侵入因子の制御遺伝子を破壊した動物モデルによる白癬菌病原性発現過程の解析

2009 Fiscal Year Annual Research Report

• Project/Area Number: 19590457
• Research Institution: Teikyo University
• Principal Investigator: 槇村 浩一 Teikyo University, 医学部, 准教授 (00266347)
• Co-Investigator(Kenkyū-buntansha): 山田 剛 帝京大学, 医真菌研究センター, 講師 (80424331) ; 西山 彌生 帝京大学, 医療技術学部, 教授 (10082231)
• Keywords: 白癬菌 / 動物モデル / 形質転換 / 病原因子 / 遺伝子破壊 / 遺伝子診断

Research Abstract

人口の20%にも上る白癬対策を講ずる上では、従来顧みられることなく放置されていた白癬菌の侵入因子(病原因子)およびその調節因子に関わる遺伝子のクローニングおよび形質転換、ならびに遺伝子破壊系の検討が必要である。そこで、白癬菌の主に侵入因子制御遺伝子破壊株と同野性株を用いた白癬動物モデル実験により、白癬の病原因子候補となっている侵入因子と感染成立とに関わる機序の解明を可能にする以下の研究ツールを研究・開発した。1)高率形質転換法としてのAgrobacterium tumefaciensを仲介とした遺伝子系質転換法(アグロバクテリウム法)と、多重形質転換体選択を可能とするために選択マーカーとして研究開発したG418を報告した。また、2)白癬菌Trichophyton mentagrophytes TIMM2789株のku80遺伝子ホモログであるTmku80遺伝子を相同組換えによって破壊したTmku80破壊白癬菌#49株を作出し、初めて任意の標的遺伝子を組み替えることが可能となった。本法によって、任意の標的遺伝子を相同組換えによりさらに破壊後、動物モデルに適用することで、この標的遺伝子破壊白癬菌と野生型白癬菌とを比較して、標的遺伝子破壊白癬菌の病原性が消失または低下した場合に、標的遺伝子がコードするタンパク質を病原因子として特定することが可能となった。3)併せて、動物モデルにおける白癬感染を定量的に評価する目的で、ヒト臨床にも応用可能な白癬菌の遺伝子診断法を併せて開発・報告した。以上の研究成果によって、国民病でもある白癬の病態、並びに「侵入因子の制御遺伝子を破壊した動物モデルによる白癬菌病原性発現過程を解析」するための基本的ツールが整備され、分子生物学的知見に基づいた白癬の予防・診断・治療に関わる新たな知と技術の創出が可能となった。

Research Products

• [Journal Article] Rapid identification of Prototheca zopfii by nested polymerase chain reaction based on the nuclear small subunit ribosomal DNA. (2009)
  • Author(s): Onozaki M
  • Journal Title: Journal of Dermatological Science 54(1) Pages: 56-59
  • Peer Reviewed
• [Journal Article] In vitro antifungal activities of luliconazole, a new topical imidazole. (2009)
  • Author(s): Koga H
  • Journal Title: Medical Mycology 47(6) Pages: 640-647
  • Peer Reviewed
• [Journal Article] Agrobacterium tumefaciens-mediated transformation of the dermato phyte, Trichophyton mentagrophytes : an efficient tool for gene transfer. (2009)
  • Author(s): Yamada T
  • Journal Title: Medical Mycology 47(5) Pages: 485-494
  • Peer Reviewed
• [Journal Article] Evaluation of a new rapid molecular diagnostic system for Plasmodium falciparum combined with DNA filter paper, loop-mediated is othermal amplification (LAMP) and melting curve analysis. (2009)
  • Author(s): Yamamura M
  • Journal Title: Japanese Journal of Infectious Diseases 62(1) Pages: 20-25
  • Peer Reviewed
• [Journal Article] Direct Colony PCR of Several Medically Important Fungi using A mpdirect(R) Plus. (2009)
  • Author(s): AlShahni MM
  • Journal Title: Japanese Journal of Infectious Diseases 62(2) Pages: 164-167
  • Peer Reviewed
• [Journal Article] Comparative study of direct PCR, microscopic examination, and culture-based morphological methods for detection and identification of dermatophytes in nail and skin samples. (2009)
  • Author(s): Uchida T
  • Journal Title: Journal of Dermatology 09236(4) Pages: 202-208
  • Peer Reviewed
• [Journal Article] Is Bjerkandera adusta important to fungus-associated chronic cough as an allergen? Eight cases report. (2009)
