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薬剤排出ポンプの生理的機能における役割の解明

米田, 智廣 大阪大学 DOI:10.18910/89570

2022.09.22

概要

細菌が有する薬剤排出ポンプは、菌自身が生存するために細胞内に取り込まれた抗菌薬を細胞外へ排出する重要な働きを担っており、薬剤耐性化を引き起こす原因の一つと位置付けられている。一方で、薬剤排出ポンプは抗菌薬が世の中に広まるよりはるか昔より細菌自身が有する細胞膜蛋白質として存在していることから、薬剤耐性における役割に加えて、細菌自身が様々な環境下において有利に生存するために必要な生理学的な機能を担っていることが明らかとなりつつある。例えば、細菌が宿主に寄生するためには、宿主細胞への接着及び侵入が必要である。また、細菌は様々な厳しい外部環境に順応しながら、自身の生存を確立し続ける必要がある。このように、細菌は外部を攻撃するための機能及び自身を守るための機能と関連して、薬剤排出ポンプの生理学的機能を発揮していると考えられる。様々な菌種の各薬剤排出ポンプの生理的な機能については、これまでに主に細菌学分野の研究者らにより報告されてきた。しかし、それぞれの知見を合わせて、各細菌の薬剤排出ポンプの生理的役割について包括的にまとめた近年の報告はないことから、私はこれまでに報告されている最新の知見を精査し、グラム陰性菌のサルモネラ、大腸菌及び緑膿菌における薬剤排出ポンプの生理的な機能について、菌が外部を攻撃するための機能との関連(宿主への細胞接着及び侵入、毒性物質の産生及び放出、クオラムセンシングによる細胞間コミュニケーションの促進、等),並びに菌が自身を守るための機能との関連(自律的運動能の獲得、バイオフィルムの形成、生理的物質の代謝、外部環境変化への適応力の獲得、等)を支持する事実を抽出・整理した。その結果、各細菌は各薬剤排出ポンプの発現を調節することで、さまざまな環境に応答・適応して菌自身の生存優位性を獲得していると考えられた。

糖への耐性における大腸菌特異的薬剤排出ポンプMdtEFの発現誘導機構の解析:大腸菌における薬剤排出ポンプMdtEF-TolCは、嫌気性環境下でのニトロソ化ストレスや酸から菌自身を守るために必要であると報告されている。また、MdtEF-TolCは生理活性物質としてインドール、糖類を認識し、環境適応に寄与する。本研究室では、糖類の代謝により、mdtEFの発現が誘導されることを報告した。さらに、糖類の一つであるN-acetyl-D-glucosamine(GlcNAc)によるmdtEFの誘導に関与する因子としてcAMP-CRP複合体が、mdtEFとオペロンを形成しているgadEのプロモーター領域に結合することによって、mdtEF遺伝子発現を負に制御していることを見出してきた。私は、CRP-cAMP複合体を介したGlucose(Glc)及びGlcNAcによるmdtEF発現誘導の詳細な機構を解明することを目的に本研究を進めた。Glc及びGlcNAcのトランスポーター欠損株を用いて、遺伝子発現及び薬剤耐性等を検討した結果、Glc及びGlcNAcによるmdtEFの発現誘導は、糖取り込みトランスポーターのⅡAGlc及びⅡANagがそれぞれ独立に制御していることが明らかとなった。MdtEFの生理的役割についてはほとんど解明されていないが、本研究よりMdtEFはその役割として、糖代謝で産出される有害な代謝産物を排出し、糖の添加による代謝亢進によって有害産物が蓄積されることを防ぐ働きをしている可能性を支持した。

脂肪酸塩への耐性における薬剤排出ポンプの役割の解明:両親媒性を有する脂肪酸塩は抗菌活性を示すことが報告されている。脂肪酸塩の抗菌活性は、主として細菌細胞膜を不安定化することで、細胞透過性の増加、細胞の増殖阻害及び溶解誘導を引き起こす。一方で、それぞれの細菌は、脂肪酸塩の抗菌作用に対して耐性を有していることが知られている。薬剤排出ポンプは、胆汁酸塩と脂肪酸塩に消化される胆汁塩が細胞内に蓄積するのを防ぐために必要であることが報告されているが、脂肪酸塩の抗菌活性に対する細菌の耐性機構及び薬剤排出ポンプの役割についてはまだ完全に解明されていない。私は、サルモネラ、大腸菌、緑膿菌における薬剤排出ポンプが脂肪酸塩耐性に及ぼす影響を調べるために、本研究を進めた。サルモネラ菌、大腸菌及び緑膿菌の各薬剤排出ポンプ変異株を用いて、炭素数6~12の脂肪酸塩に対する感受性を最小発育阻止濃度(MIC)にて測定した結果、デカン酸ナトリウム及びドデカン酸ナトリウムでは菌株ごとに感受性の差がみられた。特に、サルモネラ菌のemrABtolC両欠損株並びに大腸菌のtolC単欠損株において、高い感受性がみとめられた。また、いくつかの細胞増殖アッセイの結果、サルモネラ菌のEmrABがドデカン酸ナトリウムに対する耐性化に大きく寄与していることを示した。さらに本研究により、外膜蛋白であるTolCと共役して機能すると考えられてきたEmrABが、デカン酸ナトリウムに対する耐性においては、TolC非依存的に働くことを明らかとした。以上より、EmrABは、細胞質で毒性を示す化合物の薬剤排出ポンプとして機能するためにはEmrAB-TolC複合体として働くことが必須である一方、脂肪酸塩のような細胞膜上の膜傷害性物質に対する保護作用に対しては、TolCが必須ではないと考えられ、EmrABは、基質に依存して最適な機能を保つために、複合体形成を使い分けて環境適応に寄与していることが示唆された。今後の研究により、EmrABの生理的な意義の解明がさらに進むことを望む。

これまでの研究結果から、薬剤排出ポンプの生理的機能について多くのことが明らかとなったが、未だに機能が全く明らかとなっていない薬剤排出ポンプも存在する。薬剤排出ポンプに着目した分子標的薬は、細菌の獲得耐性や病原性発現を抑えるだけでなく、薬剤自然抵抗性の低下やバイオフィルム形成の抑制といった効果も期待できるため、今後より詳細に生理的機能を解析することで、最良の耐性菌感染症の治療薬の開発に貢献することができると考える。近年の遺伝子工学技術の発展により、より簡便に遺伝子発現系を制御した研究がおこなえるようになりつつある。今後の網羅的な解析により、遺伝子制御の引き金となる原因因子の同定、遺伝子制御メカニズムの解明などが進み、薬剤排出ポンプの生理的機能の解明につながることを期待する。

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