ISSN 1347−6920

千葉大学
真 菌 医 学 研 究センタ ー 報 告

MMRC
ANNUAL REPORT OF MEDICAL MYCOLOGYRESEARCH CENTER, CHIBA UNIVERSITY 2013千葉大学 真菌医学研究センター

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CHIBA UNIVERSITY
2013

平成
年

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平成25年

報告

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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 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 Infection 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

3
7
10
13

Project to Link Basic Sciences and Clinical Medicine

15

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

22

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

33

微生物資源分野 バイオリソース管理室

Management Unit of Microbiological Resources

35

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

38

地球規模課題対応国際科学技術協力事業（JST & JICA）

SATREPS Project（Science and Technology Research Partnership for Sustainable Development）

39

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

40

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

41

平成 24 年度 共同利用・共同研究報告2012 Fiscal Year Cooperative Research Program Report42

平成 24 年度 共同利用・共同研究研究会報告2012 Fiscal Year Cooperative Research Meetings Report48

2013 年講演会
2013 Scientific Meetings & Seminars

49

は

じ

め

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・教育に特化した公的な研究センターです .

本センターでは , 限られた人員で最大限の成果を出すべく , 平成 21 年に着任された野本明男前センター長のもとで , 組織を含め様々な改革に取り組んできました . 具体的には , センター全体を 1 部門 4 分野とし , 新たに研究プロジェクト制を導入しました . また , 各プロジェクトを担う 7 名のプロジェクトリーダー PI（principal

investigator）を任命し , 研究レベルの向上と新たな研究分野の導入を図りました . さらに平成 22 年には , 文科省
より共同利用・共同研究拠点としての認定を受け , 「真菌感染症研究拠点」のプラットフォームとして全国の真菌研究者との連携を強化しました . したがって , 本センターでは , PI 個人のプロジェクト研究とともに , 共同利
用・共同研究拠点事業を通じて , 全国の病原真菌研究者と連携した多様な共同研究を活発に行っています . さらに本センターでは , 海外との国際共同研究にも積極的に参加して大きな成果をあげています .
昨年 12 月には , PI プロジェクト及び真菌バイオリソース事業報告会を行い , 真菌感染と自然免疫応答を含
め幾つかの興味ある研究成果が報告されました . 一方 , 共同利用・共同研究拠点では , 成果が大いに期待される重点課題として 8 課題を採択しました . 今回 , その一部について , 3 月に東京大学医科学研究所と合同で成果発表会を行います . 26 年度は , 真菌感染症を含めた難治性感染症の研究とそれらの臨床への応用を目指して , 臨床および異分野との連携をさらに積極的に進める予定です .
さて , 昨年 7 月本センターは , 文科省から共同利用・共同研究拠点としての中間評価を受けました . 本セン
ターでは平成 21 年以来多くの改革に取り組みましたが , 中間評価では大変厳しい評価を頂きました . 具体的には , 研究実績 , 大学院教育への貢献 , 共同研究事業のいずれもが不十分であり , センターの研究機能強化をさらに押し進めることが要望されました . 本センターではこの評価結果を真摯に受け止め , これら課題克服へ向けたさらなる取り組みを行う所存です .
10 月 24 日 , 25 日には , 本センターが当番校として , 国立大学附置研究所・センター長会議第 2 部会（医学・生物学系）のメンバーが , 千葉市内に一同に集まり , 合同会議を開催しました . 会議二日目には ,「超高齢社会に忍び寄るカビの脅威」と題して , 公開シンポジウムを行いました . 本シンポジウムに先立ち , 新聞でもシンポジウムについて紹介され , 当日は一般市民を含む多数の参加者があり , 活発な討論が行われました . ま
た 11 月 30 日には , 本センターと山本友子薬学部教授が共催して , 「感染症グローバルネットワークフォーラ
ム 2013」を薬学部 120 周年記念講堂で開催しました . 本フォーラムでは , 国内外 , 千葉大学医学研究院 , 薬学研究院 , 附属病院から研究者を招き , 感染症の基礎 , 臨床 , 創薬に及ぶ最新の知見を紹介していただきました .
さらに 10 月 10 日には , 本センターの研究が , 日本経済新聞「大学 , 知の明日を築く」という連載特集記事で詳しく紹介されました . 昨年は , これら一連の出来事を通じて , 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 .
超高齢社会に突入した我が国では , 真菌をはじめとする難治性感染症の基盤研究を推進し , それら研究成果
を臨床や創薬開発へ繋げてゆくことが喫緊の課題となっています . 一方 , 世界規模では , 高度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今後大きな問題となります . 本センターでは , これらの現状を踏まえて , 世界に貢献できる研究センターを目指したいと願っております .
平 成 26 年 1 月
千葉大学真菌医学研究センター長

笹

川

千

尋

Preface
It is with great pleasure that I present our Annual Report 2013 for the Medical Mycology Research Center（MMRC）

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

College. The Institute was renovated and renamed the Research Institute for Chemobiodynamics in 1973. Four years
later, it was again renamed as MMRC to reflect a shift in research focus to pathogenic fungi and associated infectious

diseases. In 2001, a division of fungal resources and development was created, which was shortly followed by thecertification of MMRC to serve as a National University Cooperation in 2004. In order to further strengthen the research

activities at MMRC, it was reorganized in 2010 to a single department consisting of four research divisions － molecularbiology of pathogenic fungi, molecular immunology, clinical research, and bioresources. MMRC was certified as a Joint/
Research Center this year because of its central role in leading basic science and clinical mycology research in Japan.

The primary focus of MMRC is to achieve and maintain the highest level of research by employing multidisciplinary

approaches to infection biology including concepts and methodologies of molecular genetics, bioinformatics,
immunology, cell biology, protein chemistry, and clinical research. Currently, MMRC is going to great lengths topromote translational research that is geared towards the prevention, diagnosis, and control of fugal infectious diseases

through joint research projects with the School of Medicine, Hospital, and Faculty of Pharmaceutical Sciences at Chiba
University.

This annual report summarizes our scientific achievements during 2013. My hope is that this report can be used topromote domestic as well as worldwide collaborations with our scientists, which should ultimately result in substantial

contributions to medical fungal research and medicine.
January 22, 2014

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

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

授

准

教

授

准

教

授

助

教

技

術

職

員

客

員

教

授

特

任

助

教

技 術 補 佐 員

Professor

Associate Professor
Associate Professor
Assistant Professor

Research Technician
Guest Professor

Research Assistant Professor

Research Promotion Technician

1 ．The CRZ1 /SP1 -like gene links survival under limited

aeration, cell integrity and biofilm formation in thepathogenic yeast Cryptococcus neoformans.
1,2

1

Zuzana Moranova , Eric Virtudazob, Kristyna Hricova ,Misako Ohkusu2, Susumu Kawamoto2, Vendula Husickova1,
Vladislav Raclavsky1
1

2

Department of Microbiology, Faculty of Medicine andDentistry, Palacky University Olomouc, Czech Republic

Division of Molecular Biology, Medical Mycology Research

Susumu Kawamoto
Masashi Yamaguchi
Koji Yokoyama
Kiminori Shimizu
Misako Ohkusu
Akio Toh-e
Daisuke Hagiwara
Yumiko Nakano

aim of this study was to identify and characterize genes thatare necessary for the proliferation slowdown and G2-arrest

caused by limited aeration.

Methods. Random mutants were prepared and screened

for lack of typical slowdown of proliferation under limited

aeration. The CNAG_ 00156 . 2 gene coding for a zincfinger transcription factor was identified in mutants showing

most distinctive phenotype. Targeted deletion strain andreconstituted strain were prepared to characterize and confirm
the gene functions. This gene was also identified in a parallel

studies as homologous both to calcineurin responsive（Crz1）

Center, Chiba University, Chiba, Japanand PKC1-dependent（SP1 -like）transcription factors.

Aims. Limited aeration has been demonstrated to cause

homologue of CRZ1 /SP1 -like transcription factor in cell

slowdown in proliferation and delayed budding, resulting

eventually in a unique unbudded G2-arrest in the obligate

Results. We have confirmed the role of the cryptococcal

integrity, and newly demonstrated its role in slowdown ofproliferation and survival under reduced aeration, in biofilm

aerobic pathogenic yeast Cryptococcus neoformans. Also, theformation and in susceptibility to fluconazole.

has been identified as a virulence factor in C. neoformans. The

between slowdown of proliferation during hypoxic adaptation

ability to adapt to decreased oxygen levels during pathogenesis

千葉大学

Conclusions. Our data demonstrate a tight molecular link

真菌医学研究センター報告

第 17 巻2013

3

and maintenance of cell integrity in C. neoformans and present

a new role for the CRZ1 family of transcription factors infungi. The exact positioning of this protein in cryptococcal

3 ．Ordered Kinetochore Assembly in the Human-Pathogenic

Basidiomycetous Yeast Cryptococcus neoformans.

signalling cascades remains to be clarified.

Lukasz Kozubowski1,2, Vikas Yadav3, Gautam Chatterjee3,

2 ．A new F-actin structure in fungi: actin ring formation

Indrani Bose5, Joseph Heitman2, Kaustuv Sanyal3

around the cell nucleus of Cryptococcus neoformans.

Marie Kopecká1, Susumu Kawamoto2, Masashi Yamaguchi2
1

Shreyas Sridhar3, Masashi Yamaguchi4, Susumu Kawamoto4,

1

Division of Infectious Diseases, Department of Medicine,

Duke University Medical Center, Durham, North Carolina,
USA; 2 Department of Molecular Genetics and Microbiology,

Department of Biology, Faculty of Medicine, Masaryk

Duke University Medical Center, Durham, North Carolina,

Mycology Research Centre, Chiba University, Chiba, Japan

Biology and Genetics Unit, Jawaharlal Nehru Centre for2

University, Brno 62500, Czech Republic and Medical

The F-actin cytoskeleton of Cryptococcus neoformans

is known to comprise actin cables, cortical patches and

USA;

3

Molecular Mycology Laboratory, Molecular

Advanced Scientific Research, Bangalore, India; 4 Medical

Mycology Research Center, Chiba University, Chiba, Japan;

5

Department of Biology, Western Carolina University,

cytokinetic ring. Here, we describe a new F-actin structure

Cullowhee, North Carolina, USA

nucleus, by fluorescent microscopic imaging of rhodamine

Kinetochores facilitate interaction between chromosomes

in fungi, a perinuclear F-actin collar ring around the cell

phalloidin-stained F-actin. Perinuclear F-actin rings form

and the spindle apparatus. The formation of a metazoan

inhibitor Nocodazole or with the drug solvent dimethyl

middle, and outer layers assemble during disassembly of

in Cryptococcus neoformans treated with the microtubule

sulfoxide（DMSO）or grown in yeast extract peptone dextrose（YEPD）medium, but they are absent in cells treated withtrilayered kinetochore is an ordered event in which inner,

the nuclear envelope during mitosis. The existence of asimilar strong correlation between kinetochore assembly

Latrunculin A. Perinuclear F-actin rings may function as

and nuclear envelope breakdown in unicellular eukaryotes

intracellular funicular suspending nucleus in the central

Saccharomyces cerevisiae and Candida albicans suggest thatalong actin cables.

conserved. Here, we utilized ighresolution time-lapse

funicular cabin for the cell nucleus, and actin cables as

position in the cell and moving nucleus along the polarity axis

is unclear. Studies in the hemiascomycetous budding yeasts

an ordered kinetochore assembly may not be evolutionarily

microscopy to analyze the localization patterns of a seriesof putative kinetochore proteins in the basidiomycetous

budding yeast Cryptococcus neoformans, a human pathogen.

Strikingly, similar to most metazoa but atypical of yeasts,
the centromeres are not clustered but positioned adjacentto the nuclear envelope in premitotic C. neoformans cells.

The centromeres gradually coalesce to a single cluster ascells progress toward mitosis. The mitotic clustering ofcentromeres seems to be dependent on the integrity ofthe mitotic spindle. To study the dynamics of the nuclear

envelope, we followed the localization of two marker proteins,
Ndc1 and Nup107. Fluorescence microscopy of the nuclear

envelope and components of the kinetochore, along with

4

千葉大学

真菌医学研究センター報告

第 17 巻2013

ultrastructure analysis by transmission electron microscopy,

study, we characterized a group III histidine kinase, NikA/

reveal that in C. neoformans, the kinetochore assembles in

TcsC, in the lifethreatening pathogenic fungus, Aspergillus

opening of the nuclear envelope. Taken together, the results

production, abnormal hyphae, marked sensitivity to highan ordered manner prior to mitosis in concert with a partialof this study demonstrate that kinetochore dynamics in C.
neoformans is reminiscent of that of metazoans and shed new

fumigatus. A deletion mutant of nikA showed low conidia

osmolarity stresses, and resistance to cell wall perturbing
reagents such as congo red and calcofluor white, as well as tolight on the evolution of mitosis in eukaryotes.

fungicides such as fludioxonil, iprodione, and pyrrolnitrin.

in progeny is essential for the survival of any organism. A

SskA response regulator and SakA MAPK, which were

IMPORTANCE Successful propagation of genetic material

proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process canlead to loss or gain of chromosomes, a common feature ofmost solid cancers. Several proteins assemble on centromere
DNA to form a kinetochore. However, significant differencesin the process of kinetochore assembly exist between

unicellular yeasts and multicellular metaozoa. Here, weNone of these phenotypes were observed in mutants of thethought to be downstream components of NikA. In contrast,
in response to fludioxonil treatment, NikA was implicated in

the phosphorylation of SakA MAPK and the transcriptional

upregulation of catA, dprA, and dprB, which are regulated

under the control of SakA. We then tested the idea that
not only NikA, but also the other 13 histidine kinases

play certain roles in the regulation of the HOG pathway.

examined the key events that lead to formation of a proper

Interestingly, the expression of fos1, phkA, phkB, fhk5, andneoformans. We found that, during the progression of thea SakA-dependent manner. However, deletion mutants ofkinetochores assembled in an ordered manner concomitantunder the tested conditions. Collectively, although the signal

kinetochore in a basidiomycetous budding yeast, Cryptococcus
cell cycle, nonclustered centromeres gradually clustered andwith partial opening of the nuclear envelope in this organism.

These events have higher similarity to mitotic events ofmetazoans than to those previously described in other yeasts.

4 ．NikA/TcsC histidine kinase is involved in conidiation,

hyphal morphology, and responses to osmotic stressand antifungal chemicals in Aspergillus fumigatus.
1

1

Daisuke Hagiwara , Azusa Takahashi-Nakaguchi , Takahito
12

2

Toyotome , Akira Yoshimi , Keietsu Abe , Katsuhiko
111Kamei , Tohru Gonoi , Susumu Kawamoto1

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

2

New Industry Creation Hatchery Center,

Tohoku University, Sendai, JapanThe fungal high osmolarity glycerol（HOG）pathway iscomposed of a two-component system（TCS）and Hog1-

type mitogen-activated protein kinase（MAPK）cascade. A

group III（Nik1-type）histidine kinase plays a major rolein the HOG pathway of several filamentous fungi. In this

千葉大学

fhk6 increased by osmotic shock or fludioxonil treatment in

the histidine kinases showed no significant defects in growthtransduction network related to NikA seems complicated,

NikA plays a crucial role in several aspects of A. fumigatus

physiology and, to a certain extent, modulates the HOG
pathway.

Publications1 ）Cuia Y, Jiaa H, He D, Yu H, Gao S, Yokoyama K, Li

J, Wang L: Characterization of Edenia gomezpompae

isolated from a keratitis patient. Mycopathologia 176:
75-81, 2013.

2 ）Fukiharu T, Shimizu K, Li RY, Raut JK, Yamakoshi

S, Horie Y, Kinjo N: Coprinopsis novorugosobispora sp.

nov., an agaric ammonia fungus from Beijing, China.
Mycoscience 54: 226-230, 2013.

3 ）Hagiwara D, Takahashi-Nakaguchi A, Toyotome T,

Yoshimi A, Abe K, Kamei K, Gonoi T, Kawamoto S:

NikA/TcsC histidine kinase is involved in conidiation,
hyphal morphology, and responses to osmotic stressand antifungal chemicals in Aspergillus fumigatus. PLoS

ONE 8: e80881, 2013.

4 ）Kaitsu Y, Shimizu K, Tanaka E, Shimano S, Uchiyama

真菌医学研究センター報告

第 17 巻2013

5

S, Tanaka C, Kinjo N: Ophiocordyceps sessilis sp. nov., a9 ）Tuya W, Toyotome T, Yamaguchi M, Takahashi-

ants in Japan. Mycological Progress 12: 755-761, 2013.

A, Taguchi H, Kamei K: Effect of serum components

new species of Ophiocordyceps emerged on Camponotus

5 ）Kopecká M, Kawamoto S, Yamaguchi M: A new

F-actin structure in fungi: actin ring formation around

Nakaguch A, Muraosa Y, Yahiro M, Wang D, Watanabe
on biofilm formation of Aspergillus fumigatus and non-fumigatus Aspergillus. Jpn J Infect Dis, in press.

the cell nucleus of Cryptococcus neoformans. Microscopy10）Yoshimi A, Sano M, Inaba A, Kokubun Y, Fujioka

6 ）Kozubowski L, Yadav V, Chatterjee G, Sridhar S,

M, Yano S, Kasahara S, Shimizu K, Yamaguchi

62: 295-301, 2013.

Yamaguchi M, Kawamoto S, Bose I, Heitman J, Kaustuv

Sanyal: Ordered kinetochore assembly in the human fungal

T, Mizutani O, Hagiwara D, Fujikawa T, Nishimura
M, Kawakami K, Abe K: Functional analysis of the

α-1,3-glucan synthase genes agsA and agsB in Aspergillus

pathogen basidiomycetous yeast Cryptococcus neoformans.

nidulans: agsB is the majorα-1,3-glucan synthase in this7 ）Moráňová Z, Virtudazo EV, Pospíšilová K, Ohkusu M,11）Yamaguchi M: Ultrathin sectioning of the Myojin

mBio 4: e00614-13, 2013.

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

SP1-like gene links survival under limited aeration, cell

integrity and biofilm formation in the pathogenic yeast

Cryptococcus neoformans. Biomedical Papers, in press.

8 ）Tomita M, Kikuchi A, Kobayashi M, Yamaguchi M,

Ifuku S, Yamashoji S, Ando A, Saito A: Chracterizationof antifungal activity of the GH- 46 subclass III

chitosanase from Bacillus circulans MH-K1. Antonie van
Leeuwenhoek J Microbiol, in press.

6

千葉大学

fungus. PLoS One 8: e54893, 2013.

parakaryote discovered in the deep sea. Cytologia, inpress.

12）山口正視 : 原核生物と真核生物の中間の細胞構造をもつ生物の発見 . 顕微鏡 48: 124-127, 2013.

13）山口正視 : 子どもにきかせたい話

菌とウイルス .

こどもと保健 No. 82: 10-11, 光文書院 , 2013.

14）山口正視 : Photo Quiz: Basic mycology. Med Mycol J,
55J: J1-J2, 2014.

