidia showed significant

Here, we used next-generation sequencing（ NGS ）totrehalose content that accumulated in conidia was reduced

strains sequentially isolated from two patients, one withsensitivity to high temperature and oxidative stress. The

identify mutations that arose during infection by A. fumigatus

in the mutant conidia. Transcriptome analysis revealed thatinvasive pulmonary aspergillosis（IPA）（five isolations）andas catA, dprA, scf1, and conJ at the conidiation stage. The

isolates had identical microsatellite types, but their growth

AtfA regulated several stress-protection-related genes such

upstream high-osmolarity glycerol pathway was also involved

the other with aspergilloma（three isolations）. The serial

rates and conidia production levels were dissimilar. A whole-in conferring stress tolerance in conidia because ΔpbsB showed

genome comparison showed that three of the five isolates

a mutant lacking the SakA mitogen-activated protein kinase

including six nonsynonymous ones, were found among threestress sensitivity and reduced trehalose in conidia. However,（MAPK）produced normal conidia. We investigated another

MAPK, MpkC, in relation with SakA, and the double

deletion mutant, ΔsakA, mpkC, was defective in conidia stressfrom the IPA patient carried a mutation, while 22 mutations,
isolates from the aspergilloma patient. One aspergilloma
isolate carried the cyp51A mutation P216L, which is reported

to confer azole resistance, and it displayed an MIC indicating

tolerance. We concluded that MpkC is able to bypass SakA,

resistance to itraconazole. This isolate harbored five otherfunction of AtfA in A. fumigatus.

afyap1 and aldA genes. We further identified a large deletionand the two MAPKs redundantly regulate the conidia-related

nonsynonymous mutations, some of which were found in thein the aspergilloma isolate in a region containing 11 genes.

This finding suggested the possibility that genomic deletions

can occur during chronic infection with A. fumigatus. Overall,our results revealed dynamic alterations that occur in the

A. fumigatus genome within its host during infection andtreatment.

千葉大学

真菌医学研究センター報告

第 18 巻

2014

5

6.

Dynamics of cell components during budding of
Cryptococcus albidus yeast cells.
1

1

7.

strains isolated from patients with pulmonary aspergilloma

and chronic necrotizing pulmonary aspergillosis.

1

Masashi Yamaguchi , Kiminori Shimizu , Susumu Kawamoto ,Genome sequence comparison of Aspergillus fumigatus

Amaliya A Stepanova2, Natalya V Vasilyeva2

Azusa Takahashi-Nakaguchi 1, Yasunori Muraosa1, Daisuke

1

Watanabe1,2, Susumu Kawamoto1, Katsuhiko Kamei1, Tohru

2

Medical Mycology Research Center, Chiba University,

Hagiwara 1, Kanae Sakai 1, Takahito Toyotome 1,3, Akira

Chiba, Japan

Gonoi1, Hiroki Takahashi1

West State Medical University, St. Petersburg, Russia

1

Kashkin Research Institute of Medical Mycology of North-

Phase-contrast and freeze-substitution microscopy were used2cells（during exponential growth）varied in vacuolar contents

and there was an absence of storage compounds in the cytosol.

Inohana, Chuo-ku, Chiba, Chiba 260-8673, Japan

Division of Control and Treatment of Infectious Diseases,
Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba,

to study cellular division in the pathogenic fungus Cryptococcus

albidus in vitro. It was shown that the mother and daughter

Medical Mycology Research Center, Chiba University, 1-8-1

3Chiba 260-8673, Japan

Diagnostic Center for Animal Health and Food Safety,
Obihiro University of Agriculture and Veterinary Medicine,

Ultrastructural signs of mother cells for transitioning to bud

Nishi 2-11 Inada-cho, Obihiro, Hokkaido 080-8555, Japan

lateral, increasing the level of chromatization and nucleolus

Aspergillus fumigatus is the most important pathogenic

formation were a migration of the nucleus from distal tosize, proliferation of mitochondria and changes in topography

fungus among Aspergillus species associated with aspergillosis.

including formation of a“cover”around the nuclear envelope.

Of the various presentations of aspergillosis, one of the mostnucleolus presence. For the first time, we described theis pulmonary aspergilloma（ PA ）, which is a fungus balland daughter cells during cellular division. The formation of the

mucus, and tissue debris. Chronic necrotizing pulmonary

Specific feature of cellular division in cultures was permanent

nucleolus material being uniformly distributed between motherseptum separating the daughter cells was investigated in detail.

common forms of pulmonary involvement by A. fumigatus

composed of fungal hyphae, inflammatory cells, fibrin,aspergillosis（ CNPA ）, also known as semi-invasive orinvasive aspergillosis, is locally invasive and predominantly
seen in patients with mild immunodeficiency or with a chroniclung disease. In the present study, with the aid of a nextgeneration sequencer, we performed whole genome sequence（WGS）analysis of 17 strains isolated from patients with PA

and CNPA in Japan. A total of 99,088 SNPs were identified

by mapping the reads to A. fumigatus genome reference strain

Af 293, and according to genome-wide phylogenetic analysis,

there were no correlations between the whole genome

sequence typing, and pathological conditions. Here, we

conducted the first multi-genome WGS study to focus on

the A. fumigatus strains isolated from patients with PA and
CNPA, and comprehensively characterized genetic variationsof strains. WGS approach will help in better understanding ofmolecular mechanisms in aspergillosis caused by A. fumigatus.

6

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真菌医学研究センター報告

第 18 巻2014

8.

Dendritic cell-based immunization ameliorates

pulmonary infection with highly virulent Cryptococcus

gattii.

Keigo Ueno 1, Yuki Kinjo1, Yoichiro Okubo2, Kyoko Aki2,
Makoto Urai1, Yukihiro Kaneko1,3, Kiminori Shimizu4, Dan5

1

1

1

Ni Wang , Akiko Okawara , Takuya Nara , Kayo Ohkouchi ,
1

4

5,6

Yuki Mizuguchi , Susumu Kawamoto , Katsuhiko Kamei ,Hideaki Ohno 1,7, Yoshihito Niki 8, Kazutoshi Shibuya 2,
Yoshitsugu Miyazaki1

1

2

3

4

5

6

7

Department of Chemotherapy and Mycoses, National

Institute of Infectious Diseases, Tokyo, Japan

molecules and inflammatory cytokines. This was not observedfor BMDCs that were cultured with encapsulated strains.
When CAP60∆ -pulsed BMDCs were transferred to miceprior to intratracheal R265 infection, significant ameliorationof pathology, fungal burden, and the survival rate resulted ascompared to controls. Multi-nucleated giant cells（MGCs）

that engulfed fungal cells were significantly increased in thelungs of immunized mice. IL- 17A, IFNγ, and TNFα

-producing lymphocytes were significantly increased in the

spleens and lungs of immunized mice. Although only partially,
the protective effect of this DC vaccine was significantly
reduced in IFN γ knockout mice. These results demonstrated

Department of Surgical Pathology, Toho University Schoolthat an increase in cytokine-producing lymphocytes andDepartment of Bacteriology, Graduate School of Medicine,associated with protection against pulmonary infection with

Division of Molecular Biology, Medical Mycology Research

have been an important mediator for this vaccine-inducedof Medicine, Tokyo, Japan

Osaka City University, Osaka, Japan

Center, Chiba University, Chiba, Japan

Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University, Chiba, Japan

Division of Control and Treatment of Infectious Diseases,
Chiba University Hospital, Chiba, Japan

Department of Infectious Diseases and Infection Control,

Saitama Medical Center, Saitama Medical University,

8

CAP60∆ , which resulted in their expression of co-stimulatory

the development of MGCs that engulfed fungal cells werehighly virulent C. gattii and 1suggested that IFN γ may
protection.

9.

Ultrastructural observation of cell components during
budding in yeast Malassezia pachydermatis.

Masashi Yamaguchi 1,

Kawamoto , Amaliya A Stepanova , Natalya V Vasilyeva2Internal Medicine, Showa University School of Medicine,1

Tokyo, Japan

2

Cryptococcosis due to a highly virulent fungus, Cryptococcus

gattii, emerged as an infectious disease on Vancouver Islandand surrounding areas in 1999 , for which deaths were2

SusumuSaitama, Japan

Division of Clinical Infectious Diseases, Department of1

Kiminori Shimizu 1,

Medical Mycology Research Center, Chiba University,

Chiba, Japan

Kashkin Research Institute of Medical Mycology, NorthWestern State Medical University, St. Petersburg, Russia

The phase-contrast, scanning and transmission electron

reported among immunocompetent individuals. Previous

microscopy were used for observation of the cell components

a Canadian outbreak had immune-avoidance or immune-level of vacuolization, small number of mitochondria,

against C. gattii has not been identified. In this study, we, absence of secretory vesicles and presence of a large lipid

studies indicated that C. gattii strain R265 isolated fromsuppression capabilities. However, any protective immunity
used a gain-of-function approach to investigate protective

immunity against C. gattii infection using a dendritic cell-based（DC）vaccine. Bone marrow derived dendritic cells（BMDCs）efficiently engulfed an acapsular C. gattii mutant,

千葉大学

in exponential phase of growth in M. pachydermatis. Lower

presence of single cisterns of endoplasmic reticulum（ER）
inclusion opposite budding scar was typical for mother cells.
It was revealed the uniformity in bud formation, the numberof mitochondria, storage inclusion, and cisterns of ER

were not increased compared with the cytosol volume and真菌医学研究センター報告

第 18 巻20147

number of free ribosomes. An increase in nucleus sizes andlevel of chomatisation were observed in mother cell before

budding. The mother cell, after septum formation, differsfrom daughter cell in larger volume of cytosol and presenceof thicker cell wall. In contrast, daughter cell was typical insmaller volume of cytosol and presence of large lipid inclusion.
A diagram of organelles transition in M. pachydermatis
budding was presented.
10.

The Effects of F-Actin Inhibitor Latrunculin A on
Pathogenic Yeast Cryptococcus neoformans.

11.

Positional cloning in Cryptococcus neoformans and its

application for identification and cloning of the geneencoding methylenetetrahydrofolate reductase.

Akio Toh-e, Misako Ohkusu, Kiminori Shimizu, Susumu

Kawamoto

Medical Mycology Research Center, Chiba University,

Chiba, Japan

Cryptococcus neoformans, a basidiomycetous humanpathogenic yeast, has been widely used in research fields inMarie Kopecká1, Masashi Yamaguchi2, Susumu Kawamoto2

medical mycology as well as basic biology. Gene cloning or1

is a key step for functional analysis of a particular gene. The

2

Department of Biology, Faculty of Medicine, Masaryk

University, Brno, Czech Republic

Medical Mycology Research Centre, Chiba University,

Chuo-ku, Japan

Background: This basic research aimed to investigate theeffects of actin inhibitor latrunculin A on human pathogen

Cryptococcus neoformans by freeze-substitution and electron

identification of the gene responsible for a mutation of interest

availability therefore, of the multiple methods for cloning isdesirable. In this study, we proposed a method for a mapping-based gene identification/cloning（ positional cloning ）method in C. neoformans. To this end, we constructed a seriesof tester strains, one of whose chromosomes was labeledwith the URA5 gene. A heterozygous diploid constructed bycrossing one of the tester strains to a mutant strain of interest

microscopy to find out whether the actin cytoskeleton canloses a chromosome（s）spontaneously, which is the basis for

Methods: Cells treated by latrunculin A for 20 h in yeast

in the mitotic mapping method. Once the gene of interest

become a new antifungal target for inhibition of cell division.

extract peptone dextrose medium were investigated by phaseassigning a recessive mutant gene to a particular chromosome

is mapped to one of the 14 chromosomes, classical genetic

contrast and fluorescent microscopy, freeze-substitution

crosses can then be performed to determine its more precise

chamber and absorbance was measured. Results: The cells

used to significantly narrow down candidate genes by referringand transmission electron microscopy, counted in a Bürker
of Cryptococcus neoformans responded to the presence oflocation. The positional information thus obtained can then be

to the Cryptococcus genome database. Each candidate gene islatrunculin A, an actin inhibitor, by disappearance of actin

then examined whether it would complement the mutation.

cables and patches arrested proliferation and led to theencoding methylenetetrahydrofolate reductase as the genepatches, actin cables and actin rings. Removal of actin

production of cells that had ultrastructural disorder andirregular morphology of mitochondria and thick aberrant cell

walls. Budding cells lysed in buds and in septa. Conclusion:

We successfully applied this method to identify CNA07390

responsible for a methionie-requiring mutant in our mutant
collection.

Latrunculin A has fungistatic, fungicidal and fungilytic effects
on human pathogenic yeast Cryptococcus neoformans.

8

千葉大学

真菌医学研究センター報告

第 18 巻2014

Publications

１）de Castro PA, Chen C, de Almeida RS, Freitas FZ,

the human pathogen Cryptococcus neoformans, Fungal

Genetics and Biology 69: 13-22, 2014.

Bertoloni MC, Morais ER, Brown NA, Ramalho LN,

10）Shimizu K, Nakagawa H, Hashimoto R, Hagiwara

seq reveals a role for CrzA in the Aspergillus fumigatus

Yokoyama K, The α-oxoamine synthase gene fum8 is

Hagiwara D, Mitchell TK, Goldman GH: ChIPHigh Osmolarity Glycerol Response（HOG）signaling

pathway, Mol Microbiol, 94: 655-674, 2014.

D, Onji Y, Asano K, Kawamoto S, Takahashi H,

involved in fumonisin B2 biosynthesis in Aspergillus niger,

Mycoscience, in press.

２）Fukiharu T, Shimizu K, Utsunomiya H, Raut JK, Goto

11）Suganami A, Takase N, Sugiyama H, Virtudazo EV,

N, Coprinopsis asiaticiphlyctidospora sp. nov., an agaric

distinction between Cln 1 and Cln 2 depends on theR, Okamoto T, Kato M, Horigome R, Furuki T, Kinjo

ammonia fungus from Amami and Okinawa, southern

Japan, Mycoscience 55: 355-360, 2014.

Kawamoto S, Tamura Y: Structure based functional

ubiquitin-proteasome pathway. J. Proteomics Bioinformatics（
7 5）: 102-107, 2014.

３）Hagiwara D, Suzuki S, Kamei K, Gonoi T, Kawamoto

12）Takahashi-Nakaguchi A, Muraosa Y, Hagiwara D,

stress tolerance in conidia of Aspergillus fumigatus. Fung.

Kamei K, Gonoi T, Takahashi H: Genome sequence４）Hagiwara D, Takahashi H, Watanabe A, Takahashi-from patients with pulmonary aspergilloma and chronic

S: The role of AtfA and HOG MAPK pathway in
Genet. Biol. 73: 138-149, 2014.

Nakaguchi A, Kawamoto S, Kamei K, Gonoi T: Wholegenome comparison of Aspergillus fumigatus strains

Sakai K, Toyotome T, Watanabe A, Kawamoto S,
comparison of Aspergillus fumigatus strains isolated

necrotizing pulmonary aspergillosis. Medical Mycology,
in press.

serially isolated from patients infected with aspergillosis. J.

13）Toh-e A, Ohkusu M, Shimizu K, Kawamoto S:

５）Hagiwara D, Yoshimi A, Sakamoto K, Gomi K, Abe K:

application for identification and cloning of the gene

Clin. Microbiol. 52（12）: 4202-4209, 2014.

“Response and adaptation to cell wall stress and osmotic

stress in Aspergillus species”Stress Biology of Yeasts

Positional cloning in Cryptococcus neoformans and its

encoding methylenetetrahydrofolate reductase. Fung.
Genit. Biol., in press.

and Fungi, Eds. Takagi H and Kitagaki H, Springer, in14）Ueno K, Kinjo Y, Okubo Y, Aki K, Urai M, Kaneko Y,

６）Kawamura I, Kamei K, Yarita K, Ohkusu M, Ito K,

K, Mizuguchi Y, Kawamoto S, Kamei K, Ohno H,

press.

Tsukahara M, Honda M, Nakashima K, Akamatsu H,
Kurai H: Cryptococcus gattii genotype VGIIb infectionin Japan. Med Mycol J, 55（3）: E51-54, 2014.

７）Kopecká M, Yamaguchi M, Kawamoto S: The Effectsof F-Actin Inhibitor Latrunculin A on Pathogenic Yeast

Cryptococcus neoformans. Chemotherapy, in press.

８）Moráňová Z, Virtudazo EV, Pospíšilová K, Ohkusu M,

Shimizu K, Wang D.N, Okawara A, Nara T, Ohkouchi
Niki Y, Shibuya K, Miyazaki Y: Dendritic cell-based
immunization ameliorates pulmonary infection withhighly virulent Cryptococcus gattii. Infect Immun, in press.

15）Yamada H, Yamaguchi M, Chikamatsu, Aono A,

Mitarai S: Structome analysis of virulent Mycobacterium

tuberculosis, which survives with only 700 ribosomes at

density per 0.1 fl cytoplasm. PLoS ONE, in press.

Kawamoto S, Husičková V, Raclavský V: The CRZ1/

16）Yamaguchi M, Shimizu K, Kawamoto S, Stepanova

integrity and biofilm formation in the pathogenic yeast

budding of Cryptococcus albidus yeast cells. Problems in

SP1-like gene links survival under limited aeration, cell

Cryptococcus neoformans. Biomedical Papers 158（ 2）:

212-220, 2014.

９）Shimizu K, Imanishi Y, Toh-e A, Uno J, Chibana H,

Hull CM, Kawamoto S, Functional characterization ofPMT2, encoding a protein-O-mannosyltransferase, in

千葉大学

AA, Vasilyeva NV: Dynamics of cell components during

Medical Mycology 16: 29-35, 2014.

17）Yamaguchi M, Shimizu K, Kawamoto S, Stepanova

AA, Vasilyeva NV: Electronmicroscopic investigation of
the mother cell of the Rhodotorula minuta. Problems in

Medical Mycology 16: 153, 2014.

真菌医学研究センター報告

第 18 巻2014918）Yamaguchi M, Shimizu K, Kawamoto S, Stepanovaand MpkB independently affect micafungin sensitivity incell components during budding in yeast Malassezia21）吹春俊光 , 寶田浩太郎 , 清水公徳 , 糞に生える真菌

AA, Vasiyeva NV: Ultrastructural observation ofpachydermatis. Problems in Medical Mycology 16（4）:13-18, 2014.

19）Yamaguchi M, Worman CO: Deep-sea microorganisms

and the origin of the eukaryotic cell. Jpn J Protozool 47:

29-48, 2014.

20）Yoshimi A, Fujioka T, Mizutani O, Marui J, Hagiwara

D, Abe K: Mitogen-activated protein kinases MpkA

10

千葉大学

Aspergillus nidulans, Biosci Biotechnol Biochem, in press.

類 , 柿嶌

眞・徳増征二編 , 菌類の生物学 — 分類・, pp.263系 統・ 生 体・ 環 境・ 利 用 —（ 共 立 出 版 ）277, 2014.

22）大楠美佐子 , 亀井克彦：クリプトコックス Cryptococcus

neoformans, C. gattii.「微生物検査 イエローページ」
臨床検査 58（11）: 1417-1419, 2014.

真菌医学研究センター報告

第 18 巻2014

知花 PI（カンジダ・グラブラータ フェノーム）プロジェクト
Candida glabrata Phenome Project
研究概要（Summary）
病原性酵母カンジダ・グラブラータの全遺伝子改変株を作製し , 病原性に関する遺伝子の特定と機能解析ならびに抗真菌薬の標的探索を行う .

Using the pathogenic yeast Candida glabrata, we are systematically constructing mutants for gene
identification and functional analyses working on the pathogenisity and for screening of anti-fungal drug
targets.
准

教

授

技

術

職

員

特

任

助

教

技 術 補 佐 員
技 術 補 佐 員

1.

知花 博治
高橋
梓
佐藤美智代
大岩 真理
相田 優子

Associate Professor

Research Technician

Research Assistant Professor

Research Promotion Technician
Research Promotion Technician

Aoyama T, Nakayama H, Ueno K, Inukai T, Tanabe K,

effectively identifies transcrip- tion initiation sites and also aids

Candida glabrata. Genes Cells. 19（6）: 478-503, 2014.

prehensive polling of transcription start sites and an analysis ofToshihiro Aoyama1, Hironobu Nakayama2, Keigo Ueno3, 4,
Tatsuya Inukai2, Koichi Tanabe3, Minoru Nagi3, Martin
5

4

Bard and Hiroji Chibana

in identifying unknown genes. This study describes a comfull-length complementary DNAs derived from the genomeof the pathogenic fungus Candida glabrata. A comparisonof the sequence reads derived from a cDNA library preparedfrom cells grown under different culture conditions against thereference genomic sequence of the Candida Genome Database

Department of Electronic and Information Engineering,（ CGD:http://www.candidagenome.org/ ）revealed theMie 510-0294, Japan

start sites（TSSs）. In addition this analysis also predicted

Medical Science, 3500-3 Minami Tamagaki-cho, Suzuka,

the genome of Saccharomyces cerevisiae. a genetically close

Suzuka National College of Technology, Shiroko, Suzuka,

2

DNA sequencing of the 5́-flanking region of the transcriptome

Nagi M, Bard M, Chibana H: Genome-wide surveyof transcriptional initiation in the pathogenic fungus,

1

Hiroji Chibana
Azusa Takahashi
Michiyo Sato
Mari Ohiwa
Yuko Aida

Faculty of Pharmaceutical Sciences, Suzuka University ofMie 513-8670, Japan

expression of 4,316 genes and their acknowledged transcription

59 new genes including 22 that showed no homology torelative of C. glabrata. Furthermore. comparison of the3

National Institute of Infectious Diseases, 1-23-1 Toyama,

5́-untranslated regions（5́-UTRs）and core promoters of

4

Medical Mycology Research Center, Chiba University, 1-8-

similarities and differences among orthologous genes. Thus,

5

Shinjuku-ku Tokyo 162-8640, Japan

1 Inohana, Chuo-ku, Chiba 260-8673, Japan

Department of Biology, Indiana University-Purdue

University Indianapolis, 723 W, Michigan St, Indianapolis,

IN 46202, USA

千葉大学

C. glabrata to those of S. cerevisiae showed various global

the C. glabrata transcriptome can complement the annotationof the genome database and should provide new insights into
the organization, regulation, and function of genes of thisimportant human pathogen.

真菌医学研究センター報告

第 18 巻201411

2. Costa C, Nunes J, Henriques A, Mira NP, Nakayama H,

was further found to confer resistance to spermine,

antiporter CgTpo3（ORF CAGL0I10384g）: role in azole

the deletion of its Saccharomyces cerevisiae homologue,

Chibana H, Teixeira MC: The Candida glabrata drug:H+

drug resistance and polyamine homeostasis. Journal of

Antimicrobial Chemotherapy. 57（7）: 3159-67, 2014.
1,2

1,2

1,2

Catarina Costa , Joana Nunes , André Henriques , Nuno
1,2

3

4

P. Mira , Hironobu Nakayama , Hiroji Chibana andMiguel C. Teixeira1,2
1

2

Department of Bioengineering, Instituto Superior Técnico,
Universidade de Lisboa, Lisboa, Portugal

IBB–Institute for Biotechnology and Bioengineering,

Biological Sciences Research Group, Centre for Biologicaland Chemical Engineering, Instituto Superior Técnico,

3

4

Lisboa, Portugal

Faculty of Pharmaceutical Sciences, Suzuka University of
Medical Science, Suzuka, Japan

Medical Mycology Research Center（ MMRC ）, Chiba
University, Chiba, Japan

Objectives The ability of opportunistic pathogenic Candida

complementing the susceptibility phenotypes exhibited by

TPO3. In spermine-stressed C. glabrata cells, CgTPO3 istranscriptionally activated in a CgPdr1-dependent manner,contributing to a decrease in the intracellular concentration ofthis polyamine. Clotrimazole exposure was found to lead tothe intracellular accumulation of spermine, and pre-exposure
to this polyamine was found consistently to lead to increased

clotrimazole resistance.

Conclusions Altogether, these results point to a significant

role for CgTpo3 in azole drug resistance and in the tolerance

to high polyamine concentrations, such as those found in theurogenital tract.
Publications

１）Aoyama T, Nakayama H, Ueno K, Inukai T, Tanabe K,

Nagi M, Bard M, Chibana H: Genome-wide surveyof transcriptional initiation in the pathogenic fungus,
Candida glabrata. Genes Cells. 19（6）: 478-503, 2014.

２）Costa C, Nunes J, Henriques A, Mira NP, Nakayama

H, Chibana H, Teixeira MC: The Candida glabrataspecies to persist and invade specific niches in the human host

drug:H+ antiporter CgTpo 3（ORF CAGL0I10384g）:

antifungal therapy. This work describes the role of the Candida

Journal of Antimicrobial Chemotherapy. 57（7）: 3159-

depends on their resistance to natural growth inhibitors andglabrata drug:H+ antiporter CgTpo3（ORF CAGL0I10384g）
in this context.

Methods Deletion and cloning of CgTPO3 was achieved

using molecular biology tools. C. glabrata strain susceptibility

role in azole drug resistance and polyamine homeostasis.

67, 2014.

３）Shimizu K, Imanishi Y, Toh-e A, Uno J, Chibana H,

Hull CM, Kawamoto S: Functional characterization ofPMT2, encoding a protein-O-mannosyltransferase, in

was assayed based on growth in liquid and solid media andthe human pathogen Cryptococcus neoformans. Fungal

accumulation or HPLC were used for the assessment of４）Yamaguchi S, Sano A, Hiruma M, Murata M, Kaneshima

through MIC determination. Radiolabelled compound
the role of CgTpo 3 as a drug or polyamine transporter.

Quantitative RT–PCR was used for expression analysis.

Results CgTpo 3 was found to confer resistance to azole

drugs in C. glabrata. This protein was found to be localized

to the plasma membrane and to decrease the intracellularaccumulation of［3H］clotrimazole, playing a direct rolein its extrusion from pre-loaded C. glabrata cells. CgTPO3

12

千葉大学

Genet Biol. 69: 13-22, 2014.

T, Murata Y, Takahashi H, Takahashi S, Takahashi Y,

Chibana H, Touyama H, Ha NT, Nakazato Y, Uehara

Y, Hirakawa M, Imura Y, Terashima Y, Kawamoto
Y, Takahashi K, Sugiyama K, Hiruma M, Murakami

M, Hosokawa A, Uezato H: Isolation of dermatophytes

and related species from domestic fowl（Gallus gallus
domesticus）. Mycopathologia. 178（1-2）: 135-43, 2014.

真菌医学研究センター報告

第 18 巻2014

米山 PI（感染応答）プロジェクト
Project for Immune Response in Infections Diseases
研究概要（Summary）
感染に対する我々の生体防御は , 自然免疫と獲得免疫によって協調して行われている . 本プロジェク
トでは , 特にウイルス感染に応答した自然免疫誘導に注目し , 感染センサー分子によるウイルス由来の非自己核酸検知の分子機構の解明と , それによって引き起こされる免疫応答の生理機能を解析すること
により , ウイルス感染症に対する新たな治療戦略の開発を目指した解析を行っている .

Innate immune system plays an essential role for self-defense against infection of a variety of pathogens.
In this project, we focus on antiviral innate immunity, especially molecular machinery for detection ofviral infection and subsequent immune responses. The observations obtained from this study will help us toestablish a novel therapeutic or preventive strategy against infectious diseases by viruses.
教

授

助

教

特 任 研 究 員
非常勤技術職員
技 術 補 佐 員
技 術 補 佐 員

米山 光俊
尾野本浩司
平井 玲子
滝澤香代子
常喜 儒彦
滝沢みゆき

Professor

Assistant Professor
Project Researcher

Adjunct Research Technician

Research Promotion Technician

Research Promotion Technician

1 . Identification RNA binding proteins（RBPs）, which

are responsible for the formation of antiviral stressgranules（avSGs）.

