Osaka University

Research Institute forMicrobial Diseases

2014－2015

Contents
History & Outline

Ĳ

Organizationĳ

Former Directors & Professors

ĴDepartment Heads

ĵ

Faculty & StudentsĶ

Research & Activities
Division of Infectious Diseases……………………………………… ķ
Division of Host Defense ……………………………………………… Ĳĵ
Division of Cellular and Molecular Biology …………………………… ĳĵ
Research Center for Infectious Disease Control ……………………… Ĵĳ
Genome Information Research Center ………………………………… ĴĵInternational Research Center for Infectious Diseases ……………… ĵĳ

Office of Combined Program on Microbiology and immunology

ĶıThailand-Japan Research Collaboration Centeron Emerging and Re-emerging Infections

ĶĴ

Mahidol-Osaka Center for Infectious Diseases

Ķķ

BIKEN Endowed Department of Dengue Vaccine DevelopmentĶĸResearch Facilities

ĶĹ

Immunology Frontier Research Centerķĵ

Accounts & Building Areaķķ

Map & AccessķĹ

Photo by Hiroshi Nojima.

History & Outline

The Research Institute for Microbial Diseases was originally established as a ﬁve-department “Research

History & Outline

Research Institute for Microbial Diseases
Center for Communicable Diseases” in February, 1934 after a donation from Mr. Gendo Yamaguchi at
Dojima in Osaka City. At that time, the Institute was located on Nakanoshima campus of Osaka Medical
School, but then became a part of Osaka University in September, 1934. It was moved to its present site on
Suita campus in 1967, in accordance with the university's relocation program.
Until 1993, the Institute had grown to include nineteen departments spanning a wide range of diverse
subjects, such as infectious disease, immunology, cancer and molecular biology. Additionally, it had three
special research facilities, a central laboratory and a library. In 1997, the Research Center for Emerging
Infectious Diseases was added to the Institute. In 2005, the Institute was reorganized to constitute three
Research divisions; the “Division of Infectious Disease”, the “Division of Host Defence” and the “Division of
Cellular and Molecular Biology”, which represent 15 departments together with three attached centers for
specialized research on infectious disease and genome information. Moreover, the Research Collaboration
Center on Emerging and Re-emerging Infections was founded in Bangkok in collaboration with the National
Institute of Health, Thailand, to defend people against possible emerging and re-emerging infections.
Basic research on infectious disease, immunology, and cell biology is the Institute's principal focus. The
results of research in these ﬁelds at the Institutehave contributed considerably to the diagnosis,
prevention, and treatment of infectious diseases,
immunological diseases and cancer, as well as toprogress in basic biomedical science. The Institutewas selected as one of “the 21st century center of
excellence programs” on the theme of “combined
program on microbiology and immunology” in
2003. Also, it was certiﬁed as “joint usage /research center” by MEXT (the Ministry of education, culture, sports, science and technology) in
The copper plate recalling that the Research Institutefor Microbial Diseases was originally establishedby a donation from Mr. Gendo Yamaguchi.
(At the entrance hall of the main building)2009, and started the mission in April, 2010. Moreover, the institute newly established the “Center for
Genetic Analysis of Biological Responses” toconserve gene resources and to protect intellectual
property.

Mr. Gendo Yamaguchi
Born in Onomichi City in 1863 as the eldest
son of a physician, he moved to Osaka at theage of 15 and became one of western Japanʼsmost successful businessmen. He retired in
1917, and devoted the rest of his life toreligion and the tea ceremony. He donatedmost of the proceeds of his estate to public
enterprise, shrines and temples.

The Institute accepts and trains Master and Ph.D.
candidates in the medical and biological sciences. At
present, more than 200 full, associate, and assistantprofessors, research associates, graduate students
and research fellows pursue studies in microbiology, oncology and molecular biology in state of theart facilities at the Institute.

Ĳ

Organization

Director
Faculty Meeting

Delegate Assembly

Research Division
Division of Infectious Diseases
Department of Molecular Bacteriology
Department of Viral lnfections
Department of Molecular Virology
Department of Pharmacotherapy
Department of Immunoparasitology
Division of Host Defense
Department of Molecular Immunology
Department of Immunoregulation
Department of Host Defense
Department of Cell Biology
Department of Immunochemistry
Division of Cellular and Molecular Biology
Department of Molecular Microbiology
Department of Molecular Genetics
Department of Oncogene Research
Department of Signal Transduction
Department of Cellular Regulation

Special Research Facilities
Research Center for Infectious Disease Control
Department of Bacterial Infections
Department of Molecular Protozoology
Department of Virology
Genome Information Research Center
Department of Experimental Genome Research
Department of Genome Informatics
Department of Infection Metagenomics
International Research Center for Infectious Diseases
Department of Special Pathogens
Department of Infectious Disease Control
Pathogenic Microbes Repository Unit
Animal Resource Center for Infectious Diseases
DNA-chip Development Center for Infectious Diseases
Center for Genetic Analysis of Biological Responses

Office of Combined Program on Microbiology and Immunology
Research Promotion Group
Education Promotion Group

Research Collaboration Center in Overseas
Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections
Section of Bacterial Infections
Section of Viral Infections
Mahidol-Osaka Center for Infectious Diseases
BIKEN Endowed Department of Dengue Vaccine Development

Common Research Facilities
Central Instrumentation Laboratory
Radioisotope Laboratory
Central Laboratory for Biological Hazardous Microbes
Library

Administration
General Affairs Section
Accounting Section
Research Cooperation Section

World Premier International Research Center
Immunology Frontier Research CenterĳFormer Directors & Professors<DVKLUR.RWDNH0'3URIHVVRUí

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Organization, Former Directors & Professors

Former Directors

Former Professors
Yashiro Kotake, M.D., Professor
Sadao Yoshida, M.D., Professor
Arao Imamura, M.D., Professor
Yukichi Satani, M.D., Professor
Tenji Taniguchi, M.D., Professor
Kota Sera, M.D., Professor
Tatsunori Masayama, M.D., Professor
Shohei Otani, M.D., Professor
Teishiro Seki, M.D., Professor
Masami Suda, M.D., Professor
Kaoru Morishita, M.D., Professor
Hisashi Yamaguchi, M.D., Professor
Tsunesaburo Fujino, M.D., Professor
Masakazu Ito, M.D., Professor
Juntaro Kamahora, M.D., Professor
Shinji Nishimura, M.D., Professor
Mitsuhiko Kato, M.D., Professor
Masahiko Yoneda, M.D., Professor
Shigeru Shiba, M.D., Professor
Shozo Inoki, M.D., Professor
Mitsuo Hori, M.D., Professor
Yoshiomi Okuno, M.D., Professor
Shigeyuki Ishigami, M.D., Professor
Tsunehisa Amano, M.D., Professor
Junichi Kawamata, M.D., Professor
Yoshio Okada, M.D., Professor
Mitsuo Torii, D.Sc., Professor
Konosuke Fukai, M.D., Professor
Tatsuo Mori, M.D., Professor
Tonetaro Ito, M.D., Professor
Takeo Kakunaga, D.Pharm., Professor
Shiro Kato, M.D., Professor
Toshio Nakabayashi, M.D., Professor
Takahisa Yamanouchi, M.D., Professor
Toshio Miwatani, M.D., Professor

Michiaki Takahashi, M.D., Professor
Hajime Fujio, M.D., Professor
Tetsuo Taguchi, M.D., Professor
Aizo Matsushiro, D.Sc., Professor
Atsuo Nakata, D.Sc., Professor
Hiroto Okayama, M.D., Professor
Kumao Toyoshima, M.D., Professor
Teruo Kitani, M.D., Professor
Shin-ichiro Takai, M.D., Professor
Morihiro Matsuda, M.D., Professor
Takashi Kurimura, M.D., Professor
Koichi Yamanishi, M.D., Professor
Akira Hakura, D.Sc., Professor
Tetsu Akiyama, D.Sc., Professor
Takeshi Kurata, M.D.,D.Med.Sci., Professor
Shigeharu Ueda, M.D.,D.Med.Sci., Professor
Kazunori Shimada, M.D.,D.Med.Sci., Professor
Chihiro Sasakawa, M.D., Professor
Akio Sugino, D.Sci., Professor
Hiroshi Kiyono, D.D.S.,Ph.D., Professor
Yoshitake Nishimune, M.D., Professor
Toru Nakano, M.D., D.Med. SC., Professor
Hideo Shinagawa, D.Sc., Professor
Shin-ichi Tamura, Ph.D., Professor
Michiyuki Matsuda, M.D., D.Med. SC., Professor
Takeshi Honda, M.D., Ph. D., Professor
Naoyuki Taniguchi, M.D., Ph. D., Professor
Tamotsu Yoshimori, M.D., Ph. D., Professor
Kazuyuki Tanabe, M.D., Ph. D., Professor
Fumio Imamoto, D.Sc., Professor
Atsushi Kumanogo, M.D., Ph. D., Professor
Kazunori Oishi, M.D., Ph. D., SA Professor
Masanori Kameoka, Ph. D., SA Professor
Masaru Okabe, Ph. D., Professor
Kazuyoshi Ikuta, Ph. D., ProfessorĴDepartment Heads
2014,04,01

Director
Vice Director
Division of Infectious Diseases
Department of Molecular Bacteriology
Department of Viral Infections
Department of Molecular Virology
Department of Pharmacotherapy
Department of Immunoparasitology
Division of Host Defense
Department of Molecular Immunology
Department of Immunoregulation
Department of Host Defense
Department of Cell Biology
Department of Immunochemistry
Division of Cellular and Molecular Biology
Department of Molecular Microbiology
Department of Molecular Genetics
Department of Oncogene Research
Department of Signal Transduction
Department of Cellular Regulation

Professor Eisuke Mekada Ph.D.
Professor Yoshiharu Matsuura D. V. M., Ph.D.
Professor Yasuhiko Horiguchi D. V. M., Ph.D.
Professor Tatsuo Shioda Ph.D.
Professor Yoshiharu Matsuura D. V. M., Ph.D.
Professsor Masahiro Yamamoto Ph.D.
Professor Hitoshi Kikutani M. D., Ph.D.
Professor Taroh Kinoshita Ph.D.
Professor Shizuo Akira M. D., Ph.D.
Professor Eisuke Mekada Ph.D.
Professor Hisashi Arase M. D., Ph.D.
Professor Hiroshi Nojima Ph.D.
Professor Masato Okada Ph.D.
Professor Nobuyuki Takakura M. D., Ph.D.
Professor Hiroaki Miki Ph.D.

Research Center for Infectious Disease Control
Department of Bacterial Infections
Department of Molecular Protozoology
Department of Viorogy
Genome Infomation Research Center
Department of Experimental Genome Research
Department of Genome Informatics
Department of Infection Metagenomics
International Research Center for Infectious Diseases
Department of Special Pathogens
Laboratory of Clinical Research on Infectious Diseases
Laboratory of Infection Cell Biology
Laboratory of Viral Infection
Department of Infectious Disease Control
Laboratory of Genomic Research on Pathogenic Bacteria
Laboratory of Viral Replication
Laboratory of Combined Research on Microbiology and Immunology
Pathogenic Microbes Repository Unit

Head, Professor Toshihiro Horii Ph.D.

Animal Resource Center for Infectious Diseases
DNA-chip Development Center for Infectious Diseases
Center for Genetic Analysis of Biological Responses

Head, Professor Masahito Ikawa Ph.D.
Head, Professor Hiroshi Nojima Ph.D.
Head, Professor Masahito Ikawa Ph.D.

Office of Combined Program on Microbiology and Immunology
Research Promotion Group
Education Promotion Group

Head, Director Eisuke Mekada Ph.D.
Associate Professsor Yoshiko Murakami M. D., Ph.D.
Associate Professsor Hodaka Fujii M. D., Ph.D.

Thailand - Japan Research Collaboration Center on Emerging andRe-emerging Infections
Section of Bacterial Infections
Section of Viral Infections

Head, SA Professor Naokazu Takeda Ph.D.

Professor Toshihiro Horii Ph.D.
Head, Professor Teruo Yasunaga Ph.D.
Professor Masahito Ikawa Ph.D.
Professor Teruo Yasunaga Ph.D.
Professor Toshihiro Horii Ph.D.
Head, Professor Toshihiro Horii Ph.D.
SA Professor Yukako Fujinaga Ph.D.
SA Associate Professor Eiji Morita Ph.D.
SA Professor Tetsuya Iida Ph.D.
SA Associate Professor Takeshi Kobayashi D.V.M., Ph.D.
Associate Professor Hiroki Nagai Ph.D.
SA Professor Tetsuya Iida Ph.D.

SA Professor Shigeyuki Hamada D.D.S., Ph.D.
SA Professor Naokazu Tokeda Ph.D.

Mahidol-Osaka Center for Infectious Diseases

Head,Professor Yoshiharu Matsuura D.V.M., Ph.D.
SA Associate Professor Tamaki Okabayashi D.V.M., Ph.D.

BIKEN Endowed Department of Dengue Vaccine Development

Endowed Chair Professor Eiji Konishi Ph.D.

Central Instrumentation Laboratory
Radioisotope Laboratory
Central Laboratory for Biological Hazardous Microbes
Library

Head,Professor Hiroaki Miki Ph.D.
Head,Professor Hiroaki Miki Ph.D.
Head, Professor Tatsuo Shioda Ph.D.
Head, Professor Hiroaki Miki Ph.D.

Administration

Head, Yoshinobu Ishikura＊SA : Specially Appointed

ĵFaculty & Students

2014,04,01
Professor

16

Endowed Chair Professor

1

SA Professor

5

Associate Professor

14

SA Associate Professor

7

Lecturer1

SA Lecturer

5

Assistant Professor

28

Endowed Chair Assistant Professor

1

SA Assistant Professor

7

Educational Support Staff

3

Technical Staff

7

Administrative Staff

22

SA Researcher

48

Part-time General & Technical staff

39

Department Heads, Faculty & Students

Staff

203

Total
SA： Specially Appointed

Graduate Students
2014,04,01

Doctor Course

Master Course

Graduate School of Medicine

358Graduate School of Science1

1

Graduate School of Pharmaceutical Science22

Graduate School of Dentistry00

Graduate School of Frontier Biosciences

8

0

46

11

Total

Research Fellows & Research Students
2014,04,01

Special research students

4

Research Students

5

Visiting Research Scholars

3

JSPS Research Fellows

1

Total

13
Ķ

Research & Activities

Division of Infectious Diseases

Department of Molecular Bacteriology
Research Group

Professor
Assistant Professor
Assistant Professor
SA Researcher
SA Researcher

Yasuhiko Horiguchi, D.V. M., D. Agr. Sci.
Hiroyuki Abe, Ph.D.
Naoaki Shinzawa, Ph.D.
Aya Fukui, Ph. D.
Keiji Nakamura, D.V.M., Ph.D.

Research Projects
The objective of this department is to understand the molecular mechanisms by which pathogenic bacterial virulence factors
affect host cell functions. Our present research interests include:(1) Analysis of the structure and function of bacterial protein toxins
Bacterial protein toxins, which are the most poisonous substances on the earth, are known to act specifically on a particular celland a particular biomolecule. To understand how bacterial toxins act so powerfully and specifically, we are analyzing theireffects on the host at the systemic, tissue, cellular and molecular levels. The toxins currently under investigation are Bordetella
dermonecrotic toxin, Pasteurella toxin, Clostridium perfringens enterotoxin, and Escherichia coli cytotoxic necrotizing factor.
We are also analyzing the steric structure and molecular localization of the functional domains of the toxins. These approaches
together will help to clarify the structure and function of these bacterial toxins.
(2) Analysis of the pathogenesis of whooping coughBordetella pertussis, a pathogenic bacterium, infects the human respiratory tract and causes whooping cough, which ischaracterized by paroxysmal cough. There are two significant questions about the pathogenesis of B. pertussis infection. First,
why does B. pertussis only infect humans but not other mammals? Second, how do the bacteria cause the paroxysmal cough?
We are examining the pathology of the disease and the function of bacterial virulence factors by using an animal model of theinfection.

Fig. 1: Bacterial protein toxins with various activities that influence particular cellular functions.
Many bacterial protein toxins exert their toxicity by modifying important functions of the host cells.
The relevant physiological functions of the cells can be determined by dissecting the actions of thebacterial toxins.

ķResearch & Activities

Fig. 2: Overall structure of Clostidium perfringens enterotoxin.

Fig. 3: Bordetella pertussis, B. parapertussis, and B. bronchiseptica
are closely-related pathogenic bacteria.
B. bronchiseptica with the largest genome shows the broadest rangeof host while B. pertussis with the smallest genome shows thenarrowest host range. It is believed that B. pertussis evolved from a
lineage of B. bronchiseptica through deletion and/or translocationof a large number of genes.

Recent Publications
1. Substrate specificity of Pasteurella multocidaWR[LQIRUĮVXEXQLWVRIKHWHURWULPHULF*SURWHLQV2UWK-+)HVWHU,
Siegert P, Weise M, Lanner U, Kamitani S, Tachibana T, Wilson BA, Schlosser A, Horiguchi Y, Aktories K.
FASEB J. 2013 Feb;27(2):832-42.
2. Swine atrophic rhinitis caused by Pasteurella multocida toxin and Bordetella dermonecrotic toxin. Horiguchi Y.
Curr Top Microbiol Immunol. 2012;361:113-29.
3. Enzymatic actions of Pasteurella multocidaWR[LQGHWHFWHGE\PRQRFORQDODQWLERGLHVUHFRJQL]LQJWKHGHDPLGDWHGĮVXEXQLWRIWKHKHWHURWULPHULF*73DVH*T.DPLWDQL6$R67RVKLPD+7DFKLEDQD7+DVKLPRWR0.LWDGRNRUR.
Fukui-Miyazaki A, Abe H, Horiguchi Y. FEBS J. 2011 Aug;278(15):2702-12.
4. Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins. Kitadokoro
K, Nishimura K, Kamitani S, Fukui-Miyazaki A, Toshima H, Abe H, Kamata Y, Sugita-Konishi Y, Yamamoto S,
Karatani H, Horiguchi Y.J Biol Chem-XQ  
5. Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin. Kamitani S, Kitadokoro K,
Miyazawa M, Toshima H, Fukui A, Abe H, Miyake M, Horiguchi Y. J Biol Chem. 2010 Aug 13;285(33):25467-75.
ĸResearch & Activities

Division of Infectious Diseases

Department of Viral Infections
Research Group

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The main focus of this department is to elucidate the molecular mechanisms of viral diseases, including humanimmunodeficiency virus (HIV). The following projects are currently underway.
(1) Antiretroviral factors
HIV does not establish a productive infection in any monkey other than the chimpanzee. This is thought to be due to the fact
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75,0Į(2) Host factors that participate in the adverse effects of anti-retroviral drugs
Human genetic variations drive differences in human phenotypes. In collaboration with Thai groups, we are analyzing therelationship between human genomic variation and adverse effects of antiretroviral therapy, with the aim of establishing“tailor-made therapies” that will improve the quality of life of HIV-infected patients.
(3) Analysis of HIV-1 genome RNA dimerization
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ĺResearch & Activities

Division of Infectious Diseases

Department of Molecular Virology
Research Group

Professor
Assistant Professor
Assistant Professor
SA Researcher
SA Researcher
SA Researcher
SA Researcher
Postdoctoral Fellow

Yoshiharu Matsuura
Toru Okamoto
Takasuke Fukuhara
Chikako Ono
Sayaka Aizawa
Masami Wada
Puig-Basagoiti Francesc
Shinya Watanabe

DVM, PhD
PhD
MD, PhD
PhD
PhD
PhD
PhD
DVM, PhD

We are working on understanding the molecular mechanisms of hepatitis C virus (HCV) entry, replication, assembly, andpathogenesis, and developing a novel virus vector for gene delivery.
1. Studies on the molecular biology of HCV replication and pathogenesis
HCV infects over 130 million individuals worldwide and is one of the most common etiologic agents of chronic liver diseases,
including liver cirrhosis and hepatocellular carcinoma (HCC). Although novel innovative anti-HCV agents achieve sustained
virological responses in chronic hepatitis C patients, drug-resistant viruses emerge frequently. Therefore, host factors that are
indispensable for HCV replication are the ideal targets for the development of new broad spectrum therapeutics against chronic
hepatitis C that associate with a low possibility of emergence of breakthrough viruses against antiviral drugs. HCV internalizes
into cells by endocytosis by interacting with several candidate receptors, including hCD81, SR-BI, Claudin1, and Occludin.
After uncoating, the viral RNA is translated into a large precursor polyprotein composed of 3,000 amino acids, which iscleaved by signal peptidase, signal peptide peptidase, and virus-encoded proteases, resulting in at least 10 viral proteins. We
have shown by using transgenic mice expressing the HCV core protein, which develop hepatic steatosis and HCC, that
NQRFNLQJRXWWKHLU3$ȖJHQHLQKLELWVWKHGHYHORSPHQWRIOLYHUVWHDWRVLVDQG+&&7KLVVXJJHVWVWKDW3$ȖSOD\VDFUXFLDO
role in the development of the liver failure induced by HCV infection. Furthermore, we have shown that co-chaperones such as
FKBP8 and hBind-1 recruit Hsp90 to the HCV replication complex by interacting with NS5A protein, thereby playing animportant role in HCV replication. Liver-specific miR-122 enhances the translation of HCV RNA by interacting with its
5’ UTR. Several non-hepatic cell lines permit HCV replication when miR-122 is expressed but infectious particles are notproduced. This is probably because these cells have low expression levels of apolipoproteins such as ApoB and ApoE, unlike
hepatic cell lines such as Huh7 and Hep3B. Indeed, Huh7 cells that are deficient in both ApoB and ApoE genes (DKO cells)
exhibit severely impaired infectious particle formation, while exogenous expression of not only ApoE but also otherapolipoproteins (including ApoA1, ApoA2, ApoC1, ApoC2, and ApoC3) rescues particle formation. This suggests that ApoA,
ApoC and ApoE participate in HCV assembly in a redundant fashion. HCV belongs to the family of Flaviviridae, which
includes flaviviruses such as Japanese encephalitis virus (JEV). Since there is a robust cell culture system and a small animalmodel for JEV, we are also investigating the replication and pathogenesis of JEV as a surrogate model for HCV.

Ĳı

Research & Activities

2. Development of baculoviral vectors
Viral vectors are essential tools for studies on replication-deficient viral infectious diseases such as HCV. The development ofnovel viral vectors is also essential for future gene therapy. We are working on the baculovirus Autographa californica
nucleopolyhedrovirus (AcNPV) to develop it into a versatile viral vector for gene delivery not only in vitro but also in vivo.
AcNPV is an insect virus with a 134 kb double-stranded circular DNA genome. Due to its strong promoters, baculovirus iscommonly used for the large-scale production of recombinant protein in insect cells. Baculovirus is also capable of entering avariety of mammalian cells and expressing foreign genes under the control of mammalian promoters without replicating its
viral genome. Therefore, baculovirus is a useful viral vector, not only for the abundant expression of foreign genes in insect
cells but also for efficient gene delivery to mammalian cells. AcNPV has a number of unique beneficial properties as a viral
vector, including a large capacity for foreign gene incorporation, easy manipulation, and replication competence in insect cells
combined with incompetence in mammalian cells. Therefore, it has a significantly lower likelihood of generating
replication-competent revertants that express baculoviral gene products (which can lead to harmful immune responses against
mammalian cells) than other viral vectors that are currently in use. Furthermore, intranasal inoculation with AcNPV induces astrong innate immune response that protects mice from lethal challenges of influenza viruses. We have shown thatinternalization of viral DNA by membrane fusion of the envelope glycoprotein is required to induce inflammatory cytokines
and type I IFN viaWKH7/5GHSHQGHQWDQGLQGHSHQGHQWSDWKZD\UHVSHFWLYHO\0RUHRYHUWKHLQGXFWLRQRI,)1ȕWKURXJKWKH
STING/TBK1/IRF3 pathway attenuates the transgene expression in mammalian cells. Thus, innate immune responses induced
by infection with AcNPV attenuate its transgene expression. This characteristic means that AcNPV may be useful for selective
gene transduction into cells whose ability to evoke innate immunity is impaired by infection with various viruses.