真菌医学研究センター報告

第 17 巻2013

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 補 佐 員
技 術 補 佐 員

知花
大岩
相田

博治
真理
優子

Associate Professor

Research Promotion Technician
Research Promotion Technician

1 ．Candida glabrata drug: H＋ antiporter CgQdr2 confers

imidazole drug resistance, being activated by transcription

factor CgPdr1.
1,2

1,2

1,2

Catarina Costa , Carla Pires , Tânia R. Cabrito , Adeline
1,2

3

3

Renaudin , Michiyo Ohno , Hiroji Chibana , Isabel Sá1,2

1,2

Correia and Miguel C. Teixeira
1

Department of Bioengineering, Instituto Superior Técnico,2

Technical University of Lisbon, Lisbon, Portugal, Biological
Sciences Research Group, Institute for Biotechnology

and Bioengineering, Centre for Biological and Chemical

Engineering, Instituto Superior Técnico, Technical Universityof Lisbon, Lisbon, Portugal,

3

Medical Mycology ResearchCenter, Chiba University, Chiba, JapanThe widespread emergence of antifungal drug resistance

Hiroji Chibana
Mari Ohiwa
Yuko Aida

to confer resistance to the antifungal drugs miconazole,tioconazole, clotrimazole, and ketoconazole. By use ofa green fluorescent protein（ GFP ）fusion, the Cg Q dr 2

protein was found to be targeted to the plasma membranein C. glabrata. In agreement with these observations,CgQDR2 expression was found to decrease the intracellularaccumulation of radiolabeled clotrimazole in C. glabrataand to play a role in the extrusion of this antifungal frompreloaded cells. Interestingly, the functional heterologous

expression of Cg Q DR 2 in the model yeast Saccharomyces
cerevisiae further confirmed the role of this gene as a multidrug

resistance determinant: its expression was able to complement

the susceptibility phenotype exhibited by its S. cerevisiae
homologue, QDR2, in the presence of imidazoles and of theantimalarial and antiarrhythmic drug quinidine. In contrast to

the findings reported for Qdr2, CgQdr2 expression does notcontribute to the ability of yeast to grow under K（＋）-limiting

poses a severe clinical problem. Though predicted to play a

conditions. Interestingly, CgQDR2 transcript levels wereof the major facilitator superfamily have largely escaped

clotrimazole or quinidine. This upregulation was found torole in this phenomenon, the drug: H（＋）antiporters（DHA）

seen to be upregulated in C. glabrata cells challenged withcharacterization in pathogenic yeasts. This work describes

depend directly on the transcription factor CgPdr1, the majorreported to be involved in antifungal drug resistance, the C.

which has also been found to be a determinant of quinidine

the first DHA from the pathogenic yeast Candida glabrataglabrata QDR2（CgQDR2）gene（ORF CAGL0G08624g）.

The expression of Cg Q DR 2 in C. glabrata was found千葉大学

regulator of multidrug resistance in this pathogenic yeast,and clotrimazole resistance in C. glabrata.

真菌医学研究センター報告

第 17 巻20137

2 ．The dual role of Candida glabrata drug: H＋ antiporter

CgAqr1（ORF CAGL0J09944g）in antifungal drug
and acetic acid resistance.

Catarina Costa1,2, André Henriques1,2, Carla Pires1,2, Joana

be indirect, presumably through the transport of a stillunidentified physiological substrate. Although neither of thetested chemicals induces changes in CgA QR1 expression,

pre-exposure to flucytosine or clotrimazole was found to

make C. glabrata cells more sensitive to acetic acid stress.

Nunes 1,2, Michiyo Ohno 3, Hiroji Chibana 3, Isabel Sá-

Results from this study show that CgAqr 1 is an antifungal

1

Department of Bioengineering, Instituto Superior Técnico,multidrug resistance.

Sciences Research Group, Institute for Biotechnology

3 ．The Candida glabrata sterol scavenging mechanism,

1,2

1,2

Correia and Miguel C. Teixeira

Technical University of Lisbon, Lisbon, Portugal, 2 Biologicaland Bioengineering, Centre for Biological and Chemical

drug resistance determinant and raise the hypothesis thatit may play a role in C. glabrata persistent colonization andmediated by the ATP-binding cassette transporter

Aus1p, is regulated by iron limitation.

Engineering, Instituto Superior Técnico, Technical Universityof Lisbon, Lisbon, Portugal, 3 Medical Mycology ResearchCenter, Chiba University, Chiba, Japan

Minoru Nagi1, Koichi Tanabe1, Keigo Ueno1, Hironobu

Opportunistic Candida species often have to cope with

Yamagoe5, Takashi Umeyama5,1 Takahiro Oura2, Hideaki

inhibitory concentrations of acetic acid, in the acidic
environment of the vaginal mucosa. Given that the abilityof these yeast species to tolerate stress induced by weakacids and antifungal drugs appears to be a key factor in

Nakayama3, Toshihiro Aoyama1, Hiroji Chibana5, Satoshi

Ohno1, Susumu Kajiwara2 and Yoshitsugu Miyazaki1

1

Department of Chemotherapy and Mycoses, National
Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku2

their persistence and virulence, it is crucial to understandku, Tokyo 162 - 8640 , Japan.

antiporter CgAqr1（ORF CAGL0J09944g）, from Candida

Institute of Technology, 4259 Nagatsuta-cho, Midori-acetic acid, and also to the antifungal agents flucytosine and,Pharmaceutical Sciences, Suzuka University of Medical

to act synergistically with acetic acid against this pathogen.

4

Using a green fluorescent protein fusion, CgAqr 1 was

Mie 510-0294, Japan. 5 Medical Mycology Research Center,the underlying mechanisms. In this study, the drug: H＋

glabrata, was identified as a determinant of resistance toless significantly, clotrimazole. These antifungals were foundKajiwara Laboratory,

Graduate School of Bioscience and Biotechnology, Tokyoku, Yokohama, Kanagawa 226-8503, Japan. 3 Faculty of

Science, 3500-3 Minami Tamagaki-cho, Suzuka, Japan.

Department of Electronic and Information Engineering,

The action of CgAqr 1 in this phenomenon was analyzed.

Suzuka National College of Technology, Shiroko, Suzuka,

found to localize to the plasma membrane and to membrane

Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-

vesicles when expressed in C. glabrata or, heterologously, in

Saccharomyces cerevisiae. Given its ability to complement thesusceptibility phenotype of its S. cerevisiae homolog, ScAqr1,
CgAqr1 was proposed to play a similar role in mediating

8673, Japan.

During disseminated infection by the opportunistic

pathogen Candida glabrata, uptake of sterols such as serum

the extrusion of chemical compounds. Significantly, thecholesterol may play a significant role during pathogenesis.

accumulation of 3H-flucytosine and, to a moderate extent, of

to function as a sterol importer and in this study, we show

expression of this gene was found to reduce the intracellular

3H-clotrimazole, consistent with a direct role in antifungal

The ATP-binding cassette transporter Aus1 p is thoughtthat uptake of exogenous sterols occurred under anaerobic

drug efflux. Interestingly, no effect of CgA QR1 deletion

conditions in wild-type cells of C. glabrata but not in AUS1-

acid, suggesting that its role in acetic acid resistance may

inhibition by fluconazole was prevented in the presence ofcould be found on the intracellular accumulation of 14C-acetic

8

千葉大学

deleted mutant（aus1Δ）cells. In aerobic cultures, growth

真菌医学研究センター報告

第 17 巻

2013

serum, and AUS1 expression was upregulated. Uptake of2 ）Costa C, Pires C, Cabrito TR, Renaudin A, Ohno

and sterol alone did not reverse FLC inhibition of growth.

glabrata drug: H＋ antiporter CgQdr2 confers imidazole

limited by addition of the iron chelators ferrozine or apo-

CgPdr1. Antimicrob Agents Chemother. 57（7）: 3159-

was rescued. In a mouse model of disseminated infection,3 ）Murata M, Takahashi H, Takahashi S, Takahashi

kidney fungal burden than the wild-type strain or a strain in

T, Yamaguchi S, Miyasato H, Murakami M, Kano

sterol by azole treated cells required the presence of serum,

However, if iron availability in the growth medium wastransferrin, growth of wild-type cells, but not aus1Δ cells,

the C. glabrata aus1Δ strain caused a significantly decreased

M, Chibana H, Sá-Correia I, Teixeira MC: Candida

drug resistance, being activated by transcription factor
67, 2013.

Y, Chibana H, Murata Y, Sugiyama K, Kaneshima

which AUS1 was restored. We conclude that sterol uptake in

R, Hasegawa A, Uezato H, Hosokawa A, Sano A:

and thus may contribute to C. glabrata pathogenesis.

（Gallus gallus domesticus）in Japan. Med Mycol. 51（2）:C. glabrata can occur in iron poor environment of host tissues

Publications

1 ）Costa C, Henriques A, Pires C, Nunes J, Ohno M,

Chibana H, Sá-Correia I, Teixeira MC: The dual role

Isolation of Microsporum gallinae from a fighting cock144-9, 2013.

4 ）Nagi M, Tanabe K, Ueno K, Nakayama H, Aoyama T,

Chibana H, Yamagoe S, Umeyama T, Oura T, Ohno
H, Kajiwara S, Miyazaki Y: The Candida glabrataof Candida glabrata drug: H＋ antiporter CgAqr1（ORF

sterol scavenging mechanism, mediated by the ATP-

resistance, Front Microbiol. 26; 4: 170. 2013.

limitation, Mol Microbiol. 88（2）: 371-81, 2013.

CAGL 0 J 09944 g ）in antifungal drug and acetic acid千葉大学

binding cassette transporter Aus1p, is regulated by iron

真菌医学研究センター報告

第 17 巻20139

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 of regulatory molecule（s）, which is

responsible for formation of antiviral stress granule

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

2 ．Encephalomyocarditis virus disrupts stress granules,

the critical platform for triggering antiviral innate（avSG）.

immune responses.

Chen Seng Ng 1,2, Michihiko Jogi, Ji-Seung Yoo1,2, Koji

Koji Onomoto, and Mitsutoshi Yoneyama
Recently, we investigated intra-cellular localization of viral

RNA sensor, retinoic acid-inducible gene I（RIG-I）, in

Onomoto, Satoshi Koike3, Takuya Iwasaki3, Mitsutoshi
Yoneyama, Hiroki Kato1,2 and Takashi Fujita1,2

influenza A virus（IAV）-infected cells, and demonstrated that1

granular-like structure, which we term antiviral stress granule2

as platform for RIG-I-mediated antiviral signaling, it remains

3

infection of IAV induces RIG-I to accumulate in cytoplasmic（avSG）. Although we revealed that avSG plays a critical role

unclear how viral infection activates the signal via avSG

formation. To address this issue, we are trying to identify

regulatory molecule（s）, which is important to form avSG in
response to viral infections, using biochemical approaches.

Laboratory of Molecular Genetics, Institute for Virus
Research, Kyoto University, Kyoto, Japana

Laboratory of Molecular Cell Biology, Graduate School of
Biostudies, Kyoto University, Kyoto, Japanb

Neurovirology Project, Tokyo Metropolitan Institute ofMedical Science, Tokyo, Japan

In response to stress, cells induce ribonucleoprotein

aggregates, SGs. SGs are transient loci containing
translation-stalled mRNA, which is eventually degraded or

recycled for translation. Infection of some viruses, including

10

千葉大学

真菌医学研究センター報告

第 17 巻2013

IAV with a deletion of nonstructural protein 1（IAVΔNS1）,

adapted it for RNP. As model RNP, we prepared artificial

that IAVΔNS 1 -induced SGs are required for efficientwith expression plasmids encoding short viral RNA, His-

SG formation by different viruses using green fluorescenttagged PB2. The formed IAV RNPs in the cells were purified

induces SG-like protein aggregates. Previously, we showed

induction of type I interferon（IFN）. Here, we investigatedprotein（ GFP ）-tagged Ras-Gap SH 3 domain binding
protein 1（ GFP-G 3 BP 1）as an SG probe. HeLa cells

stably expressing GFP-G3BP1 were infected with different

viruses, and GFP fluorescence was monitored live with

time-lapse microscopy. SG formations by different viruses

was classified into 4 different patterns: no SG formation,
stable SG formation, transient SG formation, and alternate

IAV RNP generated in 293T cells, which are transfected

tagged NP and viral RNA polymerases; PA, PB1 and flag-

by affinity chromatography using Ni-conjugated and/or anti-

flag antibody-conjugated beads. Now, we are trying to detect
RIG-I activation in vitro by the artificial IAV RNPs.

4 ．Functional characterization of domains of IPS-1 using

an inducible oligomerization system.

SG formation. We focused on encephalomyocarditis virus

Shiori Takamatsu1,2, Kazuhide Onoguchi1, Koji Onomoto,We found that EMCV disrupts SGs by cleavage of G3BP1 at

Takahiro K. Fujiwara4, Mitsutoshi Yoneyama, Hiroki Kato1,2,（EMCV）infection, which exhibited transient SG formation.

late stages of infection（＞ 8 h）through a mechanism similar

to that used by poliovirus. Expression of a G3BP1 mutant

Ryo Narita 1, Kiyohiro Takahasi 1,3, Fumiyoshi Ishidate 4,

Takashi Fujita1,2that is resistant to the cleavage conferred persistent formation1

cytokines at late stages of infection. Additionally, knockdown

2

attenuated induction of IFN and potentiated viral replication.

3of SGs as well as an enhanced induction of IFN and otherof endogenous G 3 BP 1 blocked SG formation with an

Taken together, our findings suggest a critical role of SGs as

an antiviral platform and shed light on one of the mechanisms

by which a virus interferes with host stress and subsequent
antiviral responses.

3 ．Establishment of in vitro reconstitution assay system

for RIG-I-mediated signaling.

Reiko Hirai, Michihiko Jogi, Ayaha Koyama and Mitsutoshi
Yoneyama

We identified RIG-I as a sensor molecule for viral non-self RNA, however it remains unclear how RIG-I detects

Laboratory of Molecular Genetics, Institute for Virus
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

Kyoto University, Kyoto, Japan

Center for Meso-Bio Single-Molecule Imaging（CeMI）,
Institute for Integrated Cell-Material Sciences（ WPI-

iCeMS）, Kyoto University, Kyoto, Japan

The innate immune system recognizes viral nucleic acids

and stimulates cellular antiviral responses. Intracellulardetection of viral RNA is mediated by the RIG-I Like

Receptor（ RLR ）, leading to production of type I IFN

and pro-inflammatory cytokines. Once cells are infectedwith a virus, RIG-I and MDA5 bind to viral RNA andundergo conformational change to transmit a signal throughviral ribonucleoprotein complex（ RNP ）, which consists

direct interaction with downstream caspase recruitment

protein（ NP ）. In this research, we established in vitro

promoter stimulator- 1（ IPS- 1 , also referred as MAVS/

of viral genomic RNA and proteins, such as nucleocapcid
reconstitution assay system for RIG-I-mediated signaling

domain（ CARD ）-containing adaptor protein, IFN- β

VISA/Cardif）. IPS-1 is composed of N-terminal CARD,

and examined whether viral RNP could activate RIG-I in

proline-rich domain, intermediate domain, and C-terminal

et al., Cell, 2010）, we revised the published methods andanchors it to the mitochondrial outer membrane. It has been

vitro. Since in vitro assay was reported previously（Zeng千葉大学

transmembrane（TM）domain. The TM domain of IPS-1

真菌医学研究センター報告

第 17 巻201311

hypothesized that activated RLR triggers the accumulation2 ）Ng CS, Jogi M, Yoo JS, Onomoto K, Koike S, Iwasaki T,

signal proteins. However, the exact mechanisms of IPS-1-

virus disrupts stress granules, the critical platform forreveal the details of IPS- 1 signaling, we used an artificial

9511-22. 2013.

of IPS-1, which forms oligomer as a scaffold for downstream
mediated signaling remain controversial. In this study, tooligomerization system to induce oligomerization of IPS-

1 in cells. Artificial oligomerization of IPS- 1 activatedantiviral signaling without a viral infection. Using this system,

Yoneyama M, Kato H, Fujita T: Encephalomyocarditis

triggering antiviral innate immune responses. J Virol 87:

3 ）Nitta S, Sakamoto N, Nakagawa M, Kakinuma S,

Mishima K, Kusano-Kitazume A, Kiyohashi K, Murakawa

M, Nishimura-Sakurai Y, Azuma S, Tasaka-Fujita M,

we investigated the domain-requirement of IPS- 1 for its

Asahina Y, Yoneyama M, Fujita T, Watanabe M: Hepatitis

IPS- 1 could overcome the requirement of CARD and the

I-mediated type-I interferon-dependent innate immunity.

signaling. We discovered that artificial oligomerization of

TM domain. Moreover, from deletion- and point-mutant

C virus NS4B protein targets STING and abrogates RIGHepatology 57: 46-58, 2013.

analyses, the C-terminal Tumor necrosis factor Receptor-

4 ）Oarada M, Miki T, Kohno S, Sakai K, Nikawa T,

453-460）present in the intermediate domain is critical for

after a 48 hour fast elicits an inflammatory response in

IPS- 1 oligomerization is essential for the formation of a5 ）Sekai M, Tani-ichi S, Yoneyama M, Fujita T, Kina T,

activation of transcription factors, Interferon Regulatory

IL- 7 expression by interferon regulatory factors. Mol

Associated Factor（ TRAF ）binding motif of IPS- 1（ aa.

downstream signal transduction. Our results suggest thatmultiprotein signaling complex and enables downstream

Factor 3（IRF3）and Nuclear Factor-κB（NF-κB）, leading

to type I IFN and pro-inflammatory cytokine production.
Publications

Yoneyama M, Gonoi T: Refeeding with a standard dietthe mouse liver. J Nutr Biochem 24: 1314-23. 2013.

Ikuta K: Lymphocyte-stromal cell interaction induces
Immunol 54: 378-385. 2013.

6 ）Takamatsu S, Onoguchi K, Onomoto K, Narita

R, Takahasi K, Ishidate F, Fujiwara TK, Yoneyama
M, Kato H, Fujita T: Functional characterization of

1 ）Hayashi Y, Onomoto K, Narita R, Yoneyama M, Kato

domains of IPS-1 using an inducible oligomerization

expression of retinoic acid inducible gene-I and melanoma

7 ）米山光俊 : ウイルスセンサー RLR によるパターン

H, Nakagawa T, Ito J, Taura A, Fujita T: Virus-induced

differentiation-associated gene 5 in the cochlear sensory

epithelium. Microbes Infect 15: 592-8. 2013.

12

千葉大学

system. PLoS One 8: e53578, 2013.

認識とストレス顆粒形成 . 化学療法の領域 29: 107-

114, 2013.

真菌医学研究センター報告

第 17 巻2013

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
技 術 補 佐 員
技 術 補 佐 員

西城
森本
鈴木

忍
雅子
智明

Associate Professor

Research Promotion Technician
Research Promotion Technician

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

fungal infection.

