Name: 著者スポットライト:革新的な細胞ベースのアッセイを通じて新規抗生物質を明らかにするための細胞骨格ダイナミクスの探索
Uploaded: 2024-04-26T14:35:00
Description: Read this Scientific Article Protocol about Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays at JoVE.com

SMP、SR、AKSは、国立科学教育研究所の原子力部門から受けたフェローシップを認めています。RSは、原子力省からの学内資金援助を認めており、この研究は、バイオテクノロジー省(DBT)からRSへの研究助成金(BT/PR42977/MED/29/1603/2022)を通じて支援されています。著者らはまた、議定書の開発全体にわたる彼のコメント、提案、および議論に対してV Badireenath Konkimallaに感謝します。

1. S. pombeにおけるGFPタグ付き細菌細胞骨格タンパク質の発現
注:ここで使用したすべてのプラスミドおよび株に関する情報については、 表1 を参照してください。すべての培地組成については、 表2 を参照してください。
<ol><li>N末端GFP融合体(GFP-MreB)を有する大腸菌MreBおよびC末端GFP(SaFtsZ-GFP)をS.pombe発現ベクター...

細菌の細胞骨格タンパク質の重合に影響を与える薬物の同定

<ol>
	<li>Antimicrobial Resistance Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+bacterial+antimicrobial+resistance+in+2019:+a+systematic+analysis.">Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis.</a> Lancet. 399 (10325), 629-655 (2022).</li><li>Haselbeck, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+essential+gene+identification+as+a+platform+for+novel+anti-infective+drug+discovery.">Comprehensive essential gene identification as a platform for novel anti-infective drug discovery.</a> Curr Pharma Des. 8 (13), 1155-1172 (2002).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+small+molecule+that+inhibits+cell+division+by+blocking+FtsZ,+a+novel+therapeutic+target+of+antibiotics.">Discovery of a small molecule that inhibits cell division by blocking FtsZ, a novel therapeutic target of antibiotics.</a> J Biol Chem. 278 (45), 44424-44428 (2003).</li><li>Sanchez, S., Demain, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibiotics: current innovations and future trends. , (2015).</li><li>Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prokaryotic+cytoskeleton:+a+putative+target+for+inhibitors+and+antibiotics.">The prokaryotic cytoskeleton: a putative target for inhibitors and antibiotics.</a> Appl Microbiol Biotechnol. 73 (1), 37-47 (2006).</li><li>Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+bacterial+Z-ring.">Targeting the bacterial Z-ring.</a> Chem Biol. 15 (2), 93-94 (2008).</li><li>Kusuma, K. D., Payne, M., Ung, A. T., Bottomley, A. L., Harry, E. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+RND-type+efflux+pumps+confers+the+FtsZ-directed+prodrug+TXY436+with+activity+against+Gram-negative+bacteria.">Inhibition of RND-type efflux pumps confers the FtsZ-directed prodrug TXY436 with activity against Gram-negative bacteria.</a> Biochem Pharmacol. 89 (3), 321-328 (2014).</li><li>Casiraghi, A., Suigo, L., Valoti, E., Straniero, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+bacterial+cell+division:+A+binding+site-centered+approach+to+the+most+promising+inhibitors+of+the+essential+protein+FtsZ.">Targeting bacterial cell division: A binding site-centered approach to the most promising inhibitors of the essential protein FtsZ.</a> Antibiotics. 9 (2), 69 (2020).</li><li>Piddock, L. J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug-resistance+efflux+pumps+-+not+just+for+resistance.">Multidrug-resistance efflux pumps - not just for resistance.