Elucidation of local infection mechanism by genome infrastructure construction of respiratory filamentous fungal infection and application to diagnosis and treatment

• Project/Area Number: 16K09954
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Infectious disease medicine
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: Umeyama Takashi 国立感染症研究所, 真菌部, 主任研究官 (20360696)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2016: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: 真菌症 / 糸状菌 / ゲノム編集 / ゲノム解析 / 薬剤耐性 / 微生物 / 真菌 / 感染症 / アスペルギルス

Outline of Final Research Achievements

Whole genome sequencing of multi-drug resistant fungi Scedosporium prolificans and Cunninghamella bertholletiae was performed by next-generation sequencer. We constructed a plasmid for CRISPR/Cas9 genome editing in Aspergillus lentulus and Aspergillus viridinutans. We succeeded in site-directed mutation in Cyp51A of clinical isolates by transformation of ribonucleoprotein consisting of Cas9 protein and in vitro synthesized single-guide RNA. By use of this technique, we verified the contribution of amino acid mutation which might be involved in drug resistance.

Academic Significance and Societal Importance of the Research Achievements

臨床上重要な病原真菌のゲノム基盤構築により、新たな抗真菌薬の標的探索に繋がるような基礎研究が推進されることが期待できる。本研究によって呼吸器における病原糸状菌の感染成立に必要な分子が発見されれば、真菌症治療法や早期診断法の開発への応用などに新しい展開をもたらすものと期待される。また、ゲノム編集技術を導入したことにより、近年大きな問題になっている抗真菌薬耐性問題に対する原因解明につながる可能性も考えられる。