Recent Publications
1. Novel permissive cell lines for a complete propagation of hepatitis C virus. Shiokawa M, Fukuhara T, Ono C,
Yamamoto S, Okamoto T, Watanabe N, Wakita T, Matsuura Y. J Virol. 2014 Mar 5. [Epub ahead of print]
2. Innate immune response induced by baculovirus attenuates transgene expression in Mammalian cells. Ono C,
Ninomiya A, Yamamoto S, Abe T, Wen X, Fukuhara T, Sasai M, Yamamoto M, Saitoh T, Satoh T, Kawai T, Ishii
KJ, Akira S, Okamoto T, Matsuura Y. J Virol. 2014 Feb;88(4):2157-67.
3. Japanese encephalitis virus core protein inhibits stress granule formation through an interaction with Caprin-1 andfacilitates viral propagation. Katoh H, Okamoto T, Fukuhara T, Kambara H, Morita E, Mori Y, Kamitani W,
Matsuura Y. J Virol. 2013 Jan;87(1):489-502.
4. Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis
C virus. Fukuhara T, Kambara H, Shiokawa M, Ono C, Katoh H, Morita E, Okuzaki D, Maehara Y, Koike K,
Matsuura Y. J Virol. 2012 Aug;86(15):7918-33.
5. CD44 participates in IP-10 induction in cells in which hepatitis C virus RNA is replicating, through an interaction
with Toll-like receptor 2 and hyaluronan. Abe T, Fukuhara T, Wen X, Ninomiya A, Moriishi K, Maehara Y,
Takeuchi O, Kawai T, Akira S, Matsuura Y. J Virol. 2012 Jun;86(11):6159-70.
ĲĲ

Research & Activities

Division of Infectious Diseases

Department of Immunoparasitology
Research Group

3URIHVVRU（683）
0DVDKLUR<DPDPRWR3K'
$VVLVWDQW3URIHVVRU（683）
Miwa Sasai, 3K'
6SHFLDOO\$SSRLQWHG$VVLVWDQW3URIHVVRU Hironori Bando, 3K'

In our immunoparasitology laboratory, the apicomplexan protozoan parasite called Toxoplasma gondii serves as a researchmodel for analyzing host-to-pathogen interactions.
1) What is Toxoplasma?
T. gondii is a eukaryotic pathogen that causes life-threatening toxoplasmosis, a condition that includes encephalitis in
immunocompromised individuals such as those who are being treated by chemotherapy or have AIDS. It also causes
congenital diseases on primary infection during pregnancy in humans and animals. It is an obligate intracellular eukaryotic
parasite that can proliferate exclusively inside a parasitophorous vacuole, which is formed during host–cell invasion.
Taxonomically, T. gondii belongs to the phylum of Apicomplexa, which is characterized by the presence of an apical complex
with secretory organelles such as conoids, micronemes, and rhoptries (Figure 1A). The rhoptries, which are large bulb-shaped
organelles, contain a variety of proteins that are secreted into the host cytoplasm or in the forming parasitophorous vacuole
during parasite entry. These proteins then co-opt the host cell for parasite growth and survival (Figure 1B and C).

Figure 1 Toxoplasma gondii and the rhophry protein（A）A schematic figure of T. gondii. The apical complex includes rhoptries (red)。
（B）An electron microscopic picture of T. gondii residing in a parasitophorous vacuole (blue) in an infected host cell.
（C）Immunofluorescent picture of T. gondiiWRGHWHFWDUKRSWU\SURWHLQ523

2) Analysis of host cell-autonomous innate immunity against T. gondii.
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JHQHUDWLQJPLFHWKDWODFNDOOVL[*%3JHQHVRQFKURPRVRPH *ES FKU) by targeted chromosome engineering. The mice
ODFNLQJ*ES FKU were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs.
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JURZWK$OWKRXJKWKH,)1ȖLQGXFHGR[LGDWLYHUHVSRQVHDQGWKHORFDOL]DWLRQRIDXWRSKDJ\SURWHLQ$WJEZHUHQRUPDOGHOHWLRQ
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PHPEHUVRI*ESFKU restored the protective response against T. gondiiLQ*ESFKU-deleted cells. Our results suggest that the*ESFKU proteins redundantly play a pivotal role in anti-T. gondiiKRVWGHIHQVHE\FRQWUROOLQJ,)1¤ȖPHGLDWHG,UJEGHSHQGHQW
cellular innate immunity (Figure 2).
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play a critical role in cell-autonomous immunity against T. gondii. However, the involvement of other autophagy proteinsremains unknown. Here, we show that some essential autophagy proteins, but not all, participate in anti-T. gondii cellular
LPPXQLW\E\UHFUXLWLQJ,)1ȖLQGXFLEOH*73DVHV,Q$WJRU$WJ/GHILFLHQWFHOOV,)1ȖLQGXFHGVXSSUHVVLRQRIT. gondii
SUROLIHUDWLRQDQGUHFUXLWPHQWRIWKH,5*,UJEDQG*%3VDUHSURIRXQGO\LPSDLUHG+RZHYHUFHOOVODFNLQJWKHRWKHUHVVHQWLDODXWRSKDJ\SURWHLQV$WJDDQG$WJFDQPHGLDWHWKHDQWLT. gondiiUHVSRQVHDQGUHFUXLW,UJEDQG*%3VWRWKHSDUDVLWHV7KXVPRXVHDXWRSKDJ\FRPSRQHQWV$WJDQG$WJ/EXWQRW$WJDDQG$WJSDUWLFLSDWHLQWKH,)1ȖLQGXFHGUHFUXLWPHQWRILPPXQLW\UHODWHG*73DVHVWRWKHLQWUDFHOOXODUSDWKRJHQ )LJXUH )LJXUH5ROHRI,)1ȖLQGXFHG*%3VLQDQWL7JRQGLLFHOODXWRQRPRXVLPPXQLW\（A）6WUXFWXUHRI39PHPEUDQHZDVGHVWUR\HGLQ,)1ȖWUHDWHGZLOGW\SHFHOOVEXWQRWLQ（B）*%3VGHILFLHQWFHOOVRecent Publications
1. 2KVKLPD-/HH<6DVDL06DLWRK70D-6.DPL\DPD10DWVXXUD<3DQQ*KLOO6+D\DVKL0(ELVX67DNHGD
.$NLUD6<DPDPRWR05ROHRIWKHPRXVHDQGKXPDQDXWRSKDJ\SURWHLQVLQ,)1ȖLQGXFHGFHOODXWRQRPRXV
responses against Toxoplasma gondii. J Immunol
2. <DPDPRWR02NX\DPD00D-6.LPXUD7.DPL\DPD16DLJD+2KVKLPD-6DVDL0.D\DPD+2NDPRWR7
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critical role in host defense against Toxoplasma gondii. Immunity
 <DPDPRWR00D-60XHOOHU&.DPL\DPD16DLJD+.XER(.LPXUD72NDPRWR72NX\DPD0.D\DPD+
1DJDPXQH.7DNDVKLPD60DWVXXUD<6ROGDWL)DYUH'7DNHGD.$7)ȕ is a host cellular target of the
7R[RSODVPDJRQGLLYLUXOHQFHIDFWRU523J Exp Med
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WKHGLUHFWDQGVWUDLQVSHFLILFDFWLYDWLRQRI6WDWJ Exp Med
ĲĴ

Division of Host Defense

Research & Activities

Department of Molecular Immunology
Research Group

Professor
Associate Professor
Assistant Professor
SA Researcher
SA Researcher

Hitoshi Kikutani, M.D., PhD
Teruhito Yasui, Ph.D
Shuhei Sakakibara, PhD
Takeharu Minamitani, PhD
Chiau-Yuang Tsai, PhD

1) Molecular mechanisms in B cell differentiation and survival
During effective humoral immune responses, the B cell receptor (BCR)- and CD40-mediated signaling pathways are activatedand drive B cells to differentiate into antibody-secreting cells and memory B cells. Our goal is to understand the mechanismsthat regulate humoral immune responses by molecularly characterizing the signal mediators.
During our search for novel signal molecules downstream of BCR, we found that Protein Kinase N1 (PKN1) is a negative
regulator of BCR signals. PNK1 interacts with AKT kinase and inhibits its activity. In PKN1-/- mice, the selection of highaffinity B cells is impaired, although germinal center (GC) formation is accelerated. Thus, the fine tuning of the BCR signal by
PKN1 is indispensable for the quality control of humoral immune responses.
2) Molecular mechanisms in the immune pathology caused by host–pathogen interactions: Epstein-Barr virus (EBV)-encoded
latent membrane proteins (LMP) 1 and 2a affect B cell survival and selection.
EBV infects human B cells, particularly the memory B cell population. This promotes its persistent infection. Of the various
viral genes that are expressed in EBV-infected B cells, LMP1 and 2a constitutively activate and mimic the CD40 and BCR
signals, respectively. However, it has been largely unknown what impact these virus-derived molecules have on the in vivo
activation and differentiation of B cells. For this reason, we generated transgenic mice that conditionally express LMP1 or
LMP2a in GC B cells.
Although the spleen of LMP2a Tg mice has normal GCs, the titers of antigen-specific antibodies are significantly low.
Sequencing analysis of the immunoglobulin gene of the antigen-specific B cells revealed impaired affinity maturation after
immunization. In addition, LMP2a Tg mice exhibit
significantly accelerated plasma cell differentiation.
Thus, EBV LMP2a reduces the threshold for highaffinity B cell selection. This may promote the latent
infection of EBV in memory B cells (Figure 1).
Unlike LMP2a, the expression of LMP1 in B cells
strongly inhibits GC formation. Interestingly, GC
formation and antibody responses are also impaired in
the chimeric mice resulting from co-transfer of LMP1
Tg and wild-type-derived bone marrow cells. Thissuggests that LMP1 + B cells inhibit neighboring
wild-type cells. Thus, LMP1 may contribute to EBV
persistence by suppressing host humoral responses.

Figure 1. EBV LMP2a reduces the threshold for selection of high-affinity B cells
and may contribute to EBV latent infection. Activation of the BCR signaling
pathway by EBV LMP2a results in reduced threshold for selection of high-affinity B
cells. (IM: infectious mononucleosis)

3) Understanding the mechanisms behind the emergence of auto- and polyreactive B cells and their physiological and/or
pathological roles
A significant proportion of the circulating IgG+ memory B cells in healthy individuals are polyreactive clones that are reactive
to multiple self-components. Since the immunoglobulin genes of these clones have multiple somatic hypermutations (SHM), it
is expected that polyreactive IgG+ memory B cells are post-GC B cells. The inducers of polyreactive B cells have not been
extensively investigated. However, the mouse infection model of MHV68 (murLQHȖKHrpesvirus 68) provides strong evidenceĲĵResearch & Activities

that polyreactivity emerges and is selected from splenic IgG+ GC B
cells that harbor somatically mutated immunoglobulins. Therefore,
MHV68 infection is an ideal experimental system for analyzing
polyreactive B cell generation (Figure 2).
Is polyreactive antibody related to autoimmune diseases in humans?
When we analyzed the polyreactivity of plasma cells from systemic
erythematosus lupus (SLE) patients and healthy donors, both had
similar frequencies of polyreactive clones. This indicates that atleast the emergence of polyreactive clones does not correlate withSLE disease activity. By contrast, the patients with acute SLE had
high frequencies (~5%) of anti-nuclear antibody (ANA)-producing
cell clones. ANA production is generally used to diagnose SLE.
These ANA-producing cell clones are not polyreactive andrecognize each nuclear autoantigen in a specific manner. We are
currently analyzing the ANA-encoding immunoglobulin genes by
next generation sequencing (NGS) technologies to understand how

Figure 2. Polyreactive clones emerges in the splenic IgG+ germinal
center B cells during MHV68 infection. Single-cell-based cloning of
immunoglobulins followed by ELISA revealed that ~30% of thesplenic IgG+ germinal center B cells showed polyreactivity. MHV68
infection will provide ideal experimental system for study ofpolyreactive B cell generation.

autoreactive B cells in humans are generated and selected.

Recent Publications
1. Kumanogoh, A., and Kikutani H. Immunological functions of the neuropilin and plexin as receptors forsemaphorins. Nat Rev Immunol. 2013 Nov;13(11):802-14.
2. Tada S, Yasui T, Nakatsuji Y, Okuno T, Koda T, Mochizuki H, Sakoda S, Kikutani H. BAFF controls neural cell
survival through BAFF receptor. PLoS One. 2013 Jul 29;8(7):e70924.
3. Morihana T, Goya S, Mizui M, Yasui T, Prasad DV, Kumanogoh A, Tamura M, Shikina T, Maeda Y, Iwamoto Y,
Inohara H, Kikutani H. An inhibitory role for Sema4A in antigen-specific allergic asthma. J Clin Immunol. 2013
Jan;33(1):200-9.
4. Yasui T, Sakakibara-Yada K, Nishimura T, Morita K, Tada S, Mosialos G, Kieff E, Kikutani H. Protein kinase N1,
a cell inhibitor of Akt kinase, has a central role in quality control of germinal center formation. Proc Natl Acad Sci
U S A. 2012 Dec 18;109(51):21022-7.
5. Tada, S., Okuno T., Yasui T., Nakatsuji N., Sugimoto T., Kikutani H., and Sakoda S. Deleterious effects oflymphocytes at the early stage of neurodegeneration in an animal model of amyotrophic lateralsclerosis. J Neuroinflammation. 2011 Feb 23;8(1):1
ĲĶDivision of Host Defense

Research & Activities

Department of Immunoregulation
Research Group

Professor (SUP)
Associate Professor
Associate Professor (SUP)
Assistant Professor
SA Assistant Professor

Taroh Kinoshita, D. Med. Sc.
Yusuke Maeda, M.D., D. Med. Sc.
Yoshiko Murakami, M.D., D. Med. Sc.
Yuko Tashima, D. Med. Sc.
Noriyuki Kanzawa, D. Med. Sc.

Research Projects
Three research fields in diseases, immune defense, and homeostasis are our main focus. The first examines how
glycosylphosphatidylinositol-anchored proteins are synthesized, processed, transported and secreted, and how defects in theseprocesses lead to the onset and pathology of diseases. The second examines how the pH of intracellular organelles is regulated
and how these pH values regulate many biological functions. The third aims to identify the components and their functions inmembrane contact sites between closely apposing organelles where lipids and ions are transported.
1) Biogenesis, transport and remodeling of GPI-anchored proteins (GPI-APs).
Glycosylphosphatidylinositol (GPI) is a glycolipid that consists of phosphatidylinositol, glucosamine, mannoses andphosphoethanolamines. It acts as a lipid anchor for various plasma membrane proteins. GPI-APs play important roles in hostself-defense, intercellular signal transduction, and other important processes. In addition, some GPI-APs function as receptors
for certain viruses and toxins. The GPI-anchor is widely distributed and conserved in various eukaryotes, and is essential forthe development of higher animals, as well as for the growth of yeasts and protozoan parasites. The modification of proteinsdue to the attachment of the GPI-anchor functions as a protein localization and sorting signal. Our current project is to identify
and clarify the functions of all the genes involved in the biosynthesis of the GPI-anchor in the endoplasmic reticulum (ER)(PIG genes; PhosphatidylInositol Glycan), and in the sorting and localization of GPI-APs after their anchorage with GPI(PGAP genes; Post GPI-Attachment to Proteins). We expect that these studies will reveal why many proteins are modified by
the GPI-anchor.
PIG genes are involved in the biosynthesis of
the GPI-anchor in the ER. During thisbiosynthetic process, an alkyl-lipid generated
in the peroxisome is utilized. Thereafter,
GPI-APs are transported to the plasma
membrane and are enriched in rafts. PGAP
genes are involved in these latter processes.
PGAP1 and PGAP5, which localize in theER, and PGAP2 and PGAP3, which localizein the Golgi, are involved in the lipid or
glycan remodeling of the GPI-anchor.
Remodeling in the ER is necessary for theefficient transport of GPI-APs to the Golgi
via their association with p24 family
proteins. We found that this remodelingprocess affects the sorting of GPI-APs
because it alters the physical characteristicsof the GPI-anchor. The genes in yellow
boxes are currently known to be responsible
for IGD, and those in blue boxes are
responsible for PNH (see below).
2) Molecular genetics of GPI deficiencies.
In collaboration with our colleagues inEngland, we identified a disease called
inherited GPI deficiency (IGD) that is causedby the PIGM mutation. PIGM is amannosyltransferase-encoding gene thatplays an essential role in GPI biosynthesis.
The recent analysis of the exomes of patientswith IGD with the next generation sequencer
has already revealed twelve kinds of IGD toĲķ

Fig. 1 GPI-anchor biosynthesis and the transport/remodeling of GPI-APs.
PIG genes are involved in the biosynthesis of the GPI-anchor in the ER. During the biosynthesis,an alkyl-lipid generated in the peroxisome is utilized. Thereafter, GPI-APs are transported to theplasma membrane and enriched in rafts. PGAP genes are involved in these later processes.
PGAP1 and PGAP5, which localize in the ER, and PGAP2 and PGAP3, which localize in the
Golgi, are involved in the lipid or glycan remodeling of the GPI-anchor. Remodeling in the ER isnecessary for efficient transport of GPI-APs to the Golgi by association with p24 family proteins.
We found that these remodeling affects the sorting of GPI-APs because it alters the physicalcharacteristics of the GPI-anchor. The genes in yellow boxes are currently known to be
responsible for IGD and those in blue boxes are responsible for PNH.

Research & Activities

date. To further analyze IGDs, we are collaborating with many clinicians
and scientists in Japan and abroad. As complete GPI deficiency isembryonically lethal, patients with IGD due to PIG gene mutations only
have partial GPI deficiencies. The patients with IGD that is due toremodeling defects even have null mutations. The symptoms of IGD varydepending on the defective genes and the degree of defectiveness. The main
symptoms are intellectual disability and seizures that are often accompanied
by hyperphosphatasia, abnormal facial features, brachytelephalangy andsometimes other organ anomalies. IGD has been proven to be the maincause of Mabry syndrome/hyperphosphatasia mental retardation syndrome
and CHIME syndrome. IGD has also been found in Early Onset Epileptic
Encephalopathies such as Ohtahara syndrome and West syndrome. Gooddiagnostic markers for IGD are the presence of hyperphosphatasia and thedecreased flow cytometric expression of granulocyte CD16, which is a
GPI-AP . We will establish the diagnostic criteria by accumulating patient
information and seek effective therapies.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired GPI deficiency Fig. 3 pH regulation of intracellular organelles
The luminal pHs of organelles in secretory andthat is caused by somatic mutation of the X-linked PIGA gene in endocytosis pathways are acidic as indicated in the boxes
hematopoietic stem cells. However, it was recently reported that PNH can (the values are approximate).
also be caused by somatic mutation of one allele of the PIGT gene in
combination with a germline mutation in the other allele. This showed that
PIGA is not always the responsible gene in PNH, although this situation is rarely seen. PNH is a hematopoietic disease that ischaracterized by the expansion of clonal cells that are defective in GPI biosynthesis. As a result, abnormal erythrocytes lacking
the GPI-APs CD59 and DAF/CD55 (which inhibit the activation of complement) predominate, leading to massive hemolysis
due to excessive complement attack during infections and other events. We are presently analyzing the pathogenesis of PNH
by seeking the mechanism behind the clonal expansion of GPI-deficient hematopoietic stem cells.
3) pH regulation in acidic organelles and its biological significance, and analysis of membrane contact sites between organelles.
Each intracellular organelle is compartmentalized by lipid bilayers and possesses a characteristic environment, proteins andlipids that allow it to fully exercise its functions. A major environment factor is pH. The lumens of organelles in the secretoryand endocytosis pathways are known to have acidic environments. This lumen acidity is believed to play critical roles in many
biological processes in cells, including the transport, processing, and glycosylation of proteins and lipids. It is also known to
influence the morphology of organelles and to participate in the onset and pathology of diseases that are characterized by pH
regulation defects. However, the mechanisms behind the latter abnormal phenotypes are largely not known. Recently, we
reported for the first time the establishment of mutant cells in which the Golgi pH is dysregulated. Analysis of these cells led tothe identification of the gene that bore the mutations and was responsible for the deranged functions of the mutant cells. This
project aims to clarify the mechanisms and biological significance of acidic pH regulation by establishing and analyzing
additional mutant cells that are defective in organelle pH regulation and to improve our understanding about how suchmutations can lead to pathology, as this will facilitate the identification of effective drugs. As an offshoot from the above
study, we are also analyzing the components and functions of the membrane contact sites between closely apposed organelle
membranes where lipids and ions are transported.

Recent Publications
1. Implication of lipid moiety in oligomerization and immunoreactivities of GPI-anchored proteins. Seong J, Wang Y,
Kinoshita T, Maeda Y. J Lipid Res. 2013 Apr;54(4):1077-91
2. Mechanism for release of alkaline phosphatase caused by glycosylphosphatidylinositol deficiency in patients withhyperphosphatasia mental retardation syndrome. Murakami Y, Kanzawa N, Saito K, Krawitz PM, Mundlos S,
Robinson PN, Karadimitris A, Maeda Y, Kinoshita T. J Biol Chem. 2012 Feb 24;287(9):6318-25.
3. Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI. Fujita M,
Watanabe R, Jaensch N, Romanova-Michaelides M, Satoh T, Kato M, Riezman H, Yamaguchi Y, Maeda Y,
Kinoshita T. J Cell Biol. 2011 Jul 11;194(1):61-75.
4. Peroxisome dependency of alkyl-containing GPI-anchor biosynthesis in the endoplasmic reticulum. Kanzawa N,
Maeda Y, Ogiso H, Murakami Y, Taguchi R, Kinoshita T. Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17711-6.
5. GPI glycan remodeling by PGAP5 regulates transport of GPI-anchored proteins from the ER to the Golgi. Fujita M,
Maeda Y, Ra M, Yamaguchi Y, Taguchi R, Kinoshita T. Cell. 2009 Oct 16;139(2):352-65.
ĲĸDivision of Host Defense

Research & Activities

Department of Host Defense
Research Group

Professor (SUP)
Associate Professor
Assistant Professor
SA Assistant Professor (SUP)

Shizuo Akira, M.D., Ph.D.
Tatsuya Saitoh, Ph.D.
Takashi Satoh, Ph.D.
Kenta Maruyama, M.D., Ph.D.