Mitsutoshi Yoneyama
Koji Onomoto
Reiko Hirai
Kayoko Takizawa
Michihiko Jogi
Miyuki Takizawa

２ . A novel function of human Pumilio proteins incytoplasmic sensing of viral infection.

Ryo Narita1, Kiyohiro Takahasi1,2, Etsu Murakami1, Emi
Hirano1, Seiji P. Yamamoto1, Mitsutoshi Yoneyama3, Hiroki

Koji Onomoto, and Mitsutoshi Yoneyama
We demonstrated that viral infection including influenza

Kato1,4, Takashi Fujita1,4

A virus（IAV）induces RIG-I to accumulate in cytoplasmic

1（avSG（）Onomoto et al., PLoS One, 2012）. Furthermore, we

2granular-like structure, which we termed antiviral stress granulerevealed that avSG plays a critical role as platform for initiationof RIG-I-mediated antiviral signaling. To understand how

avSG is formed in response to viral infection and responsible formolecule（s）, which is responsible for avSG formation. We

4partially purified avSG and analyzed its components by mass

Research, Kyoto University, Kyoto, Japan

Institute for Innovative NanoBio Drug Discovery andDevelopment, Graduate School of Pharmaceutical Science,3

anti-viral signal activation, we are trying to identify regulatory

Laboratory of Molecular Genetics, Institute for Virus

Kyoto University, Kyoto, Japan

Division of Molecular Immunology, Medical Mycology

Research Center, Chiba University, Chiba, Japan

Laboratory of Molecular Cell Biology, Graduate School of

Biostudies, Kyoto University, Kyoto, Japan

spectrometry. As a result, we identified several RBPs in virus-

Harvard Medical School, United States of America

the RBPs in antiviral innate immunity.

RIG-I-like receptor（ RLR ）plays a pivotal role in the

induced samples. We are analyzing functional significance of

千葉大学

真菌医学研究センター報告

第 18 巻201413

detection of invading pathogens to initiate type I interferon（IFN）gene transcription. Since aberrant IFN productionis harmful, RLR signaling is strictly regulated. However,
the regulatory mechanisms are not fully understood. By

expression cloning, we identified Pumilio proteins, PUM1
and PUM 2 , as candidate positive regulators of RIG-I

signaling. Overexpression of Pumilio proteins and theirknockdown augmented and diminished IFN-β promoter

４ . DHX 36 enhances RIG-I signaling by facilitating

PKR-mediated antiviral stress granule formation.

Ji-Seung Yoo1,2, Kiyohiro Takahasi1,3, Chen Seng Ng1,2, Ryota

Ouda1,2, Koji Onomoto4, Mitsutoshi Yoneyama 4, Janice

Ching Lai5, Simon Lattmann5, Yoshikuni Nagamine5, Tadashi
Matsui6, Kuniyoshi Iwabuchi6, Hiroki Kato1,2, Takashi Fujita1,2

activity induced by Newcastle disease virus（ NDV ）,1

LGP2, but not with RIG-I or MDA5. Furthermore, all of2stress granules. Interestingly, biochemical analyses revealed3

respectively. Both proteins showed a specific association withthese components were recruited to NDV-induced antiviralthat Pumilio increased double-stranded（ds）RNA binding

affinity of LGP 2 ; however, Pumilio was absent in thedsRNA-LGP2 complex, suggesting that Pumilio facilitatesfunction. Collectively, our results demonstrate an unknown

5function of Pumilio in viral recognition by LGP2.

３. Molecular mechanism for detection of viralribonucleoprotein complex by RLRs.

Reiko Hirai, Michihiko Jogi and Mitsutoshi Yoneyama
It has remained unc lear how RIG-I detects viral

Research, Kyoto University, Kyoto, Japan

Laboratory of Molecular Cell Biology, Graduate School of

Biostudies, Kyoto University, Kyoto, Japan

Institute for Innovative NanoBio Drug Discovery andDevelopment, Graduate School of Pharmaceutical Science,4

viral RNA recognition by LGP 2 through its chaperon-likeLaboratory of Molecular Genetics, Institute for Virus

6

Kyoto University, Kyoto, Japan

Division of Molecular Immunology, Medical Mycology
Research Center, Chiba University, Chuo-ku, Chiba, JapanFriedrich Miescher Institute for Biomedical Research, Basel,
Switzerland

Department of Biochemistry I, School of Medicine,

Kanazawa Medical University, Uchinada, Ishikawa, Japan

RIG-I is a DExD/H-box RNA helicase and functions

as a critical cytoplasmic sensor for RNA viruses to initiate

antiviral interferon（IFN）responses. Here we demonstratethat another DExD/H-box RNA helicase DHX36 is a key

ribonucleoprotein complex（RNP）, which consists of viralmolecule for RIG-I signaling by regulating double-stranded（NP）. Recently, we established in vitro reconstitution assay

which has been shown to be essential for the formation ofviral RNP could activate RIG-I in vitro. As a model RNP,

PKR form a complex in a dsRNA-dependent manner. By

result, we have successfully detected RIG-I activation by viral

phosphorylation of PKR through its ATPase/helicase activity.

genomic RNA and viral proteins such as nucleocapcid protein
system for RIG-I-mediated signaling and examined whether

we prepared artificial IAV RNP generated in 293T cells. As a
RNP in vitro. Now, we are trying to visualize interaction

between RIG-I and viral RNP in vitro using Atomic force
microscope（AFM）.

RNA（dsRNA）-dependent protein kinase（PKR）activation,

antiviral stress granule（avSG）. We found that DHX36 andforming this complex, DHX36 facilitates dsRNA binding andUsing DHX36 KO-inducible MEF cells, we demonstrated

that DHX36 deficient cells showed defect in IFN production
and higher susceptibility in RNA virus infection, indicating

the physiological importance of this complex in host defense.

In summary, we identify a novel function of DHX36 as a

critical regulator of PKR-dependent avSG to facilitate viral
RNA recognition by RIG-I-like receptor（RLR）.

14

千葉大学

真菌医学研究センター報告

第 18 巻2014

Publications

hora M, Akashi K, and Yamasaki S: Dectin-2 Is a Direct

１）Narita R, Takahasi K, Murakami E, Hirano E, Yamamoto

Receptor for Mannose-Capped Lipoarabinomannan ofof human Pumilio proteins in cytoplasmic sensing of viral４）Yoo JS, Takahasi K, Ng CS, Ouda R, Onomoto２）Onomoto K, Yoneyama M, Fung G, Kato H, Fujita T:

Y, Matsui T, Iwabuchi K, Kato H, Fujita T: DHX36

SP, Yoneyama M, Kato H, Fujita T: A novel function
infection. PLoS Pathog 10: e1004417, 2014.

Antiviral innate immunity and stress granule responses.
Trends Immunol 35: 420-428, 2014.

３）Yonekawa A, Saijo S, Hoshino Y, Miyake Y, Ishikawa E,

Suzukawa M, Inoue H, Tanaka M, Yoneyama M, Oh-

千葉大学

Mycobacteria. Immunity 41: 402-413, 2014.

K, Yoneyama M, Lai JC, Lattmann S, Nagamine

Enhances RIG-I Signaling by Facilitating PKRMediated Antiviral Stress Granule Formation. PLoS
Pathog 10: e1004012, 2014.

真菌医学研究センター報告

第 18 巻201415

西城 PI（サイトカイン）プロジェクト
Project for Cytokine Research
研究概要（Summary）
生体は , 多種多様な細胞や組織が互いに時空的に作用することにより恒常性が維持される一つシス
テムであり , その維持においてサイトカインは中心的な役割を担っている . 多くの疾病は単に一つの臓器 , 組織の異常ではなく , 免疫系を始めとする種々のシステムの異常であることから , これらを統合するサイトカインの役割を知ることは非常に重要である . 本プロジェクトでは , 感染性疾患や炎症性疾患の病態形成におけるサイトカインの役割を解明し , 最終的に新たな治療薬の標的分子を見出すことを目的とする .

Cytokines play a central role in maintenance of homeostasis. Because, a disease is not caused by only one
problem of an organ, but caused by a systemic disorder, which is regulated by cytokines, it is important tostudy their functions. We aim to find new therapeutic targets for inflammatory diseases and infectious diseases
by investigating the roles of cytokines in pathogenesis.
特 任 准 教 授
特

任

助

教

技 術 補 佐 員
技 術 補 佐 員
技 術 補 佐 員

西城
矢部
森本
妹尾
鈴木

忍
力朗
雅子
彬正
智明

Associate Professor

Research Assistant Professor
Technical AssistantResearch Promotion Technician

Research Promotion Technician

1 . Dectin-1 and Dectin-2 in innate immunity against

fungal infection.

Shinobu Saijo
Rikio Yabe
Masako Morimoto
Akimasa Seno
Tomoaki Suzuki

to the ITAM and activates the caspase recruitment domain

family member 9（CARD9）–nuclear factor-κB axis, resulting

in the activation of various genes including those encoding proinflammatory cytokines. Both β-glucans and α-mannans are

Shinobu Saijo, Rikio Yabe and Akimasa Seno

major cell wall components of fungi including Candida albicans

Division of Molecular Immunology, Medical Mycology（C. albicans）and Pneumocystis carinii（P. carinii）. Recently, it

Dectin-1 and Dectin-2 are type Ⅱ transmembrane proteins

Dectin-1 and Dectin-2 play important roles in defense against

domains（ CRDs ）in their extracellular region. They are

In this review, we briefly revisit the structures, ligands, signal

Research Center, Chiba University, Chiba 260-8673, Japanof the C-type lectin family with single carbohydrate recognition
expressed mainly in dendritic cells and macrophages. Dectin-1

recognizes β-glucans with its CRD and transduces signals

through its immunoreceptor tyrosine-based activation motif

was reported that Dectin-1 is important in protection against

P. carinii by inducing reactive oxygen species, whereas bothC. albicans by preferentially inducing Th17 cell differentiation.

transduction and functional roles of Dectin-1 and Dectin-2 inhost defense against fungal infection.

（ ITAM ）-like motif in the cytoplasmic domain, whereas

Dectin-2 recognizes α -mannans and transduces its signal

through association with the ITAM-containing Fc receptor γ
chain. Upon ligand binding, spleen tyrosine kinase is recruited

16

千葉大学

真菌医学研究センター報告

第 18 巻2014

２ . C-Type Lectin Receptors in Host Defense Against

Microbial Pathogens.
1

2

2

Th17 cells by downregulating TGF-β-induced Foxp3

expression.

1

Rikio Yabe , Yoichiro Iwakura , Shinobu Saijo
1

３ . Excess IL-1 signaling enhances the development of

Ikeda S1, Saijo S2, Murayama MA3, Shimizu K3, Akitsu A3,

Division of Molecular Immunology, Medical Mycology

Iwakura Y3

Center for Animal Disease Models, Research Institute for

1

Research Center, Chiba University, Chiba 260-8673, Japan

Biological Sciences, Tokyo University of Science, Chiba
278-0022, Japan

C-type lectin receptors（CLRs）are a group of patternrecognition receptors（PRRs）that recognize carbohydrate
structures in microbes, including fungi and bacteria, as

pathogen-associated molecular patterns（PAMPs）. They are

expressed mainly in dendritic cells（DCs）and macrophages,

and among these CLRs, DC-associated C-type lectin-1

Center for Experimental Medicine and Systems Biology,
The Institute of Medical Science, The University of Tokyo,

Tokyo 108-8639, Japan

2

Division of Molecular Immunology, Medical Mycology

3

Center for Animal Disease Models, Research Institute for

Research Center, Chiba University, Chiba 260-8673, Japan

Biological Sciences, Tokyo University of Science, Chiba
278-0022, JapanIL- 1 R antagonist-deficient（ Il 1 rn -/- ）mice develop（ Dectin- 1）, DC-associated C-type lectin- 2（ Dectin- 2）,

autoimmune arthritis in which IL- 17 A plays a crucial

macrophage C-type lectin（Mcl）transduce their signalingdifferentiation is dependent on TGF-β and IL-6, we foundmacrophage-inducible C-type lectin（ Mincle ）, andthrough phosphorylation of spleen tyrosine kinase（Syk）.

role. Although many studies have shown that Th 17 cellthat Th 17 cells developed normally in Il 1 rn -/- Il 6 -/- miceOn the other hand, human DC-specific intracellularin vivo. Then, we analyzed the mechanisms of Th17 celland its mouse homologue SIGN-related gene 3（SIGNR3）,

production was increased in the lymph nodes of Il1rn-/- mice,adhesion molecule 3-grabbing non-integrin（DC-SIGN）

differentiation in Il 1rn-/-Il6-/- mice. We found that IL- 21

members of the DC-SIGN superfamily of CLRs, transduce

naive Il6-/- CD4+ T cells differentiated into Th17 cells when

In addition to pathogen recognition, Mincle, DC-SIGN,

greatly enhanced when IL-1 was added to the culture. Th17

their signaling through intracellular tyrosine-containing motif.

cultured with TGF-β and IL-21, and the differentiation wasand SIGNR3 have been shown to recognize molecules fromcell differentiation was not induced by either TGF-β or IL-1of the body. Here, we review each of these receptors in detailin naive CD4+ T cells, and IL-1 inhibited TGF-β-induced

self, suggesting pleiotropic roles of CLRs in the homeostasis
describing their expression, ligand recognition, signaling,

and associated human diseases.

alone or in combination. IL-21 induced IL-1R expression
Foxp3 expression, resulting in the promotion of Th17 celldifferentiation. Furthermore, IL-1 augmented the expressionof Th17 cell-specific transcription factors such as Nfkbiz and

Batf. These results indicate that excess IL-1 signaling canovercome the requirement of IL-6 in the differentiation of

Th17 cells by suppressing Foxp3 expression and inducing Th17
cell-specific transcription factors.

千葉大学

真菌医学研究センター報告

第 18 巻201417

４ . Rag2-deficient IL-1 Receptor Antagonist-deficient

Mice Are a Novel Colitis Model in Which Innate
Lymphoid Cell-derived IL- 17 Is Involved in the

Pathogenesis.

278-0022, Japan

2

Tokyo, 113-8657

3

Research Center, Chiba University, Chiba 260-8673, Japan4

Il1rn-/- mice spontaneously develop arthritis and aortitis by

an autoimmune mechanism and also develop dermatitis by anautoinflammatory mechanism. Here, we show that Rag2-/-/-

Il1rn mice develop spontaneous colitis with high mortality,

278-0022, Japan

Department of Biomedical Science, Graduate School of

Agricultural and Life Sciences, The University of Tokyo,

Department of Biomedical Science, Graduate School of

Division of Molecular Immunology, Medical Mycology

Center for Animal Disease Models, Research Institute for

Biological Sciences, Tokyo University of Science, Chiba

Center for Animal Disease Models, Research Institute for

Agricultural and Life Sciences, The University of Tokyo,

3

Murayama MA1, Kakuta S2, Maruhashi T1, Shimizu K1,

1

Biological Sciences, Tokyo University of Science, Chiba

2collagen-induced arthritis in mice.

Seno A1, Kubo S1, Sato N1, Saijo S3, Hattori M4, Iwakura Y1

Akitsu A 1, Kakuta S 2, Saijo S 3, Iwakura Y 1
1

５ . CTRP3 plays an important role in the development ofTokyo, 113-8657

Division of Molecular Immunology, Medical Mycology

Research Center, Chiba University, Chiba 260-8673, Japan

Department of Computational Biology, Graduate Schoolof Frontier Sciences, The University of Tokyo, Chiba 2770882, Japan

Rheumatoid arthritis（RA）is an autoimmune inflammatory

disease exhibited most commonly in joints. We found thatmaking a contrast to the suppression of arthritis in these mice.

the expression of C1qtnf3, which encodes C1q / TNF-related（ILC3s）was observed in the colon of Rag2 Il1rn mice. IL-

models with different etiology. To elucidate the pathogenic

Enhanced IL-17A expression in group 3 innate lymphoid cells
-/-

-/-

17A-deficiency prolonged the survival of Rag2 Il1rn

protein 3（CTRP3）, was highly increased in two mouse RA

mice,roles of CTRP3 in the development of arthritis, we generated

of intestinal inflammation. Although IL-17A-producing T

induced arthritis in these mice. We found that the incidence

-/-

-/-

suggesting a pathogenic role of this cytokine in the development-/-

C1qtnf 3 -/- mice and examined the development of collagen-cells were increased in Il1rn mice, these mice did not develop

and severity score was higher in C1qtnf 3 -/- mice comparedalso expanded. Thus, excess IL-1 signaling and IL-1-induced

also more severe in C1qtnf 3 -/- mice. The levels of antibodies

colitis, because CD4 Foxp3 regulatory T cell population was++

IL-17A from ILC3s cause colitis in Rag2-/-Il1rn-/- mice inwith wild-type（WT）mice. Histopathology of the joints wasagainst type II collagen and pro-inflammatory cytokine

which Treg cells are absent. These observations suggest thatmRNAs in C 1 qtnf 3 -/- mice were higher than WT mice.

important to keep the immune homeostasis of the colon.

role in the development of autoimmune arthritis, suggesting

the balance between IL-17A-producing cells and Treg cells is

18

千葉大学

These observations indicate that CTRP3 plays an important
CTRP3 as a possible medicine to treat RA.

真菌医学研究センター報告

第 18 巻2014

Publications

１）Akitsu A, Kakuta S, Saijo S, Iwakura Y: Rag2-deficient７）Nakamura Y, Sato K, Yamamoto H, Matsumura

K, Matsumoto I, Nomura T, Miyasaka T, Ishii K,

IL-1 receptor antagonist-deficient mice are a novel colitis

Kanno R, Tachi M, Yamasaki S, Saijo S, Iwakura

involved in the pathogenesis. Exp Anim 63: 235-46,

response and mucin production in the lungs aftermodel in which innate lymphoid cell-derived IL-17 Is
2014.

２） Bertuzzi M Schrettl M, Alcazar-Fuoli L, Cairns

Y, Kawakami K: Dectin- 2 deficiency promotes Th 2

pulmonary Infection with Cryptpcoccus neoformans. InfectImmun, in press.

TC, Muñoz A, Walker LA, Herbst S, Safari M,８）Norimoto A, Hirose K, Iwata A, Tamachi T, Yokota

Armstrong-James D, Munro CA, Read ND, Filler SG,

Dectin- 2 promotes house dust mite-induced Th2 andCheverton AM, Chen D, Liu H, Saijo S, Fedorova ND,
Espeso EA, Nierman WC, Haas H, Bignell EM: The

pH-responsive PacC transcription factor of aspergillus

fumigatus governs epithelial entry and tissue invasion

during pulmonary aspergillosis. PLoS Pathog. 10:e1004413, 2014.

M, Takahashi K, Saijo S, Iwakura Y, Nakajima H:
Th17 cell differentiation and allergic airway inflammationin mice. Am J Respir Cell Mol Biol. 51: 201-9, 2014.

９）Ohman T, Teirilä L, Lahesmaa-Korpinen AM, Cypryk

W, Veckman V, Saijo S, Wolff H, Hautaniemi S, Nyman

TA, Matikainen S: Dectin-1 pathway activates robust３）Chiba S, Ikushima H, Ueki H, Yanai H, Kimura Y,

autophagy-dependent unconventional protein secretion in

Iwakura Y, Taniguchi T: Recognition of tumor cells by10）Parsons MW, Li L, Wallace AM, Lee MJ, Katz HR,

Hangai S, Nishio J, Negishi H, Tamura T, Saijo S,
Dectin-1 orchestrates innate immune cells for anti-tumor
responses. eLife. 22: e04177, 2014.

４）Ikeda S, Saijo S, Murayama MA, Shimizu K, Akitsu

A, Iwakura Y: Excess IL- 1 signaling enhances thedevelopment of Th17 cells by downregulating TGF-β-induced Foxp3 expression. J Immunol 192: 1449-58,
2014.

５）Kobiyama K, Aoshi T, Narita H, Kuroda E, Hayashi

human macrophages. J Immunol 192: 5952-62, 2014.

Fernandez JM, Saijo S, Iwakura Y, Austen KF, Kanaoka

Y, Barrett NA: Dectin-2 regulates the effector phaseof house dust mite-elicited pulmonary inflammation

independently from its role in sensitization. J Immunol

192: 1361-71, 2014.

11） Yabe R, Iwakura Y, Saijo S: Host defense by C-type

lectin receptors against microbial pathogens. Glycoscience:

Biology and Medicine, 1319-1329, 2014.

M, Tetsutani K, Koyama S, Mochizuki S, Sakurai

12）Yonekawa A, Saijo S, Hoshino Y, Miyake Y, Ishikawa

S, Coban C, Ishii KJ: Nonagonistic Dectin-1 ligand

Oh-Hora M, Akashi K, Yamasaki S: Dectin-2 is a

K, Katakai Y, Yasutomi Y, Saijo S, Iwakura Y, Akira

E, Suzukawa M, Inoue H, Tanaka M, Yoneyama M,

transforms CpG into a multitask nanoparticulate TLR9

direct receptor for mannose-capped lipoarabinomannan

６）Murayama MA, Kakuta S, Maruhashi T, Shimizu K,13） Wang H, LeBert V, Hung CY, Galles K, Saijo S, Lin

Y: CTRP3 plays an important role in the development

receptors differentially induce th17 cells and vaccine

Res Commun 443: 42-8, 2014.

Immunol 192: 1107-19, 2014.

agonist. Proc Natl Acad Sci U S A 111: 3086-91, 2014.

Seno A, Kubo S, Sato N, Saijo S, Hattori M, Iwakuraof collagen-induced arthritis in mice. Biochem Biophys

千葉大学

of Mycobacteria. Immunity. 41: 402-13, 2014.

X, Cole GT, Klein BS, Wüthrich M: C-type lectin

immunity to the endemic mycosis of North America. J

真菌医学研究センター報告

第 18 巻201419

亀井 PI（臨床感染症）
プロジェクト
Project to Link Basic Sciences and Clinical Medicine
研究概要（Summary）
アスペルギルス症を中心とする難治性糸状菌感染症 , 輸入真菌症を主要な研究対象とし , 感染機構 ,新しい診断や治療法の開発を中心に研究を行っている . 並行して行っている診療活動では , 10月から正式に開始した附属病院での真菌症専門外来に加え , 学内外でのコンサルテーション活動を行なっており , 学外からの依頼は検査を含めて年間300件以上に達している . スタッフ構成では４月からこれまでの助教１名（田口）の退官に伴い准教授１名（渡辺）が着任した .
実施体制：教員２名 , 技官１名（以上常勤）, 特任助教２名 , 補助員２名およびグランドフェロー１名
で研究及び大学院生４名（博士課程）の教育指導を行なっている .

ACTIVITES: Our research focuses on the development of diagnostic / therapeutic methods for intractable
fungal diseases such as aspergillosis through an investigation into the mechanism of infection. We also takecare of patients in the speciality clinic（Fungal Disease Clinic）at the University Hospital, while providing
more than 300 consulting services on fungal diseases to physicians/clinical technologists all over the country.
STAFF: Professors（2）, technician（1）, research assistant professors（2）and research assistants（2）, grandfellow（1）are working in our group with four graduate school students.
教

授

亀井
渡辺

克彦
哲

Professor

Associate Professor

Katsuhiko Kamei
Akira Watanabe

教

田口

英昭

Assistant Professor

Hideaki Taguchi

村長
八尋
田口

保憲
真希
英昭

Research Assistant Professor

Yasunori Muraosa
Maki Yahiro
Hideaki Taguchi

鎗田
関
井上

響子
里亜
京子

Research Technician

授

准

教

（４月から着任）
助（３月で退任）

特

任

助

教

特

任

助

教

グランドフェロー

（４月から着任）
技

術

職

員

技 術 補 佐 員
技 術 補 佐 員

20

Research Assistant Professor
Grand Fellow

Research Promotion Technician
Research Promotion Technician

千葉大学

真菌医学研究センター報告

Kyoko Yarita
Rio Seki
Kyoko Inoue

第 18 巻2014

1 . Isolation and Drug Susceptibility of Candida parapsilosis

patients with invasive candidiasis since there are differences infrom Patients with Blood Stream Infections andtargeting the topoisomerase II gene based on loop-mediatedSensu Lato and other Species of C. parapsilosis Complex

Proposal of a Novel LAMP Identification Method for
the Species.
121

Plinio Trabasso , Tetsuhiro Matsuzawa , Renata Fagnani ,3

3

Yasunori Muraosa , Kenichiro Tominaga , Ribeiro Resende

Mariangela1, Katsuhiko Kamei3, Yuzuru Mikami3, Zaninelli

virulence, pathogenicity and drug susceptibility. A method

isothermal amplification（LAMP）was developed. LAMP

emerges as a promising tool for the identification of fungal

species due to the high sensitivity and specificity. LAMP canbe performed at the point-of-care, being no necessary theuse of expensive equipment. In our study, the method was

successful comparing to the DNA sequencing and proved to be

Schreiber Angelica4, Luiza Moretti Maria1

a reliable and fast assay to distinguish the three species of CPC.

1

２. Effect of Serum Components on Biofilm Formation by

Infectious Diseases Division, School of Medical Sciences,

University of Campinas, Rua Tessa´lia Vieira de Carvalho,

126 Cidade Universita´ria Zeferino Vaz, Campinas, Sao
2

3

4

Paulo 13083-887, Brazil

University of Nagasaki, 1-1-1 Manabino, Nagayo-cho,Nishi-Sonogi-gun, Nagasaki 851-2195, Japan

Medical Mycology Research Center, Chiba University, 1-81, Inohana, Chuoku 260-8673, Japan

Aspergillus fumigatus and Other Aspergillus Species.

Wuren Tuya1, Toyotome Takahito1,2, Yamaguchi Masashi3,

Takahashi-Nakaguchi Azusa4, Muraosa Yasunori1, Yahiro

Maki1, Wang Dan-Ni1, Watanabe Akira1,5, Taguchi Hideaki1,
Kamei Katsuhiko1,5

Department of Clinical Pathology, School of Medical

1de Carvalho, 126 Cidade Universita´ ria Zeferino Vaz,

2

Candida parapsilosis complex（CPC）is the third Candida3

Hospital, following C. albicans and C. tropicalis. From 2006

4

year. Records of 36 patients were reviewed. CPC were 31

5

Sciences, University of Campinas, Rua Tessa´ lia Vieira
Campinas, Sao Paulo 13083-887, Brazil

species isolated in blood cultures of patients from ourto 2010, the median annual distribution of CPC was 8 cases/（86.1 %）C. parapsilosis; 4（11.1 %）C. orthopsilosis; and 1（2.8 %）C. metapsilosis. Clinical characteristics were central

venous catheter, 34（94.4 %）; parental nutrition, 25（70 %）;Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University

Research Unit for Risk Analysis, Research Center for
Animal Hygiene and Food Safety, Obihiro University ofAgriculture and Veterinary Medicine

Division of Molecular Biology, Medical Mycology ResearchCenter, Chiba University

Division of Bio-resources, Medical Mycology ResearchCenter, Chiba University

Division of Control and Treatment of Infectious Diseases,
Chiba University Hospital

Biofilm production by microorganisms is critical forsurgery, 27（57 . 9 % ）; prior bacteremia, 20（51 . 3 % ）;their pathogenicity. Serum promotes biofilm production by

was higher in immunosuppressed patients（17 vs. 11; p ＝spp. have not been reported. We analyzed biofilm formationand 14 out 31（45.2 %）with C. parapsilosis died（p ＝0.558）.

nidulans, A. niger, and A. terreus, and examined the effects

to seven antifungal drugs, with MICs values showing

fetuin A, and bovine serum albumin（ BSA ）on hyphalmalignancy, 18（50 %）. General mortality was 47.2 %. Death. Three out four（75 %）patients with C. orthopsilosis
0.003）
Thirty-nine individual isolates were tested for susceptibility

susceptibility to all of them. Two isolates, one C. orthopsilosis

and one C. parapsilosis, had fluconazole MIC ＝4μg/mL.
Differentiation among CPC has implication in caring for

千葉大学

Aspergillus fumigatus; however, its effects on other Aspergillus

by five Aspergillus spp., i.e., A. fumigatus, A. flavus, A.

of serum/serum proteins such as fetal bovine serum（FBS）,
growth, hyphal branching, and extracellular matrix（ECM）

formation. The antifungal susceptibility of A. fumigatus

isolates that formed biofilms was also examined. All serum/

真菌医学研究センター報告

第 18 巻2014

21

serum proteins promoted the growth of all these fungal

OBJECTIVE:

followed by fetuin A and BSA. This effect was most evident

S. commune to establish a reliable serodiagnostic method.

species; growth promotion was most evident with FBS,

in case of A. fumigatus and least evident in case of A. terreus.