  • Author(s): Ogawa H
  • Journal Title: Journal of Asthma 46(8) Pages: 849-855
  • Peer Reviewed
• [Journal Article] Efficacy of itraconazole in the treatment of patients with chroniccough whose sputa yield basidiomycetous fungi; fungus-associated chronic cough (FACC) (2009)
  • Author(s): Ogawa H
  • Journal Title: Journal of Asthma 46(4) Pages: 407-412
  • Peer Reviewed
• [Journal Article] A Suggested Pathogenic Role of Trichosporon montevideense in a Case of Onychomycosis in a Japanese Monkey. (2009)
  • Author(s): AlShahni MM
  • Journal Title: Journal of Veterinary Medical Science 71(7) Pages: 983-986
  • Peer Reviewed
• [Journal Article] Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested PCR based on 28S ribosomal RNA gene sequences. (2009)
  • Author(s): Ebihara M
  • Journal Title: British Journal of Dermatology 161(5) Pages: 1038-1044
  • Peer Reviewed
• [Journal Article] Quantitative analysis of the cutaneous Malassezia microbiota in 770 healthy Japanese by age and gender using a real-time PCR assay. (2009)
  • Author(s): Sugita T
  • Journal Title: Medical Mycology 21 Pages: 1-5
  • Peer Reviewed
• [Journal Article] The importance of basidiomycetous fungi cultured from sputum of chronic idiopathic cough. (2009)
  • Author(s): Ogawa H
  • Journal Title: Respiratory Medicine 103(10) Pages: 1492-1497
  • Peer Reviewed
• [Journal Article] Polymerase chain reaction assay for specific identification of Candida guilliermondii (Pichia guilliermondii). (2009)
  • Author(s): Yamamura M
  • Journal Title: Infect Chemother. 15(4) Pages: 214-218
  • Peer Reviewed
• [Journal Article] Enhanced gene replacements in Ku80 disruption mutants of the dermatophyt e, Trichophyton mentagrophytes. (2009)
  • Author(s): Yamada T
  • Journal Title: FEMS Microbiol Lett. 298(2) Pages: 208-217
  • Peer Reviewed
• [Journal Article] Isolation and identification of Rhizomucor pusillus from pleural zygomycosis in an immunocompetent patient. (2009)
  • Author(s): Kimura M
  • Journal Title: Medical Mycology 47(8) Pages: 869-873
  • Peer Reviewed
• [Journal Article] Quantitation of fungal DNA contamination in commercial zymolyase and lyticase used in preparatin of fungi. (2009)
  • Author(s): Miyajima Y
  • Journal Title: Japanese Journal of Medical Mycology 50(4) Pages: 259-262
  • Peer Reviewed
• [Journal Article] Candida auris sp. nov., a novel ascomycetous yeast isolated from the_external ear canal of an inpatient in a Japanese hospital. (2009)
  • Author(s): Satoh K
  • Journal Title: Microbiology and Immunology 53(1) Pages: 41-44
  • Peer Reviewed
• [Journal Article] Typing of Aspergillus fumigatus and Aspergillus niger Strains by Random Amplification of Polymorphic DNA Analysis Using a Six Primer Set. (2009)
  • Author(s): Mirhendi H
  • Journal Title: Shiraz E-Medical Journal 10(4) Pages: 190-200