Research Products

• [Journal Article] Identification of a Novel Variant Form of <i>Aspergillus fumigatus</i> CalC and Generation of Anti-CalC Monoclonal Antibodies (2019)
  • Author(s): Takatsuka Shogo、Inukai Tatsuya、Kawakubo Shun、Umeyama Takashi、Abe Masahiro、Ueno Keigo、Hoshino Yasutaka、Kinjo Yuki、Miyazaki Yoshitsugu、Yamagoe Satoshi
  • Journal Title: Medical Mycology Journal Volume: 60 Issue: 1 Pages: 11-16
  • DOI: 10.3314/mmj.18-00013
  • NAID: 130007605409
  • ISSN: 2185-6486, 2186-165X
  • Peer Reviewed / Open Access
• [Journal Article] Disseminated histoplasmosis from a calcified lung nodule after long-term corticosteroid therapy in an elderly Japanese patient (2019)
  • Author(s): Kobayashi Keigo、Asakura Takanori、Kawada Ichiro、Hasegawa Hanako、Chubachi Shotaro、Ohara Kentaro、Kuramoto Junko、Sugiura Hiroaki、Fujishima Seitaro、Iwata Satoshi、Umeyama Takashi、Katano Harutaka、Uwamino Yoshifumi、Miyazaki Yoshitsugu、Kamei Katsuhiko、Hasegawa Naoki、Betsuyaku Tomoko
  • Journal Title: Medicine Volume: 98 Issue: 17 Pages: e15264-e15264
  • DOI: 10.1097/md.0000000000015264
  • Peer Reviewed / Open Access
• [Journal Article] CRISPR/Cas9 Genome Editing To Demonstrate the Contribution of Cyp51A Gly138Ser to Azole Resistance in Aspergillus fumigatus (2018)
  • Author(s): Umeyama Takashi、Hayashi Yuta、Shimosaka Hisaki、Inukai Tatsuya、Yamagoe Satoshi、Takatsuka Shogo、Hoshino Yasutaka、Nagi Minoru、Nakamura Shigeki、Kamei Katsuhiko、Ogawa Kenji、Miyazaki Yoshitsugu
  • Journal Title: Antimicrobial Agents and Chemotherapy Volume: 62 Issue: 9
  • DOI: 10.1128/aac.00894-18
  • Peer Reviewed
• [Journal Article] Cryptococcus gattii genotype VGIIa infection imported from Vancouver Island to Japan (2018)
  • Author(s): Kitaura Tsuyoshi、Takahashi Masamichi、Umeyama Takashi、Takeshita Nozomi、Katanami Yuichi、Takaya Saho、Yamamoto Kei、Kutsuna Satoshi、Hayakawa Kayoko、Kato Yasuyuki、Yamauchi Takahiro、Narita Yoshitaka、Miyazaki Yoshitsugu、Ohmagari Norio
  • Journal Title: Journal of Infection and Chemotherapy Volume: 24 Issue: 7 Pages: 573-575
  • DOI: 10.1016/j.jiac.2017.12.014
• [Journal Article] Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus (2018)
  • Author(s): Umeyama Takashi、Hayashi Yuta、Shimosaka Hisaki、Inukai Tatsuya、Yamagoe Satoshi、Takatsuka Shogo、Hoshino Yasutaka、Nagi Minoru、Nakamura Shigeki、Kamei Katsuhiko、Ogawa Kenji、Miyazaki Yoshitsugu
  • Journal Title: bioRxiv Volume: -
  • DOI: 10.1101/311712
  • Open Access
• [Presentation] Cas9/CRISPR genome editing to prove the contribution of Cyp51A G138S to azole resistance in Aspergillus fumigatus (2018)
  • Author(s): Takashi Umeyama
  • Organizer: the 7th Global Network Forum on Infection and Immunity
• [Presentation] Cas9/CRISPR genome editing to prove contribution of Cyp51A G138S to azole resistance in Aspergillus fumigatus (2018)
  • Author(s): Takashi Umeyama, Yuta Hayashi, Hisaki Shimosaka, Tatsuya Inukai, Satoshi Yamagoe, Minoru Nagi, Shigeki Nakamura, Kenji Ogawa, Yoshitsugu Miyazaki
  • Organizer: 20th Congress of the International Society for Human and Animal Mycology
  • Int'l Joint Research
• [Presentation] CRISPR/Cas9ゲノム編集技術によるAspergillus fumigatusボリコナゾール耐性変異の検証 (2018)
  • Author(s): 梅山 隆、犬飼達也、山越 智、名木 稔、中村茂樹、小川賢二、宮﨑義継
  • Organizer: 第92回日本感染症学会総会・学術講演会 第66回日本化学療法学会学術集会 合同学会
• [Presentation] 臨床分離されるアゾール耐性アスペルギルス属に対する新しい分子機構検証技術の導入 (2018)
  • Author(s): 梅山 隆、林 悠太、下坂寿希、犬飼達也、山越 智、名木 稔、中村茂樹、小川賢二、宮﨑義継
  • Organizer: 真菌症フォーラム第24回学術集会
• [Presentation] Mutations in the cyp51A gene of an azole resistant clinical isolate of Aspergillus fumigatus: direct proof using Cas9/CRISPR genome editing. (2018)
  • Author(s): T. Umeyama, Y. Hayashi, H. Shimosaka, T. Inukai, S. Yamagoe, M. Nagi, S. Nakamura, K. Ogawa, Y. Miyazaki
  • Organizer: 8th Advanecs Against Aspergillosis
  • Int'l Joint Research
• [Presentation] アスペルギルス症におけるアスペルギルス属の重複感染の検証 (2018)
  • Author(s): 梅山 隆、林 悠太、中村茂樹、中山靖子、壇辻百合香、下坂寿希、犬飼達也、名木 稔、山越 智、小川賢二、宮﨑義継
  • Organizer: 真菌症フォーラム第２３回学術集会
• [Presentation] Aspergillus fumigatus隠蔽種のNGS解析による抗真菌薬耐性の原因解明 (2016)
  • Author(s): 梅山 隆、壇辻百合香、犬飼達也、中村茂樹、山越 智、名木 稔、田辺公一、大野秀明、宮﨑義継
  • Organizer: 第65回日本感染症学会東日本地方会学術集会/第63回日本化学療法学会東日本支部総会
  • Place of Presentation: 新潟
  • Year and Date: 2016-10-26
• [Presentation] 今話題のアスペルギルス基礎研究は? (2016)
  • Author(s): 梅山 隆、犬飼 達也、山越 智、名木 稔、中村 茂樹、宮﨑 義継
  • Organizer: 第60回日本医真菌学会総会・学術集会
  • Place of Presentation: 東京
  • Year and Date: 2016-10-01
  • Invited
• [Presentation] 次世代シーケンサーを用いたAspergilus fumigatus近縁種の網羅的ゲノム解析による抗真菌薬耐性の原因解明 (2016)
  • Author(s): 梅山 隆、石川 淳、壇辻百合香、犬飼達也、中村茂樹、山越 智、名木 稔、田辺公一、大野秀明、宮﨑義継
  • Organizer: 第６４回日本化学療法学会総会
  • Place of Presentation: 神戸
  • Year and Date: 2016-06-09