Electron microscopy showed thick ECM layers surrounding

fungal cell walls after culture with FBS, particularly in A.

fumigatus. An increase in hyphal branching caused by fetuin

A was the highest in case of A. fumigatus and A. nidulans.

In this study, we attempted to identify a major antigen of

METHODS:

We used mass spectrometr y to identify an antigen

that reacted with the serum of a patient with allergic
bronchopulmonary mycosis caused by S. commune. The

protein was expressed in Escherichia coli, highly purified, andBiofilm-forming A. fumigatus showed resistance to mostthe patient sera IgG and IgE titres against the protein were

amphotericin B was suggested. Our results indicate that

RESULTS:many Aspergillus spp., particularly A. fumigatus, and that thisnamed Sch c 1; it was a homolog of glucoamylase. The IgG

antifungal agents, although a synergism of micafungin andserum promotes biofilm formation, including thick ECM, bymay be closely related to its virulence.

３. Glucoamylase is a major allergen of Schizophyllum

commune.

1,2

3

1

Toyotome Takahito , Satoh Mamoru , Yahiro Maki ,
1,4

3,5

1,4

determined by enzyme-linked immunosorbent assay.

The protein identified as a major antigen of S. commune was

and IgE titres against Sch c 1 in patient sera were significantly

higher than those in healthy volunteer sera（P ＜0.01）.

CONCLUSIONS AND CLINICAL RELEVANCE:

Sch c 1 is recognized by the host immune system ofpatients as an antigen/allergen. The purified glucoamylase Sch
c 1 is a promising candidate antigen for the serodiagnosis of

Watanabe Akira , Nomura Fumio , Kamei Katsuhiko

S. commune-induced mycosis.

1

４. Antifungal susceptibility of Aspergillus fumigatus

2

3

4

5

Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University, Chiba, Japan

Research Center for Animal Hygiene and Food Safety,

clinical isolates collected from various areas in Japan.

Obihiro University of Agriculture and Veterinary Medicine

Kikuchi Kazuyo1, Watanabe Akira2, Ito Junko1, Oku Yukio1,

Hospital

Yasunori1, Yahiro Maki1, Yaguchi Takashi1, Kamei Katsuhiko2

Clinical Proteomics Research Center, Chiba University
Division of Control and Treatment of Infectious Diseases,

Wuren Tuya1, Taguchi Hideaki1, Yarita Kyoko1, Muraosa

Chiba University Hospital

1

Medicine, Chiba University

2

Department of Molecular Diagnosis, Graduate School of

Medical Mycology Research Center, Chiba University,
Japan

Medical Mycology Research Center, Chiba University,
Japan; Division of Control and Treatment of Infectious

BACKGROUND:

Diseases, Chiba University Hospital, Japanof basidiomycosis including disorders such as allergic

Azole resistance among clinical isolates of Aspergillus

Schizophyllum commune is one of the causative agents

bronchopulmonary mycosis, allergic fungal sinusitis, andfumigatus is becoming a serious problem in Europe, but the

mucoid impaction of bronchi, the incidence of those of which

status in Japan is not yet known in detail. The aim of thisbecause only a limited number of diagnostic tools are currently

in A. fumigatus in Japan. We employed 171 clinical isolates

has been increasing. These mycoses are difficult to diagnose

study was to determine the present status of azole resistanceavailable. The biggest problem is that no specific antigens ofof A. fumigatus sensu stricto collected from 1987 to 2008 at

disease.

Japan for azole resistance determination. Identification of all

S. commune have been identified to enable serodiagnosis of the

22

千葉大学

the Medical Mycology Research Center, Chiba University,

真菌医学研究センター報告

第 18 巻2014

isolates were re-examined both from the aspect of morphology

and molecular phylogeny. The antifungal susceptibility ofthese isolates was tested based on the CLSI M38-A2 brothmicrodilution method. In our collection, only 1（0.6%）

demonstrated high sensitivity, enabling detection of one copyof 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

and 2 isolates（1.2%）showed elevated MIC to voriconazole

germinated conidia spiked in 3mL of human blood. To apply

frequency of azole resistance in A. fumigatus still remains low

of fusariosis, we evaluated its efficacy using a mouse modeland itraconazole, respectively. Our study disclosed that thein this collection.

5 . Development of cycling probe-based real-time PCRsystem to detect Fusarium species and species complex（FSSC）.

1

2

Muraosa Yasunori , Schreiber Angelica Zaninele , Trabasso
3

4

4

4

4

Plinio , Matsuzawa Tetsuhiro , Taguchi Hideaki , Moretti
3

our new real-time PCR system to the molecular diagnosis

of invasive F. solani infection. Plasma and whole blood

samples of infected mice were tested using the real-time PCRsystem. The sensitivity of the real-time PCR system wasfound 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 disseminatedMaria Luiza , Mikami Yuzuru , Kamei Katsuhikofusariosis.

1

６. Opportunistic infection in patients with acute liver

2

3

4

Medical Mycology Research Center, Chiba University,
Chiba, Japan

Department of Clinical Pathology, Faculty of Medical

failure.

Sciences, State University of Campinas, São Paulo, Brazil

Makoto Arai1, Tatsuo Kanda1, Shin Yasui1, Keiichi Fujiwara1,

Medicine, Faculty of Medical Sciences, State University of

Oda3, Osamu Yokosuka1

Medical Mycology Research Center, Chiba University,

1

Infectious Diseases Division, Department of Internal
Campinas, São Paulo, Brazil
Chiba, Japan

In the present study, we developed a new real-time PCRsystem based on the cycling probe technology（CPT）, whichis composed of two single tube real-time PCR assays: theFumio Imazeki1, Akira Watanabe2, Takeyuki Sato2, Shigeto

Department of Gastroenterology and Nephrology, Graduate
School of Medicine, Chiba University, Chiba, Japan

2

Division of Control and Treatment of Infectious Disease,3

Department of Emergency and Critical Care Medicine,

Chiba University, Chiba, Japan

Graduate School of Medicine, Chiba University, Chiba,

Fusarium genus-specific assay and the Fusarium solani speciesJapan

28 s ribosomal RNA gene. The Fusarium genus-specific

BackgroundFusarium strains with no cross-reaction with other reference

acute liver failure（ALF）, but this has increased the number

FSSC-specific assay also reacted very specifically with FSSC,

opportunistic infection.

complex（FSSC）-specific assay with primers targeting theassay was shown to be highly specific, detecting all reference

fungal strains, such as Aspergillus spp. and human DNA. The

except for a cross-reaction with Fusarium lunatum. To validate
the real-time PCR system, we tested 87 clinical isolates ofTreatment with systemic corticosteroids is often used forof profoundly immunocompromised patients and cases of

Methods

Between January 2007 and December 2012, all patients

Fusarium spp. Identification results from the real-time PCR（ n ＝51）referred to the Chiba University Hospital for

DNA sequencing of EF-1α gene. The sensitivity testing also

activity of 40 % or less of the standardized values were definedsystem were found to be 100% concordant with those from千葉大学

treatment of ALF were studied. Patients with prothrombin

真菌医学研究センター報告

第 18 巻201423

as having ALF. Patient age, sex, cause of ALF, alanine

aminotransferase and total bilirubin levels, prothrombin

activity and total amount of corticosteroid were analyzed

７. Exophiala dermatitidis pneumonia successfully treatedwith long-term itraconazole therapy.

to determine the factors associated with the occurrence of

Mukai Yutaka1, Nureki Shin-ichi1, Hata Masahiro2, Shigenaga

Resultsichi1, Yarita Kyoko4, Kamei Katsuhiko4

opportunistic infection.

Opportunistic infections occurred in 21 . 6 %（ n ＝11）

of ALF patients. Thirty-five patients underwent systemic

Takahiko2, Tokimatsu Issei1, Miyazaki Eishi3, Kadota Jun-1

corticosteroid therapy, and 31.4 % of those patients showed
opportunistic infections. Cytomegalovirus（ n ＝9 , 81 . 8% ）and Pneumocystis jiroveci（ n ＝6 , 54 . 5 % ）were the2opportunistic infection. In 7（63.6 %）of the 11 cases of3

microorganisms frequently suspected as the causes ofopportunistic infection, 2 or more species of microorganism
were detected. Seven patients（63.6 %）with opportunistic

infection were cured by treatment. Cox regression analysis forthe patients who underwent systemic corticosteroid therapy
steroid treatment revealed that age over 52 years（compared toyounger patients: odds ratio＝9.62, 95 % confidence interval＝1.22–76.9）was only the predictive factor for the occurrence ofopportunistic infection.
Conclusion

Opportunistic infections are not rare in ALF patients, andthe appropriate diagnosis and treatment of these infections are

critical during ALF treatment.

Department of Respiratory Medicine and Infectious

Diseases, Oita University Faculty of Medicine, 1 - 1
Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japan

Department of Respiratory Medicine, Oita Red Cross
Hospital, 3-2-37, Chiyo-machi, Oita 870-0033, Japan

Center for Community Medicine, Oita University Facultyof Medicine, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita
4

879-5593, Japan.

Medical Mycology Research Center, Chiba University, 1-81 Inohana, Chuo-ku, Chiba 260-8673, Japan

Exophiala dermatitidis pneumonia is extremely rare. Here

we report a case of E. dermatitidis pneumonia successfully

treated with long-term itraconazole therapy. A 63-year-old
woman without a remarkable medical history developed a

dry and chest pain. Chest radiographs revealed consolidationin the middle lobe of the lung. Cytologic examination bybronchoscopy showed filamentous fungi and E. dermatitidis

was detected in the bronchoalveolar lavage fluid. After 5
months of itraconazole therapy, her symptoms improved

and the area of consolidation diminished. Two weeks

after discontinuing the itraconazole therapy, the area ofconsolidation reappeared. Itraconazole therapy was restarted

and continued for 7 months. The abnormal shadow observedon the chest X-ray gradually diminished. Over a 27-monthfollow-up with periodic examination, there was no relapse
and the patient had a favorable clinical course.

24

千葉大学

真菌医学研究センター報告

第 18 巻2014

８. Pulmonar y mucormycosis with embolism: two

autopsied cases of acute myeloid leukemia.
1

９. GliA in Aspergillus fumigatus is required for its

tolerance to gliotoxin and affects the amount of

extracellular and intracellular gliotoxin.

1

Kogure Yasunori , Nakamura Fumihiko , Shinozaki-Ushiku
Aya 2, Watanabe Akira 3, Kamei Katsuhiko 3, Yoshizato

Wang Dan-Ni 1, Toyotome Takahito2, Muraosa Yasunori1,

Kurokawa Mineo4

Kaori 4 , Yamazaki Mami 4 , Takino Masahiko 5 , Kamei

Tetsuichi 1 , Nannya Yasuhito 1 , Fukayama Masashi 2 ,1

Department of Hematology and Oncology, Graduate

School of Medicine, The University of Tokyo Japan2

Department of Pathology, Graduate School of Medicine,3

Medical Mycology Research Center, Chiba University Japan

4

The University of Tokyo Japan

Watanabe Akira3, Wuren Tuya1, Bunsupa Somnuk4, Aoyagi
Katsuhiko3

1

Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba

2City, Chiba 260-8673, Japan

Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba

Department of Hematology and Oncology, Graduate School

City, Chiba 260-8673, Japan Research Center for Animalof Medicine, The University of Tokyo Japan; Department of

Cell Therapy and Transplantation Medicine, The University

Hygiene and Food Safety, Obihiro University of Agriculture

and Veterinary Medicine, Nishi 2-11 Inada-cho, Obihiro

of Tokyo Hospital Japan

Mucormycosis is an increasingly important cause of

Division of Clinical Research, Medical Mycology Research

3

City, Hokkaido 080-8555, Japan

Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University, 1 - 8 - 1 Inohana, Chuo-ku,

morbidity and mortality for patients with hematologicalmalignancies. The diagnosis of mucormycosis usually requires

Chiba City, Chiba 260-8673, Japan Division of Control

because the signs and symptoms are nonspecific and there are

Hospital, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba

mycological evidence through tissue biopsy or autopsy
currently no biomarkers to identify the disease. We hereinpresent two autopsied cases of acute myeloid leukemia withand Treatment of Infectious Diseases, Chiba University

4

University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba

prolonged neutropenia who developed invasive mucormycosis

accompanied by pulmonary artery embolism. Our cases werefeatured by unexplained fever and rapidly progressive dyspnea.

Computed tomography scan detected nodular lesions or

nonspecific consolidations in the lungs. Cultures, cytologicalstudy, and serum fungal markers consistently gave negative

results. Autopsy revealed embolism of the pulmonary arterywhich consisted of fibrin clots by filamentous fungi. Genomic

DNA was extracted from the paraffin-embedded clots andwas applied to polymerase chain reaction amplification,

leading to the diagnosis of infection by Rhizopus microsporus.

260-8677, Japan

Graduate School of Pharmaceutical Sciences, Chiba

5260-8673, Japan

Japan Application Center, Life Sciences and Chemical

Analysis, Agilent Technologies Japan, Ltd., Hachioji Site,
9-1, Takakura-cho, Hachioji City, Tokyo 192-8510, Japan

Gliotoxin is an important virulence factor of Aspergillus

fumigatus. Although GliA putatively belongs to the major

facilitator superfamily in the gliotoxin biosynthesis cluster,
its roles remain unclear. To determine the function of

GliA, we disrupted gliA in A. fumigatus. gliA disruption

We should carefully search for life-threatening pulmonary

increased the susceptibility of A. fumigatus to gliotoxin. The

develop pulmonary mucormycosis.

susceptibility to gliotoxin than each individual disruptant.

embolism when patients with hematological malignancies

gliT and gliA double-disrupted mutant had even higher

The extracellular release of gliotoxin was greatly decreased in

the gliA disruptant. Mice infected with the gliA disruptantof A. fumigatus showed higher survival rates than those

千葉大学

真菌医学研究センター報告

第 18 巻

201425

infected with the parent strain. These results strongly indicate

are known to be pathogens that cause fungal infections

the tolerance to gliotoxin and protection from extracellular

They are difficult to identify by culture and are identified

that GliA, in addition to GliT, plays a significant role ingliotoxin in A. fumigatus by exporting the toxin. This also

allows the fungus to evade the harmful effect of its own

gliotoxin production. Moreover, GliA contributes to thevirulence of A. fumigatus through gliotoxin secretion.

10. Detection of Mucor velutinosus in a Blood Culture

in immunodeficient patients such as those with leukemia.
at autopsy in many cases. Therefore, culture examinations

should be performed for immunodeficient patients with theconsideration of zygomycetes.

11. Fusarium napiforme systemic infection: case reportwith molecular characterization and antifungal

Af ter Autologous Per ipheral Blood Stem Cell
Transplantation: A pediatric Case Report.

Yumiko Joichi1,4, Ikue Chijimatsu2, Kyoko Yarita3, Katsuhikosusceptibility tests.

de Souza Marcela1, Matsuzawa Tetsuhiro2, Lyra Luiza3,

Busso-Lopes Ariane Fidelis 4, Gonoi Tohru 2, Schreiber

Kamei3, Mizuka Miki2, Makoto Onodera1,4, Masako Harada1,4,

Angelica Zaninele 3, Kamei Katsuhiko 2, Moretti Maria

1

1

Michiya Yokozaki4, Masao Kobayashi2, Hiroki Ohge5

2
3

4

5

Section of Infection Diseases, Laboratory Division of

Luiza4, Trabasso Plinio4

Clinical Support, Hiroshima University Hospital

University of Campinas, Campinas, São Paulo Brazil ; LIM

46 - Laboratory of Parasitology - HC/FMUSP, Kragujevac,

Department of Pediatrics, Hiroshima University Hospital

Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University

2

Hospital3

Hospital

4

Division of Laboratory Medicine, Hiroshima University
Department of Infection Diseases, Hiroshima University

Filamentous fungi were detected in the blood culture of

Department of Internal Medicine, School of Medicine,São Paulo Brazil

Medical Mycology Research Center, Chiba University,

Chiba, Japan

Department of Clinical Pathology, School of Medicine,

University of Campinas, Campinas, São Paulo Brazil

Department of Internal Medicine, School of Medicine,

University of Campinas, Campinas, São Paulo Brazil

a one-year-old boy after autologous peripheral blood stem

INTRODUCTION:

aspergillosis and received micafungin. Fungi were isolated

as a significant cause of disease in humans, especially infor 2-5 days. Grayish, cottony colonies formed. A slide

life-threatening disease. Fusarium species usually reported

cell transplantation. The patient was suspected to haveon potato dextrose agar medium and incubated at 37℃
culture showed a spherical sporangium at the tips of thesporangiophores. The fungus could have been a zygomycete.

The zygomycete was isolated from three blood cultures. The

antifungal drug was changed from micafungin to liposomal

During the last decades, Fusarium spp. has been reported

immunocompromised patients, who have high risk of invasive

as cause of human disease are F. solani, F. oxysporum and F.
verticillioides.

CASE DESCRIPTION:

We describe the second case in the literature ofamphotericin B, which resulted in an improvement in thedisseminated fusariosis caused by Fusarium napiforme, that

42℃ in a growth temperature test. Gene sequence analysis

subsequent cycles of chemotherapy.

patient ’s symptoms. Growth was observed at 37℃, but not
identified the fungus as Mucor velutinosus. To the best of

our knowledge, this is the first time M. velutinosus has been

detected in Japan, and this case is very rare. Zygomycetes

26

千葉大学

occurred in a 60-year-old woman with multiple myeloma after

DISCUSSION AND EVALUATION:

We identified the F. napiforme not only by standard

morphologic criteria by macroscopic and microscopic

真菌医学研究センター報告

第 18 巻2014

characteristics, but also confirmed by molecular biology

methods, including sequencing. The antifungal susceptibility

factor, AtfA, with special reference to stress-tolerance inconidia. The atfA deletion mutant conidia showed significantof the F. napiforme isolates were tested to seven antifungal

sensitivity to high temperature and oxidative stress. The

isolates tested.

the mutant conidia. Transcriptome analysis revealed that AtfA

drugs; the azoles were the most active drug against all thetrehalose content that accumulated in conidia was reduced in

CONCLUSIONS:

regulated several stress-protection-related genes such as catA,their profiles of low susceptibility to antifungal drugs highlighthigh-osmolarity glycerol pathway was also involved inwhat can contribute to an earlier and precise diagnosis andstress sensitivity and reduced trehalose in conidia. However,Fusarium spp. are of relevance in medical mycology, andthe importance for faster and more accurate diagnostic tests,
treatment.

12. Fatal fungemia with Scedosporium prolificans in a patientwith acute myeloid leukemia.
12

2

Nishimori Makoto , Takahashi Toshio , Suzuki Eiko , Kodaka

Taiichi1, Hiramoto Nobuhiro1, Itoh Kiminari1, Tsunemine13

3

Hiroko , Yarita Kyoko , Kamei Katsuhiko , Takegawa
Hiroshi4, Takahashi Takayuki4
1
2
3

dprA, scf1, and conJ at the conidiation stage. The upstream

conferring stress tolerance in conidia because DpbsB showed
a mutant lacking the SakA mitogen-activated protein kinase（MAPK）produced normal conidia. We investigated another

MAPK, MpkC, in relation with SakA, and the double

deletion mutant, DsakA, mpkC, was defective in conidia

stress tolerance. We concluded that MpkC is able to bypass

SakA, and the two MAPKs redundantly regulate the conidiarelated function of AtfA in A. fumigatus.

14. Identification of fungal pathogens by visible microarraysystem in combination with isothermal gene amplification.

Department of Hematology Shinko Hospital, Kobe
Laboratory Medicine, Shinko Hospital, Kobe

Sakai Kanae1, Trabasso Plinio 2, Moretti Maria Luiza 2,

Medical Mycology Research Center, Chiba University,

Mikami Yuzuru1, Kamei Katsuhiko1, Gonoi Tohru1

Laboratory Medicine, Kobe City Medical Center General1

13. The role of AtfA and HOG MAPK pathway in stress

2

4

Chiba

Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba,

Hospital, Kobe

tolerance in conidia of Aspergillus fumigatus.
1

2

1

Daisuke Hagiwara , Satoshi Suzuki , Katsuhiko Kamei ,Tohru Gonoi1, Susumu Kawamoto1
1

2

Medical Mycology Research Center（ MMRC ）, Chiba

University

National Food Research Institute（NFRI）, 2-1-12 Kan-nondai, Tsukuba, Ibaraki, Japan

Aspergillus fumigatus is a life-threatening pathogenic fungus,

Division of BioResources, Medical Mycology ResearchJapan

Departamento de Clı́nica Médica, Faculdade de Cieˆncias

Médicas, Universidade Estadual de Campinas, Campinas,

SP, Brazil

The increasing incidence of infectious diseases causedby fungi in immunocompromised patients has encouraged

researchers to develop rapid and accurate diagnosis methods.

Identification of the causative fungal species is critical indeciding the appropriate treatment, but it is not easy to getsatisfactory results due to the difficulty of fungal cultivationand morphological identification from clinical samples. In

this study, we established a microarray system that canwhose conidium is the infectious agent of aspergillosis. To

identify 42 species from 24 genera of clinically importantviability of conidia, we characterized a bZip transcription

detection process. The array uses the internal transcribed

better understand the mechanism underlying the long-term

千葉大学

fungal pathogens by using a chemical color reaction in the

真菌医学研究センター報告

第 18 巻201427

spacer region of the rRNA gene for identification of fungal

DNA at the species level. The specificity of this array wastested against a total of 355 target and nontarget fungal

carried the cyp51A mutation P216L, which is reported toconfer azole resistance, and it displayed an MIC indicating

resistance to itraconazole. This isolate harbored five otherspecies. The fungal detection was succeeded directly from 10

nonsynonymous mutations, some of which were found in the

1 ml of serum samples indicating that the array system wein the aspergilloma isolate in a region containing 11 genes.

（3）CFU/ml for whole blood samples, and 50 fg DNA per

afyap 1 and aldA genes. We further identified a large deletion

established is sensitive to identify infecting fungi from clinicalThis finding suggested the possibility that genomic deletions

in place of PCR amplification and labeling. The successful

our results revealed dynamic alterations that occur in thesample. Furthermore, we conducted isothermal amplification
identification with PCR-amplified as well as isothermallycan occur during chronic infection with A. fumigatus. Overall,A. fumigatus genome within its host during infection andamplified target genes demonstrated that our microarray

treatment.

variety of fungal species in a sample.

16. Penicilliosis marneffei complicated with interstitialsystem is an efficient and robust method for identifying a

15. Whole-Genome Comparison of Aspergillus fumigatus

Strains Serially Isolated from Patients with Aspergillosis.

Hagiwara Daisuke1, Takahashi Hiroki1, Watanabe Akira1,2,

Takahashi-Nakaguchi Azusa1, Kawamoto Susumu1, Kamei

pneumonia.

Haruhiko Furusawa1, Yasunari Miyazaki1, Shiro Sonoda1,

Kimitake Tsuchiya1, Takashi Yaguchi2, Katsuhiko Kamei2,
Naohiko Inase1

Katsuhiko1,2, Gonoi Tohru1

1

1

2

2

Medical Mycology Research Center（ MMRC ）, Chiba

Department of Respiratory Medicine, Tokyo Medical and
Dental University, Japan

Medical Mycology Research Center, Chiba University,

University, Chiba, JapanJapan

Chiba University Hospital, Chiba, Japan

A 71 -year-old man with interstitial pneumonia was

Division of Control and Treatment of Infectious Diseases,

The emergence of azole-resistant strains of Aspergillus

fumigatus during treatment for aspergillosis occurs by amutation selection process. Understanding how antifungal

resistance mechanisms evolve in the host environment duringhospitalized due to a pulmonary infection. He had been livingin Thailand and had returned to Japan three months earlier.

Antibiotic therapy initially cleared the infection; however,the patient’s condition relapsed. Pseudomonas aeruginosa and

Penicillium sp. were both detected in sputum and bronchialinfection is of great clinical importance and biological interest.

lavage fluid cultures and Penicillium sp. was identified to be

mutations that arose during infection by A. fumigatus strains

improved following treatment with itraconazole andHere, we used next-generation sequencing（NGS）to identify

P. marneffei. The infiltration observed on chest radiographs

sequentially isolated from two patients, one with invasive

tazobactam/piperacillin, and no relapse occurred. We hereinwith aspergilloma（three isolations）. The serial isolates had

infection in Japan.

pulmonary aspergillosis（IPA）（five isolations）and the otheridentical microsatellite types, but their growth rates andreport the first case of a non-HIV patient with P. marneffei

conidia production levels were dissimilar. A whole-genome

comparison showed that three of the five isolates from the IPA
patient carried a mutation, while 22 mutations, including

six nonsynonymous ones, were found among three isolates

from the aspergilloma patient. One aspergilloma isolate

28

千葉大学

真菌医学研究センター報告

第 18 巻2014

17. Cryptococcus gattii genotype VGIIb infection in Japan.
1

2

18. Pulmonary nocardiosis caused by Nocardia cyriacigeorgica

in patients with Mycobacterium avium complex lung2

disease: two case reports.

Ichiro Kawamura , Katsuhiko Kamei , Kyoko Yarita , Misako
2

3

1

4

Ohkusu , Kenta Ito , Mika Tsukahara , Masatake Honda ,Kazuhisa Nakashima5, Hiroaki Akamatsu6, Hanako Kurai1

Yagi Kazume 1, Ishii Makoto 1, Namkoong Ho 1, Asami

1

Division of Infectious Diseases, Shizuoka Cancer Center

Fumitake1, Kimizuka Yoshifumi1, Asakura Takanori1, Suzuki

Medical Mycology Research Center, Chiba University

Kamei Katsuhiko4,5, Betsuyaku Tomoko1, Hasegawa Naoki2

Daini Red Cross Hospital

1

2
3

4
5

6

Hospital

Department of Transplant and Endocrine Surgery, Nagoya

Takahiro1, Fujiwara Hiroshi2, Nishimura Tomoyasu3, Saito

Shoji1, Kamo Tetsuro1, Tasaka Sadatomo1, Gonoi Tohru4,

Keio University School of Medicine, 35 Shinanomachi,

Pathology Division, Shizuoka Cancer Center Hospital

Division of Thoracic Oncology, Shizuoka Cancer Center
Hospital

23This report describes a case of Cryptococcus gattii VGIIb

infection of the pulmonary and central nervous systems in an

4using the genotype information.