Shinobu Saijo
Masako Morimoto
Tomoaki Suzuki

resulting in the activation of various genes including those
encoding pro-inflammatory cytokines. Both β-glucans andα-mannans are major cell wall components of fungi including

Candida albicans（C. albicans）and Pneumocystis carinii（P.

Shinobu Saijo
Division of Molecular Immunology, Medical Mycology

carini）. Recently, we reported that Dectin-1 is importantin protection against P. carinii by inducing reactive oxygen

Research Center, Chiba University, Chuo-ku, Chiba, Japan

species, whereas both Dectin-1 and Dectin-2 play important

Dectin- 1 and Dectin- 2 are type II transmembrane

Th17 cell differentiation. We are now working to elucidate

roles in defense against C. albicans by preferentially inducing

proteins of the C-type lectin family with single carbohydrate

molecular mechanisms that underlying anti-fungal immunity.

They are expressed mainly in dendritic cells and macrophages.

Publications

recognition domains（CRDs）in their extracellular region.

Dectin-1 recognizes β-glucans with its CRD and transduces

signals through its immunoreceptor tyrosine-based activation

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

Dectin- 2 recognizes α-mannans and transduces its signal

1 ）Ishikawa T, Itoh F, Yoshida S, Saijo S, Matsuzawa

T, Gonoi T, Saito T, Okawa Y, Shibata N, Miyamoto
T, Yamasaki S: Identification of distinct ligands for

the C-type lectin receptors Mincle and Dectin-2 in thethrough association with the ITAM-containing Fc receptor

pathogenic fungus Malassezia. Cell Host Microbe. 13:

recruited to the ITAM and activates the caspase recruitment

2 ）Miyasaka T, Akahori Y, Toyama M, Miyamura N, Ishii

γ chain. Upon ligand binding, spleen tyrosine kinase isdomain family member 9（CARD9）-nuclear factor-κB axis,

千葉大学477-88. 2013.

K, Saijo S, Iwakura Y, Kinjo Y, Miyazaki Y, Oishi K,

真菌医学研究センター報告

第 17 巻2013

13

Kawakami K: Dectin-2-dependent NKT cell activationand serotype-specific antibody production in mice

14

千葉大学

immunized with pneumococcal polysaccharide vaccine.
PLoS One. 8. e78611. 2013.

真菌医学研究センター報告

第 17 巻2013

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当面の最大の目標としつつ , 真菌がどうやって人体に侵入するのかという観点から , 新しい診断や治療の開発を中心に研究を行っている . またこれと並行して , 附属病院における診療活動及び学内外でのコンサルテーションを行なっており , 学外からの依頼は検査を含めて年間 200－
300 件に達している .
実施体制 : 教員 2 名（＋兼務 1 名）技官 1 名 , 特任助教 2 名 , 補助員 2 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 clinic of the University Hospital, while providing consulting services on fungal diseases
to physicians/clinical technologists all over the country.
STAFF: Professors（3, including 1 assistant professor working concurrently in the University Hospital）,
technician（1）, research assistant professors（2）and research assistants（2）are working in our group withfour graduate school students.
教
助
助

亀井
教
田口
教（兼任） 渡辺
授

技

術

職

員

特

任

助

教

特

任

助

教

技 術 補 佐 員
技 術 補 佐 員
技 術 補 佐 員

鎗田
村長
八尋
奥
関
井上

克彦
英昭
哲
響子
保憲
真希
幸夫
里亜
京子

Katsuhiko Kamei
Hideaki Taguchi
Akira Watanabe

Professor

Assistant Professor
Assistant Professor（concurrent position）

Research Technician

Research Assistant Professor
Research Assistant Professor

Research Promotion Technician

Research Promotion Technician
Research Promotion Technician

千葉大学

真菌医学研究センター報告

Kyoko Yarita
Yasunori Muraosa
Maki Yahiro
Yukio Oku
Rio Seki
Kyoko Inoue

第 17 巻201315

1 . A double-blind comparative study of the safety andefficacy of caspofungin versus micafungin in thetreatment of candidiasis and aspergillosis.

Shigeru Kohno1, Koichi Izumikawa1, Minoru Yoshida2, Yoshio

Takesue3, Shinichi Oka4, Katsuhiko Kamei5, Yoshitsugu
6

7

8

Miyazaki , Tomoko Yoshinari , Nicholas A. Kartsonis ,Yoshihito Niki
1

2

9

Department of Molecular Microbiology and Immunology,

4

5

6

7

8

the caspofungin group and 10.0％（6/60）in the micafungingroup［95％ confidence interval（ CI ）for the difference:-15.9％, 5.2％］. The favorable overall response in the PPS

population for patients with esophageal candidiasis, invasive
candidiasis, and chronic pulmonary aspergillosis including

aspergilloma was 100 . 0％（6 / 6）, 100 . 0％（3 / 3）, and46.7％（14/30）in the caspofungin group, and 83.3％（5/6）,
100.0％（1/1）, and 42.4％（14/33）in the micafungin group,

respectively. In Japanese patients with Candida or Aspergillus

Fourth Department of Internal Medicine, Teikyo University

between caspofungin and micafungin. Consistent with otherJapan

micafungin was similar.

Sciences, Nagasaki, Japan

Department of Infection Control and Prevention, Hyogo
College of Medicine, Hyogo, Japan

AIDS Clinical Center, National Center for Global Healthinfections, there was no statistical difference in the safety

data on these two agents, the efficacy of caspofungin and

2 . Histopathological Study of Murine Pulmonar y

Cryptococcosis Induced by Cryptococcus gattii and
Cryptococcus neoformans.

and Medicine, Tokyo, Japan

Department of Clinical Research, Medical Mycology
Research Center, Chiba University, Chiba, Japan

Yoichiro Okubo 1, Megumi Wakayama 1, Hideaki Ohno 2,

Institute of Infectious Diseases, Tokyo, Japan

Yukihiro Kaneko2, Satoshi Yamagoe2, Takashi Umeyama2,

Department of Chemotherapy and Mycoses, National
Japan Development, Vaccine and Infectious Diseases, MSD
K. K., Tokyo, Japan

Clinical Research, Infectious Diseases, Merck ResearchLaboratories, Merck Research Institute, Upper Gwynedd,

9significant drug-related adverse events was 5.0％（3/60）in

Nagasaki University Graduate School of Biomedical

School of Medicine, Mizonokuchi Hospital, Kanagawa,3（PPS）population. The proportion of patients who developedWest Point, PA, USA

Development for Clinical Infectious Diseases, Showa
University, Tokyo, Japan

Shuhei Yamamoto 2, Naobumi Tochigi 1, Koichi Tanabe 2,
Minoru Shinozaki1, Tetsuo Nemoto1, Haruo Nakayama3,

Daisuke Sasai 1, Takao Ishiwatari 1, Kayoko Shimodaira 1,
Yoshiro Yamamoto 1 , Katsuhiko Kamei 4 , Yoshitsugu
Miyazaki2, Kazutoshi Shibuya1,51Department of Surgical Pathology, Toho University Schoolof Medicine

2

Department of Chemotherapy and Mycoses, National

micafungin in Japanese patients with fungal infections were

3

Department of Neurosurgery, Toho University Ohashi

blind study. The proportion of patients who developed

4

Division of Clinical Research, Medical Mycology Researchdrug-related adverse event or a drug-related adverse event

5

Department of Dermatology, Peking University FirstThe safety and efficacy profile of caspofungin anddirectly compared in this prospective, randomized, double-

significant drug-related adverse event（s）（defined as a serious

leading to study therapy discontinuation）was compared in

120 patients［caspofungin 50 mg, or 50 mg following a 70-

mg loading dose on Day 1（hereinafter, 70/50 mg）group:

60 patients; micafungin 150 mg: 60 patients］. The overall

response rate was primarily evaluated in the per-protocol set

16

千葉大学

Institute of Infectious Diseases

Medical Center

Center, Chiba University
Hospital

Although Cryptococcus gattii can cause life-threatening

complications, putative virulence factors of C. gattii remain

controversial. Therefore, we conducted the present study

真菌医学研究センター報告

第 17 巻2013

to elucidate the virulence factors of the yeast and found thatfor the identification of non-albicans Candida spp. wassignificantly higher than that of those infected with C. gattii

identified by Vitek 2 , the agreement between DNA

the mortality rate of mice infected with C. gattii R265 was
5815; however, no difference was found in the mortality rates

evaluated. Among 167 isolates that had previously been
microarray and sequencing results was 97.6％. This DNA

between mice infected with C. gattii R265 and Cryptococcus

microarray platform showed excellent performance.

in histopathological findings of the lungs between mice

4 . NikA/TcsC histidine kinase is involved in conidiation,

former showed alveolar expansion due to yeast proliferation

and antifungal chemicals in Aspergillus fumigatus.

showed numerous nodules in the alveolar space consisting of

Daisuke Hagiwara1, Azusa Takahashi-Nakaguchi1, Takahito

alveolar expansion was more enhanced in mice infected with

Kamei1, Tohru Gonoi1, Susumu Kawamoto1

Our study confirmed that there is a different pathophysiology

1

neoformans H99. In contrast, we found a significant difference
infected with C. gattii R265 and C. neoformans H99. The

with much lesser macrophage response, whereas the latter
macrophages and multinucleated giant cells. Furthermore,

C. gattii R265 than in those infected with C. gattii 5815.

leading to death during C. gattii and C. neoformans infections.

hyphal morphology, and responses to osmotic stressToyotome 1, Akira Yoshimi 2, Keietsu Abe 2, KatsuhikoMedical Mycology Research Center, Chiba University,
Chiba, Japan, 2 New Industry Creation Hatchery Center,

The result can provide two characteristics of C. gattii: one

Tohoku University, Sendai, Japan

via macrophage and another includes a high performance of

The fungal high osmolarity glycerol（HOG）pathway is

includes some mechanisms to escape from host recognition

pulmonary structural alteration. These characteristics may be
associated with the high virulence of C. gattii.

3 . Visual analysis of DNA microarray data for accurate

molecular identification of non-albicans Candida
isolates from patients with candidemia episodes.
11Michela De Luca Ferrari , Mariângela Ribeiro Resende ,3

3

2

Kanae Sakai , Yasunori Muraosa , Luzia Lyra , Tohru

Gonoi3, Yuzuru Mikami3, Kenichiro Tominaga4, Katsuhiko
321

Kamei , Angelica Zaninelli Schreiber , Plinio Trabasso ,1Maria Luiza Moretti

1

3

4

group III（Nik1-type）histidine kinase plays a major rolein the HOG pathway of several filamentous fungi. In thisstudy, we characterized a group III histidine kinase, NikA/

TcsC, in the lifethreatening pathogenic fungus, Aspergillus

fumigatus. A deletion mutant of nikA showed low conidia

production, abnormal hyphae, marked sensitivity to highosmolarity stresses, and resistance to cell wall perturbing
reagents such as congo red and calcofluor white, as well as tofungicides such as fludioxonil, iprodione, and pyrrolnitrin.
None of these phenotypes were observed in mutants of the

SskA response regulator and SakA MAPK, which were

Infectious Diseases Division, Department of Internal Medicine,

thought to be downstream components of NikA. In contrast,São Paulo, Brazil

the phosphorylation of SakA MAPK and the transcriptional

Faculty of Medical Sciences, State University of Campinas,

2composed of a two-component system（TCS）and Hog1-

type mitogen-activated protein kinase（MAPK）cascade. A

Department of Clinical Pathology, Faculty of Medical

in response to fludioxonil treatment, NikA was implicated in

upregulation of catA, dprA, and dprB, which are regulated

Sciences, State University of Campinas, São Paulo, Brazil

under the control of SakA. We then tested the idea thatJapan

play certain roles in the regulation of the HOG pathway.

Medical Mycology Research Center, Chiba University,

not only NikA, but also the other 13 histidine kinases

Japan International Cooperation Agency, Tokyo, Japan

Interestingly, the expression of fos1, phkA, phkB, fhk5, andThe performance of a visual slide-based DNA microarraya SakA-dependent manner. However, deletion mutants of

千葉大学

fhk6 increased by osmotic shock or fludioxonil treatment in

真菌医学研究センター報告

第 17 巻201317

the histidine kinases showed no significant defects in growth

this case report, we describe a potential refractory multidrug-

under the tested conditions. Collectively, although the signal

resistant infection following a tsunami disaster.

NikA plays a crucial role in several aspects of A. fumigatus

6 . Penicilliosis marneffei Complicated with Interstitialtransduction network related to NikA seems complicated,

physiology and, to a certain extent, modulates the HOG

pathway.

5 . Scedosporium aurantiacum brain abscess after near-drowning in a survivor of a tsunami in Japan.

Pneumonia.

Haruhiko Furusawa1, Yasunari Miyazaki1, Shiro Sonoda1,

Kimitake Tsuchiya1, Takashi Yaguchi2, Katsuhiko Kamei2,
Naohiko Inase1

Yutaka Nakamura1, Naomi Suzuki1, Yoshio Nakajima1, Yu

1Department of Respiratory Medicine, Tokyo Medical andSaito1, Nobuhito Sasaki1, Itaru Fujimura2, Yoshinobu Ogino3,

2

Medical Mycology Research Center, Chiba University, Japan

1

1

1

Utsumi , Okinori Murata , Hiromi Nagashima , Heisuke

Kanako Kato3, Yasuo Terayama3, Shinya Miyamoto4, Kyoko
Yarita5, Katsuhiko Kamei5, Toshihide Nakadate6, Shigeatsu
6

7

1

Endo , Kazutoshi Shibuya , Kohei Yamauchi
1

Division of Pulmonary Medicine, Allergy, and Rheumatology,
Department of Internal Medicine, Iwate Medical University

2

3

4

5

6

School of Medicine, Morioka, Japan

Dental University, Japan

A 71 -year-old man with interstitial pneumonia washospitalized 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 bronchial

Department of Pulmonary Medicine, Iwate Prefectural Kuji

lavage fluid cultures and Penicillium sp. was identified to be

Division of Neurology and Gerontology, Department of

improved following treatment with itraconazole andHospital, Kuji, JapanP. marneffei. The infiltration observed on chest radiographs

Internal Medicine, Iwate Medical University School oftazobactam/piperacillin, and no relapse occurred. We herein

Department of Pulmonary Medicine, Iwate Prefecturalinfection in Japan.

Medicine, Morioka, Japan

Miyako Hospital, Miyako, Japan

report the first case of a non-HIV patient with P. marneffei

Medical Mycology Research Center, Chiba University,
Chiba, Japan

Department of Critical Care Medicine, Critical Care andEmergency Center, Iwate Medical University School of

7

Medicine, Morioka, Japan

Department of Surgical Pathology, Toho University Schoolof Medicine, Tokyo, Japan

Many victims of the tsunami that occurred following theGreat East Japan Earthquake on March 11, 2011 developed

systemic disorders owing to aspiration pneumonia. Herein,

we report a case of tsunami lung wherein Scedosporium

aurantiacum was detected in the respiratory tract. A magnetic

resonance image of the patient s head confirmed multiple

brain abscesses and lateral right ventricle enlargement. In

18

千葉大学

真菌医学研究センター報告

第 17 巻2013

7 . Glucoamylase is a major allergen of Schizophyllum

commune.

1,2

3

1

Takahito Toyotome , Mamoru Satoh , Maki Yahiro , Akira
Watanabe1,4, Fumio Nomura3,5, Kamei Kamei1,41Division of Clinical Research, Medical Mycology Research2

Research Center for Animal Hygiene and Food Safety,

3

4

5

by the host immune system of patients as an antigen/allergen.

The purified glucoamylase Sch c 1 is a promising candidate
antigen for the serodiagnosis of S. commune-induced mycosis.

Publications

1 ）De Luca Ferrari M, Ribeiro Resende M, Sakai K,

Muraosa Y, Lyra L, Gonoi T, Mikami Y, Tominaga

Center, Chiba University, Chiba, JapanK, Kamei K, Zaninelli Schreiber A, Trabasso P,

Obihiro University of Agriculture and Veterinary Medicine,

Data for Accurate Molecular Identification of Non-

Hokkaido, Japan

Clinical Proteomics Research Center, Chiba University
Hospital, Chiba, Japan

Division of Control and Treatment of Infectious Diseases,

Moretti ML: Visual Analysis of DNA Microarray

albicans Candida Isolates from Patients with Candidemia

Episodes. J Clin Microbiol 51: 3826-3829, 2013.

2 ）Hagiwara D, Takahashi-Nakaguchi A, Toyotome T,

Yoshimi A, Abe K, Kamei K, Gonoi T, Kawamoto S:

Chiba University Hospital, Chiba, Japan

NikA/TcsC Histidine Kinase Is Involved in Conidiation,

Medicine, Chiba University, Chiba, Japanand Antifungal Chemicals in Aspergillus fumigatus.

Department of Molecular Diagnosis, Graduate School ofHyphal Morphology, and Responses to Osmotic Stress
PLOS ONE 8: e80881, 2013.

Background. Schizophyllum commune is one of the causative

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

bronchopulmonary mycosis, allergic fungal sinusitis, andT, Kamei K: Antifungal susceptibility of Aspergillus

has been increasing. These mycoses are difficult to diagnose

Japan. J Infect Chemother, in press.

agents of basidiomycosis including disorders such as allergic

mucoid impaction of bronchi, the incidence of those of which

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

fumigatus clinical isolates collected from various areas inbecause only a limited number of diagnostic tools are currently4 ）Kohno S, Izumikawa K, Yoshida M, Takesue Y, Oka

commune have been identified to enable serodiagnosis of the

Niki Y: A double-blind comparative study of the safety

available. The biggest problem is that no specific antigens of S.
disease.

Objective. In this study, we attempted to identify a major

antigen of S. commune to establish a reliable serodiagnostic
method.

Methods. We used mass spectrometry to identify an

S, Kamei K, Miyazaki Y, Yoshinari T, Kartsonis NA,
and efficacy of caspofungin versus micafungin in thetreatment of candidiasis and aspergillosis. Eur J Clin
Microbiol Infect Dis 32: 387-397, 2013.

5 ）Nakamura Y, Suzuki N, Nakajima Y, Utsumi Y,

Murata O, Nagashima H, Saito H, Sasaki N, Fujimura

antigen that reacted with the serum of a patient with allergic

I, Ogino Y, Kato K, Terayama Y, Miyamoto S, Yarita K,

protein was expressed in Escherichia coli, highly purified, and

K: Scedosporium aurantiacum brain abscess after near-bronchopulmonary mycosis caused by S. commune. The

the patient sera IgG and IgE titers against the protein were
determined by enzyme-linked immunosorbent assay.

Kamei K, Nakadate T, Endo S, Shibuya K, Yamauchi

drowning in a survivor of a tsunami in Japan. Respir
Investig 51: 207-121, 2013.

Results. The protein identified as a major antigen of S.