</a> Nat Rev. Microbiol. 4 (8), 629-636 (2006).</li><li>K&#228;ufer, N. F., Simanis, V., Nurse, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fission+yeast+Schizosaccharomyces+pombe+correctly+excises+a+mammalian+RNA+transcript+intervening+sequence.">Fission yeast Schizosaccharomyces pombe correctly excises a mammalian RNA transcript intervening sequence.</a> Nature. 318 (6041), 78-80 (1985).</li><li>Vyas, A., Freitas, A. V., Ralston, Z. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filament+formation+of+the+Escherichia+coli+actin-related+protein,+MreB,+in+fission+yeast.">Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.</a> Curr Biol. 17 (3), 266-272 (2007).</li><li>Srinivasan, R., Mishra, M., Wu, L., Yin, Z., Balasubramanian, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterial+cell+division+protein+FtsZ+assembles+into+cytoplasmic+rings+in+fission+yeast.">The bacterial cell division protein FtsZ assembles into cytoplasmic rings in fission yeast.</a> Gene Dev. 22 (13), 1741-1746 (2008).</li><li>TerBush, A. D., Osteryoung, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+functions+of+chloroplast+FtsZ1+and+FtsZ2+in+Z-ring+structure+and+remodeling.">Distinct functions of chloroplast FtsZ1 and FtsZ2 in Z-ring structure and remodeling.</a> J Cell Biol. 199 (4), 623-637 (2012).</li><li>Sharma, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+of+salt+bridge-mediated+resistance+to+FtsZ+inhibitor+PC190723+revealed+by+a+cell-based+screen.">A mechanism of salt bridge-mediated resistance to FtsZ inhibitor PC190723 revealed by a cell-based screen.</a> Mol Biol Cell. 34 (3), ar16 (2023).</li><li>Basi, G., Schmid, E., Maundrell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TATA+box+mutations+in+the+Schizosaccharomyces+pombe+nmt1+promoter+affect+transcription+efficiency+but+not+the+transcription+start+point+or+thiamine+repressibility.">TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility.</a> Gene. 123 (1), 131-136 (1993).</li><li>Moreno, M. B., Dur&#225;n, A., Carlos Ribas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+multifunctional+thiamine-repressible+expression+vectors+for+fission+yeast.">A family of multifunctional thiamine-repressible expression vectors for fission yeast.</a> Yeast. 16 (9), 861-872 (2000).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caring+for+Escherichia+coli.">Caring for Escherichia coli.</a> Cold Spring Harb Protoc. , (2018).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+quantification+of+DNA.">Isolation and quantification of DNA.</a> Cold Spring Harb Protoc. , (2018).</li><li>B&#228;hler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+modules+for+efficient+and+versatile+PCR-based+gene+targeting+in+Schizosaccharomyces+pombe.">Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.</a> Yeast. 14 (10), 943-951 (1998).</li><li>Pande, V., Mitra, N., Bagde, S. 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薬剤耐性(AMR)は世界的に深刻な健康上の脅威であり、新たな標的を持つ新たな抗菌薬が急務となっています。細菌の細胞骨格は、新しい抗生物質を開発するための魅力的な標的として浮上しており、TXA709などの細胞分裂タンパク質FtsZの低分子阻害剤はすでに第I相臨床試験中です30。FtsZ重合の阻害剤を同定するために、いくつかの方法が開発されている7...