Main text
We study the innate immune system, which is an evolutionally conserved host defense mechanism against microbes.
Pattern-recognition receptors (PRRs) play a critical role in the induction of innate immunity. PRRs detect various
microorganisms ranging from bacteria to fungi, protozoa and viruses, and induce the production of inflammatory factors suchas cytokines and interferons. However, PRRs also detect environmental irritants and host-derived stimulatory factors, and cancause the development of inflammatory diseases. Thus, the innate immune system can be a double-edged sword for the host.
To gain a deeper understanding of the innate immune system, we have examined a mechanism that regulates PRR-mediatedinnate immune responses and a novel function of innate immune cells.
1. Microtubule-dependent activation of the NLRP3-inflammasome
NLRP3 forms an inflammasome with its adaptor ASC, and its excessive activation can cause inflammatory diseases. However,
little was known about the mechanisms that control the assembly of the inflammasome complex. We found that microtubules
mediate the assembly of the NLRP3 inflammasome (Misawa T et al., Nat Immunol, 2013). Thus, inducers of the NLRP3
inflammasome cause aberrant mitochondrial homeostasis, which in turn diminishes the concentration of the coenzyme NAD.
7KLVWKHQLQDFWLYDWHVWKH1$'GHSHQGHQWSURWHLQĮWXEXOLQGHDFHW\ODVHVLUWXLQWKHUHE\FDXVLQJDFHW\ODWHGĮWXEXOLQWRDFFXPXODWH7KHDFHW\ODWHGĮWXEXOLQPHGLDWHVWKHG\QHLQGHSHQGHQWWUDQVSRUWRIPLWRFKRQGULDDQGWKHVXEVHTXHQWDSSRVLWLRQ
of ASC on mitochondria to NLRP3 on the endoplasmic reticulum. Therefore, the activation of the entire NLRP3LQIODPPDVRPHUHTXLUHVERWKWKHGLUHFWDFWLYDWLRQRI1/53DQGWKHFUHDWLRQE\PLFURWXEXOHVRIRSWLPDOVLWHVIRUVLJQDOtransduction (Figure 1).

Figure 1. Activation of the NLRP3-inflammasome.

2. Involvement of cathepsin D in the immunomodulatory effects of synthetic double-stranded RNA
RIG-I-like receptors (RLRs) detect double-stranded (ds) RNA and induce antiviral immune responses. RLRs also mediate theadjuvant effect of polyinosinic-polycytidylic acid (poly IC), a synthetic dsRNA. We revealed the mechanisms of the poly
IC-induced innate immune responses (Zou J et al., Immunity, 2013). Poly IC is taken up by dendritic cells (DCs) and induces
lysosomal destabilization, which causes the release of cathepsin D into the cytoplasm from the lysosome. Cathepsin D theninteracts with IPS-1, an adaptor molecule for RLRs. This interaction activates the RLR-dependent signaling pathway, as it
IDFLOLWDWHVWKHFDWKHSVLQ'PHGLDWHGFOHDYDJHRIFDVSDVHDQGWKHDFWLYDWLRQRIWKHWUDQVFULSWLRQIDFWRU1)ț%WKXVUHVXOWLQJĲĹResearch & Activities

in enhanced cytokine production. Further recruitment of the kinase RIP-1 to the IPS-1-cathepsin D complex initiates the
QHFURSWRVLVRIDVPDOOQXPEHURI'&V7KH+0*%UHOHDVHGE\WKHG\LQJFHOOVWKHQHQKDQFHVWKH,)1ȕSURGXFWLRQFDXVHGE\poly IC. Collectively, these findings indicate that cathepsin D-triggered necroptosis is a mechanism that propagates theadjuvant efficacy of poly IC (Figure 2).

Figure 2. Activation of dendritic cells by poly IC.

3. A novel role of macrophages in the maintenance of adipose tissues
Macrophages consist of at least two subgroups, M1 and M2. Whereas M1 macrophages are pro-inflammatory and play acentral role in host defense, M2 macrophages associate with anti-inflammatory reactions and tissue remodeling, among other
SURFHVVHV*HQRPHZLGHDVVRFLDWLRQVWXGLHVLQKXPDQVVXJJHVWWKDW75,%SDUWLFLSDWHVLQOLSLGPHWDEROLVP:HIRXQGWKDWTrib1 is critical for the differentiation of tissue-resident macrophages, which we term M2-like macrophages (Satoh T et al.,Nature, 2013). Trib1-deficiency results in severe reductions of M2-like macrophages in various organs, including adipose
WLVVXHV7KHVHGHIHFWVLQPDFURSKDJHGLIIHUHQWLDWLRQDUHFDXVHGE\DEHUUDQWH[SUHVVLRQRI&(%3ĮLQ7ULEGHILFLHQWERQH
marrow cells. Mice lacking Trib1 in hematopoietic cells have a diminished adipose tissue mass that is accompanied byevidence of increased lipolysis. In response to a high-fat diet, mice lacking Trib1 in hematopoietic cells develop
hypertriglyceridaemia and insulin resistance. Therefore, Trib1 is critical for adipose tissue maintenance and suppression of
metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages (Figure 3).

Figure 3. A role of M2-like macrophages.

Our future research will aim to generate a comprehensive understanding of the innate immune system and to develop an
effective treatment for immune-related inflammatory diseases.

Recent Publications
1. Uehata T, Iwasaki H, Vandenbon A, Matsushita K, Hernandez-Cuellar E, Kuniyoshi K, Satoh T, Mino T, Suzuki Y,
Standley DM, Tsujimura T, Rakugi H, Isaka Y, Takeuchi O, Akira S. Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation. Cell. 2013 May 23;153(5):1036-49.
2. Misawa T, Takahama M, Kozaki T, Lee H, Zou J, Saitoh T, Akira S. Microtubule-driven spatial arrangement of
mitochondria promotes activation of the NLRP3 inflammasome. Nat Immunol. 2013 May;14(5):454-60.
3. Zou J, Kawai T, Tsuchida T, Kozaki T, Tanaka H, Shin KS, Kumar H, Akira S. Poly IC triggers a cathepsin D- and
IPS-1-dependent pathway to enhance cytokine production and mediate dendritic cell necroptosis. Immunity. 2013
Apr 18;38(4):717-28.
4. Satoh T, Kidoya H, Naito H, Yamamoto M, Takemura N, Nakagawa K, Yoshioka Y, Morii E, Takakura N, Takeuchi
O, Akira S. Critical role of Trib1 in differentiation of tissue-resident M2-like macrophages. Nature. 2013 Mar
28;495(7442):524-8.
5. Maruyama K, Fukasaka M, Vandenbon A, Saitoh T, Kawasaki T, Kondo T, Yokoyama KK, Kidoya H, Takakura N,
Standley D, Takeuchi O, Akira S. The transcription factor Jdp2 controls bone homeostasis and antibacterial
immunity by regulating osteoclast and neutrophil differentiation. Immunity. 2012 Dec 14;37(6):1024-36.
ĲĺResearch & Activities

Division of Host Defense

Department of Cell Biology
Research Group

Professor
Associate Professor
Assistant Professor
Postdoctoral fellow
Postdoctoral fellow

Eisuke Mekada, Ph. D.
Ryo Iwamoto, Ph. D.
Hiroto Mizushima, Ph. D
Takashi Nakamura, Ph. D.
Tomoya Hikita, Ph. D.

We are studying the mechanisms of cell growth and differentiation that involve growth factors and adhesion molecules
presented at cell–cell contact sites. In particular, we are focusing on the mode of action of HB-EGF, a membrane-anchored
EGF family of growth factors, and the tetraspanin family, which are the molecules to which HB-EGF binds. These proteinsfunction in morphogenesis and tissue maintenance and repair by regulating cell proliferation, migration and adhesion. They
are also involved in the growth, invasion and metastasis of cancer cells.
1) Mode of action of HB-EGF
HB-EGF is a member of the EGF family of growth factors, and binds to and activates EGFR and ErbB4. It is synthesized as
proHB-EGF, a membrane-anchored precursor protein, and is cleaved on the cell surface to yield the soluble growth factor(sHB-EGF). The conversion of proHB-EGF into the soluble form is critical for the activity of this growth factor, andtherefore this process is tightly regulated. HB-EGF is secreted by various tissues and cells and functions in several
physiological processes. For example, it maintains heart muscle function, suppresses cell proliferation in heart valve andlung alveolar development, promotes cell migration in wound healing and eyelid closure, supports blastocyst attachment tothe uterus during implantation, and promotes cell proliferation in skin hyperplasia. ProHB-EGF is not only a precursor of
the soluble form, it is also a biologically active molecule that regulates the growth of neighboring cells in a juxtacrine
fashion. How is the conversion of the membrane-anchored form into the soluble form regulated? How does HB-EGF
function in the manifold physiological processes that are dependent on this molecule? What roles do sHB-EGF andproHB-EGF play in such physiological processes? Do they participate in pathological processes? These questions are
currently being analyzed at the molecular level.
In valve development, HB-EGF is expressed and secreted from endocardial cells. Secreted HB-EGF associates with heparansulfate proteoglycans (HSPGs) in the valve mesenchyme (upper right). When EGFR on the surface of mesenchymal cells is
activated by the HB-EGF-HSPG complex, this receptor transduces inhibitory signals for mesenchymal cell proliferation(lower left .QRFNLQPLFHH[SUHVVLQJDPXWDQW+%(*)WKDWFDQQRWELQGWR+63*V ǻ+% DVZHOODV.2PLFHGHYHORS
enlarged cardiac valves characterized by hyperproliferation of mesenchymal cells (lower right) (Iwamoto et al., 2010).

Fig. 1 The role of HB-EGF in heart valve formation.
In the valve development, HB-EGF is expressed and secreted fromendocardial cells. Secreted HB-EGF associates with heparan sulfate
proteoglycans (HSPGs) in the valve mesenchyme (upper right). When
EGFR on the surface of mesenchymal cells is activated by the
HB-EGF-HSPG complex, this receptor transduces inhibitory signals formesenchymal cell proliferation (lower left). Knock-in mice expressing a
PXWDQW+%(*)WKDWFDQQRWELQGWR+63*V ǻ+% DVZHOODV.2PLFH
developed enlarged cardiac valves with hyperproliferation of mesenchymal
cells (lower right). (Iwamoto et al., 2010)ĳı

Fig. 2. Cancer cell-stromal fibroblast interactions in the cervix.

Research & Activities

2) Role of HB-EGF in cancer malignancy
HB-EGF is expressed by cancer cells and/or cancer-derived stroma, and is involved in tumor growth, invasion andmetastasis. To develop a novel strategy for cancer therapy, we are analyzing the role of HB-EGF in cancer malignancy.

Fig.3. Cancer-derived fibroblasts enhance tumorigenesis of
cervical cancer cells in nude mice. Fluorescence imaging oftumors injected cervical cancer cells with fibroblasts (a) or
cervical cancer cells alone (b) (Murata et al. 2011).

3) Development of anti-cancer drugs targeting HB-EGF
We are developing new anti-cancer drugs that target HB-EGF. The pre-clinical and clinical studies of an anti-HB-EGF
monoclonal antibody and a non-toxic mutant protein of diphtheria toxin CRM197 are currently in progress.
4) CD9 and the tetraspanin function
CD9 is a member of the tetraspanin superfamily. It is a membrane protein with four transmembrane domains. It associateswith proHB-EGF and upregulates proHB-EGF function. CD9 is also involved in cell signaling, growth, motility, andadhesion, and in tumor cell metastasis and sperm–egg fusion. In addition, the Caenorhabditis elegans tetraspanin TSP-15 isessential for the epidermal integrity of the worm. We are analyzing the physiological activity of CD9 and other tetraspanins
by using genetically engineered mice or worms (C. elegans) lacking CD9 or other tetraspanins.

Fig. 4. Enhanced expression of HB-EGF in the ovarian cancer tissue (right) and
BK-UM, therapeutics for ovarian cancer under development in our laboratory.

Recent Publications
1. Moribe H, Konakawa R, Koga D, Ushiki T, Nakamura K, Mekada E. Tetraspanin is required for generation ofreactive oxygen species by the dual oxidase system in Caenorhabditis elegans. PLoS Genet. 2012 Sep;8(9):e1002957.
2. Miyamoto S, Iwamoto R, Furuya A, Takahashi K, Sasaki Y, Ando H, Yotsumoto F, Yoneda T, Hamaoka M, Yagi H,
Murakami T, Hori S, Shitara K, Mekada E. A novel anti-human HB-EGF monoclonal antibody with multiple
anti-tumor mechanisms against ovarian cancer cells. Clin Cancer Res. 2011 Nov 1;17(21):6733-41.
3. Murata T, Mizushima H, Chinen I, Moribe H, Yagi S, Hoffman RM, Kimura T, Yoshino K, Ueda Y, Enomoto T,
Mekada E. HB-EGF and PDGF mediate reciprocal interactions of carcinoma cells with cancer-associated fibroblasts
to support progression of uterine cervical cancers. Cancer Res. 2011 Nov 1;71(21):6633-42.
4. Iwamoto R, Mine N, Kawaguchi T, Minami S, Saeki K, Mekada E. HB-EGF function in cardiac valve development
requires interaction with heparan sulfate proteoglycans. Development. 2010 Jul;137(13):2205-14.
 ,FKLVH7$GDFKL62KLVKL0,NDZD02NDEH0,ZDPRWR50HNDGD(+XPDQL]HGJHQHUHSODFHPHQWLQPLFH
reveals the contribution of cancer stroma-derived HB-EGF to tumor growth. Cell Struct Funct. 2010;35(1):3-13.
ĳĲ

Research & Activities

Division of Host Defense

Department of Immunochemistry
Research Group

Professor（SUP）
Assistant Professor
Assistant Professor
SA Researcher

Hisashi Arase, M.D., Ph.D.
Tadahiro Suenaga, M.D., Ph.D.
Masako Kohyama, Ph.D.
Fumiji Saito, Ph.D.

Research Projects
Our department analyzes various immunological aspects in host–pathogen interactions and autoimmune diseases. In particular,
we are analyzing receptor pairs that consist of an activating and an inhibitory receptor, and that are expressed on immune cells
to test the hypothesis that these paired receptors may have evolved with pathogens. Moreover, since infections are often
followed by autoimmune diseases, we are also analyzing how infection abrogates the homeostasis of the immune system.
(1) Immune regulation by paired receptors
Immune cells express cell surface receptor pairs that consist of activating and inhibitory receptors. The inhibitory receptors
recognize self-antigens such as MHC class I molecules. We have found that these paired receptors also recognize viral proteinsand play an important role in determining host resistance to pathogens (Figure 1). We are presently analyzing the functions ofthese receptors in immune regulation and host–pathogen interactions by using various pathogens, including viruses, parasites
and bacteria.
(2) Entry mechanism of virus into cells
Several viruses that exhibit persistent infection downregulate their host’ s immune response by stimulating the host’s
inhibitory receptors. We have found that some viruses further exploit the host’ s inhibitory receptors to enter the cells. For
example, interactions between immune receptors and viral proteins play an important role in membrane fusion during theinfection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) (Figure 2). Since other viruses may also use similar
receptors to enter cells, we intend to further investigate the molecular mechanisms involved in viral entry of cells.

Figure 1. Paired receptor
Inhibitory receptors play an important role inimmune regulation (Wang et al. Nat. Immunol.
2013), whereas pathogens exploit inhibitory
receptors for immune evasion. On the other
hand, activating receptors might have evolved toprotect infections.

ĳĳ

Figure 2. Paired receptor mediated viral infection.
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membrane fusion during viral infection by associating with envelope
proteins of herpes simplex virus and varicella-zoster virus (Satoh et al. Cell
2008; Suenaga et al. Proc. Natl. Acad. Sci. USA. 2010)

Research & Activities(3) Mechanism of autoimmune pathogenesis
MHC class II allelic polymorphisms associate with susceptibility to many autoimmune diseases. However, it has been unclear
how MHC class II molecules regulate autoimmune disease susceptibility. We found that cellular misfolded autoantigens thatare rescued from protein degradation and complex with MHC class II molecules can become targets for autoantibodies inpatients with autoimmune diseases. Moreover, autoantibodies recognize misfolded proteins that are complexed with MHC
class II molecules of disease-susceptible alleles but not disease-resistant MHC class II alleles. In addition, autoantibody
binding to misfolded proteins that are transported by MHC class II molecules correlates strongly with susceptibility to
autoimmune disease. This suggests that misfolded proteins complexed with MHC class II molecules are natural autoantigens
for autoantibodies. Therefore, misfolded proteins, which normally would not be exposed to the immune system, can becometargets for autoantibodies when they avoid protein degradation (Figure 3). Notably, MHC class II molecules are both induced
by inflammation during infections and aberrantly expressed in many autoimmune diseased tissues. This suggests that the
rescue of misfolded proteins in the context of infection may promote the development of autoimmune diseases. This notion is
supported by the fact that autoimmune disease onset is often preceded by an infection. We are further analyzing how aberrant
misfolded protein/MHC class II molecule complexes are generated and how they induce autoimmune diseases.

Figure 3. Misfolded proteins complexed with MHC class II molecules are targets for autoantibodies.
Cellular misfolded proteins are transported to the cell surface without processing to peptides by associating with MHC
class II molecules at ER (Jiang et al. Int. Immunol. 2013). Furthermore, misfolded proteins complexed with MHC class
II molecules of disease-susceptible alleles are specifically recognized by autoantibodies. This suggested that misfolded
proteins complexed with MHC class II molecules are natural autoantigens for autoantibodies, which affects autoimmune
disease susceptibility (Jin et al. Proc. Natl. Acad. Sci. USA. 2014).

Recent Publications
1. Autoantibodies to IgG/HLA-DR complexes are associated with rheumatoid arthritis susceptibility.
Jin H, Arase N, Hirayasu K, Kohyama M, Suenaga T, Saito F, Tanimura K, Matsuoka S, Ebina K, Shi K,
Toyama-Sorimachi N, Yasuda S, Horita T, Hiwa R, Takasugi K, Ohmura K, Yoshikawa H, Saito T, Atsumi T,
Sasazuki T, Katayama I, Lanier LL, Arase H. Proc Natl Acad Sci USA. 2014 Mar 111(10): 3787-92.
2. Herpesvirus 6 Glycoproteins B (gB), gH, gL and gQ are Necessary and Sufficient for Cell-to-Cell Fusion.
Tanaka Y, Suenaga T, Matsumoto M, Seya T, Arase H. J Virol. 2013 Oct; 87 (19): 10900-3.
3. Transport of misfolded endoplasmic reticulum proteins to the cell surface by MHC class II molecules.
Jiang Y, Arase N, Kohyama M, Hirayasu K, Suenaga T, Jin H, Matsumoto M, Shida K, Lanier LL, Saito T, Arase H.
Int Immunol. 2013 Apr; 25(4): 235-46.
 1HXWURSKLOLQILOWUDWLRQGXULQJLQIODPPDWLRQLVUHJXODWHGE\3,/5ĮYLDPRGXODWLRQRILQWHJULQDFWLYDWLRQ
Wang J, Shiratori I, Uehori J, Ikawa M, Arase H. Nat Immunol. 2013 Jan; 14(1): 34-40.
5. Myelin-associated glycoprotein mediates membrane fusion and entry of neurotropic herpesviruses. Suenaga T, Satoh
T, Somboonthum P, Kawaguchi Y, Mori Y, and Arase H. Proc Natl Acad Sci USA. 2010 Jan 12;107(2):866-71.
ĳĴResearch & Activities

Division of Cellular and Molecular Biology

Department of Molecular Genetics
Research Group

Professor
Hiroshi Nojima, Ph.D.
Associate Professor
Norikazu Yabuta, Ph.D.
Assistant Professor（SUP） Daisuke Okuzaki, Ph.D.
SA Researcher
Satomi Mukai, Ph.D.

Research Projects:
We are studying the eukaryotic cell cycle to understand the mechanisms
underlying the observed chromosomal instability in cancer cells. Many
human cancer cells exhibit mitotic defects (such as centrosome
aberrations, abnormal spindle formation, and chromosome
missegregation). The resulting chromosomal instability is a major causeof malignant tumor progression. Our research focuses on functional
analyses of the Ser/Thr kinases Lats (large tumor suppressor) and GAK(cyclin G-associated kinase). These kinases localize to the centrosome,regulate mitotic progression in response to DNA damage, and cause
chromosome instability when their functions are disrupted. Both Lats
(Lats1 and Lats2) and GAK form complexes with Mdm2. In turn, Mdm2
controls the stability of p53, which is a transcriptional regulator of theLats2, cyclin G1, and Mdm2 genes. Thus, the Lats and GAK complexes
have intimate correlations in their regulation of cell cycle (Fig. 1).
Our research subjects are as follows:(1) Lats Group
Lats1 and Lats2, which belong to the Lats kinase family, are highly
conserved across species and localize to the centrosome during the cell
cycle. Lats1/2 plays an important role in the cell cycle checkpoint andthe Hippo pathway that mainly regulates organ size and cell growth. In
particular, Lats2 binds to Mdm2, thereby inhibiting its E3 ligase activity
and activating p53. Thereafter, p53 rapidly and selectively upregulates
Lats2 expression in G2/M cells. This positive feedback loop constitutes anovel checkpoint that plays a critical role in the maintenance of proper
chromosome numbers (Aylon et al., Gene Dev., 2006).
We have discovered the following: (A) LATS2 knockout mice areembryonic lethal, which indicates the essential role of Lats2 in thedevelopment and differentiation of mammalian germ cells. (B) Lats2-/mouse embryonic fibroblasts (MEF) display mitotic defects such as
centrosome fragmentation, abnormal chromosome segregation, andaberrant cytokinesis (Yabuta et al., J. Biol. Chem., 2007). (C) MEFs
from mice in which Lats1 is knocked out by disrupting its N-terminal
region (Lats1ǻ1ǻ1) display downregulation of Lats2 mRNA expression
and protein accumulation of Yap, a downstream factor of Lats2 in the
Hippo pathway (Ref. 4). Moreover, the MEFs also show mitotic defects
such as centrosome amplification, chromosome missegregation (Fig. 2),
cytokinesis failure, and anchorage-independent growth (Fig. 3). These
results indicate the essential role that Lats2 and Lats1 play in proper M
phase progression and chromosome stability. (D) Lats2 mediates two
novel signaling pathways: CLP (Chk1/2—Lats2—P-body) and ALB(Aurora-A—Lats1/2—Aurora-B). In the CLP pathway, Lats2
phosphorylates 14-3-3 for processing body (P-body) formation
downstream of Chk1 in response to DNA damage (Okada et al., J. Cell
Sci., 2011). More over, under severe DNA damage, Lats2 is
phosphorylated both by Chk1 and itself and fully activated, thereby
inducing caspase-dependent apoptosis through Lats2-mediatedphosphorylation of p21/CDKN1A and subsequent protein degradation of
p21 (Ref. 2, Fig. 4). In the ALB pathway, Lats2 is phosphorylated by
Aurora-A. Phosphorylated Lats2 localizes to the chromosomes and theĳĵFig. 1: The functions of the Lats and GAK complexes
correlate closely.

Fig. 2: Lats1¨1¨1MEFs exhibit chromosomal
missegregation during anaphase.

Fig. 3: Lats1¨1¨1MEFs exhibit
anchorage-independent growth.

Fig. 4: Activation of Lats2 by Chk1-mediated andauto-phosphorylation induces apoptosis (red).