  • Peer Reviewed
• [Journal Article] Malassezia属真菌の関与が疑われた外耳道炎の検討. (2009)
  • Author(s): 塩田量子
  • Journal Title: 医真菌学会誌 50(2) Pages: 109-116
  • Peer Reviewed
• [Journal Article] nested PCR法を用いた爪白癬の菌種同定と治癒判断の試み。 (2009)
  • Author(s): 佐藤公美子
  • Journal Title: 東京医科大学雑誌 67(3) Pages: 299-305
  • Peer Reviewed
• [Journal Article] 打ち抜き感染(Breakthrough infection)として発症した侵襲性ムコール症の1剖検例. (2009)
  • Author(s): 石原晋
  • Journal Title: The Japanese Journal of Antibiotics 62(1) Pages: 55-62
  • Peer Reviewed
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 病原真菌の基礎知識.特集皮膚真菌症診療ガイド-これだけは知っておきたい皮膚真菌症の知識-.MB Derma 148(全日本病院出版会) Pages: 1-5
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 宇宙:近未来のヒト生活環境と微生物(1)-真菌から見た宇宙におけるヒト生活環境-ウォストークからミールまで.The Chemical Times 211(関東化学株式会社) Pages: 22-25
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 接合菌と接合菌症.Japanese Journal of Antibiotics 62(1)(財団法人 日本抗生物質学術協議会) Pages: 61-62
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 治療の進歩新規抗真菌薬Annual Review呼吸器2009巻(中外医学社) Pages: 237-241
• [Journal Article] (2009)
  • Author(s): 山村麻倫子
  • Journal Title: 「呼吸器における薬剤の現状と開発」感染症抗真菌薬の現状。THE LUNG-perspectives 17(3)(メディカルレビュー社) Pages: 252-256
• [Journal Article] (2009)
  • Author(s): 小野崎正修
  • Journal Title: 薬剤感受性測定法と耐性菌 2特殊微生物の抗微生物薬感受性測定法真菌臨床と微生物36巻(増刊号)2(近代出版) Pages: 596-574
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 深在性真菌症 起因菌からのアプローチMedical Q平成21年7月20日号(株式会社日経ラジオ社) Pages: 1-3
• [Journal Article] (2009)
  • Author(s): 川上小夜子
  • Journal Title: 真菌検査法カラーアトラスMalassezia furfurに関連した中心静脈カテーテル感染症と微生物学的検査。深在性真菌症:SFI Forum 5(1)(メディカルレビュー社) Pages: 20-23
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 第13回クリプトコッカス症 医師から見たズーノーシス 飼い主・動物医療従事者の感染予防のためにJournal of Small Animal Medicine 11(6)(インターズー) Pages: 84-87
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 新規抗真菌薬Annual Review呼吸器2009東京(中外医学社)(工藤翔二、土屋了介、金沢実、大田健編) Pages: 237-241
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: 真菌症の診断と治療ハリソン内科学第3版Part7感染症Section 16真菌および微細藻類感染症(メディカルサイエンスインターナショナル株式会社) Pages: 1304-1306
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: その他の真菌症と微細藻類感染症ハリソン内科学第3版Part7感染症Section 16真菌および微細藻類感染症(メディカルサイエンスインターナショナル株式会社) Pages: 1325-1330
• [Journal Article] (2009)
  • Author(s): 槇村浩一
  • Journal Title: Pneumocystis感染症ハリソン内科学第3版Part7感染症Section 16真菌および微細藻類感染症(メディカルサイエンスインターナショナル株式会社) Pages: 1330-1332
• [Journal Article] Serum Galactomannan Negative Disseminated Aspergillosis In An Intensive Care Unit; A Case Report.
  • Author(s): Saito T
  • Journal Title: Infection (in press)
  • Peer Reviewed
• [Journal Article] Prototheca cutis sp. nov., a newly discovered pathogen of protothecosis isolated from inflamed human skin
  • Author(s): Satoh K
  • Journal Title: Int J Syst Evol Microbiol. (in press)
  • Peer Reviewed
• [Journal Article] 26S rDNA-based phylogenetic investigation of Japanese cattle-associated Prototheca zopfii isolates.