6 ）Okubo Y, Wakayama M, Ohno H, Yamamoto

The IgG and IgE titers against Sch c 1 in patient sera were

Umeyama T, Shinozaki M, Nemoto T, Nakayama H,

commune was named Sch c 1; it was a homolog of glucoamylase.

significantly higher than those in healthy volunteer sera（p ＜

.
0.01）

Conclusions & Clinical relevance. Sch c 1 is recognized千葉大学

S, Tochigi N, Tanabe K, Kaneko Y, Yamagoe S,
Sasai D, Ishiwatari T, Shimodaira K, Yamamoto Y,

Kamei K, Miyazaki Y, Shibuya K: HistopathologicalStudy of Murine Pulmonary Cryptococcosis Induced

真菌医学研究センター報告

第 17 巻201319

by Cryptococcus gattii and Cryptococcus neoformans. Jpn J
Infect Dis 66: 216-221, 2013.

7 ）Suzuki T, Tada Y, Tsushima K, Terada J, Sakurai

今日の治療と看護版」, 総編集 : 永井良三 , 大田
956-956, 南江堂 , 2013.

改訂第 3

健 , p. 955-956, p.

T, Watanabe A, Kasahara Y, Tanabe N, Tatsumi K:17）亀井克彦 : カンジダ症 . クリプトコッカス症 . アス

indistinguishable case from drug induced interstitial lung

第 10 版」, p. 341, p. 341-342, p. 342-343, p. 343-344,

Pneumocystis pneumonia in everolimus therapy: andisease. Respir Med 44: 450-457, 2014.

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

Nomura F, Kamei K: Glucoamylase is a major allergenof Schizophyllum commune. Clin Exp Allergy, in press.

9 ）Wuren T, Toyotome T, Yamaguchi M, Takahashi-

ペルギルス症 . ムーコル症 . 輸入真菌症 . 「内科学p. 346-347, 矢崎義雄総編集 , 朝倉書店 , 2013.

18）亀井克彦 , 渡辺

哲 : カンジダによる深在性カンジダ症 . 「微生物学・感染看護学－微生物から感染防止を考える－」, p. 65-66, 岡田
浩編 , 医歯薬出版 , 2013.

忍 , 小池和子 , 白澤Nakaguchi A, Muraosa Y, Yahiro M, Wang DN,19）亀井克彦 : 〈特集関連情報〉BSL3 対応が必要な渡航

components on biofilm formation Formation of20）亀井克彦 : サンフォード感染症治療ガイドの使い方

Watanabe A, Taguchi H, Kamei K: Effect of serum
Aspergillus fumigatus and non-fumigatus Aspergillus. Jpn J

Infect Dis, in press.

10）Furusawa H, Miayazaki Y, Sonoda S, Tsuchiya K,

Yaguchi T, Kamei K, Inase N: Penicilliosis marneffei

Complicated with Interstitial Pneumonia. Intern Med
53: 321-323, 2014.

11）町田安孝 , 福島康次 , 三好祐顕 , 小原一記 , 池田康紀 , 亀井克彦 , 宮﨑義継 , 福田健 : 経気管支鏡肺生検および気管支肺胞洗浄にて診断された慢性肺コクシジオイデス症の 1 例 . 日呼吸誌

2: 274-278,

2013.

12）永川博康 , 猪狩英俊 , 小西建治 , 加志崎史大 , 青山
眞弓 , 渡邉哲, 巽

浩一郎 , 亀井克彦 : 人工呼吸

管理中に空洞穿破により緊張性気胸を併発した肺ムーコル症の 1 剖検例 . Med Mycol J 54: 285-289,

2013.

13）山本洋輔 , 外川八英 , 岩澤真理 , 鎌田憲明 , 神戸直智 , 渡邊正治 , 渡辺

哲 , 亀井克彦 , 松江弘之 : 転倒による手の外傷を契機に発症した続発性皮膚クリ
プトコッカス症の 1 例 . 臨床皮膚科
2013.

67: 728-732,

14）亀井克彦 : 肺真菌症 . 「今日の治療指針

2013 年版, p. 286-288, 総編集 : 山口〔ポケット判〕」
原光夫 , 福井次矢 , 医学書院 , 2013.

徹, 北15）亀井克彦 : 深在性真菌症 . 「医学スーパーラーニン
グシリーズ, p. 216-221, 小野寺昭
感染症内科学」

一編集 , 丸善出版 , 2013.

16）亀井克彦 , 渡辺

20

ル症 . 「疾患症例別

哲 : アスペルギルス症 , ムーコ

千葉大学者真菌症 . 病原微生物検出情報

34: 3-4, 2013.

日和見感染症・真菌感染症 . Ther Res 34: 45-47,
2013.

21）亀井克彦 : アスペルギルスカラーアトラス Vol. 3

Aspergillus niger. Microbiological Laboratory Updates 3:

1-3, 2013.

22）亀井克彦 , 渡邉
本臨床哲 : コクシジオイデス症 . 別冊日新領域別症候群シリーズ No. 24症症候群（第 2 版）上580, 2013.

23）亀井克彦 , 渡邉
冊日本臨床

病原体別感染症編 , p. 578-

哲 : パラコクシジオイデス症 . 別

新領域別症候群シリーズ

染症症候群（第 2 版）上583-585, 2013.

24）亀井克彦 , 渡邉
臨床

哲 : ブラストミセス症 . 別冊日本

候群（第 2 版）上25）亀井克彦 , 渡邉

No. 24 感染症症

病原体別感染症編 , p. 603-605,

哲 , 豊留孝仁 : マルネッフェイ型

ペニシリウム症 . 別冊日本臨床
リーズ No. 24

No. 24 感

病原体別感染症編 , p.

新領域別症候群シリーズ

2013.

感染

新領域別症候群シ感染症症候群（第 2 版）上病原

体別感染症編 , p. 606-608, 2013.

26）亀井克彦 , 渡辺
ジーズ

哲 : 世界に広がるトロピカルディ

真菌感染症 . 化学療法の領域

1725, 2013.

29: 1718-27）亀井克彦 , 渡辺

哲 : 輸入真菌症 . 臨床と微生物

28）亀井克彦 , 渡辺

哲 : コクシジオイデス症 . 別冊日40: 611-615, 2013.
本臨床

真菌医学研究センター報告

新領域別症候群シリーズ

第 17 巻2013

No. 26 神経症

候群（第 2 版）
（I）－その他の神経疾患を含めて－ ,p. 878-881, 2013.

29）亀井克彦 , 渡辺
臨床

哲 : ヒストプラズマ症 . 別冊日本

新領域別症候群シリーズ

No. 26 神経症候（I）－その他の神経疾患を含めて－ , p.
群（第 2 版）
874-877, 2013.

30）亀井克彦 , 渡辺
臨床検査

哲 : 輸入感染症としての真菌症 .

58: 111-116, 2014.

31）渡辺 哲 , 亀井克彦 : Photo Quiz: Deep-seated mycosis

Emericella nidulans. Med Mycol J 54: 319-320, 2013.

32）渡辺哲 , 亀井克彦 : アスペルギルス . 感染症内科

1: 488-493, 2013.

33）渡辺哲 , 亀井克彦 : Cryptococcus gattii 感染症 . 検

査と技術
34）渡辺
道場
35）渡辺

41: 282-285, 2013.

候群（第 2 版）上
2013.

38）渡邉

臨床

候群（第 2 版）上2013.

40）渡邉 哲 , 亀井克彦 : Photo Quiz: Deep-seated mycosis

Pulmonary nocardiosis. Med Mycol J 54: 265-266,
2013.

41）渡辺

哲 , 亀井克彦 : 感染防止からみるやさしい

微生物学－病原体・感染症の特徴を知り , 感染対を知る

42）渡 辺哲 , 亀井克彦 : 肺アスペルギルス症 . 感染と

43）渡 辺16: 152-156, 2013.

哲 , 亀井克彦 : 肺ノカルジア症 . 別冊日本臨

千葉大学

病原体別感染症編 , p. 599-602,

Histoplasmosis. Med Mycol J 54: 103-104, 2013.

13. 旅行医学からみ抗真菌薬

No. 24 感染症症

39）渡辺 哲 , 亀井克彦 : Photo Quiz: Deep-seated mycosis

2013.

29: 730-739, 2013.

病原体別感染症編 , p. 265-267,

新領域別症候群シリーズ

哲 : 目で見る真菌と真菌症（18）診療科・基2: 56-58, 2013.

感染症症

哲 , 亀井克彦 : ヒストプラズマ症 . 別冊日本

策に役立てる－た真菌症 . 化学療法の領域

37）渡邉

新領域別症候群シリーズ No. 24

哲 : 感受性検査成績の臨床への応用 . 感染症

礎疾患から見た大切な真菌症
36）渡辺

床

第7回

第1部

病原体の特徴真菌 . Clinical Engineering 24: 1083-1088,
哲, 亀井克彦: 感染症トピックス

真菌.

MEDICAL TECHNOLOGY 41: 1268-1270, 2013.
哲, 矢口貴志: 糸状菌症. 臨床検査

104-109, 2014.

真菌医学研究センター報告

第 17 巻

2013

58:

21

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

技

術

職

員

特

任

助

教

特

任

助

教

技 術 補 佐 員

22

五ノ井 透
大荒田素子
松澤 哲宏
酒井香奈江
高橋
梓
川名 直美

Professor

Assistant Professor

Research Technician

Research 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
Tetsuhiro Matsuzawa
Kanae Sakai
Azusa Takahashi
Naomi Kawana

第 17 巻2013

1 ．Identification of fungal pathogens by visible microarray

2 . Lectin-Microarray Technique for Glycomic Profiling

Sakai K1, Trabasso P2, Moretti ML2, Mikami Y1, Kamei K1,

Shibazaki A1, Gonoi T2

system in combination with isothermal gene amplification.

Gonoi T1
1
2

of Fungal Cell Surfaces.

1

Center for Integrative Medical Science（ IMS-RCAI ）,Medical Mycology Research Center, Chiba University, Japan

Infectious Diseases Division, Department of Internal Medicine,State University of Campinas, Sãom Brazil

2

by fungi in immunocompromised patients has encouraged

Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.

Medical Mycology Research Center, Chiba University,
Chiba, Japan

The increasing incidence of infectious diseases caused

researchers to develop rapid and accurate diagnosis methods.

Laboratory for Inflammatory Regulation, RIKEN Research

Lectin microarrays are rows of lectins with different

carbohydrate-binding specificities spotted on surfaces of

Identification of the causative fungal species is critical inglass-slides. Lectin microarray technique enables glycomic

satisfactory results due to the difficulty of fungal cultivation

will describe an application of the technique in analyzing cell

this study, we established a microarray system that can

The analysis reveals genus- and species-dependent complex

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

and morphological identification from clinical samples. In
identify 42 species from 24 genera of clinically importantfungal pathogens by using a chemical color reaction in thedetection process. The array uses the internal transcribedspacer（ITS）region of the rRNA gene for identificationof fungal DNA at the species level. The specificity of thisanalyses of carbohydrate composition of fungal cell walls. We

surface glycome of yeast-form fungal cells in the living state.
cell surface carbohydrate structures of fungi, and enabled

us, therefore, to suggest that cell walls of yeast cells, whichhave been considered to have relatively simple structures,

actually have a more complex structure containing galactose

and fucose. This shows that the technique can be used to findarray was tested against a total of 355 target and non-target

new insights in the study of phylogenetic relations and in thefrom 103 CFU/ml for whole blood samples and 50 fg DNA

glycome.

fungal species. The fungal detection was succeeded directly

per 1 ml of serum samples indicate that the array system weestablished is sensitive to identify infecting fungi from clinicalsample. Furthermore, we conducted isothermal amplificationin place of PCR amplification and labeling. The successful

identification with PCR-amplified as well as isothermallyclassification of cells in the fungal kingdom based on cell wall

3 ．Refeeding with a standard diet after a 48-h fast elicits

an inflammatory response in the mouse liver.

Oarada M1, Miki T2, Kohno S3, Sakai K1, Nikawa T3,

amplified target genes demonstrated that our microarray

Yoneyama M1, Gonoi T1variety of fungal species in a sample.

1

system is an efficient and robust method for identifying a

2

3

Medical Mycology Research Center, Chiba University,
Chiba 260-8673, Japan;

Department of Medical Physiology, Graduate School of
Medicine, Chiba University, Chiba 260-8670, Japan;

Department of Nutrition, Tokushima University School of
Medicine, Tokushima 770-8503, Japan

Unhealthy eating behaviors increase the risk of metabolic

diseases, but the underlying mechanisms are not fully

千葉大学

真菌医学研究センター報告

第 17 巻2013

23

elucidated. Because inflammation contributes to thepathogenesis of metabolic diseases, it is important tounderstand the effects of unhealthy eating on the inflammatory

state. The objective of our present study was to address the

4 ．Molecular identification and antimicrobial susceptibilityof Nocardia spp. isolated from bovine mastitis in Brazil.

Condas LAZ1, Ribeiro MG 1, Yazawa K2, de Vargas APC3,

effects of a fasting-refeeding regime, a model of irregular

Salerno T 1, Giuffrida R 4, Langoni H 1, Melville PA 5,

animals were fasted for 48 h and then refed either a standard

HW7

eating, on the hepatic inflammatory responses in mouse. Theor low-carbohydrate/high-fat diet. Inflammatory gene

expression in the liver was then sequentially measured for the

Biesdorf S6, Matsuzawa T2, Gonoi T2, Kastelic JP7, Barkema

1

first 17 h after initiation of refeeding. To assess the roles of

dietary carbohydrates and toll-like receptor 2（TLR2）in theof Veterinary Medicine and Animal Science, Universidade

refeeding-induced inflammatory changes, gene expression

2

sucrose）at different doses and in TLR2-deficient mice refed3

levels in mice refed only carbohydrates（α-corn starch anda standard diet were also analyzed. Refeeding with a standard
diet increased the liver expression of Tlr2, proinflammatory

mediators（Cxcl10, Cxcl1, Cxcl2, Icam-1）and negative4

Refeeding only α-corn starch and sucrose also increased theon the doses. TLR2 deficiency significantly attenuated the6

Cxcl1, Icam-1 and A20. These findings suggest that an

7

refeeding-induced increase in the liver expression of Cxcl10,
irregular eating behavior can elicit a liver inflammatory

response, which is at least partly mediated by TLR2, and that

dietary carbohydrates play critical roles in this process. © 2013
Elsevier Inc.

Medical Mycology Research Center, Chiba University,
Chiba, Japan

Preventive Veterinary Medicine, Universidade Federal deBrazilInfectious Diseases of Animals, Veterinary Hospital,

Universidade do Oeste Paulista - UNOESTE, Presidente
5expression of these inflammatory pathway genes depending

Estadual Paulista - UNESP, Botucatu, São Paulo, Brazil

Santa Maria - UFSM, Santa Maria, Rio Grande do Sul,regulators of TLR-signaling（A20 and Atf 3）. These increases

were attenuated in mice refed a low-carbohydrate/high-fat diet.

Department of Veterinary Hygiene and Public Health, School

Prudente, São Paulo, Brazil

Department of Preventive Veterinary Medicine and Animal
Health, University of São Paulo - USP, São Paulo, Brazil

Marcos Enrietti Diagnostic Center, Secretaria do Estado da
Agricultura e Abastecimento, Curitiba, Parana, Brazil

Department of Production Animal Health, Faculty of

Veterinary Medicine, University of Calgary, Alberta,
CanadaNocardia spp. infections can cause severe damage to themammary gland due to suppurative pyogranulomatous

lesions and lack of clinical cure in response to conventional

antimicrobial therapy. Although Nocardia infections are

considered relatively uncommon in cows, there has been

an apparent worldwide increase in the incidence of bovine
mastitis caused by Nocardia spp, perhaps due to environmental

transmission of this ubiquitous pathogen. The objectives ofpresent study were to determine: （i）species distribution of

80 Nocardia isolates involved in bovine mastitis（based onmolecular methods）; and（ii）antimicrobial susceptibility

pattern of all isolates from three geographical areas in

Brazil. In this study, Nocardia nova（80％）was the mostfrequently isolated species, followed by Nocardia farcinica（9％）. Additionally, Nocardia puris, Nocardia cyriacigeorgica,

24

千葉大学

真菌医学研究センター報告

第 17 巻2013

Nocardia veterana, Nocardia africana, and Nocardia arthritidis

6 ．NikA/TcsC Histidine Kinase Is Involved in Conidiation,

the first report of N. puris, N. veterana, N. cyriacigeorgica,

and Antifungal Chemicals in Aspergillus fumigatus.

were detected using 16S rRNA sequencing. This is apparently
N. arthritidis and N. africana in association with bovine

mastitis. Based on the disk diffusion test, isolates were mostHyphal Morphology, and Responses to Osmotic StressHagiwara D 1, Takahashi-Nakaguchi A 1, Toyotome T 1,

frequently resistant to cloxacillin（75％）, ampicillin（55％）

Yoshimi A2, Abe A2, Kamei K1, Gonoi T1, Kawamoto S1

resistant to amikacin, cefuroxime or gentamicin.

1

5 ．Visual analysis of DNA microarray data for accurate2

and cefoperazone（47％）, whereas few Nocardia spp. weremolecular identification of non-Albicans Candida
isolates from patients with candidemia episodes.
1

1

3

3

De Luca Ferrari M , Ribeiro RM , Sakai K , Muraosa Y ,
2

3

3

4

3

Lyra L , Gonoi T , Mikami Y , Tominaga K , Kamei K ,2

1

1

Zaninelli Schreiber A , Trabasso P , Luiza Moretti M
1

2

3

4

Infectious Diseases Division, Department of Internal
Medicine, State University of Campinas, São Paulo, Brazil

Department of Clinical Pathology, Faculty of Medical

Medical Mycology Research Center, Chiba University,Japan

New Industry Creation Hatchery Center, Tohoku University,
Sendai, JapanThe fungal high osmolarity glycerol（HOG）pathway iscomposed of a two-component system（TCS）and Hog1-

type mitogen-activated protein kinase（MAPK）cascade. A

group III（Nik1-type）histidine kinase plays a major rolein the HOG pathway of several filamentous fungi. In thisstudy, we characterized a group III histidine kinase, NikA/

TcsC, in the life-threatening pathogenic fungus, Aspergillus

fumigatus. A deletion mutant of nikA showed low conidia

Sciences, State University of Campinas, São Paulo, Brazil

production, abnormal hyphae, marked sensitivity to highJapanreagents such as congo red and calcofluor white, as well as toMedical Mycology Research Center, Chiba University,
Japan International Cooperation Agency, Tokyo, Japan
The performance of a visual slide-based DNA microarray forosmolarity stresses, and resistance to cell wall perturbing
fungicides such as fludioxonil, iprodione, and pyrrolnitrin.
None of these phenotypes were observed in mutants of the

SskA response regulator and SakA MAPK, which were

the identification of non-Albicans Candidaspp. was evaluated.

thought to be downstream components of NikA. In contrast,

2, the agreement between DNA microarray and sequencing

the phosphorylation of SakA MAPK and the transcriptional

Among 167 isolates that had previously been identified by Vitek

results was 97.6％. This DNA microarray platform showed
excellent performance.

in response to fludioxonil treatment, NikA was implicated in

upregulation of catA, dprA, and dprB, which are regulated

under the control of SakA. We then tested the idea that not

only NikA, but also the other 13 histidine kinases play certain

roles in the regulation of the HOG pathway. Interestingly, theexpression of fos1 , phkA, phkB, fhk5 , and fhk6 increased byosmotic shock or fludioxonil treatment in a SakA-dependent

manner. However, deletion mutants of the histidine kinases
showed no significant defects in growth under the tested

conditions. Collectively, although the signal transduction
network related to NikA seems complicated, NikA plays acrucial role in several aspects of A. fumigatus physiology and,to a certain extent, modulates the HOG pathway.

