Generation of gene-knockout library in Chlamydia pneumoniae

• Project/Area Number: 19K16660
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 49050:Bacteriology-related
• Research Institution: Fukuoka University
• Principal Investigator: Shimizu Akinori 福岡大学, 医学部, 講師 (40780135)
• Project Period (FY): 2019-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2021: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2020: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2019: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 肺炎クラミジア / グループIIイントロン / 遺伝子改変 / プラスミドベクター / グループII イントロン / CRISPR/dCas9

Outline of Research at the Start

偏性細胞内寄生性細菌である肺炎クラミジアは呼吸器感染症の原因の一つである。肺炎クラミジアは感染宿主細胞の感染免疫応答やストレスシグナルを誘発することにより肺炎クラミジア自身の増殖に利用していることが報告されている。しかし、肺炎クラミジアがどのように宿主の感染応答を“ハイジャック”しているのかについては明らかにされていない。それは、肺炎クラミジアの逆遺伝学的な解析手法が未確立のままな為である。そこで本研究では肺炎クラミジアの逆遺伝学的解析手法を確立し、遺伝子破壊株のライブラリを作製して、感染分子機構の詳細解明への足掛かりを作ることを目的とする。

Outline of Final Research Achievements

I tried to establishing a gene manipulated-Chlamydia pneumoniae library previously unreported. I carried out to construct the plasmid DNA vector which I designed at first. The DNA sequences were confirmed and same as designed sequences, however the transformant clones of C. pneumoniae were not obtained. To solve this problem, previous DNA samples were check by RFLP patterns. Then, the RFLP patterns were different from re-purified plasmid DNA that were same subclones before more than a month. The reason why the RFLP patterns were changed was not uncoverd. This result shown that difficulty of establishing a gene manipulated-C. pneumoniae library by the method that I tried.

Academic Significance and Societal Importance of the Research Achievements

今回の研究結果として、グループIIイントロンを利用した遺伝子改変プラスミドベクターの構築は問題なく達成できた。しかしながら、少なくとも今回設計したグループIIイントロンのプラスミドベクターは短期間しか保存することができなかった。

Research Products

• [Journal Article] Insufficient anti-spike RBD IgA responses after triple vaccination with intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 (2024)
  • Author(s): Yoshimura Michinobu、Sakamoto Atsuhiko、Ozuru Ryo、Kurihara Yusuke、Itoh Ryota、Ishii Kazunari、Shimizu Akinori、Chou Bin、Sechi Yusuke、Fujikane Aya、Nabeshima Shigeki、Hiromatsu Kenji
  • Journal Title: Heliyon Volume: 10 Issue: 1 Pages: e23595-e23595
  • DOI: 10.1016/j.heliyon.2023.e23595
  • Peer Reviewed / Open Access
• [Journal Article] The appearance of anti-spike receptor binding domain immunoglobulin G4 responses after repetitive immunization with messenger RNA-based COVID-19 vaccines (2024)
  • Author(s): Yoshimura Michinobu、Sakamoto Atsuhiko、Ozuru Ryo、Kurihara Yusuke、Itoh Ryota、Ishii Kazunari、Shimizu Akinori、Chou Bin、Nabeshima Shigeki、Hiromatsu Kenji
  • Journal Title: International Journal of Infectious Diseases Volume: 139 Pages: 1-5
  • DOI: 10.1016/j.ijid.2023.11.028
  • Peer Reviewed / Open Access
• [Journal Article] Chlamydia pneumoniae Lung Infection in Mice Induces Fatty Acid-Binding Protein 4-Dependent White Adipose Tissue Pathology. (2023)
  • Author(s): Kurihara Y, Walenna NF, Ishii K, Soejima T, Chou B, Yoshimura M, Ozuru R, Shimizu A, Itoh R, Furuhashi M, Hotamisligil GS, Hiromatsu K.
  • Journal Title: J Immunol Volume: 210 Issue: 8 Pages: 1086-1097
  • DOI: 10.4049/jimmunol.2200601
  • Peer Reviewed / Open Access / Int'l Joint Research