Center for Infectious Diseases and Infection Control,

Shinjuku-ku, Tokyo 160-8582, Japan

Keio University Health Center, 35 Shinanomachi, Shinjukuku, Tokyo 160-8582, Japan

Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University, Inohana 1 - 8 - 1 , Chuo-ku,

immunocompetent Japanese man with a travel history, and it
hypothesizes the place where he was infected with C. gattii

Shinjuku-ku, Tokyo 160-8582, Japan

Keio University School of Medicine, 35 Shinanomachi,

Third Department of Internal Medicine, Wakayama Medical
University

Department of Medicine, Division of Pulmonary Medicine,

5

Chiba, Japan

Division of Control and Treatment of Infectious Diseases,

Chiba University Hospital, Inohana 1 - 8 - 1 , Chuo-ku,
Chiba, Japan

BackgroundPulmonar y nocardiosis frequently occurs inimmunocompromised hosts and in some immunocompetenthosts with chronic lung disease; however, few reports havedescribed pulmonary nocardiosis with nontuberculous

mycobacterial lung infection. Here we report for the firsttime two cases of pulmonary nocardiosis caused by Nocardia

cyriacigeorgica associated with Mycobacterium avium complex（MAC）lung disease caused by M. avium.

Case presentation

Case 1 is that of a 72-year-old Japanese man with untreated

MAC lung disease, who was diagnosed with rheumatoid

arthritis and initiated on methotrexate. After 3 years of

methotrexate therapy, the patient remained smear-negative andculture-positive for MAC, but also became smear-positive for

Nocardia species. He received trimethoprim/sulfamethoxazole,and his symptoms and lung infiltrates improved. Case 2 is that

千葉大学

真菌医学研究センター報告

第 18 巻201429

of an immunocompetent 53-year-old Japanese woman withother Species of C. parapsilosis Complex from Patientsof clarithromycin, rifampicin, ethambutol, and levofloxacin.

Novel LAMP Identification Method for the Species.

MAC lung disease, who was treated with a combined therapy

MAC sputum culture was negative after 1 year of combinedwith Blood Stream Infections and Proposal of a
Mycopathologia, 2014 Dec 7. [Epub ahead of print].

treatment, which was maintained for 2 years. After four６）Wuren T, Toyotome T, Yamaguchi M, Takahashi-

isolated from her sputum, although MAC was rarely isolated

Watanabe A, Taguchi H, Kamei K: Effect of Serum

treatment-free years, Nocardia species were occasionally

Nakaguchi A, Muraosa Y, Yahiro M, Wang DN,

from sputum cultures over the same period. In both cases,

Components on Biofilm Formation by Aspergillus

cyriacigeorgica by 16S ribosomal RNA gene sequencing.

67（3）: 172-179, 2014.

the Nocardia species were identified as the recently defined N.
Conclusion

We report two cases of pulmonary nocardiosis caused by N.

cyriacigeorgica associated with MAC lung disease caused by

M. avium and suggest that N. cyriacigeorgica may be a major
infective agent associated with MAC lung disease.

Publications: ogirinal articles

１）Takahashi-Nakaguchi A, Muraosa Y, Hagiwara D,

Sakai K, Toyotome T, Watanabe A, Kawamoto S,

７）Toyotome T, Satoh M, Yahiro M, Watanabe A,

Nomura F, Kamei K: Glucoamylase is a major allergenof Schizophyllum commune. Clin Exp Allergy 44（3）:450-457, 2014.

８）Kikuchi K, Watanabe A, Ito J, Oku Y, Wuren T,

Taguchi H, Yarita K, Muraosa Y, Yahiro M, Yaguchi

T, Kamei K: Antifungal susceptibility of Aspergillus

fumigatus clinical isolates collected from various areas inJapan. J Infect Chemother 20（5）: 336-338, 2014.

Kamei K, Gonoi T, Takahashi H: Genome sequence９）Furusawa H, Miyazaki Y, Sonoda S, Tsuchiya K,

from patients with pulmonary aspergilloma and chronic

Complicated with Interstitial Pneumonia. Intern Med

comparison of Aspergillus fumigatus strains isolated

necrotizing pulmonary aspergillosis. Med Mycol, inpress.

２）Tamiya H, Ochiai E, Kikuchi K, Yahiro M, Toyotome

T, Watanabe A, Yaguchi T, Kamei K: Secondary
metabolite profiles and antifungal drug susceptibilityof Aspergillus fumigatus and closely related species, A.
lentulus, A. udagawae, and A. viridinutans. J InfectChemother, in press.

３）Toyotome T, Watanabe A, Ochiai E, Kamei K:

N-acetylated α-linked acidic dipeptidase is identified as

an antigen of Histoplasma capsulatum. Biochem biophys

res commun, in press.

４）Kawakami H, Inuzuka H, Hori N, Takahashi N, Ishida

K, Mochizuki K, Ohkusu K, Muraosa Y, Watanabe

A, Kamei K: Inhibitory effects of antimicrobial agentsagainst Fusarium species. Med Mycol, in press.

５）Trabasso P, Matsuzawa T, Fagnani R, Muraosa

Y, Tominaga K, Resende MR, Kamei K, Mikami

Y, Schreiber AZ, Moretti ML: Isolation and Drug

Susceptibility of Candida parapsilosis Sensu Lato and30

fumigatus and Other Aspergillus Species. Jpn J Infect Dis

千葉大学

Yaguchi T, Kamei K, Inase N: Penicilliosis marneffei
53（4）: 321-323, 2014.

10）Muraosa Y, Schreiber AZ, Trabasso P, Matsuzawa

T, Taguchi H, Moretti ML, Mikami Y, Kamei K:
Development of cycling probe-based real-time PCRsystem to detect Fusarium species and Fusarium solani

species complex（FSSC）. Int J Med Microbiol 304（34）: 505-511, 2014.

11）Arai M, Kanda T, Yasui S, Fujiwara K, Imazeki

F, Watanabe A, Sato T, Oda S, Yokosuka O:

Opportunistic infection in patients with acute liver
failure. Hepatol Int 8（2）: 233 239, 2014.

12）Mukai Y, Nureki S, Hata M, Shigenaga T, Tokimatsu

I, Miyazaki E, Kadota J, Yarita K, Kamei K: Exophiala

dermatitidis pneumonia successfully treated with long-term itraconazole therapy. J Infect Chemother 20（7）:446-449, 2014.

13）柴景子 , 大口由香 , 青柳

哲 , 氏家英之 , 西谷

道子 , 鎗田響子 , 亀井克彦 , 清水宏：Alternaria

alternata 感染による深在性皮膚真菌症の１例 . 臨床

皮膚科

真菌医学研究センター報告

68（4）: 355-359, 2014.

第 18 巻201414）Kogure Y, Nakamura F, Shinozaki-Ushiku A, Watanabe

K, Takegawa H, Takahashi T: Fatal fungemia with

A, Kamei K, Yoshizato T, Nannya Y, Fukayama

Scedosporium prolificans in a patient with acute myeloidwith embolism: two autopsied cases of acute myeloid23）Hagiwara D, Suzuki S, Kamei K, Gonoi T, Kawamoto

M, Mineo, Kurokawa M: Pulmonary mucormycosis

leukemia. Int J Clin Exp Pathol 7（6）: 3449 - 3453 ,2014.

15）Wang DN, Toyotome T, Muraosa Y, Watanabe A,

Wuren T, Bunsupa S, Aoyagi K, Yamazaki M, Takino
M, Kamei K: GliA in Aspergillus fumigatus is requiredfor its tolerance to gliotoxin and affects the amount of

extracellular and intracellular gliotoxin. Med Mycol 52: 506-518, 2014.
（5）16）Joichi Y, Chijimatsu I, Yarita K, Kamei K, Miki M,

leukemia. Med Mycol J 55（4）: E63-E70, 2014.

S: The role of AtfA and HOG MAPK pathway in stress
tolerance in conidia of Aspergillus fumigatus. Fungal

Genet Biol 73: 138-149, 2014.

24）柴玉珠 , 山崎広子 , 渡辺

哲 , 田中内炎の１例 . 臨床眼科

68（12）: 1631-1637, 2014.

Publications: reviews, reports, books and the related
publications

Onodera M, Harada M, Yokozaki M, Kobayashi M,25）亀井克彦 , 渡辺

Culture After Autologous Peripheral Blood Stem Cell

26）渡辺

55（2）
: E43-E48, 2014.

27）渡辺Gonoi T: Identification of fungal pathogens by visible28）渡辺amplification. Mycopathologia 178（1-2）: 11-26, 2014.

29）亀井克彦：フロントエッセイ

田章人 , 亀井克彦：Curvularia lunata によるアレル

30）河野Ohge H: Detection of Mucor velutinosus in a Blood
Transplantation: A pediatric Case Report. Med Mycol J17）Sakai K, Trabasso P, Moretti ML, Mikami Y, Kamei K,

microarray system in combination with isothermal gene18）西本真由美 , 山口充洋 , 幸前朱厘 , 藤井啓嗣 , 上
ギー性気管支肺真菌症の1例 . 日呼吸誌
553-557, 2014.

3（4）:19）Kawamura I, Kamei K, Yarita K, Ohkusu M, Ito K,

Tsukahara M, Honda M, Nakashima K, Akamatsu H,

稔：眼内レ

ンズ縫着術後に生じた外傷性 Paecilomyces lilacinus 眼

臨床検査

哲：輸入感染症としての真菌症 .

58（1）: 111-116, 2014.

哲 , 亀井克彦：ムーコル症 . 呼吸器内科

25: 38-42, 2014.
（1）

哲 , 亀井克彦：我が国における輸入真菌症へのアプローチ . 感染と抗菌薬

17（1）: 43-47, 2014.

哲 , 矢口貴志：糸状菌症 . 臨床検査

104-109, 2014.

58（1）
:

真菌と病院感染 .

INFECTION CONTROL 23（6）: 1, 2014.

座談会

茂（司会）, 亀井克彦 , 二木芳人 , 宮﨑義継：
深在性真菌症の診断・治療ガイドラインを読み解く . 呼吸

31）渡 辺33（5）: 435-443, 2014.

哲 , 亀 井 克 彦： 旅 行 者 真 菌 症 .

呼吸33: 444-449, 2014.
（5）

Kurai H: Cryptococcus gattii genotype VGIIb infection in32）亀井克彦（分担）：ABPM の原因真菌に関する菌学

20）Hagiwara D, Takahashi H, Watanabe A, Takahashi-（難治性疾患等克服研究事業）（免疫アレルギー疾患

Genome Comparison of Aspergillus fumigatus Strains「アレルギー性気管支肺真菌症の診断・治療指針確J Clin Microbiol 52（12）: 4202-4209, 2014.

度終了報告書Japan. Medical Mycol J 55（3）: E51-E54, 2014.

Nakaguchi A, Kawamoto S, Kamei K, Gonoi T: Whole-

的及び血清学的解析 . 厚生労働科学研究費補助金

等予防・治療研究事業

免疫アレルギー研究分野）

Serially Isolated from Patients Infected with Aspergillosis.

立のための調査研究」平成25年度総括・分担研究年21）de Souza M, Matsuzawa T, Lyra L, Busso-Lopes

33）亀井克彦（分担）, 豊留孝仁（研究協力者）, 村長保憲

Trabasso P: Fusarium napiforme systemic infection: case

ヒストプラズマ症の迅速診断法改良・開発へ向けた

susceptibility tests. Springerplus 3: 492, 2014.

フルエンザ等新興・再興感染症研究事業）「真菌感染

Hiramoto N, Itoh K, Tsunemine H, Yarita K, Kamei

ンスに関する研究」平成25年度総括・分担研究報告

AF, Gonoi T, Schreiber AZ, Kamei K, Moretti ML,
report with molecular characterization and antifungal22）Nishimori M, Takahashi T, Suzuki E, Kodaka T,

千葉大学

p.30-37, 2014.

（研究協力者）：輸入真菌症の国内発生状況調査と基礎的研究 . 厚生労働科学研究費補助金（新型イン症の病態解明及び検査・治療法の確立とサーベイラ

真菌医学研究センター報告

第 18 巻

2014

31

書

p.48-54, 2014.

34）橋本亜希 , 渡辺

哲 , 亀井克彦：各種 Candida 属菌と治療戦略 . 感染症内科35）渡辺2（6）: 559-566, 2014.

哲 , 亀井克彦：輸入真菌症の疫学と病態

１）コクシジオイデス症の疫学と病態 . 感染症内科

2（6）: 594-598, 2014.

36）渡辺

哲 , 亀井克彦：結核重症例、合併症重症例における治療、管理の進歩 真菌感染症合併症の治療と管理 . 結核89（5）: 577-579, 2014.

37）亀井克彦 , 渡辺

哲：コクシジオイデス症 . 感染症

44（4）: 139-140, 149-153, 2014.

38）西田篤司 , 亀井克彦：アレルギー性気管支肺真菌症 . 呼と循環

62（8）: 769-775, 2014.

39）亀井克彦 , 渡辺

1068-1077, 2014.

40）渡辺

液内科
41）河野

哲：まれな真菌症 . 日胸

73（9）
:

哲 , 亀井克彦：造血器疾患とムーコル症 . 血69（4）: 584-588, 2014.

茂（座長）, 渡辺

哲（演者）：アスペルギルス症をとりまく諸問題 これからの治療戦略を考える（第62回日本感染症学会東日本地方会学術集会／第60回日本化学療法学会東日本支部総会合同学会教育. 感染症道場
セミナー2）

3（2）: 45-51, 2014.

42）亀井克彦 , 渡辺

哲：マルネッフェイ型ペニシリウ

43）亀井克彦 , 渡辺

哲：ムーコル症 . プラクティス

ム症 .

呼吸

33（10）: 1024-1027, 2014.

44）矢口貴志 , 亀井克彦：真菌 接合菌 Rhizopus oryzae,

Absidia corymbifera など . 臨床検査 58（11増）: 14201422, 2014.

45）Brummer E, Kamei K: Histoplasma capsulatum: Master

Evader of Innate Immunity. Med Mycol J 55（4）:E57-E62, 2014.

哲 , 亀井克彦：ヒストプラズマ症 . 感染症: 1224-229, 2014.
44（6）

47）渡辺

32

: 1818-1823, 2014.
46（12）科49）渡辺哲 , 亀井克彦：注目される感染症

菌症の現状と対策 . 日本内科学会雑誌

2674-2679, 2014.

50）渡辺

輸入真
103（11）:

哲 , 酒井文和 , 河端美則：血管型エーラス・

ダンロス症候群 . 呼吸33（12）: 1244-1248, 2014.

夫 , 横須賀收 , 渡辺

哲：【急性肝不全の病態と治51）新井誠人 , 安井

伸 , 神田達郎 , 藤原慶一 , 今関文

療】急性肝不全患者における日和見感染症 . 消化器内科

59（4）: 367-372, 2014.

52）大楠美佐子 , 亀井克彦：真菌
臨床検査

クリプトコックス .

58（11増）: 1417-1419, 2014.

53）亀井克彦：抗真菌薬 . 「Pocket Drugs 2014」，監修：
福井次矢 , 編集：小松康宏 , 渡邉裕司 , p. 899-900,
医学書院 , 2014. 1. 1発行 .

54）豊留孝仁 , 亀井克彦：病原真菌の病原機構と病原因子 . 「目で見る真菌と真菌症」, 編者：亀井克彦 ,p. 23-29, 医薬ジャーナル社 , 2014. 6. 20発行 .

55）亀井克彦 , 渡辺

哲：医療施設内の感染制御 . 「目で見る真菌と真菌症」, 編者：亀井克彦 , p. 122-129,
医薬ジャーナル社 , 2014. 6. 20発行 .

哲 , 亀井 克彦： 旅行医 学か ら見た真 菌症 .

「目で見る真菌と真菌症」, 編者：亀井克彦 , p. 152162, 医薬ジャーナル社 , 2014. 6. 20発行 .

57）渡辺哲 , 亀井克彦：カンジダ属の疫学 . 「侵襲性カンジダ症」, 竹末芳生 , 三鴨廣繁編 , p. 50-52, 医薬ジャーナル社 , 2014. 7. 15発行 .

58）渡邉

哲 , 亀井克彦：病原体の特徴を知る「臨床工学ライブラリーシリーズ８

真菌 .

感染防止から

, 監修：松本哲哉 , p. 134-140, 2014.
みる微生物学」

9. 25発行 .

哲 , 亀井克彦：地域流行型真菌症（いわゆる千葉大学

46（12）: 1848-1852, 2014.

48）竹内典子 , 亀井克彦：肺アスペルギルス症 . 小児内56）渡辺: 784-786, 2014.
31（6）

46）渡辺

輸入真菌症）. 小児内科

真菌医学研究センター報告

第 18 巻2014

五ノ井 PI（真菌・放線菌と宿主の分子相互作用研究）プロジェクト
Project for Host Pathogen（fungi/actinomycetes）Molecular Interaction
研究概要（Summary）
微生物資源分野では , バイオリソース管理室と協力し , 日本国内および海外のヒトや動物に由来する
病原真菌・病原放線菌を収集 , 管理 , 分譲している . これらの菌株数は , 現在約２万に達するが , 菌のマーカー遺伝子やゲノムを解析し , また薬剤感受性や電子顕微鏡による形態観察 , ２次代謝産物の解析などを行い菌株資源 , 遺伝子資源としての付加価値の向上に努めている . さらなる独自の研究テーマ（PI プロジェクト）については下記『主なテーマ』を参照してください .
主なテーマ（Research Focus）
１）ヒト・動物の病原真菌・病原放線菌の収集 , 分類 , 系統解析 , ２次代謝産物の解析 , 病原因子解析 ,２次代謝産物生合成遺伝子 , ゲノムの解析を行っている .
２）真菌・放線菌のヒトへの感染機構の解明を分子生物学的手法 , 動物モデル , ゲノム解析などを用いて行っている . 特に , 近年は , 糖鎖と糖鎖受容体を介した菌と宿主の相互作用解明に力を入れている.
３）真菌感染発症と宿主の栄養状態やストレス状態との関連を動物モデルなどを用いて研究している .
特に代謝関連分子と免疫関連分子の機能的リンクに興味を持っている .

In cooperation with Bio-Resource management office, we collect pathogenic fungi and actinomycetes inboth inside and outside of Japan. We identify pathogenic fungi and actinomycetes as a public service, andanalyze their phylogenetic relations. We store fungi and actinomycetes with the support of the National
BioResource Projects in Japan, and distribute them upon request. Currently we stock approximately 20,000
strains. We analyze sequences of marker genes and genomes, drug-sensitivities, and observe fine structuresusing electron-microscopy, to enhance biodiversity values. Other projects are listed below.
1 ）We collect, identify and phylogenetically analyze of human and animal pathogenic fungi andactinomycetes. We also analyze 2nd metabolites and their synthetic enzymes, pathogenic factors, andgenomes.
2 ）We analyze infection mechanisms of human pathogenic fungi and actinomycetes using molecularmethods, animal models, and genome analysis. In particular, we are trying to understand roles of cellsurface glycans and their receptors（lectins）of human and fungi in infection.
3 ）We study effects of diets and mental stresses on fungal infections mainly using animal models andmolecular methods. We are trying to clarify yet unknown links between metabolism and immune-related
molecules.
教

授

助

教

特

任

助

教

技 術 補 佐 員

五ノ井 透
大荒田素子
酒井香奈江
川名 直美

Professor

Assistant Professor

Research Assistant Professor

Research Promotion Technician

千葉大学

真菌医学研究センター報告

Tohru Gonoi
Oarada Motoko
Kanae Sakai
Naomi Kawana

第 18 巻

2014

33

1.

Refeeding with glucose rather than fructose elicits

greater hepatic inflammatory gene expression in mice.

Motoko Oarada 1, Azusa Takahashi-Nakaguchi1, Tomoki
Abe2, Takeshi Nikawa2, Takashi Miki3, Tohru Gonoi1

1

2

3

Medical Mycology Research Center, Chiba University,

studies should investigate the role of these effects in liver
inflammation and, consequently, liver dysfunction.

2.

Aspergillus arcoverdensis, a new species of Aspergillus

section Fumigati isolated from caatinga soil in State ofPernambuco, Brazil.

Chiba 260-8673, Japan

Tetsuhiro Matsuzawa1 , Galba M. Campos Takaki2, Takashi

Medicine, Tokushima

Yoshikazu Horie1

Department of Nutrition, Tokushima University School of
Department of Medical Physiology, Graduate School of

Medicine, Chiba University

Objective: We have previously reported that refeeding after

Yaguchi 1, Kaoru Okada2, Paride Abliz3, Tohru Gonoi1,

1

2

a 48-h fast, used as a study model of starvation and refeeding,have also demonstrated that dietary carbohydrates play criticalroles in this process. In our current study, we compared theoutcomes of refeeding with different carbohydrate sources.

Methods: Mice were fasted for 46 h and then refed with

1.5%（w/w）agar gel containing 19% carbohydrate（sources:
α -corn starch, glucose, sucrose or fructose ）. The liver

1, Inohana, Chuo-ku, Chiba, Japan

Nucleus of Research in Environmental Sciences andBiotechnology, Catholic University of Pernambuco, Rua do

Principe 526, Boa Vista, Recife, Pernambuco 50050900,

promotes acute liver inflammatory gene expression, which is
at least partly mediated by toll-like receptor 2（TLR2）. We

Medical Mycology Research Center, Chiba University, 1-8-

3BrazilDepartment of Dermatology, The First Affiliated Hospitalof Xinjiang Medical University, No. 1 Liyushan Road,
Urumqi, Xinjang 830053, China

Aspergillus arcoverdensis, a new species isolated fromsemi-desert soil in a caatinga area, State of Pernambuco,

expression of inflammatory genes and other specific genes was

Brazil, and a similar environment in the Xinjiang Uygur

initiation.

It is characterized by relatively long conidiophores forthen sequentially measured for the first 14 h after refeeding

Results: Fasting for 46 h up-regulated the liver expressionof endogenous ligands for TLRs（HspA5, Hsp90aa1, andHspd1）. Refeeding with agar gel containing α -corn starchor glucose increased the liver expression of Tlr2 , pro-inflammatory genes（Cxcl2 , Cxcl10 , Cxcl1 , Nfkb1 , Nfkb2 ,Autonomous Region, China, is described and illustrated.

Aspergillus sectionFumigati, and subglobose to broadly

ellipsoidal and smooth conidia. The delimitation of this new

species is supported further by phylogenetic analyses of the βtubulin, calmodulin and actin gene sequences.

RelB, Sectm1 a, Il1 β）, stress response genes（Atf3 , Asns,
Gadd45 a, Perk, Inhbe）, detoxification genes（Hmox1 , Gsta1 ,

Abca8 b ）, genes involved in tissue regeneration（ Gdf15 ,Krt23 , Myc,Tnfrsf12 a, Mthfd2 ）and genes involved in tumor

suppression（p53 , Txnrd1 , Btg2 ）. This refeeding also elevatedthe serum levels of alanine aminotransferase moderately but

significantly. These effects were attenuated in mice refed withagar gel containing sucrose or fructose.

Conclusion: Dietary glucose, rather than fructose, playsa critical role in refeeding-induced acute liver inflammatory
gene expression and moderate hepatocyte destruction. Further

34

千葉大学

真菌医学研究センター報告

第 18 巻2014

3.

The role of AtfA and HOG MAPK pathway in stress

tolerance in conidia of Aspergillus fumigatus.
1

2

4.

Tohru Gonoi1, Susumu Kawamoto1
1

Medical Mycology Research Center（ MMRC ）, Chiba

2

National Food Research Institute（NFRI）, 2-1-12 Kan-

University, 1-8-1 Inohana, Chuo-ku, Chiba, Japannondai, Tsukuba, Ibaraki, Japan

Aspergillus fumigatus is a life-threatening pathogenic fungus,

whose conidium is the infectious agent of aspergillosis. To

better understand the mechanism underlying the long-term

viability of conidia, we characterized a bZip transcription
factor, AtfA, with special reference to stress-tolerance instrains serially isolated from patients infected with as-

pergillosis.

1

Daisuke Hagiwara , Satoshi Suzuki , Katsuhiko Kamei ,Whole-genome comparison of Aspergillus fumigatus

Daisuke Hagiwara1, Hiroki Takahashi1, Akira Watanabe1,

Azusa Takahashi-Nakaguchi 1 , Susumu Kawamoto 1 ,Katsuhiko Kamei1, Tohru Gonoi1
1

Medical Mycology Research Center（ MMRC ）, Chiba

University, Chiba, JapanThe emergence of azole-resistant strains of Aspergillus

fumigatus during treatment for aspergillosis occurs by amutation selection process. Understanding how antifungal

resistance mechanisms evolve in the host environment during

infection is of great clinical importance and biological interest.

conidia. The atfA deletion mutant conidia showed significant

Here, we used next-generation sequencing（NGS）to identify

trehalose content that accumulated in conidia was reduced insequentially isolated from two patients, one with invasive

regulated several stress-protection-related genes such as catA,with aspergilloma（three isolations）. The serial isolates had

sensitivity to high temperature and oxidative stress. The

the mutant conidia. Transcriptome analysis revealed that AtfA

mutations that arose during infection by A. fumigatus strains

pulmonary aspergillosis（IPA）（five isolations）and the other

dprA, scf1, and conJ at the conidiation stage. The upstream

identical microsatellite types, but their growth rates andconferring stress tolerance in conidia because ΔpbsB showedcomparison showed that three of the five isolates from the IPA

high-osmolarity glycerol pathway was also involved instress sensitivity and reduced trehalose in conidia. However,a mutant lacking the SakA mitogen-activated protein kinase（MAPK）produced normal conidia. We investigated another

MAPK, MpkC, in relation with SakA, and the double

deletion mutant, Δ sakA, mpkC, was defective in conidia

stress tolerance. We concluded that MpkC is able to bypass

SakA, and the two MAPKs redundantly regulate the conidiarelated function of AtfA in A. fumigatus.

conidia production levels were dissimilar. A whole-genome
patient carried a mutation, while 22 mutations, including

six nonsynonymous ones, were found among three isolates

from the aspergilloma patient. One aspergilloma isolate
carried the cyp51A mutation P216L, which is reported toconfer azole resistance, and it displayed an MIC indicating

resistance to itraconazole. This isolate harbored five othernonsynonymous mutations, some of which were found in theafyap 1 and aldA genes. We further identified a large deletionin the aspergilloma isolate in a region containing 11 genes.

This finding suggested the possibility that genomic deletions

can occur during chronic infection with A. fumigatus. Overall,our results revealed dynamic alterations that occur in the

A. fumigatus genome within its host during infection andtreatment.

千葉大学

真菌医学研究センター報告

第 18 巻

2014

35

5.

Agelamadins C–E, bromopyrrole alkaloids compris-that amphidinins C-F（1 - 4）were 4 , 5 -seco-analogues ofsponge Agelas sp.

compounds were elucidated by the combination of J-based

ing oroidin and 3-hydroxykynurenine from a marine

Taishi Kusama1, Naonobu Tanaka1,2, Kanae Sakai3, Tohru

Gonoi 3, Jane Fromont 4, Yoshiki Kashiwada 1, Jun’ichi
Kobayashi
11Graduate School of Pharmaceutical Sciences, Hokkaido
University, Sapporo 060-0812, Japan

2

Graduate School of Pharmaceutical Sciences, The University

3

Mycology Research Center, Chiba University, Chiba 260-

4amphidinolide Q（5）. The absolute configurations of the new

configuration analysis, modified Mosher’s method, andchemical derivatization. Amphidinins D（2）and F（4）are thefirst glycosides related to amphidinolides. Amphidinins C-F（1-4）showed antimicrobial activity against bacteria and/or

fungi.
7.

of Tokushima, Tokushima 770-8505, Japan
8673, Japan

Western Australian Museum, Locked Bag 49, Welshpool

lung disease: two case reports.