千葉大学

真菌医学研究センター報告

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25

7 ．Aspergillus huiyaniae sp. nov., a teleomorphic species

in sect. Fumigati isolated from desert soil in China.
1

1

2

1

1

Matsuzawa T , Horie Y , Abliz P , Gonoi T , Yaguchi T
1

2

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

as the emergence rates of resistance mutations are different

between these two subgenotypes. In Japan, almost all of
HCV genotype 1 belongs to subgenotype 1 b. Methods

and Findings: To determine HCV subgenotype 1a or 1b inJapanese patients infected with HCV genotype 1, real-time

PCR-based method and Sanger method were used for the

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

HCV NS5B region. HCV subgenotypes were determined inof Xinjiang Medical University, No. 1 Liyushan Road,

specific probe regions for HCV subgenotypes 1a and 1b using

Department of Dermatology, The First Affiliated Hospital
Urumqi, Xinjiang 830053, China.

Aspergillus huiyaniae, a new teleomorphic species isolated

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

90％ by real-time PCR-based method. We also analyzed theultra-deep sequencing, and uncovered mutations that could

not be revealed using direct-sequencing by Sanger method.

We estimated the prevalence of HCV subgenotype 1a as 1.2-

2.5％ of HCV genotype 1 patients in Japan. Conclusions:

Although real-time PCR-based HCV subgenotyping method
seems fair for differentiating HCV subgenotypes 1 a and
1b, it may not be sufficient for clinical practice. Ultra-deepto slightly reticulate convex surfaces, and subglobose to ovatesequencing is useful for revealing the resistant strain（s）of

was supported further by the analyses of the β-tubulin,

different genotypes or subgenotypes of HCV.

or broadly ellipsoidal conidia with smooth walls. This species
calmodulin and actin gene sequences.

8 . Prevalence of hepatitis C virus subgenotypes 1a and 1b

in Japanese Patients: Ultra-deep sequencing analysis

of HCV NS5B genotype-specific region.

Wu S1, Kanda T1, Nakamoto S1,2, Jiang X1, Miyamura T1,
Nakatani SM3, Ono SK3, Takahashi-Nakaguchi, A4, Gonoi,
4

2

3

4

9 ．Hyrtimomines D and E, bisindole alkaloids from amarine sponge Hyrtios sp.

Tanaka N 1 , Momose, R 1 , Takahashi Y 1 , Kubota T 1 ,Takahashi-Nakaguchi A2, Gonoi T2, Fromont J3, Kobayashi J1
1Graduate School of Pharmaceutical Sciences, Hokkaido

2

Mycology Research Center, Chiba University, Chiba 260-

1

T , Yokosuka O
1

HCV before DAA treatment as well as mixed infection with

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

Department of Molecular Virology, Chiba University,
Graduate School of Medicine, Chiba, Japan;

Department of Gastroenterology, School of Medicine,
University of Sao Paulo（USP）, São Paulo, Brazil;

Medical Mycology Research Center, Chiba University,
Chiba, Japan

Background: Hepatitis C virus（ HCV ）subgenotypes

1a and 1b have different impacts on the treatment responseto peginterferon plus ribavirin with direct-acting antivirals（DAAs）against patients infected with HCV genotype 1,

26

千葉大学

3

University, Sapporo 060-0812, Japan;
8673, Japan;

Western Australian Museum, Locked Bag 49, Welshpool
DC, WA 6986, Australia

Two new alkaloids, hyrtimomines D（1）and E（2）,were isolated from an Okinawan marine sponge Hyrtios sp.

The structures of 1 and 2 were elucidated on the basis ofspectroscopic analysis. Hyrtimomines D（1）and E（2）arestructurally unique bisindole alkaloids possessing the canthin-

6-one skeleton with a hydroxyindole and an imidazolium

units. Hyrtimomines D（1）and E（2）exhibited antimicrobialactivity.

真菌医学研究センター報告

第 17 巻2013

10．Nagelamides U-W, bromopyrrole alkaloids from amarine sponge Agelas sp.
1

1

2

2

Tanaka N , Kusama T , Takahashi-Nakaguchi A , Gonoi T ,Fromont J3, Kobayashi J1
1Graduate School of Pharmaceutical Sciences, Hokkaido

2

Mycology Research Center, Chiba University, Chiba 260-

3

University, Sapporo 060-0812, Japan;
8673, Japan;

Western Australian Museum, Locked Bag 49, Welshpool
DC, WA 6986, Australia

pernambucoensis is characterized by its, white cleistothecia,

lenticular ascospores with two equatorial crests and irregularly

ribbed with tuberculate to verrucate convex surfaces, andovoid to broadly ellipsoidal conidia with a smooth wall. The

validation of these new species is supported further by analysesof the β-tubulin, calmodulin and actin gene sequences.

12．Nagelamides X-Z, dimeric bromopyrrole alkaloids

from a marine sponge Agelas sp.

Tanaka N1, Kusama, T1, Takahashi-Nakaguchi A2, Gonoi

T2, Fromont J3, Kobayashi J1Three new structurally unique bromopyrrole alkaloids,

1

Agelas sp. Nagelamides U（1）and V（2）possess a γ-lactam

2

while nagelamide W（3）has two aminoimidazole moieties

3

nagelamides U-W（1-3）, were isolated from a marine spongering with an N-ethanesulfonic acid and guanidino moieties,
in the molecule. The structures of 1-3 were elucidated on thebasis of spectroscopic data. Nagelamides U（1）and W（3）exhibited antimicrobial activity against Candida albicans. ©
2013 Elsevier Ltd. All rights reserved.

11．Two new species of Aspergillus section Fumigati isolated

from caatinga soil in the state of Pernambuco, Brazil.
121

Matsuzawa T , Campos Takaki GM , Yaguchi, T , Okada
2

1

K , Gonoi T , Horie Y
1

2

1

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

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

Mycology Research Center, Chiba University, Chiba 2608673, Japan;

Western Australian Museum, Locked Bag 49, Welshpool
DC, WA 6986, Australia

Three new dimeric bromopyrrole alkaloids, nagelamides

X-Z（1-3）, were isolated from a marine sponge Agelas sp.

Nagelamides X（1）and Y（2）possess a novel tricyclic skeletonconsisting of spiro-bonded tetrahydrobenzaminoimidazole

and aminoimidazolidine moieties. Nagelamide Z（3）is thefirst dimeric bromopyrrole alkaloid involving the C-8 positionin dimerization. The structures of 1-3 were elucidated on thebasis of spectroscopic data. Nagelamides X-Z（1-3）exhibited

antimicrobial activity.

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

Catholic University of Pernambuco, Rua do Príncipe, n.
526, Boa Vista, CEP 50050-900, Recife-Pernambuco,

BrazilAspergillus caatingaensis and A. pernambucoensis, isolated fromsemi-desert soil in caatinga area, the State of Pernambuco,

Brazil, are described and illustrated. Aspergillus caatingaensis

is characterized by its white cleistothecia, broadly lenticular
ascospores with four equatorial crests and irregularly ribbed toslightly reticulate with aculeate convex surfaces, and ellipsoidal

to broadly ellipsoidal conidia with a smooth wall. Aspergillus

千葉大学

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第 17 巻201327

13．Manzamenone O, new trimeric fatty acid derivativefrom a marine sponge Plakortis sp.
1

1

2

receptors mincle and dectin-2 in the pathogenic fungus
Malassezia.

2

Tanaka N , Asai M , Takahashi-Nakaguchi A , Gonoi T ,Fromont J3, Kobayashi J1
1Graduate School of Pharmaceutical Sciences, Hokkaido

2

Mycology Research Center, Chiba University, Chiba 260-3

14．Identification of distinct ligands for the C-type lectin

University, Sapporo 060-0812, Japan;

Ishikawa T1, Itoh F4, Yoshida S4, Saijo5, Matsuzawa T6,

Gonoi T6, Saito T8,9, Okawa Y4, Shibata N4, Miyamoto, T2,
Yamasaki S1.3
10856, Japan;

Western Australian Museum, Locked Bag 49, Weishpool,
WA 6986, Australia

A new structurally unique trimeric fatty acid derivative,

manzamenone O（1）, was isolated from a marine spongeof Bioregulation, Kyushu University, 3 - 1 - 1 Maidashi,

2［3.3.0］octane moieties and three long aliphatic chains. The

3

antimicrobial activity against Micrococcus luteus, Aspergillis

Department of Natural Products Chemistry, Graduate
1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan;

Research Center for Advanced Immunology, Kyushu

University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 8124

structure of 1 was elucidated on the basis of spectroscopic dataand conformational analysis. Manzamenone O（1）exhibited

Higashi-ku, Fukuoka 812-8582, Japan;

School of Pharmaceutical Sciences, Kyushu University, 3-1-

Plakortis sp. Manzamenone O（1）has a novel skeletonconsisting of C-C bonded octahydroindenone and dioxabicyclo

Division of Molecular Immunology, Medical Institute8582, Japan;

Department of Infection and Host Defense, Tohoku

Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku,
5

Sendai 981-8558, Japan;

Department of Molecular Immunology, Medical Mycology
Research Center, Chiba University, 1-8-1 Inohana, Chuo-

niger, and Trichophyton mentagrophytes.

6

Ku, Chiba 260-8673, Japan;

Department of Bio-resources, Medical Mycology ResearchCenter, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba

7

260-8673, Japan;

Precursory Research for Embryonic Science and Technology（PRESTO）, Japan Science and Technology Agency（JST）,
8

9

Saitama 332-0012, Japan;

Laboratory for Cell Signaling, RIKEN Research Center forAllergy and Immunology, Yokohama 230-0045, Japan;

WPI Immunology Frontier Research Center, Osaka
University, Suita, Osaka 565-0871, Japan

Various C-type lectin receptors（CLRs）, including Mincle

and Dectin-2 , function as pattern recognition receptors

and play a central role in immunity to fungal pathogens.

However, the precise structures of the CLR ligands invarious pathogenic fungi have yet to be completely defined.

Here we report that Malassezia, an opportunistic skinfungal pathogen, is cooperatively recognized by Mincle andDectin-2 through distinct ligands. Solvent-based fractionation

28

千葉大学

真菌医学研究センター報告

第 17 巻2013

revealed that Mincle and Dectin-2 recognize lipophilic andhydrophilic components of Malassezia, respectively. Mass
spectrometry and nuclear magnetic resonance（NMR）revealed

16．Testicular nocardiosis accompanied by cutaneous lesions

in an immunocompetent man.

glyceroglycolipid and unique mannosyl fatty acids linked to

Yamaguchi H 1, Sekimoto E1, Shirakami A1, Shibata H1,

glycoprotein was identified as a Malassezia ligand for Dectin-2.

Hirose T4, Matsuzawa T5, Gonoi T5

mannitol as two Mincle ligands. An O-linked mannobiose-richCytokine production in response to the Mincle ligands and the

Ozaki S1, Shigekiyo T1, Noda T2, Shikiji T2, Kanda, K3,

Dectin-2 ligand was abrogated in Mincle-/- and Dectin-2-/-

1

Mincle and Dectin-2 recognize distinct ligands in Malassezia

2

dendritic cells, respectively. These results demonstrate thatto induce host immune responses.

3

15．Zamamiphidin A, a new manzamine related alkaloid

from an Okinawan marine sponge Amphimedon sp.
1

1

2

Kubota T , Kamijyo Y , Takahashi-Nakaguchi A , Fromont,

J3, Gonoi T2, Kobayashi J1
1Graduate School of Pharmaceutical Sciences, Hokkaido

2

Medical Mycology Research Center, Chiba University,

3

4

5

Department of Internal Medicine, Tokushima Prefectural
Central Hospital, Japan;

Department of Dermatology, Tokushima Prefectural Central
Hospital, Japan;

Department of Urology, Tokushima Prefectural Central
Hospital, Japan;

Department of Diagnostic Pathology, Tokushima Prefectural
Central Hospital, Japan;

Division of BioResources, Medical Mycology ResearchCenter, Chiba University, Japan

We herein report the case of a 77-year-old man admitted

University, Sapporo 060-0812, Japan;

for an acute cutaneous infection and persistent fever. A

Chiba 260-0856, Japan;

scrotal swelling. He was suspected of having complications

Western Australian Museum, Locked Bag 49, Welshpool
DC, WA 6986, Australia

A manzamine related alkaloid, zamamiphidin A（1）,consisting of a new heptacyclic ring system has been isolated

from an Okinawan marine sponge Amphimedon sp. The

structure of 1 including the relative stereochemistry was
elucidated on the basis of the spectroscopic data. Compound

physical examination revealed systemic small blisters andfrom chickenpox or bullous impetigo as the initial diagnosis.
Nocardia was detected on an aspiration biopsy of the small

blisters and the surgically removed testis at a later date.
Testicular nocardiosis is a rare condition; however, we should

consider nocardiosis in the differential diagnosis becausedelay in providing treatment may worsen a patient s generalcondition.

1 showed antibacterial activity against Staphylococcus aureus（MIC, 32 μg/mL）.

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17．Manzamenones L-N, new dimeric fatty-acid derivatives

from an Okinawan marine sponge Plakortis sp.
1

1

2

Kubota T , Ishiguro Y , Takahashi-Nakaguchi A , Fromont

hepatitis for which an effective vaccine has been developed.

Here we describe ultra-deep pyrosequences（ UDPSs ）of
HAV 5’-untranslated region（5’UTR）among cases of thesame outbreak, which arose from a single source, associatedJ3, Gonoi T2, Kobayashi J1

with a revolving sushi bar. We determined the reference1

Graduate School of Pharmaceutical Sciences, Hokkaido

Sanger method. Sixteen UDPSs from this outbreak and one

2

Mycology Research Center, Chiba University, Chiba 260-

3

University, Sapporo 060-0812, Japan;
0856, Japan;

Western Australian Museum, Locked Bag 49, Welshpool
DC, WA 6986, Australia

Three new polyketides, manzamenones L-N（1-3）, havesequence from HAV-derived clone from an attendant by thefrom another sporadic case were compared with this reference.

Nucleotide errors yielded a UDPS error rate of ＜ 1％. This

study confirmed that nucleotide substitutions of this region

are transition mutations in outbreak cases, that insertion wasobserved only in non-severe cases, and that these nucleotide

substitutions were different from those of the sporadic case.

Analysis of UDPSs detected low-prevalence HAV variations

been isolated from an Okinawan marine sponge of the genus

in 5’
UTR, but no specific mutations associated with severityof spectroscopic data. Manzamenones L-N（1-3）were new

this outbreak conserved HAV IRES sequence even if we

Plakortis. The structures of 1-3 were elucidated on the basis
dimeric fatty-acid derivatives consisting of a tetrahydroindenone

in these outbreak cases. To our surprise, HAV strains in
performed analysis of UDPSs. UDPS analysis of HAVwith three carboxy groups and two hexadecanyl chains.

5’
UTR gave us no association between the disease severity ofactivity against several bacteria and fungi.

interesting to perform ultra-deep sequencing of full length

Manzamenones M（2）and N（3）showed antimicrobial

18．Ultra-Deep Sequencing Analysis of the Hepatitis A

Virus 5’
-Untranslated Region among Cases of theSame Outbreak from a Single Source.
1

2

1

1

Wu S , Shingo Nakamoto S , Tatsuo Kanda T , Jiang X ,
1

1

2

3

Nakamura M , Miyamura T , Shirasawa H , Sugiura N ,Takahashi-Nakaguchi A4, Gonoi T4, Yokosuka O1

1

2

3

Department of Gastroenterology and Nephrology, Chiba

HAV genome in order to reveal possible unknown genomic
determinants associated with disease severity. Further studies

will be needed.
Publications

1 ）Abe T, Hirasaka K, Kagawa S, Kohno S, Ochi A,

Utsunomiya K, Sakai A, Ohno A, Teshima-Kondo

S, Okumura Y, Oarada M, Maekawa Y, Terao J,

Mills EM, Nikawa T: Cbl-b is a critical regulator ofmacrophage activation associated with obesity-induced

University, Graduate School of Medicine, Chiba（260-insulin resistance in mice. Diabetes 62: 1957-1969,

Department of Molecular Virology, Chiba University,2 ）Abe T, Kohno S, Yama T, Ochi A, Suto T, Hirasaka

8677）, Japan

Graduate School of Medicine, Chiba（260-8677）, Japan

Department of Gastroenterology, National Hospital
Organization Chiba Medical Center, 4-1-2 Tsubakimori,4

hepatitis A and HAV 5’
UTR substitutions. It might be more

2013.

K, Ohno A, Teshima-Kondo S, Okumura Y, Oarada

M, Choi I, Mukai R, Terao J, Nikawa T: Soy glycinin

contains a functional inhibitory sequence against

Chiba（260-8606）, Japanmuscle-atrophy-associated ubiquitin ligase Cbl-b. Int J

Chiba（260-8673）, Japan

3 ）Condas LAZ, Ribeiro MG, Yazawa K, de Vargas

Medical Mycology Research Center, Chiba University,

Hepatitis A virus（HAV）is a causative agent of acute viral

30

千葉大学

Endocrinol, art. no. 907565, 2013.

APC, Salerno T, Giuffrida R, Langoni H, Melville

PA, Biesdorf S, Matsuzawa T, Gonoi T, Kastelic

真菌医学研究センター報告

第 17 巻2013

JP, Barkema HW: Molecular identification and

Gonoi T: Identification of fungal pathogens by visible

from bovine mastitis in Brazil. Vet Microbiol, in press.

amplification. Mycopathologia, in press.

antimicrobial susceptibility of Nocardia spp. isolated4 ）Ferrari MD, Resende MR, Sakai K, Muraosa Y, Lyra

L, Gonoi T, Mikami Y, Tominaga K, Kamei K, Schreiber

AZ, Trabasso P, Moretti ML: Visual analysis of DNA

microarray data for accurate molecular identification of non-

Albicans Candida isolates from patients with candidemia

episodes. J Cin Microbiol, in press.

5 ）Hagiwara D, Takahashi-Nakaguchi A, Toyotome T,

microarray system in combination with isothermal gene12）Shibazaki A, Gonoi T: Lectin-Microarray Techniquefor Glycomic Profiling of Fungal Cell Surfaces. Methods

in Molecular Biology, in press.

13）Matsuzawa T, Campos Takaki GM, Yaguchi T, Okada

K, Gonoi T, Horie Y: Two new species of Aspergillus

section Fumigati isolated from caatinga soil in the Stateof Pernambuco, Brazil. Mycoscience, in press.