Research & Activities

central spindle, and regulates Lats1 and Aurora-B, thereby executing proper chromosomal segregation during mitosis (Yabuta
et al., Cell Cycle, 2011). (D) Lats2 phosphorylates and regulates ASPP1/p53 complexes, thereby inducing apoptosis inmalignant tumor cells with aneuploidy and polyploidy (Aylon et al., Genes Dev., 2010). Moreover, Lats2 phosphorylates thetranscription factor Snail, thereby regulating the epithelial-mesenchymal transition (EMT) (Zhang et al., EMBO J., 2012).
(2) GAK GroupGAK is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking.
Unlike neuron-specific auxilin, which plays a similar role in neural cells, GAK has a kinase domain whose function was
XQFOHDU:HKDYHGLVFRYHUHGWKHIROORZLQJ D *$.IRUPVDFRPSOH[ZLWK33$%¶ȖDQGF\FOLQ* F\FOLQ*DQGF\FOLQ* 
which regulates the dephosphorylation activity of PP2A (Ref5, Fig. 5); (b) GAK localizes to the cytoplasm and the nucleus,
where it has two additional functions (i.e., maintenance of proper centrosome maturation and mitotic chromosome
congression; Shimizu et al., J. Cell Sci., 2009); and (c) GAK-kd-/- mice exhibit neonatal lethality with pulmonary dysfunction(Fig. 6). Since the EGFR inhibitor Gefitinib (Iressa) also efficiently inhibits the kinase activity of GAK, the side effects ofGefitinib, including interstitial pneumonia, may be due to the inhibition of GAK (Tabara et al., PLoS One, 2011). (d) Cyclin
G2 knockout (Ccng2-/-) mice are born and develop normally. However, when Ccng2-/- MEFs are irradiated with ionizing
UDGLDWLRQ ,5 WKHLUGHSKRVSKRU\ODWLRQRIȖ+$;DIWHU'1$GDPDJH i.e., the cancellation of the DNA damage response) is
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recruiting PP2A after the completion of DNA repair, thereby causing the cells to recover from DNA damage
checkpoint-dependent cell cycle arrest.

)LJ7RI33$%¶ȖE\*$.LVDQLPSRUWDQWSKRVSKRU\ODWLRQ
site for the activity of PP2A complex.

Fig. 6: Severe defects of fetal lung maturity in GAK-kd-/-mice.

Fig. 7；Cyclin G2 knockout cells show that the
GHSKRVSKRU\ODWLRQRIȖ+$;LVUHWDUGHGDIWHU,5irradiation (24 h and 32 h, green).

Recent Publications
1. Shao D, Zhai P, Del Re DP, Sciarretta S, Yabuta N, Nojima H, Lim DS, Pan D, Sadoshima J. A functional
interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response. Nat Commun. 2014
Feb 14;5:3315.
2. Suzuki H, Yabuta N, Okada N, Torigata K, Aylon Y, Oren M, Nojima H. Lats2 phosphorylates p21/CDKN1A after
UV irradiation and regulates apoptosis. J Cell Sci. 2013 Oct 1;126(Pt 19):4358-68.
3. Naito Y, Yabuta N, Sato J, Ohno S, Sakata M, Kasama T, Ikawa M, Nojima H. Recruitment of cyclin G2 to
SURP\HORF\WLFOHXNHPLDQXFOHDUERGLHVSURPRWHVGHSKRVSKRU\ODWLRQRIȖ+$;IROORZLQJWUHDWPHQWZLWKLRQL]LQJ
radiation. Cell Cycle. 2013 Jun 1;12(11):1773-84.
4. Yabuta N, Mukai S, Okamoto A, Okuzaki D, Suzuki H, Torigata K, Yoshida K, Okada N, Miura D, Ito A, Ikawa M,
Okabe M, Nojima H. N-terminal truncation of Lats1 causes abnormal cell growth control and chromosomal
instability. J Cell Sci. 2013 Jan 15;126(Pt 2):508-20.
5. Naito Y, Shimizu H, Kasama T, Sato J, Tabara H, Okamoto A, Yabuta N, Nojima H. Cyclin G-associated kinase
UHJXODWHVSURWHLQSKRVSKDWDVH$E\SKRVSKRU\ODWLRQRILWV% ȖVXEXQLWCell Cycle. 2012 Feb 1;11(3):604-16.
ĳĶResearch & Activities

Division of Cellular and Molecular Biology

Department of Oncogene Research
Research Group

Professor
Associate Professor
Associate Professor
SA Researcher

Masato Okada, D.Sc.
Shigeyuki Nada, D.Sc.
Chitose Oneyama, D.Sc.
Kentaro Kajiwara, D.Sc.

Research Projects
Cancers arise, evolve and develop progressively due to the accumulations of mutations and/or modifications in the genomic
DNA. Loss-of-function mutations in “tumor suppressor genes” induce cell immortalization, while gain-of-function mutations
in “proto-oncogenes” induce cell transformation. Cell immortalization prevents the induction of apoptosis and/or senescence,
which is a defense mechanism against cancer development. Cell transformation involves the gain of autonomous cell growth,
the loss of cell communication, morphological changes, and the elevated production of matrix proteases and growth factorsthat participate in invasion, metastasis and angiogenesis. These cellular events thus lead to the malignant conversion of cancer
cells. The primary focus of this department is to understand the molecular basis of the cell transformation that is induced by thegain-of-function mutations of proto-oncogenes. As a representative proto-oncogene, we have focused on the c-Src
proto-oncogene, which encodes a non-receptor tyrosine kinase. To date, we have analyzed its physiological roles indevelopment and the mechanisms by which its specific regulators, such as Csk and Cbp, regulate it. To obtain a full picture of
the cell signaling pathways that lead to c-Src–mediated cell transformation and to search for new therapeutic targets that will
block c-Src–mediated cancer progression, the following projects are currently in progress:
I. Function and regulation of c-Src.
In normal cells, c-Src is present as an inactive form that is phosphorylated by its negative regulatory kinase Csk. Extracellular
stimuli transiently activate it, after which it in turn activates downstream components such as the MAPK pathway and Rho
family GTPases, thereby inducing gene expression and cytoskeletal rearrangements, respectively. This promotes the cell
growth and phenotypic changes that are involved in celltransformation (Fig. 1). While the c-SRC gene is rarely mutated inhuman cancers, its protein is frequently hyperactivated andoverexpressed. This aberrant activation of c-Src is suggested tocontribute to cancer malignancy. Recently, we showed that the
oncogenic potential of c-Src is regulated by the membrane adaptor
protein Cbp, which is exclusively localized to the membrane
microdomain. We are currently analyzing the relationship between the
disruption of this regulatory system and cancer progression.
II. Mechanism of cell transformation induced by c-Src activation.
To define the signaling pathway that leads to c-Src-mediated celltransformation, we performed a comprehensive study of target
molecules by using a newly developed c-Src inducible system. To
date, proteomic analyses have found that Arhgef5 is a critical c-Src
target. Arhgef5 is a Dbl family of GEF for Rho GTPase. A miRNAarray analysis also revealed that c-Src activation alters the gene
expression of a limited number of miRNAs. We identified several
target genes for these miRNAs and analyzed their functions. Based on
the finding that many of the target molecules (e.g., FGFR3, mTOR
and ILK) are components of the c-Src signaling pathway, we proposeda new tuning system of c-Src-mediated cell transformation via
miRNA. Analysis of the regulatory mechanism for c-Src-mediatedmiRNA expression is currently in progress.

Fig. 1. Function and regulation of c-Src.

Fig. 2. A mechanism of c-Src-induced cell transformation
regulated by miRNA.

ĳķResearch & Activities

III. Regulation of cell growth signaling via the late endosome/lysosome.
During the search for c-Src targets, we recently found a new regulator of cell growth signaling, namely, p18, which is amembrane adaptor protein that localizes exclusively to late endosomes/lysosomes. p18 forms a ternary complex with thescaffolding p14/MP1 proteins and plays an essential role in the activation of mTORC1 on late endosomes/lysosomes. p18 KOmice are embryonic lethal and exhibit severe defects in lysosome biogenesis (Fig. 3). To elucidate the function of p18 inlysosome biogenesis, we are currently searching for target molecules for the p18-mTORC1 pathway in lateendosomes/lysosomes. We also found that the p18-dependent pathway is crucial for controlling cell growth andoncogene-mediated cell transformation. The elucidation of the underlying mechanism is an important objective of our project
as well.

Fig. 3. Roles played by the late endosome/lysosome-anchoredp18-mTORC1 pathway.

Recent Publications
1 : Nada S, Mori S, Takahashi Y, Okada M. p18/LAMTOR1: a late endosome/lysosome-specific anchor protein for themTORC1/MAPK signaling pathway. Methods Enzymol. 2014;535:249-63
2 : Soma-Nagae T, Nada S, Kitagawa M, Takahashi Y, Mori S, Oneyama C, Okada M. The lysosomal signaling anchor
p18/LAMTOR1 controls epidermal development by regulating lysosome-mediated catabolic processes. J Cell Sci.
2013 Aug 15;126(Pt 16):3575-84.
3 : Kajiwara K, Yamada T, Bamba T, Fukusaki E, Imamoto F, Okada M, Oneyama C. c-Src-induced activation ofceramide metabolism impairs membrane microdomains and promotes malignant progression by facilitating thetranslocation of c-Src to focal adhesions. Biochem J. 2014 Feb 15;458(1):81-93
4 : Oneyama C, Ikeda J, Okuzaki D, Suzuki K, Kanou T, Shintani Y, Morii E, Okumura M, Aozasa K, Okada M.
MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic
pathways. Oncogene. 2011 Aug 11;30(32):3489-501.
5 : Oneyama C, Hikita T, Enya K, Dobenecker MW, Saito K, Nada S, Tarakhovsky A, Okada M. The lipid raft-anchoredadaptor protein Cbp controls the oncogenic potential of c-Src. Mol Cell. 2008 May 23;30(4):426-36.
ĳĸResearch & Activities

Division of Cellular and Molecular Biology

Department of Signal Transduction
Research Group

Professor
Assistant Professor
Assistant Professor
SA Researcher
SA Researcher
SA Researcher
SA Researcher

Nobuyuki Takakura, M.D., Ph.D.
Hiroyasu Kidoya, Ph.D.
Hisamichi Naito, M.D., Ph.D.
Yinglu Han, Ph.D.
Daishi Yamakawa, Ph.D.
Weizhen Jia, Ph.D.
Taku Wakabayashi, M.D., Ph.D.

Research Projects
It is well known that the development of normal tissues and organs requires the generation of tissue-specific cells from stem
cells. The maintenance of this stem cell system also requires the generation of an appropriate microenvironment. Blood vessels
are the most essential structures in tissues and organs, as without blood vessel formation, almost all tissues cannot develop(there are some exceptions). In our research group, we are analyzing the molecular mechanisms by which blood vessels formin physiological and pathological conditions, including in cancers and inflammation. We are also elucidating the mechanismsthat cause stem cells to associate closely with blood vessels. Ultimately, we wish to employ our results to establish strategies
that will inhibit the malignant progression of various diseases. Our specific research projects are as follows:
I. Analysis of the molecular mechanism of blood vessel formation
1) Molecular analysis of angiogenesis, with a particular focus on the Tie1 and Tie2 receptors.
2) Physiologic and pathologic function of endothelial stem cells (ESCs).
3) Molecular characterization of arterio-venous patterning, with a particular focus on the apelin/APJ system.
II. Molecular analysis of self-renewal in normal and cancer stem cells
1) Analysis of cell cycle regulation in stem cells, with a special focus on Galectin-3 and the GINS complex.
2) Establishment of a strategy that can inhibit the formation of the vascular niche inhabited by cancer stem cells.

Figure 1. Maturation of blood vessels.
We elicited the concept that Angiopoietin-1 (Ang1) from mural cells (MCs) generates structurally stable blood vessels by
inducing cell adhesion between endothelial cells (ECs) and MCs (Takakura, Immunity 1998). In addition, we found that Ang1
from hematopoietic stem cells (HSCs) induces non-leaky new blood vessels during angiogenesis (Takakura, Cell 2000,
Yamada, J Exp Med 2006). Apelin produced from ECs on stimulation with Ang1 induces non-leaky and enlarged bloodvessels (Kidoya, EMBO J 2008, Blood 2000). Apelin overexpression induces larger blood vessels in tumors and promotes
effective drug delivery into tumors (Kidoya, Oncogene 2012), thereby suppressing tumor growth.

ĳĹResearch & Activities

Figure 2. Endothelial stem cells (ESCs) and angiogenesis.
During angiogenesis, the tip cells in front of new vessels determine the migration direction of the new vessels, stalk cells
produce a higher number of endothelial cells (ECs) for elongation, and finally phalanx cells induce the maturation of new
blood vessels. We recently identified ESCs in the preexisting blood vessels that may generate heterogeneous ECs as describedabove, and that play a critical regulatory role in pathological and physiological angiogenesis (Naito, EMBO J 2012).

Figure 3. Vascular niche of cancer stem cells.
By using PSF1, a component of the DNA replication factor
GINS, we succeeded in identifying cancer stem cells (CSCs)
in the vascular niche (Nagahama, Cancer Res 2010, Matsui,
Am J pathol 2013). Moreover, we found that stromal CD44
acts as a niche factor that supports the stemness of CSCs
(Kinugasa, Stem Cells 2014).

Recent Publications
1. CD44 expressed on cancer-associated fibroblasts is a functional molecule supporting the stemness and drug
resistance of malignant cancer cells in the tumor microenvironment. Kinugasa Y, Matsui T, Takakura N. Stem Cells.
2014 Jan;32(1):145-56.
2. microRNA-125b inhibits tube formation of blood vessels through translational suppression of VE-cadherin.
Muramatsu F, Kidoya H, Naito H, Sakimoto S, Takakura N. Oncogene. 2013 Jan 24;32(4):414-21.
3. A role for endothelial cells in promoting the maturation of astrocytes through the apelin/APJ system in mice.
Sakimoto S, Kidoya H, Naito H, Kamei M, Sakaguchi H, Goda N, Fukamizu A, Nishida K, Takakura N.
Development. 2012 Apr;139(7):1327-35.
4. Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels.
Naito H, Kidoya H, Sakimoto S, Wakabayashi T, Takakura N. EMBO J. 2012 Feb 15;31(4):842-55.
5. The apelin/APJ system induces maturation of the tumor vasculature and improves the efficiency of immune therapy.
Kidoya H, Kunii N, Naito H, Muramatsu F, Okamoto Y, Nakayama T, Takakura N. Oncogene. 2012 Jul 5;31
(27):3254-64.
ĳĺResearch & Activities

Division of Cellular and Molecular Biology

Department of Cellular Regulation
Research Group

Professor
Assistant Professor
Assistant Professor
SA Researcher

Hiroaki Miki, Ph.D.
Daisuke Yamazaki, Ph.D.
Yosuke Funato, Ph.D.
Yusuke Hirata, Ph.D.

Cells are equipped with the signal transduction system, which enables them to respond appropriately to the surrounding
environment, such as stimulation with various hormones/growth factors and physical interactions with other cells or the
extracellular matrix. Malfunction in the signaling system is responsible for various human diseases. A typical pathogenic
consequence of such malfunction is the abnormal proliferation of cancer cells. In our laboratory, we are investigating theintracellular signaling that regulates the proliferation, differentiation, and motility of cells at various levels, ranging frommolecules to organisms (nematodes and mice). The present main research interests are (1) oxidative stress signaling by
reversible oxidation of proteins, and (2) regulation of intracellular magnesium levels and signal transduction.
(1) Oxidative stress signaling by reversible oxidation of proteins
We discovered that thioredoxin-related protein nucleoredoxin (NRX) is a novel regulator of Wnt signaling, which plays
important roles in early development and oncogenesis. NRX binds directly to Wnt signal transducer Dishevelled (Dvl) andinhibits its function. Interestingly, the NRX-Dvl interaction is negatively regulated by the formation of intramolecular S–S
bonds in NRX. Thus, NRX regulates Wnt signaling in a redox-dependent manner. We also developed a novel method to searchfor proteins forming S–S bonds in cells by using thioredoxin mutants. With this method, we identified several proteins such as
CRMP2, which functions in Semaphorin signaling. Semaphorin treatment stimulates H2O2 generation and CRMP2 oxidation,
resulting in the formation of homodimers linked with S–S bonds. This oxidation of CRMP2 mediates the repulsive axon
guidance induced by Semaphorin stimulation (Fig. 1).

Fig. 1: CRMP2 oxidation in Semaphorin signaling(A) Repulsive guidance of axons by Semaphorin 3A (Sema3A). (B) Increase of H2 O 2 in growth cones of neurons by Sema3A. (C)Schematic illustration of the mechanism of Semaphorin signaling via CRMP2 oxidation. Sema3A stimulation generates H2O2 and oxidizes
CRMP2. Oxidized CRMP2 transiently forms protein complexes with TRX, which induces CRMP2 phosphorylation by GSK3.

ĴıResearch & Activities(2) Regulation of intracellular magnesium levels and signal transduction
We found that PRL, a tyrosine phosphatase with unknown function, is a novel S–S bond-containing protein. It has beenreported that PRL is consistently overexpressed in various human metastatic cancers and can promote experimental metastasis
in mice. We searched for novel PRL-binding proteins and identified Magnesium-Exporting protein (MagEx). MagEx regulates
the intracellular Mg2+ levels by exporting Mg2+. This function of MagEx is inhibited by the interaction with PRL. Since thisPRL-MagEx interaction is dependent on the redox state of PRL, PRL functions as a redox switch that regulates intracellular
Mg2+ levels. Endogenous MagEx is highly expressed in the intestinal epithelia and specifically localizes in their basolateral
membrane and causes hypomagnesemia. Gene disruption of MagEx in mice severely impairs the ability of their intestines toabsorb magnesium. In addition, MagEx is also highly expressed in the epithelial cells that form enamel (ameloblasts).
Indeed, MagEx-knockout mice show symptoms of amelogenesis imperfecta (Fig. 2). These results demonstrate theimportance of MagEx in the control of systemic and local magnesium levels, the dysfunction of which results in thedevelopment of various diseases.

Fig. 2: Redox regulation of the intracellular Mg2+ level by MagEx(A) MagEx stimulates Na +/Mg2+-exchange and decreases the intracellular Mg2+ level. PRL directly binds to MagEx and inhibits its
Mg2+-transporting function in a manner dependent on its redox state. Oxidized PRL is reversibly reduced by S–S bond-reducing enzymes,
such as Trx and Trp32. (B) Localization of endogenous MagEx at the basolateral membrane of the epithelial cells in the intestine.

Recent Publications
1. Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model.
Yamazaki D, Funato Y, Miura J, Sato S, Toyosawa S, Furutani K, Kurachi Y, Omori Y, Furukawa T, Tsuda T,
Kuwabata S, Mizukami S, Kikuchi K, Miki H. PLoS Genet. 2013 Dec;9(12):e1003983.
2. Oligomeric peroxiredoxin-I is an essential intermediate for p53 to activate MST1 kinase and apoptosis. Morinaka A,
Funato Y, Uesugi K, Miki H. Oncogene. 2011 Oct 6;30(40):4208-18.
3. Thioredoxin mediates oxidation-dependent phosphorylation of CRMP2 and growth cone collapse. Morinaka A,
Yamada M, Itofusa R, Funato Y, Yoshimura Y, Nakamura F, Yoshimura T, Kaibuchi K, Goshima Y, Hoshino M,
Kamiguchi H, Miki H. Sci Signal. 2011 Apr 26;4(170):ra26.
 1XFOHRUHGR[LQVXVWDLQV:QWȕFDWHQLQVLJQDOLQJE\UHWDLQLQJDSRRORILQDFWLYHGLVKHYHOOHGSURWHLQ)XQDWR<Terabayashi T, Sakamoto R, Okuzaki D, Ichise H, Nojima H, Yoshida N, Miki H. Curr Biol. 2010 Nov 9;20
(21):1945-52.
5. Par1b/MARK2 phosphorylates kinesin-like motor protein GAKIN/KIF13B to regulate axon formation. Yoshimura
Y, Terabayashi T, Miki H. Mol Cell Biol. 2010 May;30(9):2206-19.
ĴĲResearch & Activities

Research Center for Infectious Disease Control

Department of Molecular Protozoology
Research Group

Professor
SA Professor
SA Associate Professor
Assistant Professor
Assistant Professor
SA Researcher
SA Researcher

Toshihiro Horii, Ph. D.
Eisaku Kimura, Ph. D.
Nirianne M. Q. Palacpac, Ph. D.
Nobuko Arisue, Ph. D.
Takahiro Tougan, Ph. D.
Masanori Yagi, Ph. D
Jyotheeswara Reddy Edula, Ph. D

Research Projects
Malaria is a serious threat to global human health. More than 40% of the world’s population lives in malaria-endemic areas,
and two million people die from the disease annually. Controlling malaria has become more challenging since the emergenceof drug-resistant malaria parasites. This stresses the need for novel drug-targeting strategies and an effective malaria vaccine.
Our department focuses on both malaria vaccine and anti-malarial drug development. We also seek to understand themechanisms used by the malaria parasite to survive in the host.
(1) Development of a recombinant vaccine based on the malaria protein SERA
We are developing a malaria vaccine based on SE36, a recombinant protein that corresponds to the N-terminal amino acidsequence present in the serine repeat antigen (SERA) 5 of malaria parasites (Ref. 1, 2). An epidemiological study of children
living in a malaria-endemic area in Uganda revealed that the children with high antibody titers against the N-terminal domain
of SERA5 did not have fever. There was a clear negative correlation between blood parasitemia and anti-SERA IgG antibody
titers. In collaboration with The Research Foundation for Microbial Diseases of Osaka University, we produced the
GMP-grade malaria vaccine candidate BK-SE36, which is composed of SE36 and aluminum hydroxide gel. We conducted a
Phase Ib clinical trial of BK-SE36 with 140 volunteers in Uganda between 2010 and 2011. The safety of BK-SE36 wasdemonstrated in Stage 1 of the trial with Ugandan male and female adults and in the following Stage 2 with Ugandan children
aged 6–20 years. We followed up the volunteers for 1 year after vaccination and observed their malaria infection. An efficacy
rate of 72% (p=0.003) against symptomatic malaria was obtained when BK-SE36 vaccinees were compared to the control
cohort (Ref. 2). Although the polymorphism of malaria antigens often complicates malaria vaccine development, SERA genes
do not show antigenic variation (Aoki; JBC, 2002) and the polymorphism of the antigen is also limited (Ref. 4). This suggeststhat this vaccine may be effective against a wide variety of malaria parasite strains. We will conduct further clinical trials andproceed to the development of the vaccine for use in malaria-endemic areas.
(2) Approaches to understanding the evolution of malaria parasites
Malaria parasites have many unique features, such as a highly restricted host range, a complex life cycle, and host immune

Fig. 1. Patients at Apac hospital (Uganda). Of
the patients waiting at the Out-patient
Department of Apac Hospital in Northern
Uganda, majority are children under 5 years
old.
Fig. 2. Life cycle of Plasmodium falciparum and the action
point of anti-SE36 antibody induced by SE36 malaria vaccine.

ĴĳFig. 3. The SE36 malaria vaccine for clinical
trials is produced under Good Manufacturing
Practices (GMP) conditions at the Kanonji
Institute of The Research Foundation forMicrobial Diseases of Osaka University.

Research & Activities

evasion systems. To understand the molecular basis of those features, we utilized several genetic approaches, including nuclear
and organelle genome analyses, as well as population genetic analyses of the antigen-coding genes (Ref. 4, 5). The findings
from these studies will be useful for vaccine development.

Fig. 4. Kaplan-Meier plot of P. falciparum malaria episodes
after vaccination.

Fig. 5. Evolutionary tree of malaria parasites inferred from 30
protein-encoding genes from the apicoplast genome. Four humanparasites do not show a monophyletic relationship revealing
host-switching during parasite evolution.