Kazuma Yagi1, Makoto Ishii1, Ho Namkoong1, Takahiro

Asami1, Hiroshi Fujiwara2, Tomoyasu Nishimura3, Fumitake

Saito 1, Yoshifumi Kimizuka1, Takanori Asakura1, Shoji

Three structurally unique bromopyrrole alkaloids,

Katsuhiko Kamei4,5, Tomoko Betsuyaku1, Naoki Hasegawa2

Agelas sp. Agelamadin C（1）possesses a hybrid structure

Suzuki1, Tetsuro Kamo1, Sadatomo Tasaka1, Tohru Gonoi4,

1of oroidin and 3 -hydroxykynurenine connected througha dihydro-1,4-oxazine moiety. Agelamadins D（2）and E（3）are a C- 9 /C- 10 diastereomer and a 10 -epimer of 1 ,method and a TDDFT ECD calculation. Antimicrobialactivity of 1-3 was evaluated.
6.

2

3

Amphidinins C-F, amphidinolide Q analogs from ma-

1

1

Takaaki Kubota , Takahiro Iwai , Kanae Sakai , Tohru
Gonoi2, Jun’ichi Kobayashi1

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

Medical Mycology Research Center, Chiba University,

Chiba 260-0856, Japan

Four new polyketides, amphidinins C-F（1-4）, have been

isolated from the culture broth of symbiotic dinoflagellate
Amphidinium sp. The analysis of their spectral data revealed

36

千葉大学

Center for Infectious Diseases and Infection Control,
Shinjuku-ku, Tokyo 160-8582, Japan

Keio University Health Center, 35 Shinanomachi,

Shinjuku-ku, Tokyo 160-8582, Japan

Division of Clinical Research, Medical Mycology ResearchCenter, Chiba University, Inohana 1 - 8 - 1 , Chuo-ku,

52

Shinjuku-ku, Tokyo 160-8582, Japan

Keio University School of Medicine, 35 Shinanomachi,

4rine dinoflagellate Amphidinium sp.

Department of Medicine, Division of Pulmonary Medicine,
Keio University School of Medicine, 35 Shinanomachi,

respectively. The structures of 1-3 were elucidated on the basisof spectroscopic analysis as well as application of a PGME

2

gica in patients with Mycobacterium avium complex

DC, WA 6986, Australia

agelamadins C-E（1-3）, were isolated from a marine sponge

1

Pulmonary nocardiosis caused by Nocardia cyriacigeor-

Chiba, Japan

Division of Control and Treatment of Infectious Diseases,
Chiba University Hospital, Inohana 1 - 8 - 1 , Chuo-ku,

Chiba, Japan

Pulmonar y nocardiosis frequently occurs inimmunocompromised hosts and in some immunocompetenthosts with chronic lung disease; however, few reports havedescribed pulmonary nocardiosis with nontuberculous

mycobacterial lung infection. Here we report for the firsttime two cases of pulmonary nocardiosis caused by Nocardia

cyriacigeorgica associated with Mycobacterium avium complex（MAC）lung disease caused by M. avium. Case presentation

真菌医学研究センター報告

第 18 巻2014

Case 1 is that of a 72-year-old Japanese man with untreated

分離された . また , 経過中にあらたに M.avium complex

MAC lung disease, who was diagnosed with rheumatoid症（n ＝1）, 慢性肺アスペルギルス症（n ＝1）, 肺炎（n ＝4）

methotrexate therapy, the patient remained smear-negative

与されたのは９例 , うち５例では副作用のため他薬へのarthritis and initiated on methotrexate. After 3 years ofand culture-positive for MAC, but also became smear-

positive for Nocardia species. He received trimethoprim/の発症を認めた . ノカルジアの治療として ST 合剤を投変更を要した . 観察期間［中央値968日（43-2625日）］内にノカルジア症１例が再発し , ６例が死亡したが , ノカ

sulfamethoxazole, and his symptoms and lung infiltrates

ルジア症による死亡はなかった .

Japanese woman with MAC lung disease, who was treated

ST 合剤投与中に副作用の頻度が高いことに注意が必要

improved. Case 2 is that of an immunocompetent 53-year-old
with a combined therapy of clarithromycin, rifampicin,
ethambutol, and levofloxacin. MAC sputum culture wasnegative after 1 year of combined treatment, which wasmaintained for 2 years. After four treatment-free years,
Nocardia species were occasionally isolated from her sputum,

although MAC was rarely isolated from sputum cultures over

結語：肺ノカルジア症には混合感染がまれではない .

である . 肺ノカルジア症の予後については , 合併症の影
響が無視できない .
9.

Gordonia iterans sp. nov., isolated from a patient withpneumonia.

the same period. In both cases, the Nocardia species were

Ying-Qian Kang1,2 Hong Ming3, Tohru Gonoi2, Yuru Chen1,

ribosomal RNA gene sequencing. Conclusion We report two

Yuzuru Mikami2, Wen-Jun Li3

identified as the recently defined N. cyriacigeorgica by 16S

cases of pulmonary nocardiosis caused by N. cyriacigeorgica

Yu Cao4, Yan-Yan Wang1, Juan Cheng3, Takeharu Koga5,

associated with MAC lung disease caused by M. avium and1

associated with MAC lung disease.

2suggest that N. cyriacigeorgica may be a major infective agent

肺ノカルジア症の12例の臨床的検討 — 単一施設の

8.

3

後方視的研究 —日本呼吸器学会誌 .

石黒
1

1

卓 , 高柳
1

1

2

1

2

Medical Mycology Research Center（ MMRC ）, Chiba

University, Inohana, Chuo-ku, Chiba, JapanKey Laboratory of Microbial Diversity in Southwest China,

Ministry of Education, Yunnan Institute of Microbiology,

昇 , 篠原和歌子 , 田村仁樹 , 高久洋太

郎 , 鍵山奈保 , 渡邊裕1.

1

Department of Microbiology, Guiyang Medical College,

Guiyang, 550004, PR China

4

2

哲 , 五ノ井透 , 亀井克彦 , 杉田1

埼玉県立循環器・呼吸器病センター 呼吸器内科

2

千葉大学真菌医学研究センター
背景：肺ノカルジア症の臨床的な特徴についてまとめた報告は限られている .

対象と方法：2000年から2013年に当センターで診療を5

Yunnan University, Kunming, 650091, PR China

Department of Dermatology, the affiliated Hospital of
Guiyang Medical College, Guiyang, 550004, PR China

Department of Respiratory Medicine, Asakura Medical

Association Hospital, 836-0069, Raiharu, Asakura-city,
Fukuoka, JapanA second novel clinical actinobacterial strain, designatedIFM 10348（T）, was isolated from the sputum of the same

Japanese patient with bacterial pneumonia from whom thetype strain of Gordonia araii had been isolated. The strains

行った肺ノカルジア症12例を後方視的に検討した .

differed in phylogenetic position and drug-resistance profiles.

の基礎疾患は10例 , 糖尿病１例 , ステロイドおよび免疫

clarified by phenotypic, chemotaxonomic and phylogenetic

結果：平均年齢は69.0±12.4歳 , 男性10例 , 呼吸器系

The taxonomic position of strain IFM 10348（ T ）was

抑制薬の投与は１例であった . ノカルジアと同時に４例

studies. Phylogenetic analyses based on 16S rRNA gene

ルエンザ桿菌＋緑膿菌＋ Aspergillus fumigatus, A.flavus がoccupied a distinct clade within the genus Gordonia andで Mycobacterium avium complex, Streptococcus mitis, インフ

千葉大学

sequences clearly demonstrated that strain IFM 10348（T）

真菌医学研究センター報告

第 18 巻201437

was related closely to Gordonia malaquae DSM 45064（T）
and Gordonia hirsuta DSM 44140（ T ）（97 . 3 and 97 . 1 %similarities, respectively）. Strain IFM 10348（T）was also
clearly differentiated from G. malaquae DSM 45064（T）and

G. hirsuta DSM 44140（T）based on gyrB and secA1 genesequence similarity values. Strain IFM 10348（T）had MK-9

（H2）as the predominant menaquonine, contained meso-

11. Agelamadins A and B, dimeric bromopyrrole alkaloids

from a marine sponge Agelas sp.

Taishi Kusama １ , Naonobu Tanaka1,2, Kanae Sakai3, Tohru

Gonoi 3, Jane Fromont 4, Yoshiki Kashiwada 2, Jun’ichi
Kobayashi １

diaminopimelic acid, arabinose, galactose and glucosamine

1

feature 3（C16:1ω7c and/or C16:1ω6c）and C16:0 as the2

DNA G+C content of strain IFM 10348（T）was 68.0 mol%.

3

as cell-wall components, and contained C18:1ω9c, summedmajor cellular fatty acids. Mycolic acids were present. The

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

Graduate School of Pharmaceutical Sciences, The Universityof Tokushima, Tokushima 770-8505, Japan

Mycology Research Center, Chiba University, Chiba 260-

DNA-DNA relatedness data coupled with the combination

8673, Japan

10348（T）represents a novel species of the genus Gordonia,

Two structurally unique dimeric bromopyrrole alkaloids,of genotypic and phenotypic data indicated that strain IFM

for which the name Gordonia iterans sp. nov. is proposed.
The type strain is IFM 10348（T）（＝ CCTCC M2011245（T）＝ NCCB 100436（T））.

10. Nakijiquinone S（Ia）and Nakijinol C（Ib）, new mero-terpenoids from a marine sponge of the family Spongiidae.

agelamadins A（1）and B（2）, were isolated from a marine

sponge Agelas sp. Agelamadins A（1）and B（2）have astructure consisting of an agelastatin-like tetracyclic moiety

and an oroidin-like linear moiety in common. The structuresof 1 and 2 were elucidated on the basis of spectroscopic

analysis. The antimicrobial activity and cytotoxicity ofagelamadins A（1）and B（2）were evaluated.

Haruna Suzuki 1 , Takaaki Kubota 1 , Azusa Takahashi-

Nakaguchi 2 , Jane Fromont 3 , Tohru Gonoi 2 , Jun ’ichi
Kobayashi1
12
3Graduate School of Pharmaceutical Sciences, Hokkaido
University

Medivcal Mycology Research Center, Chiba University

Western Australian Museum

New meroterpenoids, nakijiquinone S（1）and nakijinol C（2）, have been isolated from an Okinawan marine spongeof the family Spongiidae. The gross structures and relative

stereochemistries of 1 and 2 were elucidated on the basis of

their spectral data. Nakijiquinone S（1）and nakijinol C（2）were new meroterpenoids consisting of a clerodane-type decalinring connected to a 2-butoxy-5-hydroxy-benzoquinone unit

or methyl 2,3,4-trihydroxybenzoate unit through a methylene,
respectively. Nakijiquinone S（1）and nakijinol C（2）showed

antimicrobial activities against several bacteria and fungi.

38

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12. Identification of Fungal Pathogens by Visible Micro-

array System in Combination with Isothermal Gene

13. Genome based analysis of type-I polyketide synthaseand nonribosomal peptide synthetase gene clusters in

Amplification.

Kanae Sakai1,3, Plinio Trabasso 2, Maria Luiza Moretti2,

Yuzuru Mikami1,3, Katsuhiko Kamei13, Tohru Gonoi1,31Division of BioResources, Medical Mycology ResearchCenter, Chiba University, Inohana 1 - 8 - 1 , Chuo-ku,

2

Hisayuki Komaki1, Natsuko Ichikawa2, Akia Hosoyama2,
Takahashi-Nakaguchi3, Tetsuhiro Matsuzawa3, Ken-Ichiro
Suzuki2, Nobuyuki Fujita2, Tohru Gonoi3

1

Departamento de Clínica Médica, Faculdade de Ciências

SP Brazil

Biological Resource Center, National Institute ofTechnology and Evaluation（NBRC）, Kisarazu, Chiba

Chiba, Japan

Médicas, Universidade Estadual de Campinas, Campinas,

3

seven strains of five representative Nocardia species.

23292-0818, Japan

NBRC, Shibuya-ku, Tokyo 151-0066, Japan

Medical Mycology Research Center（ MMRC ）, Chiba

JST/JICA, Science and Technology Research Partnership

University, Chuo-ku, Chiba 260-8673, Japan

The increasing incidence of infectious diseases caused

usually live in soil or water and play saprophytic roles, but

researchers to develop rapid and accurate diagnosis methods.

and other organs of humans and animals. Primarily because

for Sustainable Development（SATREPS）, Chiba, Japanby fungi in immunocompromised patients has encouraged

Identification of the causative fungal species is critical in

BACKGROUND: Actinobacteria of the genus Nocardia

they also opportunistically infect the respiratory system, skin,of the clinical importance of the strains, some Nocardia

deciding the appropriate treatment, but it is not easy to get

genomes have been sequenced, and genome sequences haveand morphological identification from clinical samples. In

those of Streptomyces strains, the producers of most antibiotics.

satisfactory results due to the difficulty of fungal cultivation

this study, we established a microarray system that canidentify 42 species from 24 genera of clinically important

accumulated. Genome sizes of Nocardia strains are similar to

In the present work, we compared secondary metabolite

biosynthesis gene clusters of type-I polyketide synthase

fungal pathogens by using a chemical color reaction in the（ PKS-I ）and nonribosomal peptide synthetase（ NRPS ）spacer region of the rRNA gene for identification of fungal

based on domain organization and amino acid sequence

tested against a total of 355 target and nontarget fungal

RESULTS: Draft genome sequences of Nocardia asteroides

detection process. The array uses the internal transcribed

DNA at the species level. The specificity of this array wasamong genomes of representative Nocardia species/strains
homology.

species. The fungal detection was succeeded directly fromNBRC 15531（T）, Nocardia otitidiscaviarum IFM 11049,

per 1 ml of serum samples indicating that the array system we

IFM 10847 were read and compared with published10（3）CFU/ml for whole blood samples, and 50 fg DNA

Nocardia brasiliensis NBRC 14402（T）, and N. brasiliensis

established is sensitive to identify infecting fungi from clinicalcomplete genome sequences of Nocardia farcinica IFM

in place of PCR amplification and labeling. The successful

HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0

amplified target genes demonstrated that our microarrayN. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4

sample. Furthermore, we conducted isothermal amplification
identification with PCR-amplified as well as isothermallysystem is an efficient and robust method for identifying avariety of fungal species in a sample.

10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis

Mb; N. cyriacigeorgica, 6 . 2 Mb; N. asteroides, 7 . 0 Mb;
Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/

NRPS hybrid clusters ranged between 4-11, 7-13, and 1-6,
respectively, depending on strains, and tended to increase
with increasing genome size. Domain and module structures

千葉大学

真菌医学研究センター報告

第 18 巻201439

of representative or unique clusters are discussed in the text.

CONCLUSION: We conclude the following: 1）genomesof Nocardia strains carry as many PKS-I and NRPS geneclusters as those of Streptomyces strains, 2）the number of

15. Total aflatoxin, fumonisin and deoxynivalenol con-tamination of busaa in Bomet county, Kenya.

Mary C. Kirui1, Amos E. Alakonya1, Keith K. Talam1,

PKS-I and NRPS gene clusters in Nocardia strains varies

Tohru Gonoi2, Christine C. Bii3

carry the largest numbers of clusters among the species

1

Jomo Kenyatta University of Agriculture and Technology,

work have seven common PKS-I and/or NRPS clusters, some2

Chiba University Medical Mycology Research Centre-Japan,

brasiliensis strains have some different gene clusters of PKS-I/

3

substantially depending on species, and N. brasiliensis strainsstudied, 3）the seven Nocardia strains studied in the presentof whose products are yet to be studied, and 4）different N.

Institute of Biotechnology Research, Kenya.

Japan.

Kenya Medical Research Institute, Mycology laboratories,

NRPS, although the rest of the clusters are common within

Centre for Microbiology Research, Kenya.

Nocardia strains are highly promising resources in the search

Mycotoxin contamination is a common problem in

the N. brasiliensis strains. Genome sequencing suggested thatof novel secondary metabolites.

developing countries, particularly in cereals, and this poses

14. Taurospongins B and C, new acetylenic fatty acidtraditional brew whose cereal ingredients are prone toa serious health risk to its consumers. Busaa is a Kenyan

derivatives possessing a taurine amide residue from amycotoxin contamination. This study aimed at detecting the

Takaaki Kubota 1 , Haruna Suzuki 1 , Azusa Takahashi-Busaa samples were collected from homesteads involved inmarine sponge of the family Spongiidae.

Nakaguchi 2 , Jane Fromont 3 , Tohru Gonoi 3 ,Kobayashi
1

2

3

1

Jun’
ichi

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

presence and subsequently quantifying aflatoxin, fumonisinand deoxynivalenol（DON）, in busaa in Bomet county, Kenya.
brewing in the north eastern part of Bomet East constituency.
Mycotoxins were detected in the samples using the Envirologix

QuickTox kits and quantified using the QuickScan machine

according to the manufacturer’s instructions. Among the

61 samples tested, 93, 9.8 and 23% were contaminated with

Medical Mycology Research Center, Chiba University,

aflatoxin, fumonisin and DON, respectively,（mean: 5.2±

Western Australian Museum, Locked Bag 49, Welshpool

range 280 to 4,000 µg/kg, mean 259±5.2 µg/kg, range 200

Chiba 260-0856, Japan
DC, Australia

Two new acetylenic fatty acid derivatives possessing ataurine amide residue, taurospongins B（1）and C（2）,have been isolated from an Okinawan marine sponge ofthe family Spongiidae. The gross structures of 1 and 2 were0.2 µg/kg, range: 2.8 to 11 µg/kg; mean 1,460±188 µg/kg,

to 360 µg/kg, respectively）. Although traditional brews are

not directly included in the European law on mycotoxins, it

is important to consider their mycotoxin levels. In this study,busaa is a mainly a maize product and also the European

Union（EU）guidelines on mycotoxins in maize were used as
reference. It was found out that 65.6% of busaa had aflatoxin

elucidated on the basis of their spectral data, especially 2Dlevels above the limit set in the EU guideline（4 µg/kg ）.

for 1 and 2 were established by chemical means. Taurospongin

within the set limits（fumonisins: 4,000 µg/kg; DON: 1,750

NMR and FABMS/MS data. The absolute configurations

C（2）showed inhibitor y activity against Cryptococcus
neoformans.

Although, the average levels of fumonisin and DON wereµg/kg）, studies have shown that chronic exposure to multiple
mycotoxins has detrimental health effects. Therefore, thereis need for mycotoxicological quality control of traditionallyproduced brews for public mycotoxicological safety.

40

千葉大学

真菌医学研究センター報告

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16. Lectin-microarray technique for glycomic profiling of

fungal cell surfaces.
1

Brazil.

Tetsuhiro Matsuzawa1,, Galba M. Campos Takaki2, Takashi

Laboratory for Inflammatory Regulation, RIKEN Research

Yaguchi1, Kaoru Okada2, Tohru Gonoi1, Yoshikazu Horie1

22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-1

Medical Mycology Research Center, Chiba University,

2

Center for Integrative Medical Science（IMS-RCAI）, 1-7-

2

lated from caatinga soil in the State of Pernambuco,

2

Azusa Shibazaki , Tohru Gonoi
1

17. Two new species of Aspergillus section Fumigati iso-0045, Japan

Inohana 1-8-1, Chuo-ku, Chiba, 260-8673, Japan

1 Inohana, Chuo-ku, Chiba 260-8673, Japan

Catholic University of Pernambuco, Rua do Príncipe, n.

526, Boa Vista, CEP 50050-900, Recife-Pernambuco,

BrazilLectin microarrays are rows of lectins with different

carbohydrate-binding specificities spotted on surfaces ofMedical Mycology Research Center, Chiba University, 1-8-

Aspergillus caatingaensis and A. pernambucoensis, isolated

glass slides. Lectin microarray technique enables glycomicfrom semi-desert soil in caatinga area, the State ofwill describe an application of the technique in analyzing cell

caatingaensis is characterized by its white cleistothecia, broadly

analyses of carbohydrate composition of fungal cell walls. We

surface glycome of yeast-form fungal cells in the living state.
The analysis reveals genus- and species-dependent complex

cell surface carbohydrate structures of fungi, and enabled us,

Pernambuco, Brazil, are described and illustrated. Aspergillus

lenticular ascospores with four equatorial crests and irregularly

ribbed to slightly reticulate with aculeate convex surfaces,and ellipsoidal to broadly ellipsoidal conidia with a smooth

therefore, to suggest that cell walls of yeast cells, which havewall. Aspergillus pernambucoensis is characterized by its, white

have a more complex structure containing galactose andand irregularly ribbed with tuberculate to verrucate convex

been considered to have relatively simple structures, actuallycleistothecia, lenticular ascospores with two equatorial crests

fucose. This shows that the technique can be used to find new

surfaces, and ovoid to broadly ellipsoidal conidia with aclassification of cells in the fungal kingdom based on cell wall

further by analyses of the β-tubulin, calmodulin and actin

insights into the study of phylogenetic relations and into theglycome.

smooth wall. The validation of these new species is supported

gene sequences.

18. Hikiokoshins A–I, diterpenes from the leaves of Isodon

japonicus.

Naonobu Tanaka1, Eri Tsuji1, Kanae Sakai2, Tohru Gonoi2,

Jun’
ichi Kobayashi1
12Graduate School of Pharmaceutical Sciences, Hokkaido
University, Sapporo 060-0812, Japan

Medicinal Mycology Research Center, Chiba University,

Chiba 260-0856, Japan

Diterpenes, hikiokoshins A-I, and twelve known

diterpenes were isolated from the leaves of Isodon japonicus（Burm. f.）H. Hara（Lamiaceae）. The hikiokoshins A-I

千葉大学

真菌医学研究センター報告

第 18 巻2014

41

possess various skeletons such as ternifonane {hikiokoshin A},

ent-6,7:8,15-diseco-6,8-cyclokauran-7,20-olide {hikiokoshinB}, ent- 6,7-secokauran- 7,20-olide {hikiokoshin C}, and

20. Hyrtimomines, indole alkaloids from Okinawan ma-rine sponges Hyrtios spp.

ent-7,20-epoxykaurane {hikiokoshins D-I}. Their structures

Naonobu Tanaka 1 , Rei Momose 1 , Azusa Takahashi-

Antimicrobial activities of hikiokoshins A and B were

Kobayashi1

were elucidated on the basis of spectroscopic analysis.

evaluated.

1

19. Bromopyrrole alkaloids from a marine sponge Agelas

sp.

1

1

Taishi Kusama , Naonobu Tanaka , Azusa Takahashi-

Nakaguchi 2 , Tohru Gonoi 2 , Jane Fromont 3 , Jun ’ichi

Kobayashi1
1

2
3

Nakaguchi 2 , Tohru Gonoi 2 , Jane Fromont 3 , Jun ’ichi

Graduate School of Pharmaceutical Sciences, Hokkaido

2

3Graduate School of Pharmaceutical Sciences, Hokkaido
University, Sapporo 060-0812, Japan

Medicinal Mycology Research Center, Chiba University,

Chiba 260-8763, Japan

Western Australian Museum, Locked Bag 49, Welshpool

DC, WA 6986, Australia

Six new indole alkaloids, hyrtimomines F–K（1– 6）, wereisolated from Okinawan marine sponges Hyrtiosspp. The

University

structures of 1– 6 were elucidated on the basis of spectroscopic

Western Australian Museum

alkaloid possessing an α-keto-ɛ-caprolactam ring, while

Five new bromopyrrole alkaloids, 2-bromokeramadine

Hyrtimomines H–K（3 – 6）are indole alkaloids possessing β-

Medicinal Mycology Research Center, Chiba University（1）, 2-bromo-9,10-dihydrokeramadine（2）, tauroacidins C（3）and D（4）, and mukanadin G（5）, were isolated froman Okinawan marine sponge Agelas sp. The structures ofanalysis. Hyrtimomine F（1）is a structurally unique bisindole

hyrtimomine G（2）is a symmetrical bisindole alkaloid.

carboline skeleton with an imidazolium unit. Antimicrobialactivities of hyrtimomines F–K（1– 6）were evaluated.

1-5 were elucidated on the basis of spectroscopic data andconformational analysis. Mukanadin G（5）has a tricyclic
skeleton consisting of a fused tetrahydrobenzaminoimidazoleand 2,5-dioxopyrrolidine moieties. Antimicrobial activitiesof 1-3, and 5 as well as three related known bromopyrrole

alkaloids, keramadine（6）, tauroacidin A（7）, andtaurodispacamide A（8）were evaluated.

42

千葉大学

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21. Ultra-deep sequencing analysis of the hepatitis A virus

5 -untranslated region among cases of the same out-

break from a single source.

Shuang Wu1, Shingo Nakamoto2, Tatsuo Kanda1, Xia Jiang1,1

12Masato Nakamura , Tatsuo Miyamura , Hiroshi Shirasawa ,3

Gonoi4, Osamu Yokosuka1

2

Department of Gastroenterology and Nephrology, Chiba
University, Graduate School of Medicine, Japan4

will be needed.

22. Primary brain abscess caused by Nocardia otitidiscav-

iarum.

Masaki Ishihara1, Daikei Takada2, Keiji Sugimoto2, Hiroaki

Oguro 1 , Tohru Gonoi 3 , Yasuhiko Akiyama 2 , Shuhei
Yamaguchi1

Department of Molecular Virology, Chiba University,

1

Chiba, Japan

2

Organization Chiba Medical Center, 4-1-2 Tsubakimori,3

Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku,
3

determinants associated with disease severity. Further studies

4

Nobuyuki Sugiura , Azusa Takahashi-Nakaguchi , Tohru

1

HAV genome in order to reveal possible unknown genomic

Department of Gastroenterology, National Hospital

Chiba, Japan

Medical Mycology Research Center, Chiba University, 1-8-

1 Inohana, Chuo-ku, Chiba, Japan

Hepatitis A virus（HAV）is a causative agent of acute viral

hepatitis for which an effective vaccine has been developed.

Department of Neurology, Faculty of Medicine, Shimane
University, Japan

Department of Neurosurgery, Faculty of Medicine,

Shimane University, Japan

Division of BioResources, Medical Mycology ResearchCenter, Chiba University, Japan

Diagnosing primary cerebral nocardiosis is difficult. Thiscase report describes a 79 -year-old immunocompetentJapanese woman with a primary brain abscess caused by
Nocardia otitidiscaviarum（ IFM 11321）and reviews theHere we describe ultra-deep pyrosequences（UDPSs）of HAV

findings of 11 previous patients with N. otitidiscaviarum-

outbreak, which arose from a single source, associated withours. Beta-lactams were not effective in our patient, and5’-untranslated region（5’UTR ）among cases of the same

induced brain abscesses. Four patients survived, including

a revolving sushi bar. We determined the reference sequence

the diagnosis required a pathologic analysis of the surgicalmethod. Sixteen UDPSs from this outbreak and one fromadministered to the patient. On antibiotic susceptibility

Nucleotide errors yielded a UDPS error rate of ＜ 1%. Thisbe resistant to amoxicillin-clavulanic acid, ceftriaxone,

from HAV-derived clone from an attendant by the Sanger

another sporadic case were compared with this reference.
study confirmed that nucleotide substitutions of this region

are transition mutations in outbreak cases, that insertion was

specimen. Sulfamethoxazole/trimethoprim（ ST ）was

testing, N. otitidiscaviarum（ IFM 11321）was found to

cefotaxime, cefepime, imipenem and clarithromycin, but

sensitive to amikacin, gentamicin, ST and linezolid.

observed only in non-severe cases, and that these nucleotide

Antimicrobial susceptibility patterns differ among Nocardia

Analysis of UDPSs detected low-prevalence HAV variations

Patients with suspected Nocardia infection should therefore be

substitutions were different from those of the sporadic case.

in 5’
UTR, but no specific mutations associated with severityin these outbreak cases. To our surprise, HAV strains inthis outbreak conserved HAV IRES sequence even if we

species, making species identification important for treatment.

treated empirically with ST and/or amikacin and considered
for surgical management.

performed analysis of UDPSs. UDPS analysis of HAV 5’

UTR gave us no association between the disease severity ofhepatitis A and HAV 5’
UTR substitutions. It might be moreinteresting to perform ultra-deep sequencing of full length

千葉大学

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23. Fusarium napiforme systemic infection: case report withmolecular characterization and antifungal susceptibil-

24. Lung Nocardia elegans infection diagnosed on matrix-

assisted laser desorption ionization-time of flight mass

spectrometry（MALDI-TOF MS）.

ity tests.