Abe K, Yoshimi H, Kamei K, Gonoi T, Kawamoto S:

14）Matsuzawa T, Horie Y, Abliz P, Gonoi T, Yaguchi T:

Hyphal Morphology, and Responses to Osmotic Stressin sect. Fumigati isolated from desert soil in China.

NikA/TcsC Histidine Kinase Is Involved in Conidiation,
and Antifungal Chemicals in Aspergillus fumigatus. PLoSONE DOI: 10.1371/journal.pone.0080881.

6 ）Hirasaka K, Maeda T, Ikeda C, Haruna M, Kohno

S, Abe T, Ochi A, Mukai R, Oarada M, Teshima-

Kondo S, Ohno A, Okumura Y, Terao J, Nikawa T:

Isoflavones derived from soy beans prevent MuRF 1mediated muscle atrophy in C2C12 myotubes through

SIRT1 activation. J Nutr Sci Vitaminol 59: 317-324,
2013.

7 ）Ishikawa T, Itoh F, Yoshida S, Saijo S, Matsuzawa

T, Gonoi T, Saito T, Okawa Y, Shibata N, Miyamoto

Aspergillus huiyaniae sp. nov., a teleomorphic species
Mycoscience. in press.

15）Nakano T, Matsushima-Hibiya Y, Yamamoto M,

Takahashi-Nakaguchi A, Fukuda H, Ono M, Takamura-

Enya T, Kinashi H, Totsuka Y: ADP-ribosylationof guanosine by SCO 5461 protein secreted from

Streptomyces coelicolor. Toxicon 63: 55-63, 2013.

16）Nakayama M, Hosoya K, Tomiyama D, Tsugukuni

T, Matsuzawa T, Imanishi Y, Yaguchi T: Method for

rapid detection and identification of chaetomium andevaluation of resistance to peracetic acid. J Food Protect

76: 999-1005, 2013.

T, Yamasaki S: Identification of distinct ligands for17）Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela

pathogenic fungus Malassezia: Cell Host Microbe, 13:

accelerated mTOR signaling and angiogenesis in the

the C-type lectin receptors mincle and dectin-2 in the477-488, 2013.

8 ）Kubota T, Ishiguro Y, Takahashi-Nakaguchi A, Fromont

K, Nozawa A, Nakatani Y, Furuya M: Unique mutation,

pulmonary cysts of Birt-Hogg-Dubé syndrome. Pathol

Int 63: 45-55, 2013.

J, Gonoi T, Kobayashi J: Manzamenones L-N, new18）Takahashi-Nakaguchi A, Matsumoto Y, Yamamotosponge Plakortis sp. Bioorg Med Chem Lett 23: 244-247,

K: Demonstration of Cytotoxicity against Wasps by

9 ）Kubota T, Kamijyo Y, Takahashi-Nakaguchi A, Fromont

White Butterfly. PLoS ONE 8: art. no. e60539, 2013.

dimeric fatty-acid derivatives from an Okinawan marine
2013.

J, Gonoi T, Kobayashi J: Zamamiphidin A, a newmanzamine related alkaloid from an Okinawan marine

sponge amphimedon sp. Org Lett 15: 610-612, 2013.

10）Oarada M, Miki T, Kohno S, Sakai, K, Nikawa T,

Yoneyama M, Gonoi T: Refeeding with a standard dietafter a 48-h fast elicits an inflammatory response in themouse liver. J Nutr Biochem 24: 1314-1323, 2013.

11）Sakai K, Trabasso P, Moretti ML, Mikami Y, Kamei K,

千葉大学

M, Iwabuchi K, Totsuka Y, Sugimura T, Wakabayashi

Pierisin-1: A Possible Defense Factor in the Cabbage19）Tanaka N, Asai M, Takahashi-Nakaguchi A, Gonoi T,

Fromont J, Kobayashi J: Manzamenone O, new trimericfatty acid derivative from a marine sponge Plakortis sp.
Org Lett 15: 2518-2521, 2013.

20）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

54（29）: 3794-3796, 2013.

真菌医学研究センター報告

第 17 巻20133121）Tanaka N, Kusama T, Takahashi-Nakaguchi A, Gonoi

accompanied by cutaneous lesions in an immunocompetentbromopyrrole alkaloids from a marine sponge agelas sp.

25）Wu S, Kanda T, Nakamoto S, Jiang X, Miyamura

22）Tanaka N, Momose R, Takahashi Y, Kubota T, Takahashi-

Gonoi T, Yokosuka O: Prevalence of Hepatitis C Virus

Hyrtimomines D and E, bisindole alkaloids from a marine

Deep Sequencing Analysis of HCV NS5B Genotype-

T, Fromont J, Kobayashi: J. Nagelamides X-Z, dimeric

Org Lett 15: 3262-3265, 2013.

Nakaguchi A, Gonoi T, Fromont J, Kobayashi J:

sponge Hyrtios sp. Tetrahedron Lett 54: 4038-4040, 2013.

man. Internal Med 52: 129-133, 2013.

T, Nakatani SM, Ono SK, Takahashi-Nakaguchi A,

Subgenotypes 1a and 1b in Japanese Patients: UltraSpecific Region. PLoS ONE, 8, art. no. e73615. 2013.

23）Uka D, Takahashi-Nakaguchi A, Yoshimura J, Iwabuchi26）Wu S, Nakamoto S, Kanda T, Jiang X, Nakamuraa polyembryonic wasp. Scientific Reports, 3, art. no.

Nakaguchi A, Gonoi T, Yokosuka O: Ultra-

K: Male soldiers are functional in the Japanese strain of2312, 2013.

24）Yamaguchi H, Sekimoto E, Shirakami A, Shibata H,

Ozaki S, Shigekiyo T, Noda T, Shikiji T, Kanda K,
Hirose T, Matsuzawa T, Gonoi T: Testicular nocardiosis

32

千葉大学

M, Miyamura T, Shirasawa H, Sugiura N, TakahashiDeep Sequencing Analysis of the Hepatitis A Virus

5 -Untranslated Region among Cases of the Same Outbreakfrom A Single Source. Int J Med Sci, in press.

真菌医学研究センター報告

第 17 巻2013

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

高橋

授

弘喜

1 ．Functions of the Hha and YdgT proteins in transcriptionalsilencing by the nucleoid proteins, H-NS and StpA, in
Escherichia coli.
1

2

1

1

Takeshi Ueda , Hiroki Takahashi , Ebru Uyar , Shu Ishikawa ,1

Hiroki Takahashi

Associate Professor1of hha/ydgT inactivation on the chromosomal binding of

H-NS, and conversely the effects of hns/stpA inactivation on

the chromosomal binding of Hha. Our results demonstrated

that the chromosomal binding of Hha requires H-NS/StpA,
and is necessary for the repression of a subset of genes in the

Naotake Ogasawara and Taku Oshima ,*H-NS/StpA regulon. Furthermore, the distribution of H-NS

1

genes suggests that Hha/YdgT proteins modulate formation2

Department of Microbiology, Faculty of Medicine andDentistry, Palacky University Olomouc, Czech Republic

Division of Molecular Biology, Medical Mycology Researchbinding around Hha/YdgT-dependent and -independentof the H-NS/StpA-DNA complex.

Center, Chiba University, Chiba, JapanThe Hha and YdgT proteins are suggested to modulate

the expression of horizontally acquired genes by interactingwith H-NS and StpA, which play central roles in thetranscriptional silencing of such genes. However, it is also

possible that Hha/YdgT repress gene expression independentlyof H-NS/StpA, as we have not fully understood themolecular mechanism through which Hha/YdgT modulate

H-NS/StpA activity. To gain further insight into the basic
functions of Hha/YdgT, we analysed the impact of hha/ydgTdouble inactivation on the transcriptome profile of Escherichia
coli K-12, and compared the effects with that of hns/stpA

double inactivation. In addition, we examined the effects

千葉大学

真菌医学研究センター報告

第 17 巻201333

2 ．Improved production of secreted heterologous enzyme

in Bacillus subtilis strain MGB874 via modification of
glutamate metabolism and growth conditions.

Kenji Manabe12†, Yasushi Kageyama1†, Takuya Morimoto1,

Eri Shimizu 3, Hiroki Takahashi 4, Shigehiko Kanaya 5,
6

1*

2*

Katsutoshi Ara , Katsuya Ozaki and Naotake Ogasawara
1

2

Biological Science Laboratories, Kao Corporation, 2606
Akabane, Ichikai, Haga, Tochigi 321-3497, Japan

4

5

6

suggest that this phenomenon is attributable to impaired

secretion of alkaline cellulase Egl-237 and nitrogen starvation,
caused by decreased external pH and ammonium depletion,

respectively. W ith NH 3 -pH auxostat fermentation,
production of alkaline cellulase Egl-237 in strain 874∆ rocG

was increased, exceeding that in the wild-type-backgroundstrain 168∆ rocG. Notably, in strain 874∆ rocG, high enzyme

productivity was observed throughout cultivation, possibly
due to enhancement of metabolic flux from 2-oxoglutarate

Graduate School of Biological Science, Nara Institute ofto glutamate and generation of metabolic energy through630-0101, Japan

alkaline cellulase Egl-237 obtained corresponded to about 5.5

Science and Technology, 8916-5 Takayama, Ikoma, Nara

3

Results. Transcriptome analysis and cultivation profiles

Analytical Science Laboratories, Kao Corporation, 2606
Akabane, Ichikai, Haga, Tochigi 321-3497, Japan

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

activation of the tricarboxylic acid（TCA）cycle. The level ofg l-1, the highest level reported so far.

Conclusions. We found the highest levels of productionof alkaline cellulase Egl-237 with the reduced-genome strain

1 Inohana, Chuo-ku, Chiba 260-8673, Japan874∆ rocG and using the NH3-pH auxostat. Deletion of

Science and Technology, 8916-5 Takayama, Ikoma, Naraproduction via a prolonged auxostat fermentation, possibly

Graduate School of Information Science, Nara Institute of
630-0101, Japan

Fundamental Technology Research Laboratories, Kao
Corporation, 623 Zi Ri Rd, Minhang Dist, Shanghai

200241, China

Background. The Bacillus subtilis genome-reduced

strain MGB874 exhibits enhanced production of exogenous

the glutamate dehydrogenase gene rocG enhanced enzyme

due to improved glutamate synthesis and enhanced generationof metabolism energy.

Publications

1 ）Manabe K, Kageyama Y, Morimoto T, Shimizu E,

Takahashi H, Kanaya S, Ara K, Ozaki K, Ogasawara N.

Improved production of secreted heterologous enzyme

extracellular enzymes under batch fermentation conditions.

in Bacillus subtilis strain MGB 874 via modification offunctional protein that acts as a glutamate dehydrogenase

Cell Factories. 2013 12: 18.

We predicted that deletion of the gene for RocG, a bi-

and an indirect repressor of glutamate synthesis, would

improve glutamate metabolism, leading to further increased

enzyme production. However, deletion of rocG dramaticallydecreased production of the alkaline cellulase Egl-237 instrain MGB874（strain 874∆ rocG）.

34

千葉大学

glutamate metabolism and growth conditions. Microbial

2 ）Ueda T, Takahashi H, Uyar E, Ishikawa S, Ogasawara

N, Oshima T. Functions of the Hha and YdgT Proteinsin Transcriptional Silencing by the Nucleoid Proteins,

H-NS and StpA, in Escherichia coli. DNA Research.
March 29, 2013.

真菌医学研究センター報告

第 17 巻2013

バイオリソース管理室
Management 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 with new information.

准

教

授

助
技

教
術

職

員

技 術 補 佐 員

矢口
田中
伊藤
長村

貴志
玲子
純子
由美

Associate Professor
Assistant Professor

Research Technician

Research Promotion Technician

1 ．Aspergillus waksmanii sp. nov. and Aspergillus marvanovae

sp. nov., two closely related species in section Fumigati

described using polyphasic approach.
1,2

3

4

Vit Hubka , Stephen W. Peterson , Jens C. Frisvad , Takashi
5

1

1,2

Yaguchi , Alena Kubátová , Miroslav Kolařík
1

Department of Botany, Faculty of Science, Charles University

Takashi Yaguchi
Reiko Tanaka
Junko Ito
Yumi Osamura

type isolate NRRL 179 T（＝CCF 4266 T＝Thom 4138 .
HS2T＝IBT 31900T）. Aspergillus marvanovae sp. nov. wasisolated from water with high boracic acid anions content inDukovany nuclear power station（Czech Republic）. The

sexual stage of this species is unknown, but the MAT1-1
locus was successfully amplified suggesting that the species

is probably heterothallic and teleomorphic but is represented
by only the ex-type isolate CCM 8003T（＝CCF 4037T＝in Prague, Benátská 2, 128 01 Praha 2, Czech Republic;

NRRL 62486T＝IBT 31279T＝IFM 60873T）. Both speciesof Microbiology of the AS CR, v.v.i, Víde ň ská 1083 ,section Fumigati based on morphology, maximum growth2

Laboratory of Fungal Genetics and Metabolism, Institute3

142 20 Praha 4, Czech Republic;

Bacterial Foodborne

Pathogens and Mycology Research Unit, National Center for

Agricultural Utilization Research, 1815 N. University Street,can be distinguished from all previously described species intemperature, sequence data from five unlinked loci andunique secondary metabolites profiles.

Peoria, IL 61604, USA; 4 Department for Systems Biology,
Technical University of Denmark, Soltofts Plads, Building
221, DK-2800 Lyngby, Denmark;5

Medical Mycology

Research Center, Chiba University, 1-8-1 Inohana, Chuoku, Chiba, 260-8673, Japan

Two new and phylogenetically closely related species inAspergillus section Fumigati are described and illustrated.
Homothallic Aspergillus waksmanii sp. nov. was isolated

from New Jersey soil（USA）and is represented by the ex-

千葉大学

真菌医学研究センター報告

第 17 巻201335

2 ．Genotypes of Candida albicans isolated from healthy

individuals and their distribution in patients with oral

3 ．Method for rapid detection and identification ofChaetomium and related genera and evaluation ofcandidiasis.

resistance to peracetic acid.

Yuki Takagi 1, Hideo Fukano1, Kazuo Shimozato1, Reiko

Motokazu Nakayama1, Kouichi Hosoya1, Daisuke Tomiyama1,

Kanbe5

Yaguchi2*

1

1

Tanaka2, Toshinobu Horii3, Fumihiko Kawamoto4, Toshio

Department of Maxillofacial Surgery, Aichi-Gakuin
University School of Dentistry, Nagoya, Japan;

2

MedicalTetsuhiro Matsuzawa2, Yumi Imanishi2, Jun Hitomi1, Takashi

Global R&D-Safety Science, Kao Corporation, 2606

Akabane, Ichikai-machi, Haga-Gun, Tochigi 321 -Mycology Research Center, Chiba University, Chiba, Japan;

3497, Japan; 2 Medical Mycology Research Center, Chiba

School of Medicine, Hamamatsu, Japan; 4Department ofJapan

Research, Oita University, Yufu, Japan; 5Division of

In a beverage industry, peracetic acid has been increasingly

3

Department of Infectious Diseases, Hamamatsu University

Social and Environmental Medicine, Institute of Scientific
Molecular Mycology and Medicine, Center for Neurological

Disease and Cancer, Nagoya University Graduate School of
Medicine, Nagoya, JapanFor the study of Candida albicans genotypes involved indevelopment of candidiasis, Candida albicans isolates werecollected from healthy volunteers and patients with oral

candidiasis and genotyped on the basis of 25S rDNA andmicrosatellite polymorphisms. In the microsatellite analysis

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

used as a disinfectant for the filling machinery and environment

due to merits of leaving no residue, safety to humans andits antiseptic effect against fungi and endospores of bacteria.

Recently, Chaetomium globosum and C. funicola were reported

resistant to peracetic acid, however little is known concerning
the detail of peracetic acid resistance. Therefore we assessed

the peracetic acid resistance of the species of Chaetomium andrelated genera under identical conditions and made a thoroughobservation of the microstructure of their ascospores byusing two microsatellite markers（ CDC 3 and CAI ）, 63

transmission electron microscopy. At the result, C. globosum（ allelic relations to CDC 3 alleles 1 : 2 /CAI alleles 1 :acid（the 1D antiseptic effect after 900 s and 3D antiseptic（115 : 123/18 : 27）, and V（123 : 127/32 : 41）were foundmight impede the action mechanism of peracetic acid. We

In 68 oral candidiasis isolates classified into 39 genotypes,

and C. funicola using polymerase chain reaction（PCR）tohealthy volunteer isolates were classified into 35 genotypes2）, among which genotypes II（115 : 119 / 23 : 23）, IIIat frequencies of 12.7％, 7.9％, and 7.9％, respectively.

and C. funicola showed the high resistance against peracetic

effect after 900 s）and had thick cell walls of ascospores that

also developed species-specific primers to identify C. globosum

genotypes II and III were identified in 4.4％ and 20.6％

amplify the β-tubulin gene. PCR using the primer sets

was higher in the candidiasis isolates than in the healthy

2F/2R）amplified PCR products specific for C. globosum and C.

albicans assigned by CDC3/CAI is related to the development

detect other fungi involved in food spoilage and environmentalof the isolates, respectively. The frequency of genotype III

isolates（p ＜0.05）. These results suggest that genotype III C.
of oral candidiasis.

designed for C. globosum（Chae 4F/4R）and C. funicola（Cfufunicola, respectively. PCR using these two primer sets did notcontamination. This identification method is rapid and simple
with extremely high specificity.
Publications

1 ）Fukiharu T, Shimizu K, Li RY, Raut JK, Yamakoshi

S, Horie Y, Kinjo N: Coprinopsis novorugosobispora sp.

36

千葉大学

真菌医学研究センター報告

第 17 巻2013

nov., an agaric ammonia fungus from Beijing, China,

9 ）Nakayama M, Hosoya K, Tomiyama D, Tsugukuni

2 ）Hosoya K, Nakayama M, Tomiyama D, Matsuzawa T,

rapid detection and identification of chaetomium andMycoscience 54: 226-230, 2013.

Imanishi Y, Ueda S, Yaguchi T: Risk analysis and rapid

detection of the genus Thermoascus, food spoilage fungi.
Food Cont DOI 10.1016/j.foodcont.2013.12.021

3 ）Hubka V, Peterson SW, Frisvad JC, Yaguchi T, Kubátová

T, Matsuzawa T, Imanishi Y, Yaguchi T: Method forevaluation of resistance to peracetic acid. J Food Protect

76: 999-1005, 2013.

10）Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela

K, Nozawa A, Nakatani Y, Furuya M: UniqueA, Kolarík M: Aspergillus waksmanii sp. nov. andmutation, accelerated mTOR signaling and angiogenesis

in section Fumigati described using polyphasic approach.

Pathol Int 63: 45-55, 2013.

Aspergillus marvanovae sp. nov., two closely related species

Int J Syst Evol Microbiol 63: 783–789, 2013.

4 ）Kan T, Takahagi S, Kamegashira A, Ooiwa H, Yaguchi T,

Hide M: Disseminated subcutaneous phaeohyphomycosisin the pulmonary cysts of Birt-Hogg-Dubé syndrome.