Recent Publications
1. Protective epitopes of the Plasmodium falciparum SERA5 malaria vaccine reside in intrinsically unstructured
N-terminal repetitive sequence. Yagi M, Bang G, Tougan T, Palacpac NMQ, Arisue N, Aoshi T, Matsumoto Y, Ishii
KJ, Egwang TG, Druilhe P, Horii T. PLoS One. 2014 Jun 2;9(6):e98460.
2. Phase 1b randomized trial and follow-up study in Uganda of the blood-stage malaria vaccine candidate BK-SE36.
Palacpac NM, Ntege E, Yeka A, Balikagala B, Suzuki N, Shirai H, Yagi M, Ito K, Fukushima W, Hirota Y, Nsereko
C, Okada T, Kanoi BN, Tetsutani K, Arisue N, Itagaki S, Tougan T, Ishii KJ, Ueda S, Egwang TG, Horii T. PLoS
One. 2013 May 28;8(5):e64073.
3. TLR9 adjuvants enhance immunogenicity and protective efficacy of the SE36/AHG malaria vaccine in nonhumanprimate models. Tougan T, Aoshi T, Coban C, Katakai Y, Kai C, Yasutomi Y, Ishii KJ, Horii T. Hum Vaccin
Immunother. 2013 Jan 4;9(2):283-90.
4. Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5.
Tanabe K, Arisue N, Palacpac NM, Yagi M, Tougan T, Honma H, Ferreira MU, Färnert A, Björkman A, Kaneko A,
Nakamura M, Hirayama K, Mita T, Horii T. Vaccine. 2012 Feb 21;30(9):1583-93.
5. The Plasmodium apicoplast genome: conserved structure and close relationship of P. ovale to rodent malaria
parasites. Arisue N, Hashimoto T, Mitsui H, Palacpac NM, Kaneko A, Kawai S, Hasegawa M, Tanabe K, Horii T.
Mol Biol Evol. 2012 Sep;29(9):2095-9.
ĴĴResearch & Activities

Genome Information Research Center

Department of Experimental Genome Research
Research Group

Professor
SA Associate Professor
Assistant Professor
Assistant Professor
Assistant Professor

Masahito Ikawa, Ph.D.
Ayako Isotani, Ph.D.
Yoshitaka Fujihara, Ph.D.
Haruhiko Miyata, Ph.D.
Yuhkoh Satouh, Ph.D.

In the past, naturally mutated animals were used to elucidate the mechanisms of various diseases. In the "post-genome projectera", however, genetically manipulated animals play a key role in such investigations as they are highly useful for providing
disease models. Our laboratory, in collaboration with the Animal Resource Center for Infectious Diseases, is researching the
mammalian reproductive system by genetically manipulating mice (http://www.egr.biken.osaka-u.ac.jp/).
Research Projects
We were the first group in the world to produce a genetically altered “green mouse” that glows in the dark. These
GFP-expressing mice are highly useful for many fields of research, such as stem cell transplantation and regeneration. Byutilizing these animals, we showed that it was possible to label sperm with fluorescent proteins and to visualize the fertilization
process (Fig. 1–3). By utilizing gene knockout (KO) technology, we also showed that testis-specific chaperones (CLGN,CALR3, and PDILT) are required for the quality control of the sperm membrane protein ADAM3 in the endoplasmic
reticulum. The Pdilt KO mice were found to be male infertile because their spermatozoa lack ADAM3 and cannot migrate
through the utero-tubal junction (Fig. 2, #5). The testicular germ cell-specific GPI-anchored protein TEX101 was also found to
be required for the presence of ADAM3 (#3). Finally, we were successful in visualizing the moment of sperm-egg fusion byusing transgenic mouse lines in which IZUMO1 was tagged with a fluorescent protein (Fig. 3, #4).
We are also seeking to invent new technologies for novel investigations in the field of reproduction. By transducing blastocystswith lentiviral vectors, we developed the placenta-specific gene manipulation method (Nat Biotechnol, 2007). Expression ofsoluble FLT1 in the placenta generates preeclampsia model mice (PNAS, 2011). We established rat embryonic stem (ES) cells
DQGXVHGWKHPWRJHQHUDWH0RXVHļ5DWFKLPHULFDQLPDOV )LJGenes Cells, 2011). These chimeric animals may be usefulfor generating various organs from ES or iPS cells. To examine the role(s) of miRNA, we have analyzed miRNA KO mice(#2). We also successfully generated genetically modified animals (mice and rats) by using the CRISPR/Cas system (#1). Our
recent interest is to use the CRISPR/Cas system to generate gene-modified animals for studying fertilization, implantation, andplacentation.

Fig. 1) A transgenic mouse spermatozoa
labeled with green fluorescent protein(GFP) in their acrosome and red fluorescentprotein (RFP) in their mitochondria.
Spermatozoa lose green fluorescence after
acrosome reaction.

ĴĵFig. 2) Pdilt -/- mouse spermatozoa have impaired ability to migrate from uterus into the oviduct.

Research & Activities

Fig. 3) Red-IZUMO1 transgenic spermatozoa
bind onto oocyte plasma membrane. Acrosome
reacted spermatozoa indicate altered
distribution of IZUMO1 by red fluorescence (2or 3, GFP negative). Fused sperm (4) shows
specific pattern of fluorescent lost.

)LJ 0RXVHļ5DWFKLPHULFDQLPDOVDUHJHQHUDWHGE\LQMHFWLQJUDW(6FHOOV
into mouse blastocysts (top). Contribution of mouse (white) and rat (black)cells are determined by coat color (bottom).

図 5)
A. pCAG-EGxxFP target plasmid contains
overlapping 5’ and 3’ EGFP fragments under the CAG
promoter. pX330 plasmid expresses both guide RNA(gRNA) and hCas9 endonuclease.
B.The efficiency of DSB mediated homology
dependent repair was validated by observing EGFP
fluorescence 48 hrs after the transfection (top;fluorescent field , bottom; bright field).
C. To generate gene modified mice, fertilized eggs
were injected with mRNAs coding hCas9 and gRNA
into cytoplasm or pX330 plasmid into pronuclei.
D. CRISPR/Cas9 mediated Cetn1 mutations observedin founder mice. Representative Cetn1 genomic
sequences from founder mice (top; wild-type (WT),
bottom; heterozygous 8 bp deletion (em4)).

Recent Publications
1. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.
Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M. Sci Rep. 2013 Nov 27;3:3355.
2. MiR-200b and miR-429 Function in Mouse Ovulation and Are Essential for Female Fertility. Hasuwa H, Ueda J,
Ikawa M, Okabe M. Science. 2013 Jul 5;341(6141):71-3.
3. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa. Fujihara Y,
Tokuhiro K, Muro Y, Kondoh G, Araki Y, Ikawa M, Okabe M. Proc Natl Acad Sci U S A. 2013 May 14;110(20):8111-6.
4. Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1. Satouh Y, Inoue N,
Ikawa M, Okabe M. J Cell Sci. 2012 Nov 1;125(Pt 21):4985-90.
5. Testis specific PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility.
Tokuhiro K, Ikawa M, Benham AM, Okabe M. Proc Natl Acad Sci U S A. 2012 March 6;109 (10):3850-3855.
ĴĶResearch & Activities

Genome Information Research Center

Laboratory of Genome Research
Research Group$VVRFLDWH3URIHVVRU

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We are investigating the molecular biological mechanisms involved in human diseases, especially cardiovascular diseases, byusing animal models.
1) We have established a diastolic heart failure model using Dahl salt-sensitive rats. This model showed that left ventricular(LV) fibrosis and stiffening play crucial roles in the development of heart failure with preserved ejection fraction (HFpEF).
Digitalis-like factors and the subsequently activated Na+ /Ca 2+ exchanger entry mode may play important roles in thedevelopment of hypertensive HFpEF and the effect of carnitine when it is administered to the HFpEF model (3). In addition,
serum interleukin-16 (IL-16) levels are elevated in patients with HFpEF and the rat model (1). Enhanced cardiac expression of
IL-16 in transgenic mice induces cardiac fibrosis and LV myocardial stiffening that is accompanied by increased macrophage
infiltration (Figure 1).
2) To understand the cellular and molecular aspects of vascular smooth muscle (SM) cell growth in atherosclerotic plaques, we
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and its functions.

Figure 1. Enhanced cardiac expression of IL-16 under theĮ0+&SURPRWHUFDXVHVLQFUHDVHGP\RFDUGLDOILEURVLVDQG
stiffness in mice. (left) Four-chamber view of the hearts from
non-transgenic and transgenic mice. (right) Sirius Red-stained
heart LV sections of fibrosis areas.

Figure 2. The human vascular smooth muscle Į-actin
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including -1M (center) and 4M (right) point mutants in thetranscriptional nuclear factor binding regions specifically did
not.

Recent Publications
1. Tamaki S, Mano T, Sakata Y, Miwa T, et.al. Interleukin-16 promotes cardiac fibrosis and myocardial stiffening inheart failure with preserved ejection fraction. PLoS One. 2013 Jul 19;8(7):e68893.
 7VXNDPRWR<0DQR76DNDWD<0LZD7HWDO$QRYHOKHDUWIDLOXUHPLFHPRGHORIK\SHUWHQVLYHKHDUWGLVHDVHE\angiotensin II infusion, nephrectomy, and salt loading. Am J Physiol Heart Circ Physiol. 2013 Dec 1;305(11):H1658-67.
3. Kamimura D, Ohtani T, Miwa T, et al.Ca2+ Entry mode of Na+/Ca2+ exchanger as a new therapeutic target forheart failure with preserved ejection fraction. European Heart J. 2012, 11, 1408-1416.
ĴķResearch & Activities

ĴĸGenome Information Research Center

Research & Activities

Department of Genome Informatics
Research Group

Professor
Professor (SUP)
Assistant Professor
Assistant Professor
Assistant Professor (SUP)

Teruo Yasunaga, Ph.D.
Tatsuya Takagi, Ph.D.
Naohisa Goto, Ph.D.
Shota Nakamura, Ph.D.
Norihito Kawashita, Ph.D.

Research Projects
Our group studies the genome information of various organisms by using high-performance computers to identify new
biological phenomena and to understand how organisms evolve. In addition, we develop software tools for bioinformatics andmolecular biology. Our laboratory operates a computer system for genome sequence data analyses that is made available toresearchers in our university. We also hold training courses for genome analysis at least once every year.
(1) Large-scale genome analysis
The complete genome sequences of more than 1,000 organisms are currently available. By using bioinformatics and molecular
evolution techniques, we intend to analyze this enormous body of genome data. We are developing software and algorithms forlarge-scale genome analysis. In particular, we developed CONSERV, a conserved sequence finder. By analyzing the complete
genome sequences of 266 organisms, we identified invariant sequences that may have been present in the last common
ancestor of all extant life forms (Fig. 1). By comprehensively analyzing the genome of the influenza virus, we also intend to
elucidate the evolutionary pathways this virus has taken (Fig. 2).

Figure 1. Large-scale genome analysis of 266 organisms. Identificationof sequences conserved in almost all known genomes.

ĴĹ

Figure 2. Comprehensive analysis of the influenza virus genome.

Research & Activities(2) Next generation sequencer data analysis
The recently developed “next generation sequencing” technology has enabled sequencing of the whole genome of any
microorganism in one sequencer run, thus producing massive amounts of nucleotide sequence data in each run. We have beendeveloping software to handle these data and have set up an analysis system for collaborative sequencing projects of
microorganisms with other laboratories (Fig. 3).
(3) Operation of a computer system for genome information analysis at Osaka University
We provide computer resources for researchers in our university. We also provide mirrored access to major nucleotide, protein,
and genome databases through our servers (Fig. 4), which are fully synchronized with the mother servers and are kept up todate at all times.

Figure 3. Next generation sequencing enables
sequencing of a whole genome in one run.

Figure 4. Genome Information Research Center Computer System:
Analysis servers (left) and 1.8PB storage system (right).

Recent Publications
1. Tachibana S, Sullivan SA, Kawai S, Nakamura S, Kim HR, Goto N, Arisue N, Palacpac NM, Honma H, Yagi M,
Tougan T, Katakai Y, Kaneko O, Mita T, Kita K, Yasutomi Y, Sutton PL, Shakhbatyan R, Horii T, Yasunaga T,
Barnwell JW, Escalante AA, Carlton JM, Tanabe K. Plasmodium cynomolgi genome sequences provide insight into
Plasmodium vivax and the monkey malaria clade. Nat Genet. 2012 Sep;44(9):1051-5.
2. de Silva UC, Tanaka H, Nakamura S, Goto N, Yasunaga T. A comprehensive analysis of reassortment in influenza A
virus. Biol Open. 2012 Feb 23;1(4):385-90.
3. Yamashita A, Kawashita N, Kubota-Koketsu R, Inoue Y, Watanabe Y, Ibrahim MS, Ideno S, Yunoki M, Okuno Y,
Takagi T, Yasunaga T, Ikuta K. Highly conserved sequences for human neutralization epitope on hemagglutinin of
influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition. Biochem
Biophys Res Commun. 2010 Mar 19;393(4):614-8.
4. Nakamura S, Yang CS, Sakon N, Ueda M, Tougan T, Yamashita A, Goto N, Takahashi K, Yasunaga T, Ikuta K,
Mizutani T, Okamoto Y, Tagami M, Morita R, Maeda N, Kawai J, Hayashizaki Y, Nagai Y, Horii T, Iida T, Nakaya
T. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput
sequencing approach. PLoS One. 2009;4(1):e4219.
5. Yamashita A, Goto N, Nishiguchi S, Shimada K, Yamanishi H, Yasunaga T. Computational search forover-represented 8-mers within the 5'-regulatory regions of 634 mouse testis-specific genes. Gene. 2008 Dec 31;427(1-2):93-8.
ĴĺResearch & Activities

Genome Information Research Center

Department of Infection Metagenomics
Research Group

Professor (SUP)
Professor (SUP)SA Professor (SUP)
Assistant Professor (SUP)
Assistant Professor (SUP)

Toshihiro Horii, Ph.D.
Teruo Yasunaga, Ph.D.
Tetsuya Iida, Ph.D.
Naohisa Goto, Ph.D.
Shota Nakamura, Ph.D.

1. RAPID (Robotics Assisted Pathogen IDentification)
Under the aegis of the Program of Research Centers for Emerging and Re-emerging Infectious Diseases of MEXT, Japan, andin collaboration with the Omics Science Center, RIKEN, we are constructing a framework called “RAPID” that will facilitate
the emergency diagnosis of infectious diseases. We are also cooperating with the research centers of eight countries in Asia
and Africa in an effort to identify the causative agents in naturally occurring outbreaks.
2. Metagenomic Diagnosis of Infectious Diseases
Metagenomic analysis allows us to diagnose many of the major human infectious diseases (including respiratory tract
infections, enteric infections, and blood-borne infections) by using a single common protocol. In addition, to pre-empt zoonotic
disease outbreaks, we are seeking to identify new pathogenic microorganisms in animal-derived samples that may have
zoonotic potential.
3. Metagenomic Analysis of the Intestinal Microbiome
The intestinal microbiome plays an important role in protecting the host from pathogen invasion. We are currently analyzing
the intestinal microbiome of patients with diarrheal diseases to elucidate how the human host, the intestinal microbiome, andpathogenic microorganisms interact. This analysis will help us to understand the changes that occur in the intestinal
microbiome during the course of infection.
4. Development of Novel Methods for Pathogen Detection
To develop more efficient and comprehensive methods of identifying pathogens, we are studying the efficacy of different
methods for amplifying the genome of pathogenic microorganisms and subtracting the host genome.

Extract DNA or RNA

Next-generation sequencer

DATA analysis

Fig. 1. Metagenomic diagnosis of infectious diseases using a next-generation sequencer.

Fig. 2. The next-generation sequencers installed in our department, Roche 454 GS Junior Bench Top System, illumina MiSeq Personal Sequencer,
Pacific Biosciences RS System.

ĵıResearch & Activities

Fig. 3. Distribution of detected organisms
in viral infection cases

Fig. 4. Standard operating protocol for RAPID

Recent Publications
1. Masahata K, Umemoto E, Kayama H, Kotani M, Nakamura S, Kurakawa T, Kikuta J,Gotoh K, Motooka D, Sato S,
Higuchi T, Baba Y, Kurosaki T, Kinoshita M, ShimadaY, Kimura T, Okumura R, Takeda A, Tajima M, Yoshie O,
Fukuzawa M, Kiyono H,Fagarasan S, Iida T, Ishii M, Takeda K. Generation of colonic IgA-secreting cellsin the
caecal patch. Nat Commun. 2014 Apr 10;5:3704.
2. Yonogi S, Matsuda S, Kawai T, Yoda T, Harada T, Kumeda Y, Gotoh K, Hiyoshi H, Nakamura S, Kodama T, Iida
T. BEC, a novel enterotoxin of Clostridium perfringensfound in human clinical isolates from acute gastroenteritis
outbreaks. InfectImmun. 2014 Mar 24.
3. Imai A, Gotoh K, Asano Y, Yamada N, Motooka D, Fukushima M, Kanzaki M, Ohtani T, Sakata Y, Nishi H, Toda
K, Sawa Y, Komuro I, Horii T, Iida T, Nakamura S, Takashima S. Comprehensive metagenomic approach fordetecting causativemicroorganisms in culture-negative infective endocarditis. Int J Cardiol. 2014 Mar 15;172(2):e288-9.
4. Seki M, Gotoh K, Nakamura S, Akeda Y, Yoshii T, Miyaguchi S, Inohara H, Horii T, Oishi K, Iida T, Tomono K.
Fatal sepsis caused by an unusual Klebsiellaspecies that was misidentified by an automated identification system.
J Med Microbiol. 2013 May;62(Pt 5):801-3
5. Monira S, Nakamura S, Gotoh K, Izutsu K, Watanabe H, Alam NH, Nakaya T, Horii T, Ali SI, Iida T, Alam M.
Metagenomic profile of gut microbiota in childrenduring cholera and recovery. Gut Pathog. 2013 Feb 1;5(1):1.
ĵĲResearch & Activities

International Research Center for Infectious Diseases
Department of Infectious Disease Control

Laboratory of Clinical Research on Infectious Diseasesř
SA Associate Professor Wataru Kamitani, PhD.
SA Researcher Yusuke Sakai, DVM, PhD.

Research Group

Research projects: Our group studies the molecular biology and host cell–virus interactions of Severe Acute Respiratory
syndrome (SARS) coronavirus (SARS-CoV). SARS-CoV is the etiological agent of a newly-emerged human respiratory
disease that originated in southern China in 2002 and spread worldwide in the 2003 epidemic. Coronavirus is an enveloped
virus that carries a long, single-stranded, positive-sense genomic RNA. On infection, the genomic RNA is translated to
produce two large polyproteins that are then proteolytically processed by two viral proteinases into 16 mature viral proteins
called nsp1 to nsp16. Currently, our group is studying the effect of SARS-CoV nsp1 protein on host cells. SARS-CoV nsp1
protein inhibits host gene expression by first binding to the 40S ribosome and then inactivating the translational machinery. In
addition, the 40S ribosome-bound nsp1 induces RNA cleavage of host mRNA. The nsp1 protein also binds to the
5’ untranslated region (UTR) of SARS-CoV RNA, which confers resistance to the nsp1-mediated shutoff of translation. Ourgroup is also studying various SARS-CoV–host cell interactions to understand SARS-CoV pathogenesis at the molecular level.
More recently, another coronavirus called the Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in the
Middle East in April of 2012. It too causes acute respiratory distress syndrome. Our group is presently studying the molecularbiology and host cell–virus interactions of MERS-CoV.

Figure: Model of SARS-CoV nsp1-mediated shutoff. SARS-CoV nsp1 causes the translational shutoff bybinding to the 40S ribosome. The nsp1-40S ribosome complex also induces the cleavage of host mRNA.
SARS-CoV nsp1 also binds to the 5’ UTR of its own RNA. This latter interaction is crucial for theevasion by the coronavirus of its nsp1-mediated shutoff.

Recent Publications
1. Severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specifictranslational shutoff of host mRNA. Tanaka T, Kamitani W, DeDiego ML, Enjuanes L, Matsuura Y. J Virol. 2012
Oct;86(20):11128-37.
2. A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1 protein. Kamitani W, Huang
C, Narayanan K, Lokugamage KG, Makino S. Nat Struct Mol Biol. 2009 Nov;16(11):1134-40.
3. Suppression of host gene expression by nsp1 proteins of group 2 bat coronaviruses. Tohya Y, Narayanan K,
Kamitani W, Huang C, Lokugamage K, Makino S. J Virol. 2009 May;83(10):5282-8.
4. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits
eIF2alpha phosphorylation. Ikegami T, Narayanan K, Won S, Kamitani W, Peters CJ, Makino S. PLoS Pathog. 2009
Feb;5(2):e1000287.
ĵĳResearch & Activities

International Research Center for Infectious Diseases
Department of Infectious Disease Control/DERUDWRU\RI&OLQLFDO5HVHDUFKRQ,QIHFWLRXV'LVHDVHVŚ
Invited Professor
SA Associate Professor
SA Researcher

Research Group

Kazunori Oishi M.D., Ph.D.
Yukihiro Akeda Ph.D.
Dan Takeuchi M.D., Ph.D.

The research activities in our group include 1) studies on the diagnosis, pathogenesis, and prevention of invasive bacterial
diseases such as pneumococcal and Haemophilus influenzae infection; 2) studies on the epidemiology and pathogenesis of the
zoonotic pathogen Streptococcus suis; 3) the development of novel vaccine antigens; 4) the development of diagnostic test kits
for infectious diseases, including drug-resistant bacteria, and for clinical research using these test kits; and 5) studies on theprotein secretion systems of pathogenic bacteria. Our research has been conducted with foreign collaborators to clarify thepathogenic mechanisms of infectious diseases, with the hope that the research outcomes will eventually be translated tosuccessful clinical interventions. Our most recent achievements include the proposal of a surrogate marker for the
immunogenicity of current pneumococcal vaccines and breakthrough research on the mechanism by which Streptococcus suiscauses meningitis (Figure).
In addition, our research group is conducting a training course in tropical infectious diseases for young medical doctors under
the Research Institute for Microbial Diseases and the Graduate School of Medicine, Osaka University. This successful training
course develops the skills of many young doctors working in the field of infectious diseases.

Figure. Mechanism of Streptococcus suis infection in Thailand (Emerg Infect Dis 2011, J Infect Dis 2014)

Recent Publications
1. Hamaguchi S, Hirose T, Matsumoto N, Akeda Y, Irisawa T, Seki M, Hosotsubo H, Yamamoto K, Tasaki O, Oishi K,
Shimazu T, Tomono K. Neutrophil extracellular traps in bronchial aspirates: a quantitative analysis. Eur Respir J.
2014 (In press)
2. Tamura K, Matsubara K, Ishiwada N, Nishi J, Ohnishi H, Suga S, Ihara T, Chang B, Akeda Y, Oishi K; Japanese
IPD Study Group. Hyporesponsiveness to the infecting serotype after vaccination of children with seven-valentpneumococcal conjugate vaccine following invasive pneumococcal disease. Vaccine. 2014 (In press).
3. Takeuchi D, Akeda Y, Nakayama T, Kerdsin A, Sano Y, Kanda T, Hamada S, Dejsirilert S, Oishi K. The
Contribution of Suilysin to the Pathogenesis of Streptococcus suis Meningitis. J Infect Dis. 2014 (In press).
4. Kerdsin A, Dejsirilert S, Sawanpanyalert P, Boonnark A, Noithachang W, Sriyakum D, Simkum S, Chokngam S,
Gottschalk M, Akeda Y, Oishi K. Sepsis and spontaneous bacterial peritonitis in Thailand. Lancet. 2011 Sep 3;378(9794):960.
5. Kerdsin A, Dejsirilert S, Puangpatra P, Sripakdee S, Chumla K, Boonkerd N, Polwichai P, Tanimura S, Takeuchi D,
Nakayama T, Nakamura S, Akeda Y, Gottschalk M, Sawanpanyalert P, Oishi K. Genotypic profile of Streptococcus
suis serotype 2 and clinical features of infection in humans, Thailand. Emerg Infect Dis. 2011 May;17(5):835-42.
ĵĴResearch & Activities

International Research Center for Infectious Diseases
Department of Special Pathogens

Laboratory for Infection Cell Biology
Research GroupSA Professor
SA Assistant Professor
SA Assistant Professor
SA Researcher

Yukako Fujinaga, Ph.D.
Yo Sugawara, Ph.D.
Takuhiro Matsumura, Ph.D.
Masahiro Yutani, Ph.D.