Marcela de Souza1, Tetsuhiro Matsuzawa2, Lyra L3, Busso-

Yukimasa Ooi1, Hideyuki Shiba2,3, Koji Nagai2,3, Tomohiro

Lopes AF 4, Tohru Gonoi 2, Schreiber AZ 3, Katsuhiko

Higashiyama 4, Toyofumi Nakanishi 4, Takashi Nakano 1,

1

Department of Internal Medicine, School of Medicine,1

Medical Mycology Research Center, Chiba University,

2

Division of Rheumatology, Osaka Medical College Hospital,

Department of Clinical Pathology, School of Medicine,3

Department of the First Internal Medicine, Osaka Medical

Kamei2, Maria Luiza Moretti4, Plinio Trabasso4

2

3

University of Campinas, Campinas, São Paulo Brazil
Chiba, Japan

University of Campinas, Campinas, São Paulo Brazil

During the last decades, Fusarium spp. has been reported

as a significant cause of disease in humans, especially inimmunocompromised patients, who have high risk of invasive

life-threatening disease. Fusarium species usually reported

as cause of human disease are F. solani, F. oxysporum and F.
verticillioides.

CASE DESCRIPTION: We describe the second case in

the literature of disseminated fusariosis caused by Fusarium

napiforme, that occurred in a 60 -year-old woman with

Akira Ukimura1, Tohru Gonoi5

45Infection Control Office, Osaka Medical College Hospital,
Japan

Japan

College, Japan

Department of Clinical and Labolatory Medicine, Osaka
Medical College, Japan

Medical Mycology Research Center, Chiba University,Japan

A 73-year-old man with adultonset Still’s disease developed

a high fever, coughing, dyspnea and bloody sputum and wastherefore admitted to our hospital. Thoracic X-ray and CT
scans revealed oval lesions in the bilateral lungs. A bacterial

isolate from the sputum was identified to be Nocardia elegans

multiple myeloma after subsequent cycles of chemotherapy.

（ N. elegans ）on comparative 16 S rRNA gene sequencing

the F. napiforme not only by standard morphologic criteria

Flight Mass Spectrometry（MALDI-TOF MS）. The patient

DISCUSSION AND EVALUATION: We identified

and Matrix-Assisted Laser Desorption Ionization-Time ofby macroscopic and microscopic characteristics, but also

recovered following treatment with imipenem/cilastatin andsequencing. The antifungal susceptibility of the F. napiforme

of nocardiosis caused by N. elegans identified on MALDI-

confirmed by molecular biology methods, including

isolates were tested to seven antifungal drugs; the azoles werethe most active drug against all the isolates tested.

amikacin. To the best of our knowledge, this is the first case

TOF MS.

CONCLUSIONS: Fusarium spp. are of relevance in

medical mycology, and their profiles of low susceptibility toantifungal drugs highlight the importance for faster and moreaccurate diagnostic tests, what can contribute to an earlier
and precise diagnosis and treatment.

44

千葉大学

真菌医学研究センター報告

第 18 巻2014

25. Aspergillus huiyaniae sp. nov., a teleomorphic species

in sect. Fumigati isolated from desert soil in China.
1

1

2

Tetsuhiro Matsuzawa , Yoshikazu Horie , Paride Abliz ,Tohru Gonoi1, Takashi Yaguchi1
1

2

Medical Mycology Research Center, Chiba University, 1-81 Inohana, Chuo-ku, Chiba 260-8673, Japan

Department of Dermatology, The First Affiliated Hospital

Cheng J, Koga T, Mikami Y, Li W-J: Gordonia iterans

sp. nov., isolated from a patient with pneumonia. Int J

Syst Evol Microbiol. 64: 3520-2525, 2014.

６）Kirui MC, Alakonya AE, Talam KK, Gonoi T, Bii

CC: Total aflatoxin, fumonisin and deoxynivalenol con-tamination of busaa in Bomet county, Kenya. African
Journal of Biotechnology 13（26）: 2675-2678, 2014.

７）Komaki H, Ichikawa N, Hosoyama A, Takahashi-Na-

kaguchi A, Matsuzawa T, Suzuki K-I, Fujita N, Gonoi

of Xinjiang Medical University, No. 1 Liyushan Road,

T: Genome based analysis of type-I polyketide synthase

Aspergillus huiyaniae, a new teleomorphic species isolated

BMC genomics 15: 323, 2014.

Urumqi, Xinjiang 830053, China

from desert soil in Xinjiang, China, was described andillustrated. Aspergillus huiyaniae is characterized by its

yellowish white to pale yellow cleistothecia, broadly lenticular

ascospores with two equatorial crests and irregularly ribbed

and nonribosomal peptide synthetase gene clusters inseven strains of five representative Nocardia species.

８）Kubota T, Iwai T, Sakai K, Gonoi T, Kobayashi J: Am-

phidinins C-F, amphidinolide Q analogs from marine
dinoflagellate Amphidinium sp. Org Lett16（21）: 5624-

5627, 2014.

to slightly reticulate convex surfaces, and subglobose to ovate９）Kubota T, Suzuki H, Takahashi-Nakaguchi A, Fromont

was supported further by the analyses of the β -tubulin,acetylenic fatty acid derivatives possessing a taurine am-or broadly ellipsoidal conidia with smooth walls. This species
calmodulin and actin gene sequences.

J, Gonoi T, Kobayashi J: Taurospongins B and C, newide residue from a marine sponge of the family Spongi-

idae. RSC Advances 4（22）: 11073-11079, 2014.

Publications（アルファベット順）

１）de Souza M, Matsuzawa T, Lyra L, Busso-Lopes AF,

Gonoi T, Schreiber AZ, Kamei K, Moretti ML, Trabasso P: Fusarium napiforme systemic infection: case10）Kusama T, Tanaka N, Sakai K, Gonoi T, Fromont J,

Kashiwada Y, Kobayashi J: Agelamadins A and B, dimeric bromopyrrole alkaloids from a marine sponge Age-las sp. Org Lett 16（15）: 3916-3918, 2014.

report with molecular characterization and antifungal11）Kusama T, Tanaka N, Sakai K, Gonoi T, Fromont J,２）Hagiwara D, Suzuki S, Kamei K, Gonoi T, Kawamoto

pyrrole alkaloids comprising oroidin and 3-hydroxykyn-susceptibility tests. Springer Plus 3: 492, 2014.

S: The role of AtfA and HOG MAPK pathway in stress
tolerance in conidia of Aspergillus fumigatus. Fungal

Genet Biol 73: 1389-149, 2014.

３）Hagiwara D, Takahashi H, Watanabe A, Takahashi-

Nakaguchi A, Kawamoto S, Kamei K, Gonoi T:
Whole-genome comparison of Aspergillus fumigatus

Kashiwada Y, Kobayashi J: Agelamadins C–E, bromo-

urenine from a marine sponge Agelas sp. Org Lett16（19）:5176-5179, 2014.

12）Kusama T, Tanaka N, Takahashi-Nakaguchi A, Gonoi T,

Fromont J, Kobayashi J: Bromopyrrole alkaloids from amarine sponge Agelas sp. Chem Pharm Bull（Tokyo）62: 499-503, 2014.
（5）strains serially isolated from patients infected with asper-13）Matsuzawa T, Campos Takaki GM, Yaguchi T, Okada

４）Ishihara M, Takada D, Sugimoto K, Oguro H, Gonoi T,

sis, a new species of Aspergillus section Fumigati isolated

gillosis. J Clin Microbiol 52（12）: 4202-4209, 2014.

Akiyama Y, Yamaguchi S: Primary brain abscess causedby Nocardia otitidiscaviarum. Intern Med 53（17）: 2007-2012, 2014.

５）Kang Y, Ming H, Gonoi T, Chen Y, Cao Y, Wang Y,

千葉大学

K, Abliz P, Gonoi T, Horie Y: Aspergillus arcoverdenfrom caatinga soil in State of Pernambuco, Brazil. My-

coscience doi: 10.1016/j.myc.2014.04.006, 2014.

14）Matsuzawa T, Horie Y, Abliz P, Gonoi T, Yaguchi T:

Aspergillus huiyaniae sp. nov., a teleomorphic species in

真菌医学研究センター報告

第 18 巻201445

sect. Fumigati isolated from desert soil in China. Myco-21）Tanaka N, Momose R, Takahashi-Nakaguchi A, Gonoi

15）Matsuzawa T, Takaki GMC, Yaguchi T, Okada K,

alkaloids from Okinawan marine sponges Hyrtios spp.

science 55（3）: 213-220, 2014.

Gonoi T, Horie Y: Two new species of Aspergillus sec-tion Fumigati isolated from caatinga soil in the State ofPernambuco, Brazil. Mycoscience 55（2）: 79–88, 2014.

16）Oarada M. Abe T, Nikawa T, Miki T, Gonoi T:

T, Fromont J, Kobayashi J: Hyrtimomines, indole
Tetrahedron 70: 832-837, 2014.

22）Tanaka N, Tsuji E, Sakai K, Gonoi T, Kobayashi J:

Hikiokoshins A–I, diterpenes from the leaves of Isodon
japonicus. Phytochemistry 102: 205-210, 2014.

Refeeding with glucose rather than fructose elicits greater23）Wu S, Nakamoto S, Kanda T, Jiang X, Nakamuration DOI: http://dx.doi.org/10.1016/j.nut.2014.11.014,

Nakaguchi A, Gonoi T, Yokosuka O: Ultra-deep se-

hepatic inflammatory gene expression in mice. Nutri-

2014.

17）Ooi Y, Shiba H, Nagai K, Higashiyama T, Nakanishi T,

Nakano T, Ukimura A, Gonoi T: Lung Nocardia elegans
infection diagnosed on matrix-assisted laser desorption

ionization-time of flight mass spectrometry（MALDITOF MS）Intern Med 53（18）: 2111-2113, 2014.

18）Sakai K, Trabasso P, Moretti ML, Mikami Y, Kamei

K, Gonoi T: Identification of Fungal Pathogens by Vis-

ible Microarray System in Combination with Isothermal

Gene Amplification. Mycopathologia 178（1-2）: 11-26,
2014.

19）Shibazaki A, Gonoi T: Lectin-microarray technique forglycomic profiling of fungal cell surfaces. Methods in
Molecular Biology 1200: 287-294, 2014.

20）Suzuki H, Kubota T, Takahashi-Nakaguchi A, Fromont

J, Gonoi T, Kobayashi J: Nakijiquinone S（ Ia ）and

M, Miyamura T, Shirasawa H, Sugiura N, Takahashi-

quencing analysis of the hepatitis A virus 5’-untranslated

region among cases of the same outbreak from a single

source. Int J Med Sci 11（1）: 60-64, 2014.

24）Yagi K, Ishii M, Namkoong H, Asami T, Fujiwara H,

Nishimura T, Saito F, Kimizuka Y, Asakura T, Suzuki

S, Kamo T, Gonoi T, Kamei K, Tasaka S, Betsuyaku T,

Hasegawa N: Pulmonary nocardiosis caused by Nocar-

dia cyriacigeorgica in patients with Mycobacterium avium
complex lung disease: two case reports. BMC Infect Dis
14（1）: 684, 2014.

25）石黒

卓 , 高柳洋太郎 , 鍵山奈保 , 渡邊

杉田

一施設の後方視的研究 ―日本呼吸器学会誌 . 印刷
中 , 2014.

sponge of the family Spongiidae. Chem Pharm Bull（To-kyo）62（2）: 209-212.

千葉大学

哲 , 五ノ井透 , 亀井克彦 ,裕 . 肺ノカルジア症の12例の臨床的検討 ―単Nakijinol C（Ib）, new meroterpenoids from a marine46

昇 , 篠原和歌子 , 田村仁樹 , 高久

真菌医学研究センター報告

第 18 巻2014

高橋 PI（微生物創生）プロジェクト
Project for Systems Biology of Microorganisms
研究概要（Summary）
当分野では , 計算機を駆使して新たな生物学的知見の発見を目指しています . 一つは、膨大な実験
データを対象にデータ処理技術の開発を通した生命の理解を目指した「バイオインフォマティクス」の研究を行っています . また、生命を真にシステムとして理解することを目的とした「システムズバイ
オロジー」の研究も進めています .

Our research areas are Systems Biology and Bioinformatics. Our Bioinformatics approach aims to deeply
and clearly understand massive biological experiment data, e.g., sequence data by next generation sequencers.
Systems Biology aims to understand how biological systems work and help the experimental design mainly bymathematical modelling approach.

准

教

授

高橋

弘喜

1 . Whole-Genome Comparison of Aspergillus fumigatus

Strains Serially Isolated from Patients with Aspergillosis.

Daisuke Hagiwara a, Hiroki Takahashia, Akira Watanabea,b,aaAzusa Takahashi-Nakaguchi , Susumu Kawamoto , Katsuhikoa,baKamei and Tohru Gonoi

1

2

Hiroki Takahashi

Associate Professor

Medical Mycology Research Center（ MMRC ）, Chiba

comparison showed that three of the five isolates from the IPA

patient carried a mutation, while 22 mutations, including six

nonsynonymous ones, were found among three isolates fromthe aspergilloma patient. One aspergilloma isolate carried the

cyp51A mutation P216L, which is reported to confer azole

resistance, and it displayed an MIC indicating resistance toitraconazole. This isolate harbored five other nonsynonymous

mutations, some of which were found in the afyap1 andUniversity, Chiba, Japan

aldA genes. We further identified a large deletion in the

Chiba University Hospital, Chiba, Japanfinding suggested the possibility that genomic deletions can

The emergence of azole-resistant strains of Aspergillus

our results revealed dynamic alterations that occur in the

Division of Control and Treatment of Infectious Diseases,

fumigatus during treatment for aspergillosis occurs by amutation selection process. Understanding how antifungal

resistance mechanisms evolve in the host environment during

aspergilloma isolate in a region containing 11 genes. Thisoccur during chronic infection with A. fumigatus. Overall,A. fumigatus genome within its host during infection andtreatment.

infection is of great clinical importance and biological interest.

Here, we used next-generation sequencing（NGS）to identify

mutations that arose during infection by A. fumigatus strains

sequentially isolated from two patients, one with invasive
pulmonary aspergillosis（IPA）（five isolations）and the otherwith aspergilloma（three isolations）. The serial isolates had

identical microsatellite types, but their growth rates andconidia production levels were dissimilar. A whole-genome

千葉大学

真菌医学研究センター報告

第 18 巻201447

2 . Antisense Transcription Regulates the Expression ofintestines. In this study, we explored the role of iron in LEE

the Enterohemorrhagic Escherichia coli Virulence（locus for enterocyte effacement）virulence gene expression

Iron Concentration.

genes, the expression of LEE genes was greatly reduced in fur

Regulatory Gene ler in Response to the Intracellular

1

2

3

4

Toru Tobe , Hilo Yen , Hiroki Takahashi , Yoko Kagayama ,4

4in EHEC. In contrast to the expression of Fur-regulated

mutants irrespective of the iron concentration. The expressionof the ler gene, the LEE-encoded master regulator, was

Naotake Ogasawara , Taku Oshima

affected at a post-transcription step by fur mutation. Further

1of the ler gene by increasing the intracellular concentration

2

3

4

Department of Biomedical Informatics, Graduate School of

Medicine, Osaka University, Suita, Osaka, Japan.

Department of Microbiology and Immunology, Graduate

School of Medicine, Osaka University, Suita, Osaka, Japan.

Medical Mycology Research Center, Chiba University,

Chiba, Chiba, Japan.

Graduate School of Information Science, Nara Institute of
Science and Technology, Ikoma, Nara, Japan.

Enteric pathogens, such as enterohemorrhagic E. coli

（EHEC）O157: H7, encounter varying concentrations ofiron during their life cycle. In the gastrointestinal tract, theanalysis showed that the loss of Fur affected the translationof free iron, and the transcription of the antisense strand wasnecessary for regulation. The results indicate that LEE gene

expression is closely linked to the control of intracellular freeiron homeostasis.
Publications1 ）Hagiwara D, Takahashi H, Watanabe A, Takahashi-

Nakaguchi A, Kawamoto S, Kamei K, Gonoi T:
Whole-genome comparison of Aspergillus fumigatus

strains serially isolated from patients with aspergillosis. J

Clin Microbiol. Dec; 52（12）: 4202-9, 2014.

amount of available free iron is limited because of absorption

2 ）Tobe T, Yen H, Takahashi H, Kagayama Y, Ogasawara

developed sophisticated iron-responsive systems to utilize

expression of the enterohemorrhagic Escherichia

by host factors. EHEC and other enteric pathogens have

N, Oshima T: Antisense transcription regulates thelimited iron resources, and these systems are primarilycoli virulence regulatory gene ler in response to the

could be a signal that controls gene expression in thee101582, 2014.

regulated by the Fur repressor protein. The iron concentration

48

千葉大学

intracellular iron concentration. PLoS ONE Jul 9; （9 7）:

真菌医学研究センター報告

第 18 巻2014

バイオリソース管理室
Management of Unit of Microbiological Resources
研究概要（Summary）
病原真菌・放線菌の「保存・管理・提供」体制を整備し , 最新情報が付加された信頼できる菌株の提供を通じて , 真菌症ならびにその原因菌の研究・教育の基盤を支援している .

We are developing a system for preservation, management and distribution of pathogenic fungi andactinomycetes. We support the base of research and education of mycoses and their pathogens in order tosupply reliable strains that are added new information.

准

教

授

助
技

教
術

職

員

技 術 補 佐 員
技 術 補 佐 員

1.

矢口
田中
伊藤
長村
山中

貴志
玲子
純子
由美
美花

Associate Professor
Assistant Professor

Research Technician

Research Promotion Technician
Research Promotion Technician

New species in Aspergillus section Fumigati fromreclamation sites in Wyoming（U.S.A.）and revision of
A. viridinutans complex.
1

1,2

2

3

A. Nováková , V. Hubka , Z. Dudová , T. Matsuzawa ,A. Kubátová2, T. Yaguchi3, M. Kolařík2
1

2

3

Department of Botany, Faculty of Science, Charles

Takashi Yaguchi
Reiko Tanaka
Junko Ito
Yumi Osamura
Mika Yamanaka

calmodulin（ caM ）genes, macro- and micromorphology（optical and scanning electron microscopy）, maximum growthtemperatures and mating experiments. In addition, RNApolymerase II gene（RPB2）, actin（act1）and ITS sequenceswere deposited for the ex-type isolates of newly described
species. The mating experiment results, phylogenetic analyses

and ascospore morphology suggested the presence of fivespecies in the A. viridinutans complex. Aspergillus aureolus

University in Prague, Czech Republic（syn. Neosartorya aureola）was the only homothallic species.

Microbiology of the AS CR, Czech Republicand A. wyomingensis sp. nov., were heterothallic and theirLaboratory of Fungal Genetics and Metabolism, Institute ofMedical Mycology Research Center, Chiba University,Japan

The Aspergillus viridinutans complex inc ludes

morphologically similar, soil-inhabiting species. Although

its species boundaries have not been fully defined, many

Three species, A. felis, A. udagawae（syn. N. udagawae）

morphologically distinguishable teleomorph was inducedby 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 wasamplified. Aspegillus viridinutans and A. aureolus were typified
in accordance with the rules of the new botanical code. Other

isolates from the complex have been isolated as opportunistic

species outside the A. viridinutans complex isolated from thespecies were dominant in spoil sites subjected to various types

well as two new sister species, A. brevistipitatus sp. nov. andhuman and animal pathogens. In the present study, thesereclamation sites were A. fumigatiaffinis and A. lentulus asof reclamation management after coal mining. These species

A. conversis sp. nov. which were closely related each to othermethods, sequence data from the β-tubulin（benA）and

distant from all anamorphic species and resemble A. brevipes,

were characterised using two different PCR-fingerprinting

千葉大学

and to N. papuensis. Both new species are phylogenetically

真菌医学研究センター報告

第 18 巻201449

A. duricaulis and A. unilateralis in micromorphology and are

detection and molecular and metabolic characterization of

distinguishable from each other by the slower growth of A.

heat-resistant fungi and their risk to human health.

the reclamation sites represented A. fumigatus s. str. which

3.

conversis on all tested media. Interestingly, no isolate fromis usually reported as the dominant species from the section

Fumigati in soil.
2.

Occurrence, detection and molecular and metabolic
characterization of heat-resistant fungi in soils andplants and their risk to human health.
12

Tetsuhiro Matsuzawa 2, Yumi Imanishi 3, Seiichi Ueda 4,

Takashi Yaguchi2*

M. Frąc , S. Jezierska-Tys , T. Yaguchi

2

1Institute of Agrophysics, Polish Academy of Sciences,3

Molecular and Environmental Microbiology, Poland

4

Department of Plant and Soil System, Laboratory of

2

3

Department of Environmental Microbiology, University ofLife Sciences, Poland

Medical Mycology Research Center, Chiba University,

Thermoascus, food spoilage fungi.

Kouichi Hosoya1, Motokazu Nakayama1, Daisuke Tomiyama1,

1

3

Risk analysis and rapid detection of the genus

Global R&D-Safety Science, Kao Corporation, Japan

Medical Mycology Research Center, Chiba University,
Japan

Department of Public Health, School of Medicine, Kurume
University, Japan

Graduate School of Human Health Science, University ofNagasaki, Japan

Recently the numbers of spoilage accidents in food industry

Japan

by the species of Thermoasus are increasing, but the risk of

Heat-resistant fungi are often factors causing spoilage ofthat their heat-resistances were higher than those of otherheat-processed products, especially fruit. Contamination offood spoilage have never been evaluated. It became obvious

heat-resist fungi, Byssochlamys, Hamigera and Neosartorya

agricultural raw materials is often as a result of their contact

by our analyses. On the other hand, T. aurantiacus andresistant fungi can be a risk to consumers’health by toxic

production in Potato dextrose broth or Czapek-glucosewith the soil. Materials contaminated by spores of heat-metabolites（mycotoxins）produced by these microorganisms.

Due to the resistance of the fungus to high temperatures

they are able to survive the industry pasteurization process.

B. verrucosa had the idh gene, but they showed no patulin
medium. Therefore, Thermoascus must be discriminated fromother fungi in the food industry. We developed a rapid andhighly-sensitive method of detecting Thermoascus in the genus

Therefore, the only way to prevent the development of theselevel by using PCR. This method is expected to be extremely

by conducting tests for the presence of heat-resistant fungi.

production process.

microorganisms in the product is suitable selecting material

The use of traditional culture methods is long and, therefore,

beneficial for the surveillance of raw materials in the fooddoes not apply in the selecting raw materials for production.

However, time is a critical factor in assessing the acceptanceor rejection of a given batch of raw material, due to thenecessity of processing the raw material fresh, which is very

important especially in the case of fruit. Due to the sparse
literature on rapid detection techniques for heat-resistantfungi in agricultural raw materials in recent years researchers

are looking for methods, effective in the detection of thesepathogens. This review includes characterization of occurrence,

50

千葉大学

真菌医学研究センター報告

第 18 巻2014

4.

Phylogeny, identification and nomenclature of thegenus Aspergillus.
1

1

1

R.A.Samson RA , C.M.Visagie , J.Houbraken , S.-B.

Hong2, V. Hubka3, C.H.W. Klaassen4, G.Perrone5, K.A.

Seifert6, A. Susca5, J.B. Tanney6, J. Varga7, S. Kocsubé7, G.
7

8

9arguments for this decision. We introduce new combinations

for accepted species presently lacking an Aspergillus nameand provide an updated accepted species list for the genus,
now containing 339 species. To add to the scientific value ofthe list, we include information about living ex-type culturecollection numbers and GenBank accession numbers for

Szigeti , T. Yaguchi , J.C. Frisvad

available representative ITS, calmodulin, β-tubulin and1working technique for Aspergillus and propose calmodulin as a

2

3

4

5

6

CBS-KNAW Fungal Biodiversity Centre, The Netherlands

Korean Agricultural Culture Collection, National Academy

secondary identification marker.

Department of Botany, Charles University in Prague, Czech

Publicationsof Agricultural Science, RDA, South Korea
Republic

Medical Microbiology & Infectious Diseases, The

Netherlands

Institute of Sciences of Food Production National ResearchCouncil, Italy

Biodiversity（ Mycology ）, Eastern Cereal and Oilseed

Research Centre, Agriculture & Agri-Food Canada,
7

8

9

RPB2 sequences. In addition, we recommend a standard

Canada１）Fr ą c M, Jezierska-Tys S, Yaguchi T: Occurrence,

detection and molecular and metabolic characterizationof heat-resistant fungi in soils and plants and their risk tohuman health. Advanc Agronomy（in press）.

２）Furusawa H, Miyazaki Y, Shiro Sonoda S, Tsuchiya

K, Yaguchi T, Kamei K, Inase N: Penicilliosis marneffei

complicated with interstitial pneumonia. Intern Med 53:

321-323, 2014.

Department of Microbiology, Faculty of Science and３）Furuya M, Hong S-B, Tanaka R, Kuroda N, Nagashima

Medical Mycology Research Center, Chiba University,

Distinctive expression patterns of GPNMB and folliculin

Informatics, University of Szeged, Hungary
Japan

Department of Systems Biology, Technical University ofDenmark, Denmark.

Y, Nagahama K, Suyama T, Yao M, Nakatani Y:

in renal tumors in patients with Birt-Hogg-Dubé
syndrome. Cancer Science（in press）.

４）Hayashi Y, Eguchi H, Toibana T, Mitamura Y, Yaguchi

T: Polymicrobial sclerokeratitis caused by Scedosporium

Aspergillus comprises a diverse group of species based on

apiospermum and Aspergillus cibarius. Cornea 33: 875-7,

which significantly impact biotechnology, food production,５）Hosoya K, Nakayama M, Tomiyama D, Matsuzawa T,

traditionally associated with nine teleomorph genera,

detection of the genus Thermoascus, food spoilage fungi.

morphological, physiological and phylogenetic characters,indoor environments and human health. Aspergillus was
but phylogenetic data suggest that together with genera

such as Polypaecilum, Phialosimplex, Dichotomomyces andCristaspora, Aspergillus forms a monophyletic clade closely

related to Penicillium. Changes in the International Code of

Nomenclature for algae, fungi and plants resulted in the moveto one name per species, meaning that a decision had to bemade whether to keep Aspergillus as one big genus or to split

2014.

Imanishi Y, Ueda S, Yaguchi T: Risk analysis and rapid
Food Cont 41: 7-12, 2014.

６）Iribe Y, Kuroda N, Nagashima Y, Yao M, Tanaka R,

Gotoda H, Kawakami F, Imamura Y, Nakamura Y,
Ando M, Araki A, Matsushima J, Nakatani Y, Furuya

M: Immunohistochemical characterization of renal

tumors in patients with Birt-Hogg-Dubé syndrome.