11）Novakova A, Hubka V, Dudova Z, Matsuzawa T,

Kubatova A, Yaguchi T, Kolarik M: New species inAspergillus section Fumigati from reclamation sites incaused by Exophiala ologosperma in a patientwith Wegener s

Wyoming（ U.S.A. ）and revision of A. viridinutans

5 ）Matsuzaki Y, Ota K, Sato K, Nara S, Yaguchi T, Nakano12）Takagi Y, Fukano H, Shimozato K, Tanaka R, Horii T,

caused by Trichophyton rubrum in a patient with myasthenia

isolated from healthy individuals and their distribution in

granulomatosis. Acta Derm Venereol 93: 356-357, 2013.

H, Sawamura D: Deep pseudocystic dermatophytosis
gravis. Acta Derm Venereol 93: 358-359, 2013.

6 ）Matsuzawa T, Campos Takaki GM, Yaguchi T, Okada

complex. Fungal Diversity, in press.

Kawamoto F, Kanbe T: Genotypes of Candida albicans

patients with oral candidiasis. J Infct Chemother DOI
10.1007/s10156-013-0626-5, 2013.

K, Gonoi T, Horie Y: Two new species of Aspergillus13）矢口貴志 : 食品危害カビにおける分類 , 同定の動向 .

of Pernambuco, Brazil. Mycoscience, in press.

14）矢口貴志 , 伊藤純子 , 矢澤勝清 : 同定の難しいノ

section Fumigati isolated from caatinga soil in the State
7 ）Matsuzawa T, Horie Y, Abliz P, Gonoi T, Yaguchi T:

Aspergillus huiyaniae sp. nov., a teleomorphic species

in sect. Fumigati isolated from desert soil in China.
Mycoscience, in press.

8 ）Mochizuki K, Sawada A, Suemori S, Kawakami H,

Niwa Y, Kondo Y, Ohkusu K, Yamada N, Ogura
S, Yaguchi T, Nishimura K, Kishino S: Intraocularpenetration of intravenous micafungin in inflamed

human eyes. Antimicrob Agents Chemother 57: 40274030, 2013.

千葉大学

日本食品微生物雑誌 30: 75-81, 2013.

カルジア－分類・検査・薬剤感受性を整理する－

Medical Technology 41: 1219-1225, 2013.

15）矢口貴志 : 真菌 . 新 GMP 微生物試験法（佐々木次雄 , 棚元憲一 , 川村邦夫編）じほう . 東京 . pp. 136157, 2013.

16）矢口貴志 : 凍結乾と乾燥による系統保存
装第 6 版

冷凍空調便覧

Ⅳ巻

酵母 . 新食 品・ 生 物 編 .

（社）日本冷凍空調学会 . pp. 312-314, 2013.

17）渡辺哲 , 矢口貴志 : 糸状菌症 . 臨床検査 58: 104-

109, 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
ソースプロジェクト（NBRP）を開始し , 国が戦略的に整備することが重要なものについて体系的に収集 , 保

bioresources, with a focus on those that required strategic
development by the national government. After the reviewing

the NBRP of every five years, in FY2012, the third phase has

存 , 提供などを行うための体制を整備してきた . その後

started.

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

5 年ごとの見直しを行い , 2012 年度より第 3 期が開始さ

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 , 相互の機関の連携を図り , これらの病原微生物株の収集・保存・提供

体制を整備して , 高度情報を賦与した信頼できる病原微生物株として提供し , 感染症と病原体の教育・研究をす

Chiba University s Medical Mycology Research Centerand this project is carried out by MMRC（pathogenic fungi/

Microbial Diseases（pathogenic bacteria）, Gifu University s

Graduate School of Medicine（pathogenic bacteria）, andNagasaki University s Institute of Tropical Medicine（pathogenic

protozoa）. Working together, they cooperate in various efforts to

support education and research pertaining to infectious diseases

る人々を支援している .

and pathogens. Specifically, they are developing a system

対応できる病原微生物コレクションを目指している .

microorganisms, and they supply reliable strains of pathogenic

本プロジェクトは , 今後いかなる感染症が発生しても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

38

千葉大学

for collection, preservation, and distribution 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.

真菌医学研究センター報告

第 17 巻2013

地球規模課題対応国際科学技術協力事業（JST & JICA）「AIDS 患者及びその他の免疫不全患者における新規診断法による真菌症対策」プロジェクト
SATREPS project（Science and Technology ResearchPartnership for Sustainable Development）

Diagnostic Approaches in the Management of Fungal Infections in
AIDS and Other Immunocompromised Patients

独立行政法人国際協力機構（JICA）および独立行政

Increasing numbers of fungal infections caused by yeasts

法人科学技術振興機構（JST）の地球規模課題対応国際and moulds are a serious threat not only to the Japanese

りブラジル連邦共和国サンパウロ州立カンピーナス大学

These infections hamper the patients quality of life and

科学技術協力事業（SATREPS）により , 平成 22 年度よ医学部感染症科との間で協力事業を実施し , 平成 25 年

society, but also to many other countries, including Brazil.

sometimes claims their lives. In this SATREPS project,

3 月で終了した . 本プロジェクトの目的は AIDS を始め

sponsored by JST and JICA, we worked together with

研究を推進・実用化し , その成果をブラジルに提供してof UNICAMP（State University of Campinas, Brazil）toとした免疫不全患者における真菌症の診断法・治療法のthe Division of Infectious Diseases, University Hospital

両国の医療の向上に役立てることにある .

develop accurate, rapid, and sensitive diagnostic methods for

年間の実施期間中に主要なテーマ（独自の DNA チップ

applied these methods to clinical cases in Brazil.

による真菌症の診断 , リアルタイム PCR 及び LAMP 法

and the results were very promising. Our project was highly

検討等）を無事完了することができた . 本プロジェクト

completion of the project term. Since these new technologies

びブラジルの影響下にあるポルトガル語圏アフリカ諸

particularly in South America and Portuguese-spoken African

途中 , 東日本大震災など予期せぬ事態が生じたが , 3による病原真菌の同定及び真菌症の診断 , β- グルカンによる真菌症の診断・原因菌の同定 , 薬剤最適投与法のの研究成果はブラジルを始めとして周辺中南米諸国およ国の医療レベルにも影響を与える大規模なものであり ,本プロジェクトは終了時評価で「A＋」の高評価を得た . なおこの終了時評価については , http://www.jst.go.jp/

fungal infections, such as DNA chips for the infection, andThe project was performed during FY 2010 to FY2012,

rated by the evaluation committee with the score of A＋ on

can be applied to diagnosis of patients in other countries,
countries, the fruit of our study is expected to give substantial
beneficial ripple effects to these societies.

globallhyouka/index.html にて閲覧可能である .

千葉大学

真菌医学研究センター報告

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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 の維持にも , 将来大きく貢献するはずです .

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.

A new project for epidemiological study of Cryptococcal fungi in
HIV-infected patients is launched in collaboration with Kenya

Medical Res. Insti.（KEMRI）and doctors from UCSF, USA.

40

千葉大学

真菌医学研究センター報告

第 17 巻2013

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
真菌症の中でもアスペルギルス症は特に多彩な病型を

aspergillosis（ CNPA ）is one example: the activity of the

もち , 重症度 , 経過 , 治療とも大きく異なっている．そ

disease varies among the patients, and sometimes an acute性が異なるばかりか , 同一症例においても経過中に急性the causative fungi isolated from the patients of various forms/の中でも慢性肺アスペルギルス症では , 症例ごとに活動

増悪がみられることがあり , これらのさまざまな病原性をもった菌株のゲノムを比較することにより , 病原因子

や病原機構の解明に結びつくことが期待される．

exacerbation results in serious outcome. Genetic analyses ofactivities are expected to help clarify the mechanism of theirpathogenicity.

To this end we performed the whole genome sequence

慢性肺アスペルギルス症（CPA: 菌球型を含む）及（WGS）analysis of eight strains isolated from patients with

例 か ら 得 ら れ た Aspergillus fumigatus に つ い て genome

with the aid of a next-generation sequencer. 81 826 SNPs

び侵襲性肺アスペルギルス症（IPA）のそれぞれ 4 症

sequencer（Illumina, MiSeq）を用いて菌のゲノム配列を比較したところ , ゲノム株（Af 293）のゲノム配列と比較して 81 826 の SNPs が見つかり , このうち CPA 間で共通の非同義置換は 7 個 , IPA では同じく 27 個であった . また機能不明遺伝子 Afu3g05880 に内在する tandem

IPA（invasive pulmonary aspergillosis）and CNPA in Japan
were identified by mapping the reads to A. fumigatus genomereference strain Af293. Of the identified SNPs, seven and 27

non-synonymous substitutions were specific to strains isolated

from patients with IPA and CNPA, respectively. Moreover,
the tandem repeat sequence in 5 -UTR of Afu3g05880 was

repeat は , CPA では , より少ない一定の繰り返し数を

found as a novel microsatellite region for differentiating

遺伝子の機能を探るとともに , ゲノム配列の解析を進IPA and CNPA. We expect comprehensive characterization

迫 り た い . 並 行 し て A. fumigatus の group sIII histidine

IPA and CNPA helps better understanding of molecularfumigatus L-Fucose specific lectin: AFL など）, A. fumigatus

We also performed molecular and cellular biological

示し , IPA とは異なることが示された . 今後 , これらのめ , 菌が CPA と IPA という異なる病態をとる原因にkinase（NikA）の機能解析 , 新しいレクチンの発見（A.

に感染する mycovirus 等の新しい知見を得た . 病原機構

between A. fumigatus strains isolated from patients withof genetic variations of strains isolated from patients withmechanisms in aspergillosis.

research of A. fumigatus, and reported in a paper a novelの解明 , 治療法開発に向けて研究を進める予定である．

pathway of group III histidine kinase（NikA）. We have

Aspergillosis is known to take various forms, and the

mycoviruses which infect the fungus. These results will beseverity, clinical course, and treatments differ significantly

depending on the forms. Chronic necrotizing pulmonary

千葉大学

also obtained new results on lectins of A. fumigatus, andpublished soon.

真菌医学研究センター報告

第 17 巻201341

平成 24 年度 共同利用・共同研究報告2012 Fiscal Year Cooperative Research Program Report
研究課題 ’
12－1

な偏性好気性真菌であり , 本菌は , 肺で感染後 , 脳血液クリプトコックスの低酸素ストレス応答機構
解析～低酸素応答遺伝子として見出した「転写因子 A」の分子機能解析を中心として～
川本進・清水公徳・大楠美佐子・萩原大祐（千葉大学真菌医学研究センター）

関門を越え脳髄膜へ移行して病原性を発揮して行く際 ,高酸素環境から低酸素環境への酸素欠乏ストレス条件に打ち勝ってはじめて増殖し , 病原性を発揮して行く . す
なわち , 本菌の低酸素環境ストレス応答は , 本菌感染の病原因子の一つと言える . 我々は , 本菌の細胞周期制御機構を研究中 , 低酸素環境条件下では細胞周期制御がflexible になるという , 本菌のユニークな低酸素ストレス

Raclavsky, Vladislav（チェコ・パラツキー大学）
Sipiczki, Matthias（ハンガリー・デブレツェン大学）

応答現象を見出した . そして , 我々は , Agrobacerium を用（東京大学大学院薬学系研究科）

これまでに「転写因子 A」を見出して来たが , 平成 24

関水和久・松本靖彦
田村
三浦裕（千葉大学大学院医学研究院）
恵・園田智子構築してスクリーニングし , 低酸素応答遺伝子として ,年度には , 「転写因子 A」の分子機能解析を更に詳細に進めた .

（横浜市立大学大学院医学研究科）

畑

いた , ゲノムランダム挿入遺伝子変異体ライブラリーを邦彦（鹿児島大学大学院農学研究科）

C. neoformans の低酸素応答遺伝子「転写因子 A」は ,カルシニューリン応答（Crz1）転写因子 , 及び , PKC1-

依存性（Sp1）転写因子とに homologous な分子であり ,Hypoxia signaling analysis of Cryptococcus neoformans

C 末端近くに , いわゆる , Zinc-finger を 3 つ持つ転写因

Susumu Kawamoto, Kiminori Shimizu, Misako
Ohkusu, Daisuke Hagiwara

本菌が G2 arrest 状態に入るために必須な遺伝子として（Chiba University, Medical Mycology Research Center）

Vladislav Raclavsky（Palacky University, Czech）
Matthias Sipiczki（University of Debrecen, Hungary）
Kazuhisa Sekimizu, Yasuhiko Matsumoto

子の一つ「Crz1/Sp1」であった . また , 低酸素環境下 ,同定した本分子は , 低酸素環境下での増殖の slowdownの他 , 本菌の細胞壁の integrity の維持 , 莢膜合成の抑制
的な制御 , バイオイルム生成の促進 , フルコナゾール感

受性 , カルシウム感受性など , 本菌の感染にも関連した
種々の細胞生理機能に重要な役割を持つ分子であるこ（University of Tokyo, Graduate School of Pharmaceuticalとを明らかにした . 真菌の低酸素応答シグナリングに転Yutaka Tamura

我々が C. neoformans で初めて見出し報告したが , 更に ,Satoshi Miura, Tomoko Sonoda

ル伝達下流分子の探索などを行って , C. neoformans の低Sciences）（Chiba University, Graduate School of Medicine）（Yokohama City Univerersity, Graduate School of

Medicine）

Kunihiko Hata

（Kagoshima University, Graduate School of Agriculture）

研究成果
病原酵母 Cryptococcus neoformans は , 生存に酸素が必須

42

千葉大学

写因子「Crz1/Sp1」が重要な役割を担っていることは ,本菌における低酸素応答転写因子「Crz1/Sp1」のシグナ

酸素ストレスに対する環境応答シグナリング機構全体の解明を目指すとともに , 本菌病原性への本分子の寄与を検討 , 考察しつつある .

発表論文1 ）Moráňová Z, Virtudazo EV, Pospíšilová K, Ohkusu M,
Kawamoto S, Husičková V, Raclavský V: The CRZ1 /真菌医学研究センター報告

第 17 巻2013

SP1 -like gene links survival under limited aeration, cell型転写因子 AtrR を世界で初めて見出し , A. fumigatus にCryptococcus neoformans. Biomedical Papers, Publishedていることを明らかにした . A. fumigatus の atrR 破壊株

integrity and biofilm formation in the pathogenic yeast

online 22 April 2013（DOI: 10.5507/bp.2013.024）.

の RNA-seq 解析を行ったところ , AtrR はトランスポー

ター遺伝子だけでなく , エルゴステ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 . 非常に研究課題 ’
12－2

興味深いことに , AtrR 制御下のエルゴステロール生合

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
五味勝也・阿部敬悦（東北大学大学院農学研究科）
川本

おいてもこのオーソログが同様にアゾール耐性に関与し進・清水公徳・萩原大祐

成酵素遺伝子は , すでに報告のある bHLH 型転写因子

SrbA 制御下のものと一致していたことから , エルゴス
テロール生合成酵素の発現は 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 . また , アゾール系薬剤耐性に関与していると考えられる ABC トランスポーター（AbcC/Cdr1B）の遺伝子は SrbA 制御下になく , AtrR 単独による制御を（千葉大学真菌医学研究センター）

小山泰二（野田産業科学研究所（現キッコーマン））

Molecular analyses of mechanisms of drug
resistance and signal transduction leading tonovel antifungal drug discovery in Aspergillus
fungi
Katsuya Gomi, Keietsu Abe

受けていると考えられた .

一方 , A. nidulans の二成分性情報伝達系の生育必須因子であるリン酸基中間因子遺伝子 ypdA の発現制御株（CypdA）と , 下流 response regulator 遺伝子 sskA および srrAの欠損を加えた CypdAsskAΔ , CypdAsrrAΔ , CypdAsskAΔ

srrAΔ 株を育種した . ypdA の発現抑制により , ypdA の転写量と蛋白質発現量が野生株比 5％以下となった . ypdAの発現抑制は , フルジオキソニル処理と類似する致死をもたらしたが , sskAΔ , srrAΔ の導入は生育を部分的に回（Graduate School of Agricultural Science, Tohoku

復させ , sskAΔ srrAΔ の導入は生育を完全に回復させた .

Susumu Kawamoto, Kiminori Shimizu, Daisuke
Hagiwara

なる sskAΔ 変異の形質を残していた .

University）

CypdAsskAΔ srrAΔ の生育は回復したが , 隔壁間長が長く（Medical Mycology Research Center, Chiba University）

Yasuji Koyama（Noda Institute for Scientific Research）

研究成果
A. fumigatus の薬剤耐性化機構については , 薬剤の標的分子や排出系の変異によることが示唆されているものの , 遺伝子レベルでの詳細な解析がほとんどなされていない . 一方 , A. fumigatus はヒト粘膜上での感染と生育を示すことから , このような固相表面における生育に関わる環境応答系が重要な機能を持っていると考えられる .

そこで , 本研究では A. fumigatus に対する効果的な新規創薬開発に資するため , 薬剤耐性化の分子機構の解明ならびに浸透圧シグナル伝達系の機構解明を目指した .

我々は麹菌においてアゾール系薬剤の排出に関与する

ABC トランスポーターの発現を制御する新規 Zn2Cys6

千葉大学

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研究課題 ’
12－3の餌にテトラサイクリンを添加することによって Tet 株

新規性の高い抗真菌薬標的の探索と阻害剤のアッセイ系の開発中の当該遺伝子の発現を抑制することができた . 最終的に抗真菌薬の標的として 5 つに絞ることができた . 今後

これらの株を用いてマウスを用いた感染実験検証 , 組換えタンパク等を用いて阻害剤のアッセイ系を開発して行知花博治（千葉大学真菌医学研究センター）
吉田 稔・松山晃久（理化学研究所）
青山俊弘（鈴鹿工業高等専門学校）

Searching for anti-fungal drug targets having
high novelty and developing of an assay system
for screening the repressors
Hiroji Chibana（Medical Mycology Research Center, Chiba University）

Minoru Yoshida, Akihisa Matsuyama

きたい .

発表論文
1 ）Nagi M, Tanabe K, Ueno K, Nakayama H, Aoyama T,
Chibana H, Yamagoe S, Umeyama T, Oura T, Ohno
H, Kajiwara S, Miyazaki Y: The Candida glabratasterol scavenging mechanism, mediated by the ATPbinding cassette transporter Aus1p, is regulated by ironlimitation, Mol Microbiol. 88: 371-81, 2013.