Research Projects:
Many bacterial toxins can severely damage the host, even at very low concentrations. Most bacterial toxins are enzymes thatact catalytically and with high specificity on functional host cell molecules, thereby markedly modulating host homeostasis.
The toxins are also often highly efficient in accessing their target molecule in the host. The ingenious transport systems
involved often exploit the fundamental host machinery of membrane trafficking and the functions of intracellular organelles.
Therefore, studies seeking to elucidate toxin trafficking could provide us with valuable information about basic cellularfunction, as well as aiding our understanding of the pathology induced by these toxins and helping us to develop effective
therapeutic strategies against them. We are currently engaged in studying the structure and function of the botulinum
neurotoxin complex, which must pass down the digestive tract and cross the epithelial barrier lining the intestine to cause
food-borne botulism.

Hypothetical model for intestinal absorption of botuliunum neurotoxin complex (type B 16S toxin). We propose a three-stepmechanism by which the botulinum neurotoxin complex breaches the intestinal epithelial barrier. First, apically located 16S toxin
binds to intestinal epithelial cells via the interaction of HA (hemagglutinin) component and galactose containing-glycoconjugates,
and is transcytosed. Then, once located in the basolateral compartment, HA component binds to E-cadherin, and thereby disrupts
the paracellular barrier. Finally, a large amount of toxin accumulates in the basolateral area via paracellular movement.

Recent Publications
1. Amatsu S1, Sugawara Y1, Matsumura T, Yutani M, Kitadokoro K, and Fujinaga Y. Crystal structure of botulinum
whole hemagglutinin reveals a huge triskelion-shaped molecular complex. J Biol Chem. 2013; 288; 49: 35617-25.
1These authors contributed equally.
2. Fujinaga Y, Yo Sugawara, Takuhiro Matsumura. Uptake of Boulinum Neurotoxin in the Intestine. Curr Top
Microbiol Immunol. 2013; 364: 45-59 [Review]
3. Sugawara Y, Fujinaga Y. The botulinum toxin complex meets E-cadherin on the way to its destination. Cell Adh
Migr. 2011; 5(1): 34-36. [Review]
4. Sugawara Y, Matsumura T, Takegahara Y, Jin Y, Tsukasaki Y, Takeichi M, Fujinaga Y. Botulinum HA disrupts theintercellular epithelial barrier by directly binding E-cadherin. J Cell Biol. 2010; 189 (4), 691-700
5. Jin Y1, Takegahara Y1, Sugawara Y, Matsumura T, Fujinaga Y. Disruption of the epithelial barrier by botulinum
hemagglutinin (HA) proteins - Differences in cell tropism and the mechanism of action between HA proteins oftypes A or B, and HA proteins of type C. Microbiology. 2009; 155(Pt 1): 35-45. 1These authors are contributed
equally.
ĵĵResearch & Activities

International Research Center for Infectious Diseases
Department of Special Pathogens

Research GroupSA Associate Professor

Eiji Morita, Ph. D.

Research & Activities

Laboratory of Viral Infection
Positive-strand RNA viruses can dramatically rearrange the intracellular membranes of the host cell and produce unusualorganelle-like structures called ‘replication complexes’ or ‘membranous webs’ . These membrane structures appear in close
proximity to the endoplasmic reticulum, and probably serve as a scaffold for the assembly of virus replication machinery byproviding an organization and an environment that facilitate viral propagation. These structures are also known to serve asshells that protect the viruses against various cellular stress responses and allow persistent viral replication in the cytoplasm.
Our group has been focusing on the molecular mechanisms involved in the formation of these replication complexes todetermine the dynamic state of viral and/or host factors during the viral propagation cycle. Recently, we succeeded in purifying
replication complexes from cells infected with hepatitis C virus or flavivirus. Quantitative mass spectrometry analyses led tothe identification of several cellular factors that are specifically recruited to these viral replication complexes. We are currently
focusing on the molecular functions of these newly identified cellular factors on the presumption that they are involved in thebiosynthesis of the viral replication complexes. We are also studying the molecular mechanisms of viral particle formation andthe mechanisms of autophagy induction seen in virus-infected cells. These studies may contribute to the development of novel
antiviral therapies.

Figure: Fluorescent (A-D) and electron (E and F) microscope images of viral replication complexes in flavivirus-infected Vero cells.
Twenty-four hours after infection, the cells were fixed and stained with antibodies against viral non-structural (anti-NS2B, magenta),
structural (anti-E, green), and nuclear (DAPI, cyan) proteins. Unusual membrane structures, 5 to 10 nm in diameter and positive for viral
antigens, are detected adjacent to the nucleus in the virus-infected cells. The endoplasmic reticulum is observed at the periphery of thisstructure, and many virus-like particles are detected inside of the lumen.

Recent Publications
1. Fujita, N.†, Morita, E. †, Itoh, T., Tanaka, A., Nakaoka, M., Osada, Y., Umemoto, T., Saitoh, T., Nakatogawa, H.,
Kobayashi, S., Haraguchi, T., Guan, J.L., Iwai, K., Tokunaga, F., Saito, K., Ishibashi, K., Akira, S., Fukuda, M.,
Noda, T., Yoshimori, T. Recruitment of the autophagic machinery to endosomes during infection is mediated byubiquitin. J Cell Biol. 2013 †These authors contributed equally
2. Morita, E., Arii J, Christensen D, Votteler J, Sundquist WI. Attenuated protein expression vectors for use in siRNA
rescue experiments. Biotechniques. 2012 Aug;0(0):1-5.
3. Morita, E., Sandrin, V., McCullough, J., Katsuyama, A., Hamilton, IB., Sundquist, WI. ESCRT-III protein
requirements for HIV-1 budding. Cell Host Microbe. 2011, 3:235-42.
4. Morita, E., Colf, LA., Karren, MA., Sandrin, V., Rodesch, CK., Sundquist, WI. Human 5. ESCRT-III and VPS4
proteins are required for centrosome and spindle maintenance. Proc Natl Acad Sci U S A. 2010 107(29):12889-94.
5. Bajorek, M. †, Morita, E. †, Skalicky, JJ, Morham, SG., Babst, M., Sundquist, WI. Biochemical analyses of human
IST1 and its function in cytokinesis. Mol Biol Cell. 2009, 20(5):1360-73. †These authors contributed equally.
ĵĶResearch & Activities

International Research Center for Infectious Diseases
Department of Special Pathogens

Laboratory of Genomic Research on Pathogenic Bacteria
Research GroupSA Professor
SA Assistant Professor
SA Researcher

Tetsuya Iida, Ph.D.
Shigeaki Matsuda, Ph.D.
Chonchanok Theethakaew, Ph.D.

This research group is studying pathogenic bacteria from the genomic point of view.
Our main research targets are as follows:
1. Characterization of the mechanism(s) used by bacterial pathogens to infecthost organisms by identifying infection-related changes in pathogen
genome expression: To understand the molecular mechanisms by which
bacterial pathogens infect host organisms, we are using DNA microarrays
and other molecular methods to investigate the changes in the genome
expression pattern of various bacterial pathogens that occur during theirinteraction with their target host.

Figure 1. Whole genome sequence ofVibrio parahaemolyticus

2. Analysis of the mechanism(s) that lead to the emergence of new infectious
diseases: The unique features of various newly emerged bacterial pathogens
are being studied by analyzing their genomes and comparing them withthose of other bacterial strains.
3. Investigation of the life cycles of bacterial pathogens in their naturalenvironments: Based on what is currently understood about various
bacterial pathogens, we are seeking to characterize their life cycles in theirnatural habitats.

Figure 2. Characterization and comparison of the
genomes of pathogenic bacteria by using DNA
microarrays

4. Development of new methods for the rapid identification of bacterial
pathogens based on genomic information: To rapidly diagnose bacterial
infections, a novel system for identifying bacterial pathogens by
high-throughput DNA sequencing is being developed.

Recent Publications
1. Okada R, Zhou X, Hiyoshi H, Matsuda S, Chen X, Akeda Y, Kashimoto T, Davis BM, Iida T, Waldor MK, Kodama
T. The Vibrio parahaemolyticus effector VopC mediates Cdc42-dependent invasion of cultured cells but is not
required for pathogenicity in an animal model of infection. Cell Microbiol. 2014 Jun;16(6):938-47.
2. Matsuda S, Okada N, Kodama T, Honda T, Iida T. A cytotoxic type III secretion effector of Vibrio parahaemolyticus
targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes. PLoS Pathog. 2012 Jul 19;8(7):e1002803.
3. Hiyoshi H, Kodama T, Saito K, Gotoh K, Matsuda S, Akeda Y, Honda T, Iida T. VopV, an F-actin-binding type III
secretion effector, is required for Vibrio parahaemolyticus-induced enterotoxicity. Cell Host Microbe. 2011 Oct
20;10(4):401-9.
4. Monira S, Nakamura S, Gotoh K, Izutsu K, Watanabe H, Alam NH, Endtz HP, Cravioto A, Ali SI, Nakaya T, Horii
T, Iida T, Alam M. Gut microbiota of healthy and malnourished children in Bangladesh. Front Microbiol. 2011 Nov
21;2:228.
5. Gotoh K, Kodama T, Hiyoshi H, Izutsu K, Park KS, Dryselius R, Akeda Y, Honda T, Iida T. Bile acid-induced
virulence gene expression of Vibrio parahaemolyticus reveals a novel therapeutic potential for bile acid sequestrants.
PLoS One. 2010 Oct 13;5(10):e13365.
ĵķResearch & Activities

International Research Center for Infectious Diseases
Department of Special Pathogens

Research GroupSA Associate Professor
SA Associate Professor

Takeshi Kobayashi, D.V.M., Ph.D.
Yuta Kanai, D.V.M., Ph.D.

Research projects
1) Oncolytic viral therapy using reovirus
Mammalian orthoreoviruses (reoviruses) are members of the family
Reoviridae and contain a genome consisting of 10 segments ofdouble-stranded (ds) RNA. Reoviruses are highly tractable experimental
models for studies of dsRNA virus replication and pathogenesis. In thelast decade, the potential of reoviruses as oncolytic agents against
various tumors, including head and neck, colon, breast, and pancreaticcancers, has been investigated in animal models and humans. The
putative oncolytic potential of reoviruses is based on the observationthat reoviruses induce cell death and apoptosis in tumor cells with anactivated Ras signaling pathway. However, while wild-type
reovirus-based oncolytic therapies have been safe, the efficacy so far islimited. We are thus using genetic modification to develop safer andmore effective reovirus-based cancer therapeutics.

Research & Activities

Laboratory of Viral Replication

Fig1 Strategy to generate reovirus from cloned cDNA2) Highly pathogenic bat reovirus
Bats are a natural reservoir for many important zoonotic viruses,
including Hendra virus, Nipah virus, and potentially SARS coronavirus and Ebola virus. In 1968, the Pteropine orthoreovirus(PRV) was isolated from flying fox. While it was not associated with any disease, Melaka virus, which is genetically similar to
PRV, was recently isolated from a human patient in Malaysia with acute respiratory tract infection (RTI). Subsequently, otherrelated strains of bat-associated orthoreoviruses have been isolated in Malaysia, Indonesia, and China. We also isolated andcharacterized another new PRV, Miyazaki virus, from a patient with acute RTI after returning to Japan from Indonesia in 2007.
These isolates have given rise to increasing concern about bat-transmitted orthoreovirus infections in humans. We areinvestigating how PRV replicates and causes disease by using a combination of genetic, biochemical and biophysicalapproaches. Our aim is ultimately to develop vaccines, diagnostics, and therapeutics for bat reovirus-related diseases.

Recent Publications
1. A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNApolymerase. Komoto S, Kawagishi T, Kobayashi T, Ikizler M, Iskarpatyoti J, Dermody TS, Taniguchi K. J Virol
Methods. 2014 Feb;196:36-9.
 1RQVWUXFWXUDOSURWHLQıVPHGLDWHVUHRYLUXVLQGXFHGFHOOF\FOHDUUHVWDQGDSRSWRVLV%RHKPH.:+DPPHU.
Tollefson WC, Konopka-Anstadt JL, Kobayashi T, Dermody TS. J Virol. 2013 Dec;87(23):12967-79.
3. The reovirus sigma1s protein is a determinant of hematogenous but not neural virus dissemination in mice. Boehme
KW, Frierson JM, Konopka JL, Kobayashi T, Dermody TS. J Virol. 2011 Nov;85(22):11781-90.
4. An improved reverse genetics system for mammalian orthoreoviruses. Kobayashi T, Ooms LS, Ikizler M, Chappell
JD, Dermody TS. Virology. 2010 Mar 15;398(2):194-200.
5. Identification of functional domains in reovirus replication proteins muNS and mu2. Kobayashi T, Ooms LS,
Chappell JD, Dermody TS. J Virol. 2009 Apr;83(7):2892-906
ĵĸResearch & Activities

International Research Center for Infectious Diseases
Department of Infectious Disease Control

Laboratory of Combined Research on Microbiology and Immunology
Research Group

Associate Professor
SA Associate Professor
SA Researcher
SA Researcher
SA Researcher
SA Researcher

Hiroki Nagai, Ph.D.
Tomoko Kubori, Ph.D.
Andree Marie Hubber, Ph.D.
Xuan Thanh Bui, Ph.D
Akiko Okura
Masayo Yamada

Research Projects
Protein secretion is a process of fundamental importance for bacterial pathogenesis. Whether they deliver toxins or directly
inject effector proteins into the cytoplasm of host cells, bacterial protein secretion systems play a central role in modulating
eukaryotic cell functions. Legionella pneumophila are Gram-negative bacteria that are found ubiquitously in soil andfreshwater environments. Once inhaled by humans, Legionella infections can result in a severe form of pneumonia known as
Legionnaires’ disease. Legionella use a type IV secretion system (T4SS) to deliver effector proteins, which mediates theestablishment of a replicative niche in host cells.
The goal of our research is to understand, at the molecular level, how Legionella subverts host cellular functions to accomplish
successful intracellular replication. To this end, the following research projects are currently in progress.
(1) Analysis of the structure and function of the type IV secretion apparatus.
Essentially nothing is known about the substrate transfer across eukaryotic and bacterial membranes that occurs via type IV
secretion systems. Furthermore, the macromolecular structure of the type IV secretion apparatus is largely unknown. To
address these questions, we are analyzing the structure and function of the type IV secretion apparatus of Legionella.
(2) Analysis of effector proteins that translocate from Legionella to host cells.
We previously demonstrated that RalF is translocated by the type IV secretion
system of Legionella into host cells and is required for the recruitment of hostARF proteins to Legionella-containing vacuoles. We also recently demonstrated
that the effector LubX acts as an E3 ligase and targets another effector forproteasomal degradation within host cells. LubX is the first effector protein thathas been shown to target and regulate another effector within host cells.

Fig. 1. T4SS core component DotD represents
a conserved structural motif of the periplasmic
ring.

(3) Interaction of Legionella and its natural hosts.
The ability of Legionella to survive and replicate within eukaryotic cells has
been acquired during its history of interactions with natural hosts such as
free-living amoebae. Taking advantage of the recent development of the NGS
technique, we are analyzing the molecular interactions between amoebae andamoeba symbionts/pathogens, including Legionella.

Fig. 2 Discovery of metaeffector.

Recent Publications
1. Hubber A, Kubori T, *Nagai H. Modulation of the ubiquitination machinery by Legionella. Curr Top Microbiol
Immunol. 2014:376, 227-247
2. Hori JI, Pereira MS, Roy CR, *Nagai H, Zamboni DS. Identification and functional characterization of K(+)
transporters encoded by Legionella pneumophila kup genes. Cell Microbiol. 2013: 15, 2006-2019
3. Nagai H and Kubori T. Dot/Icm type IVB secretion systems of Legionella and other gram-negative bacteria. Front
Microbiol.,2011: 2,136.
4. Kubori T, Shinzawa N, Kanuka H, Nagai H. Legionella metaeffector exploits host proteasome to temporally regulate
cognate effector. PLoS Pathog. 2010;6(12),e1001216.
5. Nakano N, Kubori T, Kinoshita M, Imada K, Nagai H. Crystal structure of Legionella DotD: insights into therelationship between type IVB and type II/III secretion systems. PLoS Pathog. 2010;6(10),e1001129.
ĵĹResearch & Activities

International Research Center for Infections Diseases
Pathogenic Microbes Repository Unit

Head

SA Professor (SUP)
Associate Professor

Tetsuya Iida, Ph.D.
Toshio Kodama, Ph.D.

Research & Activities

Pathogenic Microbes Repository Unit

In this facility, as a part of the activity of the National BioResource Project (NBRP) directed by the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and as a member of the Japan Society for Culture Collections (JSCC),
pathogenic bacterial strains are being collected and preserved. Those strains will be distributed upon request to investigators inand outside this country. The list of the collection is available at the websites of this facility and NBRP.

ĵĺOffice of Combined Program on
Microbiology and Immunology

Research & Activities

Office of Combined Program on Microbiology and Immunology
Research promotion group
Education promotion group

Associate Professor
Associate Professor

Yoshiko Murakami, M.D., Ph.D
Hodaka Fujii, M.D., Ph.D.

Office activities
Our institute and the Immunology Frontier Research Center are world-class institutes for microbiological and immunologicalresearch, respectively. To take maximal advantage of this situation and to promote the cross-fertilization of research between
these institutes, we have developed the combined microbiology and immunology program.
Research promotion
To promote combined microbiological and immunological research, we have the following plans.
1. Organization of the Awaji international forum on infection and immunology, which is held in September annually.
2. Organization of the research progress report, which is held every 2 months within the institute.
3. Organization of the large research presentation program and the competition in research presentation, which are held annually.
The aim of these activities is to facilitate microbiological and immunological research by promoting research collaboration,
information exchange, and personal exchange between laboratories, and by generating a conducive research environment.
Education promotion
To facilitate seamless research on microbiology and immunology, we direct a multidisciplinary graduate program on
microbiology and immunology, including designing the curriculum and its contents. We also organize Open House sessions of
the institute and give guidance to new students. In addition, we organize the combined program on clinical microbiology andimmunology in close cooperation with other research centers, in particular, the Thailand–Japan Research Collaboration Centeron Emerging and Re-emerging Infections.

Research group

Associate Professor

Yoshiko Murakami, MD. PhD

Research projects
I have an additional appointment in the Department of Immunoregulation as the leader of the PNH group.
(See details on the Department page).
1. Investigation of the pathogenesis of acquired glycosylphosphatidylinositol (GPI) deficiency, paroxysmal nocturnal
hemoglobinuria (PNH).
2. Investigation of the pathogenesis of inherited GPI deficiency.

Recent Publications
1. PIGA mutations cause early-onset epileptic enceohalopathies and distinctive features. M. Kato* H. Saitsu* Y.
Murakami* K. Kikuchi, Watanabe, M. Iai, K. Miya, R. Matsuura, R. Takayama, C. Ohba, M. Nakashima,Y.
Tsurusaki, N. Miyake, S. Hamano, H. Osaka, K. Hayasaka, T. Kinoshita, N. Matsumoto. Neurology. 2014 in press(*equally contribution).
2. Mutations in PGAP3 impair GPI-anchor maturation and lead to intellectual disability with hyperphosphatasia andadditional phenotypic features. Howard MF* Murakami Y* Pagnamenta AT, Haas CD, Fischer B, Hecht J, Keays
DA, Knight SJL, Kölsch U, Krüger U, Leiz S, Maeda Y, Mitchell D, Mundlos S, Philipps JA, Robinson PN, Kini U,
Taylor JC, Horn D, Kinoshita T, Krawitz PM. Am J Hum Genet.2014;94(2)278-87(*equally contribution).
3. Glycosylphosphatidylinositol (GPI) anchor deficiency caused by mutations in PIGW is associated with Westsyndrome and hyperphosphatasia with mental retardation syndrome. Chiyonobu T, Inoue N, Morimoto M, KinoshitaT, Murakami Y. J Med Genet. 2014; 51(3): 203-7.
4. Case report with vitamin B6 responsive epilepsy due to inherited GPI deficiency. Kuki, I., Y. Takahashi, Okazaki,
Ebara, N. Inoue, T. Kinoshita, Y. Murakami. Neurology. 2013;81(16):1467-9.
5. Deregulated expression of HMGA2 is implicated in clonal expansion of PIGA deficient cells in paroxysmal
nocturnal haemoglobinuria. Murakami Y, Inoue N, Shichishima T, Ohta R, Noji H, Maeda Y, Nishimura J,
Kanakura Y, Kinoshita T. Br J Haematol. 2012 Feb;156(3):383-7.
ĶıResearch & Activities

Office of Combined Program on
Microbiology and Immunology

Research Group

Associate Professor
Assistant Professor

Hodaka Fujii, M.D., Ph.D.
Toshitsugu Fujita, Ph.D.

Research & Activities

Chromatin Biochemistry Research Group

Locus-specific biochemical analysis of genome functions
A comprehensive understanding of the mechanisms behind genome functions such as transcription and epigenetic regulation
requires the identification of the molecules that bind to the genomic regions of interest in vivo. However, there are few
non-biased methods for identifying such molecules. To facilitate the biochemical and molecular biological analysis of specificgenomic regions, we developed the locus-specific chromatin immunoprecipitation (ChIP) technologies to purify the genomic
regions of interest. The ChIP tech nologies consist of inser tional ChIP (iChIP) and engineered DNA-binding
molecule-mediated ChIP (enChIP).
The scheme of iChIP is as follows (left panel): (i) The recognition sequence(s) of an exogenous DNA-binding protein such as
LexA are inserted into the genomic region of interest in the cell that is to be analyzed. (ii) The DNA-binding domain (DNADB) of the exogenous DNA-binding protein is fused with a tag(s) and a nuclear localization signal(s), and then expressed in
the cell that is to be analyzed. (iii) The resulting cell is stimulated and crosslinked with formaldehyde or other crosslinkers, if
necessary. (iv) The cell is lysed and the crosslinked DNA is fragmented by sonication. (v) The complexes, including thecomplexes bearing exogenous DNA DB, are immunoprecipitated with an antibody against the tag.
T he scheme of enCh I P is as follows (r ig ht pa nel): (i) A
DNA-binding molecule/complex (DB) that recognizes the DNA
sequence in a genomic region of interest is generated. This canbe achieved by utilizing technologies such as engineeredzinc-finger proteins, transcription activator-like (TAL) proteins,
and the CRISPR system consisting of dCas9 together with
gRNA. The engineered DB is fused with a tag(s) and an NLS(s),and then expressed in the cell that is to be analyzed. (ii) The
resulting cell is stimulated and crosslinked with formaldehyde orother crosslinkers, if necessary. (iii) The cell is lysed, and the
DNA is fragmented by sonication or digested with nucleases suchas restriction enzymes. (iv) The complexes, including thosecont ai ni ng the engi neered DB, are subjected to aff i nit ypurification methods such as immunoprecipitation.
Thus, the complexes isolated by iChIP and enChIP retain
molecules interacting with the genomic region of interest.
Reverse crosslinking and subsequent purification of DNA, RNA,
proteins, or other molecules allow these molecules to be
identified and characterized.
We will apply locus-specific ChIP to elucidate the molecularmechanisms behind important epigenetic phenomena, including:(a) the lineage commitment of lymphocytes, (b) the regulation ofodorant receptor expression, (c) the insulator function, (d) the
detection and repair of DNA double-strand break, and (e) the
Figure. A scheme of locus-specific ChIP consisting of
epigenetic suppression of the expression of tumor suppressor
iChIP and enChIP
genes.
Recent Publications
1. Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin
immunoprecipitation (enChIP). Fujita T, Asano Y, Ohtsuka J, Takada Y, Saito K, Ohki R, Fujii H. Sci Rep. 2013
Nov 8;3:3171.
2. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding
molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Fujita T, Fujii H. Biochem Biophys
Res Commun. 2013 Sep 13;439(1):132-6.
3. Direct identification of insulator components by insertional chromatin immunoprecipitation. Fujita T, Fujii H. PLoS
One. 2011;6(10):e26109
4. Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. Hoshino A, Fujii H.
J Biosci Bioeng. 2009 Nov;108(5):446-9.
5. Regulation of Fas-mediated immune homeostasis by an activation-induced protein, Cyclon. Saint Fleur S, Hoshino
A, Kondo K, Egawa T, Fujii H. Blood. 2009 Aug 13;114(7):1355-65.
ĶĲResearch & Activities

Germ Cell Group
Research Group

Associate Professor

Masami Nozaki, Ph. D.