Pathol Int（in press）.

it into several smaller genera. The International Commission７）Kano R, Yoshida E, Yaguchi T, Hubka V, Anzawa

instead of using smaller genera. In this paper, we present thetype gene（ MAT 1 - 2）of Trichophyton verrucosum.

of Penicillium and Aspergillus decided to keep Aspergillus

千葉大学

K, Mochizuki T, Hasegawa A, Kamata H: Mating

真菌医学研究センター報告

第 18 巻2014

51

Mycopathologia 177: 87-90, 2014.

８）Kawakami H, Inuzuka H, Mochizuki K, Muto T,16）Shigemura T, Nakazawa Y, Matsuda K, Sano

K, Yaguchi T, Motobayashi M, Saito S, Noda S,

Ohkusu K, Yaguchi T, Yamagishi Y, Mikamo H: Case

Kobayashi N, Agematsu K, Honda T, Koike K: Serial

Infect Chemother 20: 57-60, 2014.

of a patient with disseminated mucormycosis after

Yaguchi T: Aspergillus tubingenesis endophthalmitis

100: 206-209, 2014.

of keratitis caused by Streptomyces thermocarboxydus. J９）Kokuzawa S, Suemori S, Mochizuki K, Hirose Y,

monitoring of Mucorales DNA load in serum samples

allogeneic bone marrow transplantation. Int J Hematol

after cataract surgery with implantation of preloaded17）Umeda S, Tateno M, Miyagi E, Sakurai K, Tanaka

10）Matsuzawa T, Takaki GMC, Yaguchi T, Okada K,

Uterine tumors resembling ovarian sex cord tumors

intraocular lens. Semin Ophthalmol 29: 218-221, 2014.

R, Tateishi Y, Tokinaga A, Ohashi K, Furuya M:

Abliz P, Gonoi T, Horie Y: Aspergillus arcoverdensis,（UTROSCT）with metastasis: clinicopathological study

from caatinga soil in State of Pernambuco, Brazil.

18）Visagie CM, Houbraken J, Frisvad JC, Hong S-B,

11）Novakova A, Hubka V, Dudova Z, Matsuzawa T,

Yaguchi T, Samson RA: Identification and nomenclature

a new species of Aspergillus section Fumigati isolated

Mycoscience（in press）.

of two cases. Int J Clin Exp Pathol 7: 1051-1059, 2014.

Klaassen CHW, Perrone G, Seifert KA, Varga J,

Kubatova A, Yaguchi T, Kolarik M: New species inof the genus Penicillium. Stud Mycol 78 : 343 - 371 ,Wyoming（ U.S.A. ）and revision of A. viridinutans

19）Wakana D, Itabashi T, Kawai K, Yaguchi T, Fukushima12）Ono K, Hayashi H, Tateno M, Tanaka R, Suzuki R,

glycosides, malsterosides A-C, from Malbranchea

serous carcinomas and extensive serous endometrial

20）矢口貴志（他34名）：新菌類用語集（日本菌学会編

of 6 patients. Int J Clin Exp Pathol 7（11）: 7979-7988,

21）矢口貴志：真菌の新分類 . 目で見る真菌と真菌症

13）Onuki T, Goto Y, Kuramochi M, Inagaki M, Bhunchet22）矢口貴志：Neosartorya. ウィルス・細菌・真菌・寄Aspergillus section Fumigati from reclamation sites incomplex. Fung Diver 64: 253-274, 2014.

Maruyama Y, Miyagi Y, Furuya M: Uterine superficial

intraepithelial carcinomas: clinicopathological analysis
2014.

E, Suzuki K, Tanaka R, Furuya M: Radiologically
Indeterminate Pulmonary Cysts in Birt-Hogg-DubéSyndrome. Ann Thorac Surg 97: 682-685, 2014.

14）Samson RA, Visagie CM, Houbraken J, Hong S-B,

K, Goda Y, Hosoe T: Cytotoxic anthrasteroid

filamentosa. J Antibiot 67: 585-588, 2014.

集）日本菌学会 : p199, 2014.

（亀井克彦編）. 医薬ジャーナ社: pp 10-15, 2014.

生虫同定便覧～医薬品 , 医療関係 , 食品 , 水産 , 畜

産 , 農業 , 工業分野～ . 技術情報協会 , 2014（CD出版）.

23）矢口貴志：微生物図鑑

培養・同定と汚染制御

Hubka V, Klaassen CHW, Perrone G, Seifert KA,

Vol. 1 真菌 . サイエンス＆テクノロジー: pp 1-250,

Yaguchi T, Frisvad JC: Phylogeny, identification and24）矢口貴志：菌類と動物・ヒト . 菌類のふしぎ 第２版

Susca A, Tanney JB, Varga J, Kocsubé S, Szigeti G,

nomenclature of the genus Aspergillus. Stud Mycol 78:
141-173, 2014.

15）Sato T, Aoki M, Aoki T, Kubota M, Yaguchi T,

Uzuhashi S, Tomioka K: Fungi isolated from soiled

bean sprouts in Japan. Jpn Agri Res Quar 48: 317-329,

2014.

52

2014.

千葉大学

2014.

形とはたらきの驚異の多様性（国立科学博物館編）東海大学出版: pp 138-140, 2014.

25）矢口貴志 , 西村和子：ヒトの病原真菌類 . 菌類の生
物学

分類・系統・生態・環境・利用（日本菌学会

企画）共立出版 : pp 296-310, 2014.

真菌医学研究センター報告

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文部科学省 ナショナルバイオリソースプロジェクト「病原微生物」
Ministry of Education, Culture, Sports, Science and Technology
National BioResource Project Pathogenic Microorganisms

文部科学省では2002年度からナショナルバイオリソー

development by the national government. After the reviewing

スプロジェクト（NBRP）を開始し , 国が戦略的に整備

the NBRP every five years, in FY2012, the third phase has

供などを行うための体制を整備してきた . その後５年ご

Chiba University ’s Medical Mycology Research Center

することが重要なものについて体系的に収集 , 保存 , 提stared.

との見直しを行い , 2012年度より第３期が開始された .

（MMRC）is the“NBRP Center”for pathogenic microorganism,究センター（病原真菌・放線菌）, 大阪大学微生物病研

actinomycetes）, Osaka University’s Research Institute for

NBRP 病原微生物中核機関である千葉大学真菌医学研究所および岐阜大学大学院医学研究科（病原細菌）と長崎大学熱帯医学研究所（病原性原虫）は , 相互の機関のand this project is carried out by MMRC（pathogenic fungi/

Microbial Diseases（pathogenic bacteria）, Gifu University’sGraduate School of Medicine（pathogenic bacteria ）, and連携を図り , これらの病原微生物株の収集・保存・提供

Nagasaki University’s Institute of Tropical Medicine（pathogenic

生物株として提供し , 感染症と病原体の教育・研究をす

support education and research pertaining to infectious diseases

本プロジェクトは , 今後いかなる感染症が発生してもfor collection, preservation, and distribution of pathogenic

体制を整備して , 高度情報を賦与した信頼できる病原微る人々を支援している .

対応できる病原微生物コレクションを目指している .

In FY2002, the Ministry of Education, Culture, Sports,

Science and Technology（MEXT）implemented the National

BioResource Project（NBRP）to construct the frameworkfor systematic collection, preservation, and distribution of

protozoa）. Working together, they cooperate in various efforts toand pathogens. Specifically, they are developing a system
microorganisms, and they supply reliable strains of pathogenic

microorganisms that are backed by high-level information.
The project aims to establish a reliable and sufficient at

the collection to deal with infectious diseases carried by any

pathogenic microorganisms.

bioresources, with a focus on those that required strategic

千葉大学

真菌医学研究センター報告

第 18 巻201453

長崎大学熱帯医学研究拠点特定領域共同研究「熱帯地域 , 特にアフリカおよびベトナムで発生している真菌症・放射菌症の原因菌の収集と形態学的 , 生理学的 , 分子生物学的解析」プロジェクト

Cooperative Research of Priority Areas with NEKKEN, Nagasaki University
Project for Morphological, Physiological and Molecular Biological Analysis of
Pathogenic Fungi and Actinomycetes Collected in Africa and Vietnam.
長崎大学熱帯医学研究所ケニア拠点を中心に , 上記プ

ロジェクトを展開しています . 現在までにケニア全土の主要穀物（トウモロコシ , 小麦）やミルクなどを汚染するカビ毒（発がん性アフラトキシン他）とその生産菌の解析を進め , 現地食物の多くが , 世界の安全基準値を大きく上回るカビ毒で汚染されていることを明らかにしま
した . 結果は昨年度 , 現地のマスコミにも取り上げられ ,大きな反響を呼び起こしました . また新たに現地で , エ

イズ患者の命を奪う主な原因である真菌感染症 , 特にク

リプトコッカス属菌による感染を中心に疫学的調査を計画しています . 海外での研究は , 現地の研究者や監督官

庁と信頼関係を築き , 許可を得るなど多くの問題を解決

2012 年 2 月

ケニア国キスム市の病院・研究施設問Under assistance of Kenya Research Station, Inst. NEKKEN,

しなければ前進できません . しかし , 現地の医療に貢献

Nagasaki univ., we are analyzing toxins contaminating majorとの友好を深めるために努力を重ねています . 一方これ

We found the local foods are contaminated by the toxins at

し , 人々の生活の質（QOL）の向上を図り , さらに日本らの研究は地球のグローバル化 , 温暖化 , 環境・食糧事

情の悪化が進む中で , 日本の人々の医療や QOL の維持

local grains（maze, wheat）and milks, and also producer fungi.
concentrations far above the international standards. The result

has been announced in newspapers, and received large attention.

にも , 将来大きく貢献するはずです .

A new project for epidemiological study of Cryptococcal fungi in

祭時に愛飲される地ビールが , 原料となるトウモロコ

Medical Res. Insti.（KEMRI）and doctors from UCSF, USA.

体に有害なレベルまで汚染されていることを発表しましof the domestic beer, busaa, which is very popular in Kenyan

特に平成26年度には , ケニアの地域に伝わり冠婚葬

シ , ヒエなどに生息するカビの産生する毒によって , 人. 同年10月からた（論文参照 . また地元の新聞に掲載）
は , ケニア中央研究所の研究員 Olga 氏を日本に招き , ケ

ニアの食糧を汚染するカビとカビ毒 , 皮膚真菌症 , エイズ患者のクリプトコッカス症などについて共同で研究を続けています .

HIV-infected patients is launched in collaboration with Kenya
In 2014 we published on the high risk mycotoxin contamination

local areas and served in several types of celebrations. The results

were published in an academic journal and also announced innationwide papers. In October 2014, we invited Mr. Olga,a research officer of Kenya Medical Research Institute to

Medical Mycology Research Center, Chiba University, andstarted collaborative works on food contaminating fungi andmycotoxins, human pathogenic Cryptococcus in HIV patients,

and dermatophytes in Kenya.

54

千葉大学

真菌医学研究センター報告

第 18 巻2014

アスペルギルス症を中心とした新興真菌症制圧プロジェクト
The Project on Controlling Aspergillosis and the Related Emerging Mycoses
アスペルギルス症はわが国を始めとする先進諸国で最その後（投与終了後７日以降）に肺胞領域の散在性の肉も多くの患者が失われている真菌症であり , いずれの国

芽腫様病変の形成が認められるなど興味深い知見が得らでも大きな問題となっている . 様々な病型を取るが , 侵襲性肺アスペルギルス症と慢性肺アスペルギルス症が ,頻度 , 予後などの点からも早急な解決を求められている疾患といえる . このような病態の相違に加え , 同じ慢性

肺アスペルギルス症の間でも症例により様々な活動性の違いがあることや同一症例でありながら急速な活動性の憎悪を見ることなどの点がアスペルギルス症の大きな特徴であり , これらは本菌による感染機構を理解する上でれた .

Aspergillosis takes various forms such as acute invasive

aspergillosis and chronic necrotizing pulmonary aspergillosis（ CNPA ）, and is the most serous and important fungal

infection in developed countries,. However, little is know as

to its mechanism of infection.

To learn the mechanism of pathogenicity of A. fumigatus,

重要と考えられる . これらの病態における菌株の比較解

we performed comparative genome analysis of A. fumigatus

構の解明 , さらには治療法開発への進展が期待される .

aspergillosis）and CNPA（chronic necrotizing pulmonary

析や動物モデルによる解析などにより病原因子や感染機
我々は昨年度 , 慢性肺アスペルギルス症と侵襲性肺ア

スペルギルス症それぞれの患者（計８人）から単離した原因菌を比較ゲノム学的に解析し , ゲノムには病態にリンクする特徴を見いだせないことを報告した . 今回は ,さらにこの研究を発展させ , 慢性肺アスペルギルス症と侵襲性肺アスペルギルス症各１例の患者から , 経時的に単離したアスペルギルス・フミガータス計８菌株を用

い , 感染中あるいは治療中に同菌に起こる遺伝子突然変異について解析した . 今回の我々の症例では , 治療期間

中に菌の交替が見られなかったにも拘わらず , 単離した菌はそれぞれ様々に異なる遺伝子変異を獲得しており ,strains isolated from total eight IPA（invasive pulmonary
aspergillosis）patients, and reported there was no genomic

difference found which links to the pathological conditions.
In the last year, we developed the research and compared

genome sequences of five and three strains isolated sequentiallyfrom an IPA and CNPA patients, respectively. Based on

MultiLocus Microsatellite typing, we found no alteration of

causative agents within the patients during the observation.
However, we found the eight isolates have their intrinsic

genetic mutations of their own, including a mutation

conferring resistance to an azole-drug, which was used
in a treatment. Our results indicate A. fumigatus strains

その中には , 治療に用いたアゾール薬に対する耐性を与continuously produce several mutant strains even after

ルギルス症原因菌が , 感染後も患者体内で様々な遺伝子of the exacerbation frequently seen in CNPA patients, we

える突然変異なども見いだされた . この結果は , アスペ

変異を獲得し多様化して生存することを示唆する .

また , 慢性肺アスペルギルス症の経過中に出現することがある急性増悪の原因解明を目的とし動物実験モデルの作成を試みている . マウスに菌体由来物質を経気道的に反復投与を行うことによって投与終了後早期（72時間まで）では肺動脈周囲への炎症細胞の集簇が観察され ,千葉大学

dwelling in human body. To better understand the mechanism
made an analysis of mice, which were repeatedly given

fungal extracts intratracheally. After repeated instillation,

pathological examinations showed infiltrate of inflammatory

cells around arteries in 72 hs, and the development of
granulomatous changes in alveolar area in 7days, which may

be related to the exacerbation process in patients.

真菌医学研究センター報告

第 18 巻201455

平成 25 年度 共同利用・共同研究報告2013 Fiscal Year Cooperative Research Program Report
研究課題 ’
13－1

atrR/srbA 二重破壊株と同等の感受性を示したことから ,新規抗真菌薬開発を目指したアスペルギルス
属糸状菌の薬剤耐性機構とシグナル伝達機構の解析

ランスポーターも直接的に制御していることが示された . また , 両末端に GFP を連結させた AtrR と SrbA の細胞内局在解析を行ったところ , AtrR は構成的に核に局在し , SrbA は核膜または小胞体周辺に局在していた .

阿部敬悦・五味勝也（東北大学大学院農学研究科 生物産業創成科学専攻）

川本

AtrR はエルゴステロール生合成のみならず薬剤排出ト進・清水公徳・萩原大祐

一 方 , A. fumigatus の AtrR 破 壊 株 で は 麹 菌 と 同 様 ア

ゾール系薬剤に超感受性を示し , 標的分子 Cyp51A の発現が著しく低下していることが明らかになった . さらに , アゾール耐性に重要であると報告されている ABC（千葉大学真菌医学研究センター）

Molecular mechanisms of drug-resistance and signaltransduction in Aspergilli and their application todevelopment of new antifungal drugs
Keietsu Abe, Katsuya Gomi

（Graduate School of Agricultural Science, Tohoku

University）

Susumu Kawamoto, Kiminori Shimizu, Daisuke
Hagiwara（Medical Mycology Research Center, Chiba University）

研究成果（1）ア スペルギルス属糸状菌のアゾール系薬剤耐性に関与する ABC トランスポーターおよびエルゴステ
ロール生合成系を制御する転写因子の機能解析
麹菌において見出されたアゾール系薬剤耐性に関す

トランスポーター Cdr1B の発現も低下しており , 感染治療において重要なアゾール系薬剤の応答に AtrR が中心的な機能を果たしていることが示唆された . また , AtrR

破壊株の低酸素条件下における生育が著しく悪くなるとともに , マウスを用いた感染実験の結果から病原性も顕著に低くなっていることが明らかとなったことから , 本
菌の病原性にも深く関与していると推察された .

（2）ア スペルギルス属糸状菌の浸透圧シグナル伝達系の機能解析と浸透圧シグナル伝達系下流致死因子の探索

Aspergillus nidulans を中心に二成分性情報伝達系（TCS）-

HOG MAP キ ナ ー ゼ（MAPK） 経 路 が 浸 透 圧 応 答 シ
グナル伝達系として解析されてきた . ゲノム情報からAspergillus 属糸状菌においては , 浸透圧シグナル伝達系が保存されている . TCS は , ヒスチジンキナーゼ（HK）→
リン酸基中間因子（YpdA）→レスポンスレギュレーター

る新規 Zn2Cys6型転写因子 AtrR の Aspergillus fumigatus の（RR）からなり , 下流 HOG-MAPK 経路を負に調節し排出 ABC トランスポーターだけでなく , エルゴステ

創薬標的である . TCS の阻害によるシグナル伝達の遮断

オーソログ破壊株の RNA-seq 解析により , AtrR は薬剤

ロール生合成酵素遺伝子の発現にも関わっていることが示された . 興味深いことに , エルゴステロール生合成酵

素遺伝子は bHLH 型転写因子 SrbA 制御下のものと一致していたことから , これらの遺伝子発現が AtrR と SrbAの２種類の転写因子によって協調的に制御されている可能性が強く示唆された . 麹菌の atrR 破壊株は srbA 破壊株よりもアゾール系薬剤に対して高い感受性を示し ,

56

千葉大学

ている . そのうち TCS の YpdA は必須因子であり , 新規

は , 下流経路を構成的に活性化して致死性を示すと考えられている . これまでに A. nidulans の sskA と srrA 遺伝子の単独および二重欠損株を作出し , さらにそれら欠損株
において ypdA 遺伝子の条件発現株の造成を行った . 本

年度は , これら ypdA 遺伝子条件発現株の ypdA 発現抑制時の表現型を詳細に解析した . ypdA 遺伝子の発現抑制は死形質を示し , ypdA 遺伝子発現抑制下に srrAΔ と srrAΔ

真菌医学研究センター報告

第 18 巻2014

の単独破壊導入で生育が部分回復し , sskAΔ srrAΔ 二重研究課題 ’
13－2産物は非抑制時の10% 以下に低下し , 抗 YpdA ペプチド

Cryptotoccus neoformans の特異なゲノム安定化機構の分子基盤－それを標的とした新規治療戦略を目指して

破壊で完全回復した . 転写抑制により ypdA 遺伝子転写
抗体での Western 解析からも YpdA 蛋白質量が10% 以下に低下した . YpdA 発現量の低下に伴い , HogA MAPKの構成的リン酸化が確認された . sskAΔ は隔壁間長が長くなる形質を示すが , ypdA 遺伝子発現抑制下での sskAΔ

松浦

並に回復した .

川本

srrAΔ 導入では , 隔壁間長が長いままで菌体量が野生型

彰・高田実里（千葉大学大学院融合科学研究科）

進・東江昭夫・高橋弘喜（千葉大学真菌医学研究センター）

発表論文
１）Hagiwara D., Takahashi-Nakaguchi A.,ToyotomeT., Yoshimi A., Abe K., Kamei K., Gonoi T., S.
Kawamoto S.: NikA/TcsC histidine kinase is involved
１ in conidiation, hyphal morphology, and responses

to osmotic stress and antifungal chemicals in Aspergillus

fumigatus. PLoS ONE; 8（12）:e80881（2013）.

２）Hagiwara D.: Yoshimi A., Sakamoto K., Gomi K., and

Abe K.: “Response and adaptation to cell wall stressand osmotic stress in Aspergillus species.”Stress Biology

Molecular basis for specific regulation of
genome integrity in Cryptococcus neoformans
and its application to the development of novel
therapeutic strategies
Akira Matsuura, Misato Takada（Graduate School of Advanced Integration Science, Chiba

University）

Susumu Kawamoto, Akio Toh-e, Hiroki Takahashi

of Yeasts and Fungi: Application for Industrial Brewing（Medical Mycology Research Center, Chiba University）

Elsevier,（2014）In press.

研究成果
Cryptococcus neoformans は環境に常在する担子菌酵母であり , 主に免疫機能の低下した人に感染し重篤なクリプ

and Fermentation. Takagi H. and Kitagaki H. edts,

トコックス症を引き起こす日和見感染真菌として知られている . 本菌は , 環状プラスミドが維持できない , 遺伝子ターゲティングの効率が悪く , 導入された直鎖状DNA 断片の末端に高頻度でテロメア反復配列が付加される , など DNA 修復に関連するユニークな性質をもつ

ことが明らかにされている（Edman, 1992）. 本研究では ,染色体末端の維持機構という観点から C. neoformans 特有のゲノム維持機構を明らかにし , さらにこのような特有のゲノム維持機構が C. neoformans の生活環とどのように関連し , あるいはどのように制御されているかを明らかにすることを目的とした .

本年度はまず , C. neoformans 一倍体細胞にテロメア

DNA 伸長酵素テロメラーゼの相同遺伝子 CnEST2 の遺伝子破壊コンストラクトを導入することで , CnEST2 欠損細胞の作製を試みた . 得られた CnEST2 欠損細胞では染色体末端の構造が大きく変化していることが見いださ
れ , このことから , 本菌においても他生物種と同様 , 染

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色体末端の維持にはテロメラーゼの活性が必須であることが明らかになった . CnEST2 欠損細胞におけるゲノム
再編成の分子機構を明らかにするため , 真菌医学研究センター所蔵の次世代シークエンサーを用いて全ゲノム

シークエンスを行ったところ , 野生型株において複数の研究課題 ’
13－3
病原真菌における一酸化窒素（NO）の生成
機構と生理的役割

テロメア末端近傍に特異的に存在するレトロトランスポ

高木博史

ゾン様配列 Cnl1 が , CnEST2 欠損細胞で高頻度に増幅し ,（奈良先端科学技術大学院大学バイオサイエンス研究科）

タンデムに並んで存在することが明らかになった . しかしながら , Cnl1 が自身の活性により別の染色体座位に転川本進 , 知花博治 , 東江昭夫 , 萩原大祐

（千葉大学真菌医学研究センター）移したことにより生じる新たな連結点の数は染色体末端の数に比べて少ないため , トランスポゾン様配列がゲノ
ム中でコピー数を増加する要因として , 主として組換えを介した機構が関与している可能性が考えられる .

C. neoformans における特異な DNA 修復経路選択の分子機構をさらに解析するため , 相同組換え , 非相同末端結合に関わる進化的に保存された遺伝子の探索を行った
ところ , ゲノム中に RAD52 , RAD51 , MRE11 , DMC1 の相同遺伝子を見いだした . それらのゲノム修復における役割を破壊株を作製することにより検討した結果 , 出芽酵母の相同遺伝子欠損の場合とは異なる表現型が観察さSynthetic mechanism and physiological role ofnitric oxide in pathogenic fungus
Hiroshi Takagi（Graduate School of Biological Sciences, Nara Instituteof Science and Technology）

Susumu Kawamoto, Hiroji Chibana, Akio Toh-e,
Daisuke Hagiwara（Medical Mycology Research Center, Chiba University）

れた . 今後は , テロメラーゼ活性の制御に必要な分子の研究成果を含めた DNA 末端修復機構の選択制の違いが生じる機

ける劇的な環境変化（温度 , 酸素濃度 , 栄養など）によ探索をさらに進めつつ , テロメラーゼ , 組換え関連因子

病原真菌はヒトに感染する際 , 自然環境と生体内にお構を分子の側から解析することで , この生物種の特異なるストレスに曝されている . 真菌はこのようなストレスたいと考えている .

すことから , 一酸化窒素（NO）がシグナル分子として ,DNA 損傷修復のメカニズムと生理的意義を明らかにしに応答し , 耐性を獲得することで , 増殖し , 病原性を示

真菌のストレス耐性や病原性の発揮に関わる可能性が考えられる . 本研究では , 酵母 Saccharomyces cerevisiase に見出した NO の生成機構と生理的役割について , NO 生成に関与する酵素（Mpr1, Tah18）のオルソログが存在す
る病原真菌を用いて解析する . 平成25年度には , 以下の研究成果が得られた .

ま ず , Aspergillus fumigatus や Candida glabrata に つ い ては , プロモーター部位を改変した Tah18オルソログ遺伝

子の発現抑制株をそれぞれ作製し , 解析を行った . その結果 , C. glabrata の Tah18発現抑制株を用いたカイコ感染実験では , 野生型株と比較して毒性が顕著に低下していることを見出した . また , Cryptococcus neoformans については , Mpr1オルソログ遺伝子破壊株を作製し , 解析を行った . その結果 , 50℃の熱ショックに対し , 野生型株

より高い感受性を示すことを見出した .

今後 , Mpr1, Tah18の遺伝子破壊株 , 発現抑制株 , 過剰

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発現株などを用いて , 細胞内 NO レベルを定量するとと発現系を利用して , 活性中心領域を明らかにする研究を原真菌においても Mpr1や Tah18が NO の生成と生理機

ク質を C. neoformans に発現させてたり , 異種発現させたもに , ストレス耐性などの表現型を観察することで , 病能に関与するかどうか考察を行う .

ORF4タンパク質を外部から作用させた時の生育抑制効
果などを検討していきたい .

研究課題 ’
13－4

アスペルギルス症の原因となり , 重篤化をもたらす病

マイコウイルス由来新規抗菌性タンパク質の単離とそれを利用した抗病原性真菌剤の開発
森山
川本

行っており , 今後は , 短縮など加工された ORF4タンパ

裕充（東京農工大学大学院農学府研究院）
進・五ノ井 透・東江 昭夫（千葉大学真菌医学研究センター）

原真菌 Aspergillus fumigatus に対する新たな薬剤開発を目的として , 五ノ井教授の研究グループは , A. fumigatus を弱毒化する新規なマイコウイルスの探索とその応用研究
を , 高橋梓博士を中心として行っているが , 本研究課題においては , マイコウイルス探索や同定に関して共同研究を行っている . これまでの探索研究の結果 , ４種のマイコウイルス由来の２本鎖 RNA ゲノムが同定されておScreening for antifungal proteins in mycoviruses
and development of antifungal agents
Hiromitsu Moriyama（Graduate School of Agriculture, Tokyo University ofAgriculture and Technology）

Susumu Kawamoto, Tohru Gonoi, Akio Toh-e（Medical Mycology Research Center, Chiba University）

研究成果

り , このうち , ２種に関しては , 宿主菌に生育阻害をもたらすことなどが確認できており , マイコウイルスである Partitiviridae 属と Chrysoviridae 属に分類されるが , 既報のウイルスとは類似性は示すが同一ではなく , 新種であることが確認されている . マウス感染を用いた実験でも病原性の抑制効果が示されており , 今後 , 宿主菌に対する作用機作などについての検討や , 上述した MoCV1-A

ORF4の効能などについても , 共同研究を継続させることで実施していくことが望まれる .