（Chemical Genomics Research Group, RIKEN）

研究課題 ’
12－4（Suzuka National College of Technology）

真菌感染防御における IL-17 産生機構に関する研究Toshihiro Aoyama

研究成果
Saccharomyces cerevisiae, C. albicans, Aspergillus fumigatus,
Cryptococcus neoformans の真菌類およびヒトゲノムのデー

岩倉洋一郎に相同性がないか , あるいは極めて低いこと , 2 ）ゲノ

西城

タベースを用いて , 最適な標的の条件として 1 ）ヒト（東京大学医科学研究所・システム疾患モデル研究センター・細胞機能研究分野）

忍（千葉大学真菌医学研究センター）

ム配列が公開されている病原真菌に高く保存されていること , 3 ）in vivo で必須であること , 以上の 3 点に条件として薬剤標的に探索を進めている . まず , 条件 1 ）と 2 ）を満たす遺伝子を Candida glabrata のゲノムから抽Studies on the production of IL-17 in immune
responses against fungal infection

出した . C. glabrata は , 病原真菌の中で遺伝子操作が最Yoichiro Iwakura

生物と位置づけ , ゲノムワイドな遺伝子機能解析を進めTokyo）

も容易であるため , 我々は本菌を病原真菌の実験モデルている . C. glabrata のゲノムから抽出した遺伝子のうち100 遺伝子について Tet 株を構築することができた . Tet

株は , テトラサイクリンを添加した培地において , Tet（The Institute of Medical Science, The University of

Shinobu Saijo（Medical Mycology Research Center, Chiba University）

がって , Tet 株が , テトラサイクリンに対して高い感受研究成果
IL-17A は主に活性化 CD4＋ T 細胞から産生され , 細

必須か否かを判定することができる . さらに標的分子あおいて中心的な役割を担っている . 申請者らは , マウ

であることを検証することは標的分子を絞り込む上で極albicans）で刺激し , その培養上清が naïve T 細胞を Th17

promoter が挿入された遺伝子の転写が抑制される . した性を示すことを基準にすることにより各遺伝子が生育にるいはその遺伝子が感染時に C. glabrata にとって必須

めて重要である . Tet 株を感染させた場合には , 蚕幼虫

44

千葉大学

胞外寄生菌に対する感染防御や自己免疫疾患の発症にス 骨 髄 由 来 樹 状 細 胞（BMDC） を Candida albicans（C.
細胞に分化させることを見出した . 一方 , IL-17A ノッ

真菌医学研究センター報告

第 17 巻2013

ク ア ウ ト マ ウ ス に C. albicans を 感 染 さ せ る と 野 生 型（WT）マウスと比較し生存率が有意に低下することを＋研究成果
気道や腸管上皮 , また , 皮膚表皮では , 細胞と細胞の明らかにした . しかし最近 , CD4 細胞以外にも IL-17A

間が様々な接着分子を介して連結され（tight junction）,

胞との機能の違いが注目されている .

破 壊 に , 外 来 お よ び 内 在 性 の protease が 関 与 し て いを産生する細胞が見出され , Th17 と他の IL-17 産生細そこで , まずマウスに C. albicans を感染させ in vivo で

外 来 抗 原 の 侵 入 を 防 い で い る . そ の tight junction のることが示されつつある . 真菌 Aspergillus による肺炎

も IL-17A が産生されるかどうかを検討したところ , 感

は , Aspergillus 自体の感染による免疫応答だけでなく ,なった . 今後は , ノックアウトマウスやノックインマウ

破壊が発症及び症状の重症化に関わっている可能性が強染後比較的早期に IL-17A が産生されることが明らかとスと細胞移植の系などを組み合わせ , どの細胞から分泌される IL-17A が感染防御に重要な役割を担っているのかを明らかにする予定である .

発表論文1 ）Ikeda S., Saijo, S., Murayama MA, Shimizu K., AkitsuA., and Iwakura, Y. Excess IL-1 signaling enhances thedevelopment of Th17 cells by downregulating TGF-binduced Foxp3 expression. J. Immunol., in press.

く示唆されている . 実際に , マウスに Aspergillus 由来のprotease を吸入させると , 組織破壊と好中球の浸潤を伴う強い気道炎症が認められた . さらに , 各種欠損マウスを用いた結果 , Aspergillus protease による好中球性気道炎症は , T 細胞 , B 細胞 , NKT 細胞 , マスト細胞非依存的であることが明らかになった . Aspergillus protease による

好中球性気道炎症局所では , IL-17, IL-17F, IL-21, IL23, IL-6, TNF といった Th17 型サイトカインおよび誘導因子の発現の亢進が認められ , これらサイトカインが

好中球性気道炎症の誘導に関与することが期待された .

しかしながら , これらのサイトカイン欠損マウスでも研究課題 ’
12－5

Aspergillus protease による好中球性気道炎症は正常マウスと同程度に誘導されることが明らかになった . 現在 , こ

真菌性肺炎の発症機序の解明
中江Aspergillus が産生する protease による宿主の気道上皮のれらサイトカイン以外に , 好中球性気道炎症の誘導に関わる因子の同定を進めている .

進（東京大学医科学研究所・システム疾患モデル研究センター・システムズバイオロジー研究分野）

新江
西城賢（杏林大学保健学部・免疫学教室）
忍（千葉大学真菌医学研究センター）

Studies on the pathogenesis of fungal pneumonia
Susumu Nakae（Laboratory of Systems Biology, The Institute ofMedical Science, The University of Tokyo）

Ken Arae（Department of Immunology, Faculty of Health

Sciences, Kyorin University）

Shinobu Saijo（Medical Mycology Research Center, Chiba University）

千葉大学

真菌医学研究センター報告

第 17 巻201345

研究課題 ’
12－6

特異的 IgE 陽性であることが明らかとなった . SC 特異的抗体陽性喘息患者はアスペルギルスにも感作されてい真 菌 に よ る 重 症 喘 息 発 症 機 構 の 解 明:
Schizophyllum commune 応答を中心としてる傾向が見出されたが , SC 特異的抗体価はアスペルギ

ルス特異的抗体価との相関性は認められなかった . これ
らの結果から喘息患者の一部は SC に感作が成立してい廣瀬晃一（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）
亀井克彦・豊留孝仁

ることが明らかとなった .

研究課題 ’
12－7（千葉大学真菌医学研究センター）

病原性担子菌酵母糖タンパク質糖鎖の構造解析

Roles of Fungi in the Development of Severe
Asthma: Possible roles of the immune responses
against Schizophyllum commune
Koich Hirose（Department of Allergy and Clinical Immunology,

Graduate School of Medicine, Chiba University）

Akira Watanabe

大橋貴生・藤山和仁（大阪大学生物工学国際交流センター）

五ノ井透（千葉大学真菌医学研究センター）

Structural analysis of glycoprotein sugar chainsin pathogenic Basidiomycete yeast.

（Department of Control and Treatment of Infectious

Takao Ohashi, Kazuhito Fujiyama

Hiroshi Nakajima

Gonoi Toru

Diseases, Chiba University Hospital）（Department of Allergy and Clinical Immunology,

Graduate School of Medicine, Chiba University）

Katsuhiko Kamei, Takahito Toyotome（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 . スエヒロタケ（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（International Center for Biotechnology, Osaka University）（Medical Mycology Research Center, Chiba University）

研究成果
病原性担子菌酵母を含む真核生物において多くのタンパク質には糖鎖が付加しており , その糖鎖構造は多様である . 非病原性モデル子嚢菌酵母である出芽酵母や分裂酵母では糖鎖構造および糖鎖生合成関連遺伝子が報告されている . また , 病原性子嚢菌酵母である Candida

albicans および Aspergillus fumigatus では糖鎖生合成関連

遺伝子の機能不全変異により病原性の低下が報告され率的なスクリーニング方法の報告は無く , 喘息患者におている . 一方 , 病原性担子菌酵母においては Cryptococcus

よる SC 特異的抗体のスクリーニング方法を確立し , 喘

ろか , 糖鎖構造情報はほとんど蓄積されていない . 前年

ける SC 感作率は不明である . 本研究では ELISA 法に息患者における SC 感作率を検討するとともに , 喘息重neoformans を除いて , 糖鎖生合成関連遺伝子の解析どこ

度までに代表的な病原性担子菌酵母である C. neoformans,症度との相関を明らかにすることを目的とした .

Rhodotorula mucilaginosa および Malassezia furfur の糖鎖構探索・同定した . この主要抗原を用いて SC 特異的抗を得ていた . 本年度はさらに上述の 3 種の病原性担子菌

まず SC による ABPM 患者血清を用いて主要抗原を造解析に着手し , 特に O- 結合型糖鎖について構造情報

体測定 ELISA 法を確立し , 喘息患者における感作率を

酵母の N- 結合型糖鎖の構造解析を行った .

Step4 12 名）中 , 4 名が SC 特異的 IgG 陽性 , 6 名が SC

ジルアミノ化法を用いて , ピリジルアミノ化糖鎖を調検討した . 47 名の喘息患者（Step2 6 名 , Step3 29 名 ,

46

千葉大学

各病原性担子菌酵母を培養後 , ヒドラジン分解－ピリ

真菌医学研究センター報告

第 17 巻2013

製後 , サイズ分画 HPLC, 逆相 HPLC および MALDI-

TOF-MS による解析を行った . その結果 , C. neoformans

においては Hex7-9PentGlcNAc2 の糖鎖が主要な糖鎖として検出された . R. mucilagionsa については , サイズ分
画 HPLC による分析が終了し , Hex5-7GlcNAc2 の糖鎖がDevelopment of method for classification andidentification of fungi based on the profilingof ribosomal proteins as obser ved by mass
spectrometry

主要な糖鎖として検出された . M. furfur についても同

Takashi Yaguchi, Reiko Tanaka

ミノ化糖鎖の分画を行い精製糖鎖を得た後 , LC-ESI-

Hiroaki Sato, Masaki Torimura

様にサイズ分画 HPLC, 逆相 HPLC によるピリジルア

MS/MS による分析を行った . 主要な糖鎖について , 標

準糖鎖と両 HPLC にて溶出位置が一致する糖鎖が検出され , LC-ESI-MS/MS の結果を考慮し以下の様に構造（Medical Mycology Research Center, Chiba University）（National Institute of Advanced Industrial Science andTechnology）

決定することができた ; Manα1,2Manα1,6（Manα1,3）

研究成果

β1 , 4 GlcNAc（ M 8 A ）, Manα1 , 2 Manα1 , 6（ Manα

め , A. fumigatus 基準株（IFM 47795＝ Af293）の精製リ

β1 , 4 GlcNAc（ M 7 A ）, Manα1 , 2 Manα1 , 6（ Manαソームタンパク質の正確な分子量リストを作成すること

1,4GlcNAcβ1,4GlcNAc（M8B）, Manα1,2Manα1,6（Man（真菌医学研究センター所有）を用いてリボソームを抽Manα1,6（Manα1,2Manα1,2Manα1,3）Manβ1,4GlcNAc
1 , 3）Manα1 , 6（ Manα1 , 2 Manα1 , 3）Manβ1 , 4 GlcNAc

1 , 2 Manα1 , 3）Manα1 , 6（ Manα1 , 2 Manα1 , 3）Manβ

生物種の同定・分類は基準株との比較で行われるたボソームタンパク質の質量分析を行い , 基準株のリボを目的とした . 試料菌株をビーズ破砕し , 超遠心分離機

α1,2Manα1,3）Manα1,6（Manα1,2Manα1,2Manα1,3）

出し , マトリックス支援レーザー脱離イオン化質量分析

より , 本 3 種病原性担子菌酵母糖鎖の基礎情報が取得でムタンパク質のマススペクトルを観測した . 各リボソー

素遺伝子の同定 , 本酵素の阻害剤の探索の効率化が達成列から計算される分子量との差異を比較して翻訳後修飾

Manβ1,4GlcNAcβ1,4GlcNAc（M9A）. 本研究の結果にきた . これらの情報を元に将来的には糖鎖生合成関連酵
され , 効果的な抗真菌剤の開発へと発展することが期待される .

ムタンパク質の正確な分子量を解析し , 翻訳アミノ酸配の有無を明らかにした . その結果 , 全ゲノム解読された

A. fumigatus および類縁の Neosartorya fischeri の計 3 株について , 分類の基準となるリボソームタンパク質の正しい分子量リストを作成した . さらに , ゲノム解読株間で ,研究課題 ’
12－8

一部のリボソームタンパク質に変異が生じていることをリボソームタンパク質をバイオマーカーとした質量分析法による真菌類の同定・分類法の開発
矢口貴志（千葉大学真菌医学研究センター）
佐藤浩昭・鳥村政基（（独）産業技術総合研究所

計（MALDI-TOFMS, 産総研所有）を用いて各リボソー

環境管理技術研究部

実証し , A. fumigatus 類縁菌の同定及び株レベルでの識別が , 本法によって可能であることを見出した .

今後 , DNA 解析法で得られた分類結果や形態および薬剤耐性などの特徴と比較することによって , 本法の妥当性を実証するとともに , 得られたマススペクトルを

データベース化し , 真菌の新しい分類指標を提示することが期待できる .

門）

千葉大学

真菌医学研究センター報告

第 17 巻201347

平成 24 年度 共同利用・共同研究研究会報告2012 Fiscal Year Cooperative Research Meetings Report
研究会－1

薬学部 , 附属病院 , 融合科学研究科などに数多くの研究者がおり , それぞれが活発に研究を進めている . しかし

千葉大学感染症研究ネットワーク
第 1 回研究会

それらの連携は意外なほど少なく , ネットワーク化が期
待されていた . そこで , これら千葉大学の学内各部局における感染症研究の統合 , さらには活性化を進めること

山本友子・高屋明子・佐藤慶治を目的として今年度から本センターの共同利用・共同研（千葉大学大学院薬学研究院）

米山光俊・川本究会として「千葉大学感染症研究ネットワーク」を企画

進・亀井克彦した . 第 1 回目となる今回は平成 24 年 6 月 23 日（土）（千葉大学真菌医学研究センター）に千葉大学薬学部大会議室（医薬系総合研究棟Ⅱ地下

北潔（東京大学大学院医学研究科）

1 階）にて開催した . 学内の感染症研究グループから計

Chiba University Research Network on Infectious
Diseases授（東大医学研究科）から「感染症研究が結ぶ世界の

Tomoko Yamamoto, Akiko Takaya, Yoshiharu 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）

8 題の一般演題が発表され , これに引き続き , 北

絆」と題した特別講演をいただいた . この種の学内ネットワークを目指した会合は今回が初めての試みであったが , 参加者は 63 名に達した .
全体を通じて非常に活発な議論が行われ , 学内各部局

における感染症研究の統合 , 活性化という目的は十分に達成されたものと考えられた。今後更に発展させるべく計画を進めている .

Kiyoshi Kita

（Graduate School of Medicine and Faculty of Medicine,

The University of Tokyo）

研究成果
千葉大学の学内には , 感染症研究にかかわる医学部 ,

48

千葉大学

潔教

真菌医学研究センター報告

第 17 巻2013

2013 年講演会
2013 Scientific Meetings & Seminars
1 ）第 125 回

真菌医学研究センター講演会

Dr. Jan Schmid（Massey University, Palmerston North,
New Zealand）

The biological significance of sex and protein-coding

DNA repeats in Candida albicans

日時 : 8 月 27 日（火） 15 時〜

場所 : 真菌医学研究センター B1 講堂
2 ）第 3 回感染免疫応答セミナー（主催 : COE スター
トアッププログラム「感染免疫応答」, 共催 : 博士

課程教育リーディングプログラム）
猪原直弘博士4 ）第 2 回感染症研究グローバルネットワークフォー

Naohiro Inohara（University of Michigan Medical School）

ラム 2013（主催 : 千葉大学真菌医学研究センター

割〜マウスモデルからの示唆

細菌の自然免疫克服戦略の解明とその応用」, 基盤

C. difficile 感染症重症化を防ぐ薬剤と自然免疫の役

日時 : 平成 25 年 9 月 20 日（金）18 時 30 分〜
場所 : 医学部本館 2 階

大カンファレンスルーム

共同利用・共同研究拠点事業 , 特別推進研究「病原研究 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」）

3 ）平成 25 年国立大学附置研究所・センター長会議

特別講演 :

第 2 部会シンポジウム（主催 : 国立大学附置研究金城雄樹

所・センター長会議第 2 部会 , 後援 : 千葉市 , 日本感染症学会 , 日本医真菌学会）
講演 : 亀井克彦教授（国立感染症研究所室長）

Yuki Kinjo

感染免疫における iNKT 細胞の役割

Katsuhiko Kamei

カビによる病気と診断・治療の現状

常世田好司

西城

（German Rheumatism Research Centre Berlin, Department

忍特任准教授Head）

Shinobu Saijo

カビの病気を防ぐ免疫のしくみ

Koji Tokoyoda

高橋弘喜准教授

感染を記憶する骨髄Hiroki Takahashi

ゲノム情報から読み解くカビの病気

日時 : 平成 25 年 10 月 25 日（金）10 時 〜 12 時
場所 : 京成ホテルミラマーレ 6 階ローズルーム

千葉大学

飯田哲也
（大阪大学微生物病研究所教授）

Tetsuya Iida

次世代 DNA シーケンサを用いた感染症研究

真菌医学研究センター報告

第 17 巻

2013

49

荒川宜親

神田達郎（千葉大学医学研究院（名古屋大学大学院医学系研究科

教授）

講師）

Tatsuo Kanda

Yoshichika Arakawa

新型多剤耐性グラム陰性菌が獲得した耐性機構とB 型肝炎ウイルスに対する肝細胞自然免疫応答

西城

それらの地球規模での拡散

忍（千葉大学真菌医学研究センター

一般講演 :

准教授）

Shinobu Saijo

石和田稔彦（千葉大学医学部附属病院

C 型レクチンと真菌感染防御機構

講師）

西田篤司

Naruhiko Ishiwada

小児細菌性髄膜炎予防ワクチンと日本への導入効（千葉大学薬学研究院

果に関する研究

Atsushi Nishida

教授）

アレルギー性気管支肺真菌症患者から単離された

西村基（千葉大学医学部附属病院／医学研究院

助教）

Motoi Nishimura

臨床細菌検査の動向－質量分析計による最近同定5 ）第 4 回感染免疫応答セミナー（主催 : COE スター

助教）

Kazumi Norose

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－

トアッププログラム「感染免疫応答」, 共催 : 博士

課程教育リーディングプログラム）

Glen N. Barber 博士（University of Miami, School of Medicine）

星野忠次

Cytosolic DNA sensors, STING and Innate immunity

准教授）

Tyuji Hoshino

日時 : 平成 25 年 12 月 6 日（金）17 時〜

2 価金属原子対を標的とした抗ウイルス薬の開発

50

日時 : 平成 25 年 11 月 30 日（土）場所 : 千葉大学薬学部 120 周年記念講堂

野呂瀬一美（千葉大学薬学研究院

する化学物質

9 時 30 分 〜 18 時 10 分など－

（千葉大学医学研究院

Schizophyllum commune（和名 : スエヒロタケ）が生産

千葉大学

場所 : 医学部本館 1 階

真菌医学研究センター報告

第 17 巻2013

第 1 講義室

平成 26 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 2014
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

千葉大学

真菌医学研究センター報告

第 17 巻2013

51

ISSN 1347−6920

千葉大学
真 菌 医 学 研 究センタ ー 報 告

MMRC
ANNUAL REPORT OF MEDICAL MYCOLOGYRESEARCH CENTER, CHIBA UNIVERSITY 2013千葉大学 真菌医学研究センター

17

第
巻

17

CHIBA UNIVERSITY
2013

平成
年

25

平成25年

報告