Research Projects(1) Epigenetics in germ cells
Many testicular germ cell-specific genes are retroposons, most ofwhich contain a CpG-rich region within their ORFs. We discoveredthat in somatic cells methylation of CpG dinucleotides within the
ORF represses its promoter and that demethylation is necessary forgene expression in spermatogenic cells. We are currently
examining the molecular basis of epigenetic modifications,
including DNA methylation and histone methylation, that occur
within a distinct genomic region in germ cells.

Figure legend. Neural crest cells derived from ES cells
differentiated into osteoblasts.

(2) Unique structure of sperm chromatin
The haploid genome in the mammalian sperm nucleus is packaged into a highly compact structure containing protamines andsome remaining histones. We are currently analyzing the physiological importance of the somatic-like, histone-containing
regions of sperm chromatin.
(3) Establishment of neural crest stem cell lines from ES cells and application of these cell lines in regenerative medicine

Recent Publications
Inoue H, Ohnishi Y, Nakajima M, Kakudo M, Nozaki M. A novel function of EpCAM in oral squamous cell carcinoma
cells under anchorage-independent conditions. Int J Oncol. 2011 Dec; 39 (6): 1401-5.

ĶĳResearch Collaboration Center in Overseas

Director

SA Professor

Naokazu Takeda, Ph.D.

Research Collaboration Center in Overseas

It was believed until recently that infectious diseases could be conquered through the development of chemotherapies andvaccines; however, the recent worldwide emergence of new infectious diseases and reemergence of infectious diseases thatwere once considered to be controlled has seriously challenged this notion. Under these circumstances, intensive research thatclosely monitors and rapidly analyzes emerging and re-emerging infections is urgently required. Since a variety of infectious
diseases can spread rapidly across national borders, it is obvious that these diseases cannot be controlled by the independent
efforts of individual nations.

Research & Activities

Thailand – Japan Research Collaboration Center on
Emerging and Re-emerging Infections

Campus of Ministry of Public HealthTo this end, Osaka University founded the Research Collaboration Center on Emerging and Re-emerging Infections(RCC-ERI) in the Thai National Institute of Health (NIH), Department of Medical Sciences, Ministry of Public Health ofThailand in 2005. The second phase of the program (2010-2014), which is now named the Japan Initiative for Global Research
Network on Infectious Diseases ( J-GRID), is on-going.
The facility consists of P2 and P3 biohazard containment laboratories and various other equipment and facilities in 600 m2
floor space. Previously, most research projects conducted abroad were short-term, with the researchers only staying for at mosta few months to complete their experiments. Due to the installation of the RCC-ERI, researchers are now able to stay for
longer periods of time. The RCC-ERI aims to carry out research projects on both emerging and re-emerging infections in close
collaboration with the researchers at the NIH, while at the same time developing the talents of young scientists from Japan andSoutheast Asian countries in the field of infection.
To conduct basic and applied research and to develop human resources, the RCC-ERI consists of two sections that are devoted
to bacterial and viral infection research. In addition, we aim to establish an effective system that would (i) provide informationthat would help to prevent the emergence of emerging and re-emerging infections, and (ii) promptly activate a variety ofcountermeasures should such a disease emerge, including developing therapeutics or vaccines. Finally, we wish to begin
collaborations with laboratories from the nations that neighbor Thailand so that we can be at the frontline with the capacity toquickly respond to any globally spreading infectious disease.

BSL-2 Laboratory

BSL-3 LaboratoryĶĴResearch Collaboration Center in Overseas

Section of Bacterial Infections
Research GroupSA Professor
SA Assistant Professor
SA Researcher
Postdoctoral Fellow
Postdoctoral Fellow

Shigeyuki Hamada, D.D.S. Ph.D.
Kazuhisa Okada, Ph.D.
Kentaro Inoue, Ph.D.
Mathukorn Na-Ubol, Ph.D.
Warawan Wongboot, Ph.D.

In collaboration with the National Institute of Health, Department of Medical Sciences, Ministry of Public Health of Thailand, theSection of Bacterial Infections pays special attention to emerging and reemerging bacterial diseases that are prevalent or break out in
Asian countries. We conduct molecular epidemiology studies on enteric or systemic bacterial infections. Moreover, we also develop
detection and identification techniques for the
diagnosis of bacterial diseases described below.
It is reported that pneumonia, tuberculosis, andacute diarrheal diseases are associated with highmorbidity and mortality rates in Thailand.
Consequently, in the Program of Promotion of
Research Network for Emerging and Reemerging
Infectious Diseases during the 2010–2014 fiscal
years, we prioritized research on cholera in Thailand
and its causative agent, Vibrio cholerae.
In addition, Streptopcoccus suis, which isoccasionally pathogenic and frequently isolated
from diseased pigs, has been found to cause several
systemic (zoonotic) infectious diseases in humans,
namely meningitis, infective endocarditis, and toxic
shock-like syndrome. This has mainly been
observed in Asian countries, including Northern
Thailand. We will examine this emerging zoonotic
Geographical spread and temporal changes of Vibrio cholerae O1 during the 2007–2010
infection closely, and elucidate the molecularcholera outbreaks in Thailand.
Yellow squares indicate the major MLVA type(s) in each outbreak site shown in the map.
pathogenesis of S. suis infections.

Recent Publications
1. Okada K, Roobthaisong A, Swaddiwudhipong W, Hamada S, Chantaroj S. Vibrio cholerae O1 Isolate with Novel
Genetic Background, Thailand-Myanmar. Emerg Infect Dis. 2013 19(6):1015-17.
2. Takeuchi D, Akeda Y, Nakayama T, Kerdsin A, Sano Y, Kanda T, Hamada S, Dejsirilert S, Oishi K. The
Contribution of Suilysin to the Pathogenesis of Streptococcus suis Meningitis. J Infect Dis. 2013 first published online.
3. Okada K, Roobthaisong A, Nakagawa I, Hamada S, Chantaroj S. Genotypic and PFGE/MLVA analyses of Vibrio
cholerae O1: geographical spread and temporal changes of isolates during the 2007-2010 cholera outbreaks in
Thailand. PLoS One. 2012 7(1):e30863.
4. Takeuchi D, Kerdsin A, Pienpringam A, Loetthong P, Samerchea S, Luangsuk P, Khamisara K, Wongwan N,
Areeratana P, Chiranairadul P, Lertchayanti S, Petcharat S, Yowang A, Chaiwongsaen P, Nakayama T, Akeda Y,
Hamada S, Sawanpanyalert P, Dejsirilert S, Oishi K. Population-Based Study of Streptococcus suis Infection in
Humans in Phayao Province in Northern Thailand. PLoS One. 2012 7(2):e31265.
5. Okada K, Chantaroj S, Taniguchi T, Suzuki Y, Roobthaisong A, Puiprom O, Honda T, Sawanpanyalert P. A rapid,
simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal
swab samples. Diagn Microbiol Infect Dis. 2010 66(2):135-9.
ĶĵResearch Collaboration Center in Overseas

Research GroupSA Professor
SA Associate Professor
SA Associate Professor
Postdoctoral Fellow
Research Fellow
Research Fellow
Research Fellow

Naokazu Takeda, Ph.D.
Kazushi Motomura, M.D., Ph.D.
Atsushi Tanaka, V.M.D., Ph.D.
Nitchakarn Noranate, Ph. D.
Chris Verathamjamras, M. Sc.
Uranan Tumkosit , M. Sc.
Michittra Boonchan, M.Sc.

Research Collaboration Center in Overseas

Section of Viral Infections

The Section of viral Infections focuses on three emerging and reemerging viral diseases that are prevalent in Asian countries
including Thailand in collaboration with the National Institute of Health, Department of Medical Sciences, Ministry of Public
Health of Thailand. The first one is mosquito-borne infectious diseases, and we study chikungunya fever fromepidemiological, molecular biological and immunological aspects. The second one is blood-borne infectious diseases (HIVdiseases/AIDS), and we perform virological and immunological characterization of the HIV-1 CRF01_AE strains prevalent inSoutheast Asian countries including Thailand. In addition, the studies to elucidate the mechanism of the anti-retroviral drug
resistance are in progress. The last one is enteric viral diseases including norovirus and hepatitis A virus infections, in which
we perform molecular epidemiological studies using specimens from patients with diarrhea as well as environmental water.
We also perform genetic analysis of the viruses and develop control measures of the diseases.

Figure 1. Genome organization of chikungunya virus (CHIKV) andresearch projects on chikungunya fever. CHIKV (BSL3) is apositive-sense single stranded RNA virus with 60-70 nm in diameter.
When the subgenomic region encoding entire structural proteins was
expressed, they self-assembled into virus-like particles, which weremorphologically and antigenically similar to those of native CHIKV.
Expression vectors encoding envelop proteins, E3, E2, 6k and E1,
lentivirus packaging vector, and lentivirus vector encoding luciferase
were used to transfect 293T cells. The resultant lentivirus pseudotyped
with CHIKV envelop proteins (BSL2) was applicable to the CHIKV
neutralization test (Ref.3).

Recent Publications
1. Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected
Thai individuals. Chaitaveep N, Utachee P, Nakamura S, Chuenchitra T, Ekpo P, Takeda N, Pattanapanyasat K,
Kameoka M. Microbes Infect. 2014 Feb;16(2):142-52.
2. CRF01_AE-specific neutralizing activity observed in plasma derived from HIV-1-infected Thai patients residing in
northern Thailand: comparison of neutralizing breadth and potency between plasma derived from rapid and slow
progressors. Sapsutthipas S, Tsuchiya N, Pathipavanich P, Ariyoshi K, Sawanpanyalert P, Takeda N,
Isarangkura-na-ayuthaya P, Kameoka M. PLoS One 2013; 8: e53920.
3. Development of a Pseudotyped Lentiviral Vector--Based Neutralization Assay for Chikungunya Virus Infection.
Kishishita N, Takeda N, Anuegoonpipat A, Anantapreecha S. J Clin Microbiol 2013; 51: 1389-95.
4. Poly (I:C), an agonist of toll-like receptor-3, inhibits replication of the Chikungunya virus in BEAS-2B cells. Li YG,
Siripanyaphinyo U, Tumkosit U, Noranate N, A AN, Pan Y, Kameoka M, Kurosu T, Ikuta K, Takeda N,
Anantapreecha S, Virol J 2012; 9: 114.
5. Two N-linked glycosylation sites in the V2 and C2 regions of human immunodeficiency virus type 1 CRF01_AE
envelope glycoprotein gp120 regulate viral neutralization susceptibility to the human monoclonal antibody specificfor the CD4 binding domain. Utachee P, Nakamura S, Isarangkura-Na-Ayuthaya P, Tokunaga K, Sawanpanyalert P,
Ikuta K, Auwanit W, Kameoka M. J Virol 2010; 84: 4311-20.
ĶĶResearch Collaboration Center in Overseas

Mahidol-Osaka Center for Infectious Diseases (MOCID)
Research Group

Director Professor (SUP)
Associate Professor
Postdoctoral Fellow
Research Fellow
Research Fellow

Yoshiharu Matsuura, DVM, Ph. D
Tamaki Okabayashi, DVM, Ph. D.
Orapim Puiprom, Ph.D.
Panjaporn Chaichana, M.Sc.
Nantarat Chantawat, M. Sc.

The MOCID interest focuses on several tropical infectious diseases that are of human health importance in Thailand, especiallymosquito-borne viral infectious diseases such as dengue fever/dengue hemorrhagic fever and chikungunya fever. We focus on
the development of rapid diagnosis kits for the viral diseases, and the studies of the prevalence of the viral infections in humanand mosquitoes and the pathogenesis of the viruses. We would like to raise young scientists’ interest and research skills on theinfectious diseases through our collaborating with Mahidol University by using clinical samples.
Our main research project1) Epidemiology of mosquito-borne virus infections in human and mosquitoes in Thailand.
2) Generation and characterization of human and mouse monoclonal antibodies against dengue virus and chikungunya virus.
3) Development of diagnosis kits for the viral diseases.

Recent Publications
1. Low levels of antibody-dependent enhancement in vitro using viruses and plasma from dengue patients.Chaichana P,
Okabayashi T, Puiprom O, Sasayama M, Sasaki T, Yamashita A, Ramasoota P, Kurosu T, Ikuta K.PLoS One. 2014
Mar 18;9(3):e92173.
2. Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus.
Pitaksajjakul P, Benjathummarak S, Pipattanaboon C, Wongwit W, Okabayashi T, Kuhara M, Misaki R, Fujiyama
K, Ramasoota P. Biochem Biophys Res Commun. 2014 Mar 14.
3. Detection and characterization of enteric viruses in flood water from the 2011 thai flood. Ngaosuwankul N,
Thippornchai N, Yamashita A, Vargas RE, Tunyong W, Mahakunkijchareon Y, Ikuta K, Singhasivanon P,
Okabayashi T, Leaungwutiwong P. Jpn J Infect Dis. 2013;66(5):398-403
4. Characterization of chikungunya virus infection of a human keratinocyte cell line: role of mosquito salivary glandprotein in suppressing the host immune response.
Puiprom O, Morales Vargas RE, Potiwat R, Chaichana P, Ikuta K, Ramasoota P,Okabayashi T. Infect Genet Evol.
2013 Jul;17:210-5.
5. Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies
derived from dengue patients at acute phase of secondary infection.Sasaki T, Setthapramote C, Kurosu T, Nishimura
M, Asai A, Omokoko MD, Pipattanaboon C, Pitaksajjakul P, Limkittikul K, Subchareon A, Chaichana P,
Okabayashi T, Hirai I, Leaungwutiwong P, Misaki R, Fujiyama K, Ono K, Okuno Y, Ramasoota P, Ikuta K.
Antiviral Res. 2013 Jun;98(3):423-31.
ĶķResearch Collaboration Center in Overseas

Research Group

Endowed Chair Professor Ph.D.
Endowed Assistant Professor Ph.D.

Eiji Konishi
Atsushi Yamanaka

BIKEN Endowed Department of Dengue Vaccine Development was established in Faculty of Tropical Medicine, Mahidol
University, Thailand, in 2011 by endowment from The Research Foundation for Microbial Diseases of Osaka University,
Osaka, Japan to Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Dengue fever is the most important mosquito-borne viral disease, which is distributed in tropical regions and producing an
estimated 300,000 patients daily. Dengue hemorrhagic fever is its severer form and has a mortality up to 20% if anappropriate treatment is not done. Unfortunately, no approved vaccines or specific antivirals have been developed.
Our department will carry out basic research studies on (1) mechanisms involved in pathogenesis of dengue fever anddengue hemorrhagic fever, (2) virulence, transmission and evolution of dengue viruses, and (3) dengue vaccine
development using several strategies.

World-first efficacy trial for evaluating a candidate dengue vaccine
suggested a need to improve the current neutralization test using Verocells recommended by WHO. We have established an antibody assay
V\VWHPXVLQJ.FHOOVEHDULQJ)FȖUHFHSWRUVWRVHHNPRUHDFFXUDWH
antibody levels required for disease protection. A patient sample showedneutralizing activity with dengue type 1 virus-infected cell counts <10 3
PFU/ml and enhancing activity with infected cell counts >103 PFU/ml
depending on dilution factor as shown above. The assay also indicated thepresence of both complement-dependent and independent antibodies.

Research Collaboration Center in Overseas

BIKEN Endowed Department of Dengue Vaccine Development

Most mouse monoclonal antibodies generated using dengue type 1 virus
antigen displayed both neutralizing and enhancing activities depending on
concentration (NEAb), whereas some showed only enhancing (EAb) or
neutralizing activities (NAb). One obstacle hampering dengue vaccine
development is a concern of deterioration caused by vaccine-induced
enhancing antibodies. An antigen possessing epitopes that can effectively
induce NAb should contribute to development of a safe and efficacious
dengue vaccine.

Recent Publications
1. A mouse monoclonal antibody against dengue virus type 1 Mochizuki strain targeting envelope protein domain II
and displaying strongly neutralizing but not enhancing activity. Yamanaka A, Kotaki T, Konishi E. J Virol. 2013
Dec;87(23):12828-37.
2. Genetic and evolutionary analysis of cell-fusing agent virus based on Thai strains isolated in 2008 and 2012.
Yamanaka A, Thongrungkiat S, Ramasoota P, Konishi E. Infect Genet Evol. 2013 Oct;19:188-94.
3. Comparison of infection-neutralizing and -enhancing antibody balance induced by two distinct genotype strains ofdengue virus type 1 or 3 DNA vaccines in mice. Sjatha F, Takizawa Y, Kotaki T, Yamanaka A, Konishi E. Microbes
Infect. 2013 Nov;15(12):828-36.
4. Memory B cells: a proposed new immunological correlate for protective efficacy of Japanese encephalitis vaccine.
Konishi E. Expert Rev Vaccines. 2013 Aug;12(8):871-3.
5. A review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet
available. Ishikawa T, Yamanaka A, Konishi E. Vaccine. 2014 Mar 10;32(12):1326-37.
ĶĸSpecial Research Facilities
Common Research Facilities

Research Facilities

Animal Resource Center for Infectious Diseases
Research Group

Head, Professor
SA Associate Professor
Assistant Professor
Assistant Professor
Assistant Professor

Masahito Ikawa, Ph.D.
Ayako Isotani, Ph.D.
Yuhkoh Satouh, Ph.D.
Yoshitaka Fujihara, Ph.D.
Haruhiko Miyata, Ph.D.

To study microbial diseases, it is important to analyze the interactions between the host and pathogenic organisms. Animalmodels are indispensable in current microbiological and immunological research, particularly since through the use ofmolecular biology and biotechnology, we can generate gene-manipulated mice that can aid our understanding of themechanisms of infection. For these purposes, experimentally infected animals should be managed in a suitable, safe andcontrolled manner. Animal Resource Center for Infectious Diseases is a unique facility that was established in 1967 to meetthese requirements. The center is separated into three areas: one for animal experiment for microbial disease model (BSL2 andBSL3), an SPF area. The section for animal experiment for disease model is completely air-conditioned and maintained at anegative air pressure to minimize the risk of contamination. Each sub-area has an individual pass-through type autoclave tosterilize all materials before their removal. The exhaust air is filtered to avoid exterior dissemination of pathogenic microbes.
These measures ensure that disease model animals can be handled safely without accidental cross-contamination.
Before gaining access to this restricted facility, researchers must take an official orientation and submit a research plan forcommittee review. The condition of the animals is regularly inspected.
Services such as the generation of gene-manipulated animals, in vitro fertilization, and cryopreservation of mouse strains areavailable at our facility. (Table 1)

Table 1) No. of mouse lines produced/preserved

Figure 1: Biosafety level 3 room (Building A)
The room for disease model animal andexperimentation at biosafety level 3. Hemorrhageic
fever with renal syndrome-causing virus (HFRSV)
was isolated in this area. In addition, animal
experiments for Creutzfeldt-Jakob Disease (CJD),
severe acute respiratory syndrome (SARS) and
Acquired Immune Deficiency Syndrome (AIDS) canbe handled in this facility.

ĶĹ

period1995-2000
2001-2003
2004-2006
2007-20092010-20112012-2013
total

Tg mice
228
104
43
22
31
41
469

KO mice
50
57
69
74
79
159
488

Cryopreservation
261
443
331
216
220
377
1848

Tg, transgenic; KO, knock-out

Special Research Facilities
Common Research Facilities

Research Facilities

Research Group

Head, Professor
Assistant Professor
Associate Professor (SUP)

Hiroshi Nojima, Ph.D.
Daisuke Okuzaki, Ph.D.
Norikazu Yabuta, Ph.D.

Research Facilities

DNA-chip Development Center for Infectious Diseases

Facility Management: The establishment of infectious diseases is driven by the gene expression of pathogenic organisms
within the infected host cells. To understand parasite pathogenesis and pathophysiology and to develop new methods toprevent and treat infectious diseases, it is necessary to identify the pathogenic genes that are expressed in the infected host cells
and to determine how they induce disease at the genetic level. This process requires an analysis of the transcriptional patternsof both the genes of the pathogenic organism and the responsive genes of the host genome.
The DNA-chip Development Center for Infectious Diseases is a unique facility that was established in 2004 to analyze thetranscriptional dynamics and variations involved in infectious diseases. Two research approaches are employed in this facility:(1) Transcriptome analysis using DNA-chip analyzers
The high-density DNA microarray system in this facility permits
comprehensive transcriptional analysis of gene expression in the human ormouse host, and in various pathogenic organisms. Two DNA microarraysystems (Agilent- and Affymetrix-type) are available in this center. Our
real-time PCR analysis system (ABI, PRISM7900HT-2) and Nano-counterare useful for more accurate quantitative analysis of the transcriptional levelsof particular genes. In addition, a novel DNA microarray system (Genopal of
Mitsubishi Rayon Co. Ltd.) is currently being used in this center. An example
of its practical use is the establishment of a blood RNA-based system for the
diagnosis of autoimmune diseases such as vasculitis. This system employs a“focused microarray” that examines the expression of ~200 bloodcell-specific and disease-related genes.
(2) Proteome analysis using mass spectrometry
Comprehensive translational analyses are also very important in furthering
our understanding of infectious diseases. The MS/MS spectrometer installedin this facility allows the analysis of the expression, interactions, andmodifications of proteins from humans, mice, and pathogenic organisms. Thiscenter is also capable of recent technical innovations, such as the mass
spectrometric detection of pathogenic organisms to facilitate the developmentof novel diagnostic systems for infectious diseases.

Fig. 1: High-density DNA microarray system.

Fig. 2: MS/MS spectrometer.