して , 菌糸生育抑制 , 異常な色素沈着や分生子形成抑発表論文
１）Urayama S, Fukuhara T, Moriyama H, Toh-e A,
Kawamoto S: Heterologous expression of a gene of
Magnaporthe oryzae chrysovirus 1 strain A disrupts

MoCV1-A ウイルスの遺伝子がコードするタンパク質のneoformans. Microbiology and Immunology 58: 294-302

我々はイネいもち病菌に感染するマイコウイルス

Magnaporthe oryzae chrysovirus, MoCV1-A が 宿 主 菌 に 対制などの生育阻害現象をもたらすことを見出しており ,
うち , パン酵母 Saccharomyces cerevisiae の遺伝子発現系の利用により ORF ４が抗菌性タンパク質をコードするこ

growth of the human pathogenic fungus Cryptococcus（2014）.

とを明らかにしてきた .

本研究においては , MoCV1-A ORF4完全長（820残基）が , ヒト病原性酵母 Cryptococcus neoformans に対しても生育阻害効果を有するか否かの検討を行った . 川本進
教授の研究室で開発された C. neoformans 発現ベクターに

MoCV1-A ORF4タンパク質を発現させたところ , パン

酵母 ADH1プロモーターの共役により , MoCV1-A ORF4タンパク質発現が確認され , ORF4発現の C. neoformans では ,異常な液胞化や生育速度の減少 , そして莢膜多糖形成の.
抑制が確認することができた（原著論文１）

現在 , MoCV1-A ORF4に関しては , パン酵母遺伝子

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研究課題 ’
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が国の国民病として認知されている今日 , 人に大量かつ長期の感作をもたらす真菌のアレルゲン性の有無を

病原性を有するAspergillus niger 及び醸造黒
麹菌のアレルゲン遺伝子の検索検証する研究は意義深いと考える . 現在まで , Aspergillus

fumigatus によるアレルギー性肺炎が真菌が原因のアレルギーとして広く認知されているが , 上述した産業利用さ鎌田洋一

れている真菌群については , 研究がなされていない . 本（国立医薬品食品衛生研究所 , 現岩手大学農学部）

研究では Aspergillus niger および醸造黒麹菌が保有する可（千葉大学真菌医学研究センター）

同定するための第一段階として , カビアレルゲンデータ

横山耕治・髙橋治男

山田 修・高橋 徹（酒類総合研究所）
橋本ルイ子（千葉県衛生研究所）
渡辺麻衣子（国立医薬品食品衛生研究所）
川上裕司・橋本一浩（エフシージー総合研究所）

Systematic screening of allergen genes ofpathogenic Aspergillus niger and domestic
Aspergillus fungi
Yoichi Kamata（National Institute of Health Sciences, present address:

Iwate University）

Koji Yokoyama, Haruo Takahashi

能性のあるアレルゲン遺伝子を , ゲノム情報を利用してベースの作製とゲノム研究への応用手法を検討した .

国際免疫学会が保持するアレルゲンデータベースから , カビアレルゲン部分を抽出し , リスト化した . その
際 , 各アレルゲンのアレルゲン性の強弱を評価し , リス

トに追加した . 具体的には , アレルゲン遺伝子の同定

後 , 組換えアレルゲンタンパク質が作製され , あるいは , 粗抽出物から精製され , 当該真菌アレルギーと臨床診断された患者血清中の IgE 抗体が結合することが確かめられたアレルゲン , および同アレルゲンタンパク質が臨床診断にまで応用できているアレルゲン , 不純物を含んだ粗抽出標品を用いた臨床成績によってのみ同定されたアレルゲンとして , その強弱をランキングした . こ
れらの情報を総括し , インターネット上での検索に適（Medical Mycology Research Center, Chiba University）

合するよう , カビアレルゲンデータベースを構築した .

（National Research Institute of Brewing）

公開されている . 作製したカビアレルゲンデータベース

Osamu Yamada, Toru Takahashi
Ruiko Hashimoto（Chiba Prefecture Institute of Public Health）

Maiko Watanabe（National Institute of Health Sciences）
Yuji Kawakami, Kazuhiro Takahashi（FCG Research Institute）

Aspergillus 属真菌については , その２種のゲノム情報が中のアレルゲン遺伝子に相同性のある遺伝子群が , ２種の Aspergillus 属真菌中にあるかどうか , BLAST 検索を行

い , その結果をデータベース内に追加し , ゲノム中の遺伝子にアクセスできるようにした . 構築したカビアレル

ゲンデータベースは , 一般サーバーを利用しての公開を経て , 現在は岩手大学中のサーバー中に保持し , 運営を研究成果
発酵や醸造は古来より人類が行ってきたもので , 食品

続けている .

の保存性の向上 , 味覚の向上 , 有用化学物質の生成など

公開データベース業へ非常に大きな貢献をしている . 発酵・醸造技術が産

agr.iwate-u.ac.jp/

をもたらし , 人類の発展 , 食文化の発展 , また , 食品産

業化することは , 発酵・醸造食品由来の真菌の操作が大・カビアレルゲンデータベース : http://fungusallergen.

規模に行われることであり , 作業者が大量の真菌の暴露を受けることを意味する . また , 食品処理施設内に , 当

該の真菌が定着することを示す . すなわち真菌を利用する産業分野においては , 単一種の真菌に , 大量かつ長期的に暴露を受けることとなる . アレルギー性疾患が , 我

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研究課題 ’
13－6

37℃でのバイオフィルム形成に比べ39℃のバイオフィル

病原真菌の mild heat stress 応答分子の探索と診断マーカーへの応用
長環（福岡歯科大学）
永尾潤一（福岡歯科大学）
中山浩伸（鈴鹿医療科学大学）
知花博治（千葉大学真菌医学研究センター）

The search of mild heat shock response molecules
and application to a diagnosis marker for thepathogenic fungi
Tamaki Cho（Fukuoka Dental College）
Junichi Nagao（Fukuoka Dental College）
Hironobu Nakayama（Faculty of Pharmaceutical Sciences, Suzuka Universityof Medical Science）

Hiroji Chibana（Medical Mycology Research Center, Chiba University）

研究成果
体外から39℃～ 40℃の温和な加温（mild heat stress）

をして骨折や癌の治療効果を上げる温熱療法がある . 一

方で , ヒトが病原体に感染すると体温は39℃～40℃に上昇し , これによる宿主免疫の活性化が亢進されることが知られている . すなわち , mild heat stress はヒト細胞に重要な生物活性を誘導すると考えられる . 病原真菌

Candida albicans がカテーテルなど医療器具にバイオフィ

ルムを形成しやすく , さらにバイオフィルムという増殖形態により薬剤に抵抗性を示すことが臨床現場で問題視されている . 我々は , C. albicans のバイオフィルム形成時の抗真菌薬効果を37℃（通常体温相当）と39℃, 41℃（発熱体温相当）を比較検討した . その結果 , 細胞壁合成阻

害薬のミカファンギンは16倍 , エルゴステロール合成阻

害薬のフルコナゾールは４倍と37℃と比較して感受性が. さらにこの温度それぞれ増加した（Cho T et al, 2012）
下では菌糸形優位のバイオフィルムを形成する傾向が観察された . C. albicans のバイオフィルム形成に対する

37℃と39℃の温度の影響を調べるために , マイクロアレ

イ法による網羅的な遺伝子発現解析を行った . その結果 ,千葉大学

ム形成では , ２℃の温度差で酵母形増殖にかかわる蛋白遺伝子が強く抑制され , 一方で39℃において菌糸に特異的な表層タンパク遺伝子の発現が強くなった . 本研究における mild heat stress は宿主の発熱刺激を想定したもの

で , そのような環境下で変動する病原真菌の細胞表層蛋
白質は , 宿主応答の有力な候補になると思われる .

研究課題 ’
13－7

Schizophyllum commune による気管支喘息重症化メカニズムの解明
廣瀬晃一（千葉大学大学院医学研究院）
渡邉 哲（千葉大学医学部附属病院）
中島裕史（千葉大学大学院医学研究院）
豊留孝仁（帯広畜産大学）
亀井克彦（千葉大学真菌医学研究センター）

Roles of sensitization to Schizophyllum commune
in the development of severe asthma
Koich Hirose（Department of Allergy and Clinical Immunology,

Graduate School of Medicine, Chiba University）

Akira Watanabe（Department of Control and Treatment of Infectious

Diseases, Chiba University Hospital）

Hiroshi Nakajima（Department of Allergy and Clinical Immunology,

Graduate School of Medicine, Chiba University）

Takahito Toyotome（Obihiro University of Agriculture and Veterinary

Medicine）

Katsuhiko Kamei（Medical Mycology Research Center, Chiba University）

研究成果
真菌への暴露 , 感作は喘息の重症化に関与することが知られている . スエロタケ（SC）は喘息 , アレルギー性

気管支肺真菌症（ABPM）の発症に関与することが報告されている真菌だが , これまでに SC 特異的抗体の効率

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的なスクリーニング方法の報告は無く , 喘息患者における SC 感作率は不明である . 我々はこれまでの共同研究により SC による ABPM 患者血清を用いて主要抗原を探より単離したマンザミンアルカロイド Zamamiphidin A,

Hyrtios 属の海綿より単離したインドールアルカロイド

Hyrtimomine 類 , Plakortis 属の海綿より単離したオキシリ

索・同定し , この主要抗原を用いて SC 特異的抗体測定

ピン Manzamenone 類 , Spongiidae 科の海綿より単離したにおける感作率を検討した結果 , 47名の喘息患者（Step2

びにアセチレン脂肪酸誘導体 Taurospongin 類に , 抗菌おELISA 法を確立した . この ELISA 法を用いて喘息患者

６名 , Step3 29名 , Step4 12名）中 , ４名が SC 特異的 IgG

陽性 , ６名が SC 特異的 IgE 陽性であることが明らかと

メロテルペノイド Nakijiquinone S および Nakijinol C ならよび抗真菌活性が認められた .

今後は , 特異性の高い抗真菌活性を示す化合物の探索なった . さらに SC 特異的抗体陽性喘息患者と陰性喘息を継続して行う予定である .

患者は陰性喘息患者に比較し１秒率（FEV1.0）が有意たため , 本研究ではアスペルギルス , カンジダ , アルテ

発表論文１）Kubota, T.; Ishiguro, Y.; Takahashi-Nakaguchi, A.;
Fromont, J.; Gonoi, T.; Kobayashi, J: “Manzamenones
L-N, dimeric fatty-acid derivatives from an Okinawan
marine sponge of the genus Plakortis ”. Bioorg. Med.

の結果 , SC 特異的 IgE 抗体陽性喘息患者は , 陰性喘息患

２）Kubota, T.; Kamijyo, Y.; Takahashi-Nakaguchi, A.;

いることが明らかとなった . 今後は SC 感作が喘息患者

A, a new manzamine related alkaloid from an Okinawan

患者の呼吸機能を比較した結果 , SC 特異的 IgE 陽性喘息に低下していることが明らかとなった .

これまでの報告でも真菌感作が生じている喘息患者においては , 複数の真菌に感作が生じている傾向が見られルナリアに対する特異的 IgE 抗体の有無を検討した . そ
者に比較し有意にアスペルギルス感作が高率に成立してにおける呼吸機能低下の独立した因子であるか否か , 症

例数を増やした多変量解析が必要と考えられた .

研究課題 ’
13－8

Chem. Lett. 2013, 23 : 244-247.

Fromont, J.; Gonoi, T.; Kobayshi, J: “Zamamiphidinmarine sponge Amphimedon sp.”Org. Lett. 2013, 15 :

610-612.

３）Tanaka, N.; Asai, M.; Takahashi-Nakaguchi, A.; Gonoi,T.; Fromont, J.; Kobayashi, J: “Manzamenone O, new

海洋生物を素材とした抗真菌剤の開発
五ノ井透（千葉大学真菌医学研究センター）
小林淳一 , 久保田高明（北海道大学大学院薬学研究院）

Development of antifungal agents from marine
microorganisms
Tohru Gonoi

trimeric fatty-acid derivative from a marine sponge
Plakortis sp.”Org. Lett. 2013, 15 : 2518-2521.

４）Tanaka, N.; Kusama, T.; Takahashi-Nakaguchi, A.;

Gonoi, T.; Fromont, J.; Kobayashi, J: “Nagelamides

U-W, bromopyrrole alkaloids from a marine sponge

Agelas sp.”Tetrahedron Lett. 2013, 54 : 3794-3796.

５）Tanaka, N.; Momose, R.; Takahashi, Y.; Kubota, T.;

Takahashi-Nakaguchi, A.; Gonoi, T.; Fromont, J.;
Kobayashi, J: “ Hyrtimomines D and E, bisindole

alkaloids from a marine sponge Hyrtios sp.”Tetrahedron
Lett. 2013, 54 : 4038-4040.

（Medical Mycology Research Center, Chiba University）

６）Tanaka, N.; Kusama, T.; Takahashi-Nakaguchi, A.;（Graduate School of Pharmaceutical Sciences, Hokkaido

X-Z, dimeric bromopyrrole alkaloids from a marine

Jun’ichi Kobayashi, Takaaki Kubota
University）

sponge Agelas sp.”Org. Lett. 2013, 15 : 3262-3265.

７）Tanaka, N.; Momose, R.; Takahashi-Nakaguchi, A.;

研究成果
沖縄で採取した Agelas 属の海綿より単離したブロモピ

ロールアルカロイド Nagelamide 類 , Amphimedon 属の海綿

62

Gonoi, T.; Fromont, J.; Kobayashi, J: “Nagelamides

千葉大学

Gonoi, T.; Fromont, J.; Kobayashi, J: “Hyrtimomines,

indole alkaloids from Okinawan marine sponges Hyrtios

spp.”Tetrahedron 2014, 70 : 832-837.

真菌医学研究センター報告

第 18 巻2014８）Suzuki, H.; Kubota, T.; Takahashi-Nakaguchi, A.;

dimeric bromopyrrole alkaloids from a marine sponge

S and nakijinol C, new meroterpenoids from a marine

10）Kubota, T.; Suzuki, H.; Takahashi-Nakaguchi, A.;

Fromont, J.; Gonoi, T.; Kobayashi, J: “Nakijiquinone

sponge of the family Spongiidae”Chem. Pharm. Bull.
2014, 62 : 209-212.

９）Kusama, T.; Tanaka, N.; Sakai, K.; Gonoi, T.; Fromont,J.; Kashiwada, Y.; Kobayashi, J:“Agelamadins A and B,

千葉大学

Agelas sp”Org. Lett . 2014, 16(15): 3916-3918.

Fromont, J.; Gonoi, T.; Kobayashi, J:“Taurospongins B

and C, new acetylenic fatty acid derivatives possessing ataurine amide residue from a marine sponge of the family
Spongiidae”RSC Advances 2014, 4 : 11073-11079.

真菌医学研究センター報告

第 18 巻201463

平成 25 年度 共同利用・共同研究研究会報告2013 Fiscal Year Cooperative Research Meetings Report
研究会－1

活発な議論が行われた . なお , プログラムは以下のとお

感染症研究
グローバルネットワークフォーラム2013りである .

開会の挨拶
山本友子（薬学研究院 教授）

山本友子（千葉大学大学院薬学研究院）
高屋明子（千葉大学大学院薬学研究院）
佐藤慶治（千葉大学大学院薬学研究院）
米山光俊（千葉大学真菌医学研究センター）
川本 進（千葉大学真菌医学研究センター）
亀井克彦（千葉大学真菌医学研究センター）

セッション１
座長：松田和洋（エムバイオテック（株） マイコプラズマ感染症
研究センター長）
石和田稔彦（医学部附属病院

講師）「小児細菌性髄膜炎予防ワクチンと日本への導入効果にInfectious diseases research network forum 2013
Tomoko Yamamoto, Akiko Takata, Keiji Sato

（Department of Microbiology and Molecular Genetics,

Graduate School of Pharmaceutical Sciences, Chiba

University）,

Mitsutoshi Yoneyama, Susumu Kawamoto,Katsuhiko Kamei（Medical Mycology Research Center, Chiba University）

研究成果

関する研究」
西村基（医学部附属病院 / 医学研究院「臨床細菌検査の動向―質量分析計による細菌同定など―」
野呂瀬一美（医学研究院

助教）「トキソプラズマ性網脈絡膜炎の病態解析―ケモカインと T 細胞の動態―」
特別講演１
座長：西城

忍（真菌医学研究センター

金城雄樹（国立感染症研究所

千葉大学学内で進められている数多くの感染症研究についてこれらをネットワーク化し ､ 更なる研究のグレー

助教）

准教授）

室長）

「感染免疫における iNKT 細胞の役割」

ドアップを目指して ､ 昨年度から開始された【千葉大学

特別講演２

感染症研究ネットワーク】であるが , 第２回目となる今

座長：伊藤素行（薬学研究院

年度は平成25年11月30日（土） に千葉大学薬学部120周

常世田好司

年記念講堂（医薬系総合研究棟Ⅱ（German Rheumatism Research Centre Berlin,１階）にて開催された . 前回の「学内の研究ネットワーク化」からさらに進展させ ､ 全国さらに国際的な感染症研究のネットワーク

教授）

Department Head）「感染を記憶する骨髄」

化を目指し ､ 会の名称も「感染症研究グローバルネット
ワークフォーラム」と変更された . 内容は学内の感染症

セッション２

研究グループによる一般演題（計８題）に加え , 第一線

座長：高橋弘喜（真菌医学研究センター

で活躍しておられる国内外の４名の先生方による特別講

星野忠次（薬学研究院

演が行われ , まさにグローバルの名称にふさわしい内容「２価金属原子対を標的とした抗ウイルス薬の開発」となった .

64

参加者は118名に達し ､ 全体を通じて非常に

千葉大学

真菌医学研究センター報告

第 18 巻

2014

准教授）

准教授）

神田達郎（医学研究院

講師）

特別講演４

「B 型肝炎ウイルスに対する肝細胞自然免疫応答」

座長：高屋明子（薬学研究院

特別講演３「新型多剤耐性グラム陰性菌が獲得した耐性機構と座長：八尋錦之助（医学研究院准教授）

飯田哲也（大阪大学微生物病研究所准教授）

荒川宜親（名古屋大学大学院医学系研究科

教授）

それらの地球規模での拡散」

教授）「次世代 DNA シーケンサを用いた感染症研究」

閉会の挨拶

セッション３（真菌医学研究センター長 , 東京大学名誉教授 , 日本

座長：米山光俊（真菌医学研究センター
西城

忍（真菌医学研究センター

教授）

笹川千尋
生物科学研究所常務理事）

准教授）「C 型レクチンと真菌感染防御機構」
西田篤司（薬学研究院

教授）「アレルギー性気管支肺真菌症患者から単離された

schizophyllum commune（和名：スエヒロタケ）が生産する化学物質」

千葉大学

真菌医学研究センター報告

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65

2014 年講演会
2014 Scientific Meetings & Seminars
1 ）東京大学医科学研究所－千葉大学真菌医学研究セン
ター 共同利用・共同研究拠点事業 平成25年度
成果報告会

久保田高明
（北海道大学大学院薬学研究院准教授）

Takaaki Kubota“海洋生物を素材とした抗真菌剤の開発”【午後の部】
医科学研究所と千葉大学真菌医学研究センターとの合同成果報告会

感染症・免疫共同研究領域：
猪腰淳嗣
（北里大学

特別講演：
亀井克彦
（千葉大学真菌医学研究センター

准教授）

Junji Inokoshi

教授）

Katsuhiko Kamei“真菌症の現状と展望”“新規抗ウイルス剤をめざした RNA 干渉制御物質のスクリーニング”

長谷耕二（東京大学医科学研究所

稲田健一（藤田保健衛生大学

Kenichi Inada

客員教授）

Hase Koji“腸内細菌による免疫エピゲノム修飾作用”
千葉大学真菌医学研究センター成果報告：
廣瀬晃一（千葉大学大学院医学研究院

准教授）“ウイルス感染に伴い形成される遺伝子制御ネット
ワークの解明とそれに基づく新規診断マーカーと治療標的の同定”江下優樹 , Lucky Ronald Runtuwene（大分大学 准教授 , 大分大学 大学院生）

Yuki Eshita, Lucky Ronald Runtuwene

准教授）“ゲノム情報を利用した蚊媒介性疾患制圧のための網羅的発現遺伝子解析”

Koichi Hirose“スエヒロタケ（Schizophyllum commune）特異的 IgE抗体測定 ELISA 法による喘息患者における感作率
の検討”

小川 遼
（東京大学医科学研究所

森山裕充（東京農工大学大学院“Molecular Structure of Herpesviruses by Cryo-Electron
Microscopy”

大学院生）

Ryo Ogawa

准教授）

Hiromitsu Moriyama“マイコウイルス由来新規抗菌性タンパク質の単離とそれを利用した抗病原性真菌剤の開発”

呉羽 拓（沖縄科学技術大学院大学

松浦 彰（千葉大学大学院融合科学研究科“胸腺上皮細胞における CCR4-NOT 複合体の生理学的意義の解明”

Taku Kureha

教授）

Akira Matsuura“Cryptococcus neoformans の特異なゲノム安定化機構の分子基盤－それを標的とした新規治療戦略を目指
して－”

66

千葉大学

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第 18 巻

2014

研究員）

牧野晶子
（京都大学ウイルス研究所

特別講演：
特定助教）
長谷耕二
（慶応義塾大学薬学部大学院薬学研究科 教授 , 東大医科学研究所国際粘膜ワクチン開発研究セン
ター粘膜バリア学分野 客員教授）

Akiko Makino“内在性フィロウイルスの機能解析”小澤 真（鹿児島大学

准教授）

Makoto Ozawa“A 型インフルエンザウイルス感染動態の生体内ラ
イブイメージング”

伊藤量基（関西医科大学

脇田隆字（国立感染症研究所・ウイルス第二部
准教授）

Itou Tomoki“ヒト Toll 様受容体の発現と機能の解析”
日時 : ３月７日（金）午後の部 13時30分～
場所 : 東京大学医科学研究所 講堂
2 ）第33回知の拠点セミナー（国立大学共同利用・共同研究拠点セミナーシリーズ）
講

Koji Hase“腸内細菌による工ピジェネティクス制御を介したTreg 分化誘導機構の解明”

Takaji Wakita“DAA 時代における C 型肝炎ウイルス研究”
山崎 晶（九州大学生体防御医学研究所感染ネットワーク研
究センター免疫制御学分野 教授）

Sho Yamasaki“結核菌を認識する受容体と免疫応答”
荒瀬 尚（大阪大学免疫学フロンティア研究センター免疫化学研究室 教授 , 大阪大学微生物病研究所 免疫
化学分野 教授）演：

亀井克彦（千葉大学真菌医学研究センター

部長）

教授）

Hisashi Arase“ペア型レセプターを介した宿主病原体相互作用”

Katsuhiko Kamei“生活環境におけるカビと健康被害”
日時：平成26年６月20日（金）17：30～
場所：京都大学東京オフィス
3 ）第３回感染症研究グローバルネットワークフォーラ
ム2014（共催：千葉大学真菌医学研究センター共同
利用・共同研究拠点事業 , 千葉大学 COE スタート
アップ“病原体感染と免疫応答の統合的解析拠点”,
東京大学医科学研究所社会連携研究部門 , 特別推進
研究“病原細菌の自然免疫克服戦略の解明とその応用”, 基盤研究 A“エフェクターと宿主標的分子間相互作用を基盤としたサルモネラ感染分子機構の解明”, 厚生労働科学研究費補助金“肝炎等克服実用化研究事業”（B 型肝炎創薬実用化等研究事業）“Ｂ型肝癌における自然免疫の機能解明とその制御による発癌抑止法開発”）

中山俊憲（千葉大免疫発生学

教授）

Toshinori Nakayama“Pathogenic 記憶と Th2細胞の形成と維持機構”
一般講演：
相馬亜希子
（千葉大園芸学研究科

助教）

Akiko Soma

“病原性大揚菌 O157株の宿主感染に関わる低分子
RNA の同定と機能解析”
高屋明子（千葉大薬学研究院

准教授）

Akiko Takaya,“グラム陽性菌の内因性 rRNA 修飾と薬剤耐性”

千葉大学

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2014

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村田武士
（千葉大理学研究科

６）真菌医学研究センター Monthly セミナー
（東京大学医科学研究所 細菌感染生物学社会連携研
究部門共催）

教授）

Takeshi Murata“創薬標的膜蛋白質の体系的構造解析技術の確立に
向けて”
猪狩英俊
（医学部附属病院感染症管理治療部

教授）

Shoichiro Kurata

部長）

Hidetoshi Igari,“Non-communicable Oiseases が日本の塗抹陽性結核
治療成績へ及ぼす影響について”

加藤直也（東京大学医科学研究所先端ゲノム医科学分野
教授）

倉田祥一朗
（東北大学大学院薬学研究科

准Naoya Kato

“ショウジョウバエ自然免疫における病原細菌の認
識と排除”
日時：９月30日（火）15時～
場所：真菌医学研究センター

大会議室７）真菌医学研究センター Monthly セミナー
（東京大学医科学研究所 細菌感染生物学社会連携研
究部門共催）“肝炎ウイルス制御後の肝発がん抑止戦略”

平原 潔（千葉大学大学院医学研究院

日時：平成26年11月15日（土）９：30～16：50
場所：千葉大学医学部記念講堂

Kiyoshi Hirahara“CD４ T 細胞を介した免疫恒常性の制御およびその准教授）

破綻による慢性真菌感染症について”

４）真菌医学研究センター Monthly セミナー
（東京大学医科学研究所 細菌感染生物学社会連携研
究部門共催）
石和田稔彦（千葉大学医学部附属病院

講師）

Naruhiko Ishiwada“小児細菌性髄膜炎予防ワクチンと日本への導入効
果に関する研究”
日時：６月24日（火）16時～
場所：真菌医学研究センター

日時：10月７日（火）16時～
場所：真菌医学研究センター

８）真菌医学研究センター Monthly セミナー
（東京大学医科学研究所 細菌感染生物学社会連携研
究部門共催）
尾野本浩司（千葉大学真菌医学研究センター 助教）

Koji Onomoto,

大会議室５）真菌医学研究センター Monthly セミナー
（東京大学医科学研究所 細菌感染生物学社会連携研
究部門共催）
豊留孝仁（帯広畜産大学 動物・食品検査診断センター 講師）

Takahito Toyotome“細胞質内ウイルス感染センサー RLR の機能解析”
矢部力朗（千葉大学真菌医学研究センター

68

日時：12月９日（火）10時～
場所：真菌医学研究センター

大会議室

千葉大学

特任助教）

Rikio Yabe
“抑制性 C 型レクチン受容体による骨代謝機構”“真菌関連疾患から考える真菌と宿主のかかわり”
日時：７月22日（火）16時～
場所：真菌医学研究センター

大会議室

真菌医学研究センター報告

第 18 巻2014

大会議室9） 真菌医学研究センター Monthly セミナー

（東京大学医科学研究所 細菌感染生物学社会連携研
究部門共催）
大荒田素子（千葉大学真菌医学研究センター

助教）

Motoko Oarada“現代人の食と炎症”
清水公徳（千葉大学真菌医学研究センター

助教）

Kiminori Shimizu“Aspergillus niger のフモニシン産生に関する遺伝子の機能解析”

日時：12月17日（水）10時～
場所：真菌医学研究センター

大会議室

千葉大学

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第 18 巻201469

平成 27 年 3 月発行
発行者

千葉大学真菌医学研究センター
センター長

笹川

〒260-8673

千葉市中央区亥鼻 1－8－1

電話千尋

043－222－7171（代表）March 2015
Published by

Chihiro Sasakawa, Ph. D.

Director, Medical Mycology Research Center
Chiba University

1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan

TEL: 81-43-222-7171
印刷

株式会社

正文社

Printed by Seibunsha, Ltd. Chiba, Japan

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1

CHIBA UNIVERSITY
2014