  • Author(s): Kishimoto Y
  • Journal Title: The Journal of Veterinary Medical Science (in press)
  • Peer Reviewed
• [Journal Article] Nourseothricin acetyltransferase : a new dominant selectable marker for the dermatophyte Trtchophyton mentagrophytes.
  • Author(s): Alshahni M
  • Journal Title: Medical Mycology (in press)
  • Peer Reviewed
• [Journal Article] Human External Ear Canal as the Specific Reservoir of Malassezia slooffiae.
  • Author(s): Kaneko T
  • Journal Title: Medical Mycology (in press)
  • Peer Reviewed
• [Presentation] 日常検査における酵素基質を用いた黄色ブドウ球菌(MSSA/MRSA)スクリーン培地の有用性 (2010)
  • Author(s): 菅原恵子
  • Organizer: 第21回日本臨床微生物学会総会(P-127)
  • Place of Presentation: 東京ドーム
  • Year and Date: 2010-01-31
• [Presentation] カビから見る深在性真菌症 (2010)
  • Author(s): 槇村浩一
  • Organizer: 第21回日本臨床微生物学会総会イブニングセミナー1
  • Place of Presentation: 東京ドームホテル
  • Year and Date: 2010-01-30
• [Presentation] 抗真菌化学療法 -その可能性- (2010)
  • Author(s): 槇村浩一
  • Organizer: 第2回新都心血液学セミナー(大学院特別講義)
  • Place of Presentation: 京王プラザホテル 新宿
  • Year and Date: 2010-01-21
• [Presentation] Phylogenic analysis and molecular detection and identification of Protothecha. (2009)
  • Author(s): Koichi Makimura
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Specific detection and identification of fungal DNA using quantitative PCR and loop-mediated isothermal amplification; their advantages and limitations. (2009)
  • Author(s): Koichi Makimura
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Rapid direct colony PCR from fungi by ampdirect plus. (2009)
  • Author(s): Alshahni M
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Real-time PCR quantitation of DNA contamimant in recent β-glucanase products used for fungal preparations (2009)
  • Author(s): Miyajima Y
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Development of Prototheca zopfii detecting system with TaqMan MGB probe and resolight dye. (2009)
  • Author(s): Onozaki M
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Clinical isolates of Aspergillus fumigatus and A. niger from transplant recipients in Japan. (2009)
  • Author(s): Moe Asano
  • Organizer: The International Society for Human and Animal Mvcoloev 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Antifungal activity of luliconazole in a guinea pig seborrheic dermatitis model with Malassezia restricta. (2009)
  • Author(s): Yukimi Munechika
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Preliminary identification and typing of pathogenic and toxigenic fusarium species based on restriction digestion of ITS1-5.8S rDNA-ITS2 region. (2009)
  • Author(s): Hossein Mirhend
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Identification of pathogenic yeasts speies based on PCR-fragment size polymorphism (PCR-FSP) by using normal agarose gel electrophoresis. (2009)
  • Author(s): Hossein Mirhendi
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] A new species of the genus Malassezia based on the sequence analysis of 26S (D1/D2) and internal transcribed spacer 1 in Ribozomal DNA. (2009)
  • Author(s): Hossein Mirhendi
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] A newly pathogen of protothecosis as a novel achlorophyllic alga isolated from the inflamed skin caused by protothecosis. (2009)
  • Author(s): Satoh K
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Development of analyzing system for microbial flora on board International Space Station and astronauts. (2009)