Recent Publications
1. Nagi-Miura N, Okuzaki D, Torigata K, Sakurai MA, Ito A, Ohno N, Nojima H. CAWS administration increases the
H[SUHVVLRQRILQWHUIHURQȖDQGFRPSOHPHQWIDFWRUVWKDWOHDGWRVHYHUHYDVFXOLWLVLQ'%$PLFHBMC Immunol.
2013 Sep 24;14:44.
2. Muso E, Okuzaki D, Kobayashi S, Iwasaki Y, Sakurai MA, Ito A, Nojima H. Ficolin-1 is up-regulated in leukocytes
and glomeruli from microscopic polyangiitis patients. Autoimmunity. 2013 Dec;46(8):513-24.
3. Okuzaki D, Kimura S, Yabuta N, Ohmine T, Nojima H. LeukoCatch, a quick and efficient tool for the preparation ofleukocyte extracts from blood. BMC Clin Pathol. 2011 Aug 17;11:9.
4. Okuzaki D, Fukushima T, Tougan T, Ishii T, Kobayashi S, Yoshizaki K, Akita T, Nojima H. Genopal™: a novel
hollow fibre array for focused microarray analysis. DNA Res. 2010 Dec;17(6):369-79.
5. Tougan T, Okuzaki D, Nojima H. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell
quantity of mRNA without PCR amplification. Nucleic Acids Res. 2008 Sep;36(15):e92.
ĶĺSpecial Research Facilities
Common Research Facilities

Research Facilities

Center for genetic analysis of biological responses
Research Group<Production laboratory for genetically-manipulated animals>Head, Professor
Masahito Ikawa, Ph.D.
SA Associate Professor Kazuo Yamagata, Ph.D.
SA Assistant Professor Jun Ueda, Ph.D.
<Resource laboratory for genetically-manipulated animals>
Guest Professor
Kenichi Yamamura, M.D., Ph.D.
SA Associate Professor Ayako Isotani, Ph.D.
Assistant Professor
Haruhiko Miyata, Ph.D.
<Laboratory for analysis of genetically-manipulated animals>
Professor
Shizuo Akira, M.D., Ph.D.
Professor
Hisashi Arase, M.D., Ph.D.
Professor
Taroh Kinoshita, Ph.D.

<Laboratory for analysis of genetically-manipulated animals>
Professor
Hitoshi Kikutani, M.D., Ph.D.
Professor
Hiroshi Nojima, Ph.D.
Professor
Nobuyuki Takakura, M.D., Ph.D.
Professor
Masato Okada, Ph.D.
Assistant Professor
Yoshitaka Fujihara, Ph.D.
<Laboratory for promotion of collaborative research>
Guest Professor
Yoichiro Iwakura, Ph.D.
Guest Professor
Nobuaki Yoshida, M.D., Ph.D.
Assistant Professor
Naohisa Goto, Ph.D.
Assistant Professor
Yuhkoh Satouh, Ph.D.

Our bodies are kept homeostatically stable by functions of proteins produced from many genes. In other words, our health isbasically maintained in accordance with the balance of our gene products. Many diseases can therefore be traced to a defect in,or malfunction of, various genes. In order to find and develop new drugs or new therapy, it is very important to identify thefunction of each gene in vivo. However, at present, we do not have enough information about the function of various genes toclarify their relationships to each other, or to analyze the relationships to specific diseases systematically.
Gene-disrupted animals can be a powerful tool in helping us understand the role of certain genes in vivo. Such animals can beproduced and used as a model for various human diseases as well as to screen new drugs. With this in mind, preparation ofgene-disrupted mouse lines of entire genes is planned and progressing as research projects on a national level in many
countries. The production of gene-manipulated animals may have another aspect. These animals are considered to be variableanimal resources protected by patents to develop new drugs and therapeutic methods. It is highly important for Japan to make areasonable contribution in this area. Under these circumstances, we created a consortium of three research institutes from three
universities (The Research Institute for Microbial Diseases,
Osaka University: The Institute of Medical Science,
University of Tokyo: Center for Animal Resources andDevelopment, Kumamoto University), placing theheadquarters at Kumamoto University. With this consortium,
we are sharing our specialties with each other and aiming to
produce many gene-manipulated animals closely focused onhuman diseases. In our center, we are mainly focusing on
genes related to reproduction, infection and allergy, taking
advantage of an existing disease screening system in our
university, including features such as fluorescent coloredsperm and eggs (Figure 1). Through these gene-manipulated
animals, we aim to perform translational research for theestablishment of new therapeutic systems and to aid in the
Figure 1. Strategy for elucidating sterility
discovery of new drugs.
Recent Publications
1. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.
Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M. Sci Rep. 2013 Nov 27;3:3355.
2. MiR-200b and miR-429 Function in Mouse Ovulation and Are Essential for Female Fertility. Hasuwa H, Ueda J,
Ikawa M, Okabe M. Science. 2013 Jul 5;341(6141):71-3.
3. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa. Fujihara Y,
Tokuhiro K, Muro Y, Kondoh G, Araki Y, Ikawa M, Okabe M. Proc Natl Acad Sci U S A. 2013 May 14;110(20):8111-6.
4. Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1. Satouh Y, Inoue N,
Ikawa M, Okabe M. J Cell Sci. 2012 Nov 1;125(Pt 21):4985-90.
5. Testis specific PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility.
Tokuhiro K, Ikawa M, Benham AM, Okabe M. Proc Natl Acad Sci U S A. 2012 March 6;109 (10):3850-3855.
ķıResearch Facilities

Special Research Facilities
Common Research Facilities

Head, Professor

Professor

Research Facilities

The Biken History Museum
Hiroshi Nojima, Ph.D.

The Research Institute for Microbial Diseases (RIMD) was established in 1934 by combining two institutions, namely the
Takeo Tuberculosis Institute and the Osaka Leprosy Institute. To commemorate the 70th anniversary of its establishment, aplan of the Biken History Museum was proposed, and it was opened on December 17, 2010.
The Research Institute for Microbial Diseases (RIMD) originated from three institutions, namely Osaka Medical School, theTakeo Tuberculosis Institute (donated by Mr. Jiemon Takeo), and the Osaka Leprosy Institute (donated by an anonymous
benefactor). In 1929, Dr. Chozaburo Kusumoto (the president of Osaka Medical School) and Dr. Tenji Taniguchi (professor of
Bacterial Serology, Osaka Medical School) were concerned about thefrequent introduction of non-native infectious diseases like cholera andthe plague into the Kansai district via the Kobe port, a majorinternational port of Japan at that time; given its central location of the
Kansai district, it readily served as a gateway from which these diseases
could spread throughout the rest of Japan. The concerns of Dr.
Chozaburo Kusumoto and Dr. Tenji Taniguchi were heightened by theirexperiences with the Great Kanto Earthquake in 1923, which had
revealed to them how rapidly infectious diseases could spread.
These concerns led them to strongly encourage the Governor of the
Osaka Prefecture, Mr. Zenzaburo Shibata, and the Osaka business
community to establish public institutions on infectious diseases in theFig.1. A picture of Biken History Museum before theopening ceremony.
Kansai district, particularly in Osaka. In response to this campaign, thebusinessman Mr. Gendo Yamaguchi donated 200,000 yen (theequivalent now of several hundred million yen), which made it possible
to construct the main building of RIMD at the Nakanoshima campus of
Osaka Medical School (Dojima, Osaka City) in February 1934. After its
construction, the researchers of the three institutions mentioned above
moved into the RIMD building and continued their active research.
Both members and non-members of Osaka University can visit theMuseum free of charge from 9:00 a.m. to 5 p.m. on working days. A
gorgeous pamphlet will be presented by writing the visitors name at the
information desk of RIMD which is located near the Museum entrance.
Inside the museum, you will find the portrait bust of Mr. Jiemon
Takeo, 10th (left) and 11th (right), Koch’ s microscope donated by theGerman government, influenza and SARS virus model made by Kaiyodo
Fig. 2. The portrait bust of Mr. Jiemon Takeo, 10th (left)
Co. Ltd. (Kadoma, Osaka).
and 11th (right).

Fig. 3. Koch’ s microscope.

Fig. 4. A plastic model of In fluenza virus
made by Kaiyodo Co. Ltd.

Fig. 5. A plastic model of Severe Acute Respiratory
Syndrome (SARS) virus made by Kaiyodo Co. Ltd.

ķĲSpecial Research Facilities
Common Research Facilities

Research Facilities

Central Instrumentation Laboratory
Head

Professor
Associate Professor
Assistant Professor
Specially Appointed Assistant Professor

Hiroaki Miki, Ph.D.
Shinji Higashiyama, Ph.D.
Kazunobu Saito, Ph.D.
Miki Morimatsu, Ph.D.

The Central Instrumentation Laboratory was established in the ResearchInstitute for Microbial Diseases in 1959 for the mutual advantage of
researchers, by bringing machines from each laboratory when equipment
was lacking. Nowadays, various precise and high performance machines
are available in the laboratory at all times. There are ultracentrifuges,
transmission and scanning electron microscopes, a Biacore, cell
analyzer/sorters, an automatic plasmid DNA separation device, DNA
sequencers, mass spectrometers etc. Also, cell storage large tanks
equipped with automatic liquid nitrogen supply system and a specified
chemical treatment room are also present. In addition, professional
technicians are employed to maintain and manage the devices, as well as
to provide service, education and training for newcomers. In-house services such as flow cytometer cell sorting, mass
spectrometry protein identification, electron microscope image capture, and DNA sequencing services are available at theinstitute in response to requests from researchers. These services play a very important role in the institute since experimental
machines become more and more complicated and researchers have difficulty handling the variety of experimental machines
by themselves.

Radioisotope Laboratory
Head

Professor
Hiroaki Miki, Ph.D.
Associate Professor Shinji Higashiyama, Ph.D.

The Radioisotope (RI) Laboratory is adjacent to the main building of the Institute in1967, and was extended by branch laboratories with a combined space of about 600 sq.
m. during the establishment of the North building in 1979 and the Central Laboratory
for Biological Hazardous Microbes in 1983. In 1998, a radiation exposure room wasestablished on the first basement level of the South building. The Genome Information
Research Center radioisotope laboratory joined in 2007. The main RI Laboratory, theNorth building RI Laboratory and the Genome Information Research Centerradioisotope laboratory were closed in 2010 and 2011. The new RI Laboratory wasestablished in the Immunology Frontier Research Center building in 2011.
The RI Laboratory is designed for biomedical experiments with radioisotopes andplays an important role in the Institute. Its facilities include an RI stockroom, a
distribution room, a tissue culture room, and an area for RI measuring equipment.
Safety requirements are met by a stringent security system that involves the use of ID
cards and the computerized management of radioisotopes. About 200 researchers use
this laboratory every year.

ķĳResearch Facilities

Special Research Facilities
Common Research Facilities

Head

Professor

Tatsuo Shioda, D. Med. Sc.

Research Facilities

Central Laboratory for Biological Hazardous Microbes
This laboratory was set up in 1983 to ensure the safe handling of biologicallyhazardous microbes, such as hemorrhagic fever with renal syndrome (HFRS) virus.
Since then, all experimental studies using such microbes, including the humanimmunodeficiency virus (HIV), have been carried out in this laboratory. The
laboratory is a three-story building that is 550 m2 in area. The first floor is reservedfor experiments using radioisotopes. The facilities are designed to protect
researchers from pathogenic infection and to prevent the spread of biohazardous
pathogens outside the building.
The supply of fresh air is regulated to keep the room interiors at a negative pressure.
Air is released from the facility through high-quality outlet filters to minimize
contamination of the outside environment. Each room is equipped with safetycabinets and autoclaves for the sterilization of used materials before their disposal.
The entire laboratory was renovated from 2005 to 2007 to increase number ofpathogens simultaneously used in this laboratory.
In 2012 and 2013, 45 and 53 researchers, respectively, were approved by the
Biosafety Committee to use this laboratory. The microbes used included HIV,
human and avian influenza viruses, SARS corona virus, and scrapie agent.

Library
HeadProfessor

Hiroaki Miki , Ph.D.

The RIMD library collects academic books and jounals on microbiology and immunology, as well as work on related scientificfields such as cell biology, genetics, histology, developmental biology, biochemistry, pharmacology pathology, microbiology
and oncology. In particular, we have collected rare books on parasitology that cannot be found at other institutes. These books
are frequently accessed by visitors to the RIMD library. Due to the construction of a new building for the Integrated Life
Science Building, the RIMD library was moved to a temporary library room set up on the 1st floor of the south building of
RIMD on December 2007. At July 2010, the RIMD library was moved to a new space located at the 1st floor of the new
RIMD main building. Since the temporary library space was quite small, we were forced to discard most of the old books andjournals. For this reason, we now only hold journals published after 1991, all of the back issues of Biken Journal, and 14,000
books. We now purchase 18 and 23 journals published in English and Japanese, respectively. Most of the books and journals
are kept in the stock room set up on the 1st floor of the north building, but textbooks and newly arrived journals after 2010 are
arranged on the front shelf of the bureau. Most of the materials in the RIMD library are registered on the online index at themain Library of Osaka University, which can also be accessed by libraries throughout Japan via the Interlibrary Loan (ILL)
system. One librarian handles the RIMD library together with two professors, two associate professors who act as members of
RIMD library committee.

ķĴRelated Facility
World Premier International Research Center

Immunology Frontier Research Center
ɜUniqueness and Objectives
Immunology has always been a scientific strength of Japan. In
particular, Osaka University has been historically known for its
leading immunology researchers including Prof. Shizuo Akira.
The Osaka University Immunology Frontier Research Center(IFReC), directed by Prof. Akira, is an example of this reputation.
IFReC was selected by the Japanese Ministry of Education, Culture,
Sports, Science and Technology (MEXT) as one of the nation's
exclusive World Premier International (WPI) Research Center
Initiative Programs. Established on October 1st of 2007, the centeris expected to engage in high level research that will make it aninternational leader in the field of immunology. Immunology
investigates the mechanisms that protect the body against microbial
infection. Because the immune system is essential for eliminating
infectious pathogens from the body, its malfunction gives rise tovarious disorders such as autoimmune diseases, allograft rejection
during transplantation, and allergies.
The scientific aim of IFReC is to unveil a comprehensive
understanding of the dynamic immune system by employing avariety of imaging technologies and Bioinformatics to immunology.

International Scientiﬁc Advisory Board
Director

Deputy Director
Management Committee
Board of Representatives
Laboratories
Immunology Groups
Host Defense（Shizuo Akira）

Experimental Immunology（Shimon Sakaguchi）

Immunoglycobiology（Taroh Kinoshita）

Cell Signaling（Takashi Saito）

Immunopathology（Atsushi Kumanogoh）

Lymphocyte Diﬀerentiation（Tomohiro Kurosaki）

Immunochemistry（Hisashi Arase）

Lymphocyte Development（Fritz Melchers）

Immune Regulation（Tadamitsu Kishimoto）

Malaria Immunology（Cevayir Coban）

Immune Regulation（Tsuneyasu Kaisho）

Vaccine Science（Ken Ishii）

Mucosal Immunology（Kiyoshi Takeda）

Immune Network（Rikinari Hanayama）

Molecular Immunology（Hitoshi Kikutani）

Immunoparasitology（Masahiro Yamamoto）

Biochemistry & Immunology（Shigekazu Nagata）
Imaging Groups
Single Molecule Imaging（Toshio Yanagida）

Chemical Imaging Techniques（Kazuya Kikuchi）

Biofunctional Imaging（Yoshichika Yoshioka）

Biophotonics（Nicholas Isaac Smith）

Immunology and Cell Biology（Masaru Ishii）

Immune Response Dynamics（Kazuhiro Suzuki）

Nuclear Medicine（Jun Hatazawa）

Brain-Immune Interaction（Ben Seymour）

Bioinformatics Groups
Information Systems（Yutaka Hata）

Systems Immunology（Daron M. Standley）

Research Planning and Management Oﬃce
Administrative Director / Director of RPMO
Head of Administrative Oﬃce

ķĵ

General Aﬀairs Section

Accounting Section

Related Facility

ɜResearch fields / Expected Achievements
To date, research in immunology has either been carried out by isolating immune cells from the body and examining these cells
in vitro or by using in vitro cultured cell lines. Although such studies have provided many new insights on the immunology
system, we still do not understand the system well enough to predict how it will respond when a certain pathogen invades. One
approach to resolving this problem is to study immune responses in a spatiotemporal manner. Given the importance of thespatiotemporal organization of the lymphoid organs in an immune response and the importance of understanding how single
immune cells behave, combining immunology techniques with imaging techniques is vital for advancing immunology to a newfrontier.
To meet our needs and expectations, new imaging techniques will need to be developed by applying an interdisciplinary effort
that combines people from disparate fields like physics, computer science, and immunology.
By integrating the immunology and imaging fields, we will be able to understand the dynamic interactions of immune cells andtheir activation. This will lead to new and more efficient development strategies for vaccines and immune therapies when
combating infectious diseases, cancers and autoimmunity disorders.

ķĶAccounts
Management Expenses Grants（unit : thousand yen）
Classification

2009

2010

2011

2012

2013

Personnel

859,673

887,150

863,168

877,581

932,979

Non-Personnel

548,947

704,408

567,143

477,809527,783

Total

1,408,620

1,591,558

1,430,311

1,355,390

1,460,762

Other Grants（unit : thousand yen）
Classification

2009

2010

2011

2012

2013

Contract Research

1,040,180

908,861

711,772

634,565

601,976

Donations for Research

343,772

689,654765,777

730,868

262,716

Miscellaneous

2,090

4,506

4,082

4,283

4,426

1,386,042

1,603,021

1,481,631

1,369,716

869,118

Total

Grants-in-Aid for Scientific Research（unit : thousand yen）

ķķClassification

2009

2010

2011

2012

2013

MEXT Research
Grants688,999453,744466,212439,280

436,020

Health and Labor
Sciences Research Grants

118,789

107,632

87,913

130,669

245,040

Health and Laborother Research Grants

13,98800

00

Global COE
Program Grants

1,149,599
120,037

1,149,599
85,441

1,149,599
74,992

1,149,599
76,651

1,149,599
0

Total

941,813646,817629,117646,600

681,060

Building Areaӳӷ

Building Area …… 8,702㎡

Accounts, Building Area

Site Area …… 36,036㎡

Gross Floor Area …… 39,945㎡

ӽ

Ӽ

Ӷ

ӹ
ӹ

Ӹ

Ӵ

ӵ

Ӻ

① Main building（left）and⑩ Integrated Life Science building（right）

ӱӻ② South buildingӲ⑦ Central Laboratory for Biological Hazardous Microbes
and⑤⑥ Animal Resource Center

Building name

Total floor numbers①Main building

7

1,706

6,397

②South building

2

409

945

③North building

3

492

1,252

④Annex

2

768

1,548

⑤Animal Resource Center A

2

640

1,391

⑥Animal Resource Center B

4

355

1,425

⑦Central Laboratory for Biological Hazardous Microbes

3

241

550

⑧Central Instrumentation Laboratory

2

378

504

⑨Depository for dangerous chemicals

1

160

160

⑩Integrated Life Science building

10

1,072

9,258

⑪Cutting-edge Research Building for Infectious Diseases

9

973

7,448

⑫Animal Resource Center C（belonging to IFReC）

4

738

2,482

⑬IFReC building

9

770

6,585

Building area（㎡）

Total floor area（㎡）ķĸ

Map & Access
LocationJapan

To Saito Nishi

Kyoto
Tokyo
Osaka
NagoyaTo MinohKayanoOnoharaImamiya

Route 171

Shimizud Ibaraki-Stural Roa
PrefecTo
Ikeda

To Kyoto

YukoukaiGeneralHospital

National
Cardiovascular Center
Senri Kinran University

To
Osaka
(Itami)
Airport

Shiga

Hyogo

Ibaraki-shi Stn.

HankyuSenri Line

Senri-Chuo
Stn.

Nara
To
Umeda

To
Kawaramachi
Stn.

Meishin
Expressway

Handai Byoin Mae Stn.
MonorailNational
Museumof Ethnology
Expo Park

Chugoku Expressway
Exp

Kita-Osaka
Kyuko Line

Mie

Osaka

Osaka
University
Suita
CampusKita-Senri
Stn.

YamadaStn.

Koen
Higashiguchi
Stn.

Suita
I.C.

Banpaku Kinen
oO
ute
Koen Stn.
r Lo
op R oute

HankyuKyoto Line

Osaka Monorail
Unobe Stn.

Kinki
Expressway

Minami-IbarakiStn.
To Umeda
Stn.

To Awaji Stn.

To Umeda Stn.

To Matsubara
To
Kadoma-shi Stn.

Paciﬁc Ocean

Transportation access
To Saito Nishi
Osaka University Suita Campus

Chugoku Expressway
To Takarazuka

Handai Byoin Mae Stn.
Banpaku Kinen
Koen Stn.

To
To
Kyoto Stn. Kyoto Stn.

Osaka (Itami) Airport

SubwayMidosuji Line

Meishin Expressway

HankyuSenri Line

Kita-Osaka
Kyuko LineTo Shin-Kobe Stn.

Yamada Stn.

ine

oL

JR

d
kai

To

en

JR

ns
nka

To
Kadoma-shi Stn.

Shi

Kinki Expressway

Shin-Osaka Stn.

To Kobe Stn.

JR Osaka
Loop Line

Osaka(Umeda)Stn.

Tsuruhashi Stn.

Namba Stn.

Kintetsu Line

To
Nara

JR Lines
Nankai Line

Tennoji Stn.
JR Hanwa Line

JR Shinkansen
Monorail

To Kansai International Airport and WakayamaķĹ

Ibaraki-shi
Stn.

JRTokaidoLine

Chuo Loop
Highway

Wakayama

Kita-Senri Stn.
Senri-Chuo Stn.

To Kyoto Stn.

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Shin-Mido Suji

Kyoto

Ibaraki I.C.

ettsu Ro

Paciﬁc Ocean

Toyokawa Stn.

●Train : 12-minute walk from “Kita-Senri” Station on Hankyu Senri Line.
●Monorail : 20-minute walk from “Handai Byoin Mae” Station on Osaka Monorail Saito Line.
●Bus : From Senri-Chuo Station :5-minute walk from “Handai-Guchi” Bus Stop on Hankyu Buses heading to “Onohara Higashi”, “Toyokawa-Eki”, “Fujikasai”.
12-minute walk from “Handai Honbu Mae” Bus Stop on Hankyu Buses heading to “Handai Honbu Mae” or “Ibaraki Mihogaoka”.
●Bus : From Hankyu Ibaraki-shi Station:
12-minute walk from “Handai Honbu Mae” Bus Stop on buses heading to “Handai Honbu Mae” (via JR Ibaraki Station).

Map & Access

Suita Campus

ResearchInstitutefor
MicrobialDiseasesĂŵƉƵƐDĂƉ

8
BusStop
HandaiͲGuchi

2
1

OsakaMonorail
HandaiͲByoinmae Sta.

3

Senri Gate

EastGate

WestGate

9BusStop
HandaiͲHonbuͲ
MaeHankyuKitasenri Sta.

4

7

6
5

MainGate
ExpoPark

1 ResearchInstituteforMicrobialDiseases

6 OsakaUniversityHospital

2 ImmunologyFrontierResearchCenter

7 AdministrationBureau

3 GraduateSchoolofEngineering

8 TheInstituteofScientific

4 GraduateSchoolofMedicine
5 GraduateSchoolofFrontierBiosciencesandIndustrialResearch

9 OsakaUniversityDentalHospital

Published by : RIMD, Osaka University／Edited By : Hiroshi Nojima, the chair of RIMD Public Relations Committee
RIMD, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Tel: +81-6-6877-5111, Fax: +81-6-6879-8266 URL: http://www.biken. osaka-u. ac. jpķĺ