  • Author(s): Makimura K
  • Organizer: The International Society for Human and Animal Mcology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Enhanced gene replacements in Ku80 disruption metants of the dermatophyte, Trichophyton mentagrophytes. (2009)
  • Author(s): Tsuyoshi Yamada
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Detection of dermatophytes and nondermatophytes from onychomycosis patients after systemic antifungal treatment : Evaluation of cure by nested PCR. (2009)
  • Author(s): Mayuko Muro
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Karyotype diffrence of the Czech and Japanese Candida glabrata bloodstream isolates. (2009)
  • Author(s): Petr Hamal
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] Acase of onychomycosis in a Japanese monkey. (2009)
  • Author(s): Alshahni M
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] In vitro susceptibility of Prothothotheca Zopfil genotype 1 and Z (2009)
  • Author(s): Hideto Sobukawa
  • Organizer: The International Society for Human and Animal Mycology 2009
  • Place of Presentation: Tokyo
  • Year and Date: 20090525-20090529
• [Presentation] 長期ステロイド剤外用後に生じた皮膚フサリウム症の一例 (2009)
  • Author(s): 山田陽三
  • Organizer: 第108回日本皮膚科学会総会
  • Place of Presentation: 福岡
  • Year and Date: 20090424-20090426
• [Presentation] Lamp-mediated isothermal amplification (LAMP法)を用いて診断した熱帯熱マラリアの1例. (2009)
  • Author(s): 山村麻倫子
  • Organizer: 第83回日本感染症学会総会
  • Place of Presentation: 京王プラザ
  • Year and Date: 20090423-20090424
• [Presentation] 深在性真菌症と起因菌 (2009)
  • Author(s): 槇村浩一
  • Organizer: 第119回山梨臨床検査医学研究会
  • Place of Presentation: 甲府富士屋ホテル
  • Year and Date: 2009-12-04
• [Presentation] 真菌症遺伝子診断のアップデート (2009)
  • Author(s): 槇村浩一
  • Organizer: 第三十回関東医真菌懇話会
  • Place of Presentation: ヤンセンファーマ 本社三階会議室 神田・東京
  • Year and Date: 2009-11-21
• [Presentation] 真菌症遺伝子診断のアップデート (2009)
  • Author(s): 槇村浩一
  • Organizer: 第30回関東医真菌懇話会 シンポジウム
  • Place of Presentation: ヤンセンファーマ(株)西神田
  • Year and Date: 2009-11-21
• [Presentation] 医真菌領域における菌種同定。 (2009)
  • Author(s): 槇村浩一
  • Organizer: 日本菌学会・日本防菌防黴学会合同シンポジウム
  • Place of Presentation: 東京医科歯科大学歯学部特別講堂
  • Year and Date: 2009-11-07
• [Presentation] Microbes in space (2009)
  • Author(s): 槇村浩一
  • Organizer: グルカン研究会
  • Place of Presentation: 大手町サンケイプラザ
  • Year and Date: 2009-10-13
• [Presentation] Microbe in space. (2009)
  • Author(s): Makimura, Koichi
  • Organizer: Glucan Workshop 2009.
  • Place of Presentation: Otemachi Sankei Plaza Sankei Bldg, Tokyo.
  • Year and Date: 2009-10-13
• [Presentation] Microbiological research on board International Space Station/ "KIBO" (2009)
  • Author(s): Koichi Makimura
  • Organizer: The 23^rd Annual Meeting of the Japanese Society for Biological Sciences in Space.
  • Place of Presentation: JAXA Tsukuba Space Center
  • Year and Date: 2009-10-02
• [Presentation] 病原真菌と検査法のアップデート (2009)
  • Author(s): 槇村浩一
  • Organizer: 真菌の検査法と病原性に関する研究会
  • Place of Presentation: 札幌市教育文化会館
  • Year and Date: 2009-07-17
• [Presentation] 病原真菌と健康障害のup to date. (2009)
  • Author(s): 槇村浩一
  • Organizer: 第326回八戸地区病院薬剤師会薬学例会
  • Place of Presentation: 八戸パークホテル
  • Year and Date: 2009-04-16
• [Presentation] 深在性真菌症と起因菌のアップデート (2009)
  • Author(s): 槇村浩一
  • Organizer: 重症感染症セミナー
  • Place of Presentation: 熊本大学医学部付属病院山崎記念館
  • Year and Date: 2009-04-15
• [Remarks] 1.帝京大学医真菌研究センター ウェブサイト
  • URL: http://timm.teikyo-u.ac.jp
• [Remarks] 2.病原真菌データーベース
  • URL: http://www.pfdb.net/