細菌感染症と戦うために現在採用されているほぼすべての抗生物質に対する広範な耐性により、新しいカテゴリーの抗生物質がすぐに必要になりました。2019年の報告によると、抗生物質耐性感染症は127万人の命を落とし、耐性菌感染症による合併症を考慮すると、全体で495万人が死亡したとのことです1。臨床診療では依然として有効ですが、現在の抗生物質は、主に細胞壁、DNA、およびタンパク質合成に焦点を当てた、狭い範囲の細胞プロセスを主に標的としています。過去半世紀にわたって、新しい抗菌薬の開発の標的として商業的に利用されたタンパク質は30未満です2,3。このように、実行可能な標的の範囲が限られているため、抗生物質耐性菌と戦うための新しい抗生物質やその誘導体の発見に大きな制約が生じます。したがって、新たな抗生物質耐性の問題を克服するためには、新しい標的と作用機序を持つ新しい抗生物質の開発が必要です。
抗菌標的は、理想的には細菌細胞増殖の必須成分であり、系統発生的に多様な種全体で保存され、真核生物の相同性が最も低く、抗生物質へのアクセスが可能である必要があります4。細胞分裂や細胞形状維持に関与する細菌の細胞骨格タンパク質が発見されて以来、抗菌化合物の開発に有望な焦点として浮上しています5。これらのタンパク質は、細菌の生存能力に不可欠であり、真核細胞の細胞骨格と同様に、細胞の形状(MreB、CreS)、分裂(FtsZ、FtsA)、およびDNA分離(ParM、TubZ、PhuZ、AlfA)の維持に極めて重要な役割を果たします。特に、FtsZは広範囲の原核生物にわたって著しく高いレベルの保存性を示し、MreBはほぼすべての棒状細菌に見られます。このような幅広い分布と細胞生存率の関連性により、これらのタンパク質は抗生物質研究において魅力的な標的となっています5,6,7。
in vivo観察、in vitro相互作用、および酵素実験を組み合わせた多面的なアプローチを採用して、潜在的な阻害剤の主要な標的として細菌の細胞骨格タンパク質を徹底的に検証することが重要です7。面倒な手順や実質的なコストへの影響は、この目的のために利用可能な多くのアッセイを妨げます。これらは、細菌の細胞骨格に影響を与える可能性のあるリード化合物のスクリーニングにおける広範な利用に対する顕著な障害です。中でも顕微鏡法は、細胞形態の変化を直接調べることで化合物の有効性を評価するための非常に効率的かつ迅速な方法として際立っています。しかし、標的タンパク質と他の細胞骨格複合体とのヘテロ会合、オフターゲット結合および膜電位の変化による間接的な影響、細胞への効率的な浸透の困難さ、および特にグラム陰性菌における排出ポンプの存在により、細菌細胞の変形の正確な原因を特定することは集合的に複雑になります8,9,10。
Schizosaccharomyces pombe、または一般に知られている分裂酵母は、棒状の単細胞真核生物です。分裂酵母は、細胞周期と分裂、細胞組織化、ヒトを含む高等真核生物との染色体複製などの細胞プロセスにおける並外れた保存により、細胞生物学および分子生物学のモデル生物として広く使用されています11,12。さらに、Erringtonらは、極に局在する細菌細胞骨格タンパク質DivIVAを分裂酵母で発現させ、DivIVAが負の曲面に蓄積することを実証しました13。ここでも、Balasubramanianらは、MreB14やチューブリン相同体FtsZ15のような大腸菌アクチン細胞骨格タンパク質のメカニズム、アセンブリ、およびダイナミクスに新たな洞察をもたらすために、細胞モデルシステムとして分裂酵母を最初に確立しました。また、酵母14で発現した場合、落射蛍光顕微鏡でMreBの重合を効率的に妨げるA22の能力も実証されています。これに続いて、他のグループも分裂酵母を使用して、葉緑体FtsZ1およびFtsZ2タンパク質の集合特性を研究することに成功しました16。最近では、2つの病原菌、すなわち黄色ブドウ球菌とヘリコバクターピロリに由来する3つの既知のFtsZ阻害剤(サンギナリン、ベルベリン、およびPC190723オンFtsZタンパク質)の影響を包括的に評価することにより、細菌細胞骨格阻害剤を特異的にスクリーニングするための細胞プラットフォームとしての分裂酵母の使用の実行可能性の概念実証を確立しました17.さらに、このシングルステップの細胞ベースのアッセイは、真核細胞に潜在的に毒性がある可能性のある化合物を特定するリスクを最小限に抑えるのに役立つことが証明されています。
本報告では、分裂酵母システムを利用して、 黄色ブドウ球菌 由来のFtsZおよび 大腸菌由来のMreBを標的とする低分子阻害剤の効果を半自動スクリーニングおよび定量するための標準的な96ウェルプレートを用いた体系的なワークフローを提案します。ここでは、FtsZとMreBをそれぞれ特異的に標的とする確立された阻害剤PC190723とA22を使用して、半自動ワークフローを設定し、最適化します。このワークフローでは、電動式高精度ステージを備えた落射蛍光顕微鏡と、標準的な96ウェルプレートでの自動画像取得を使用して、現在の標準化を改善しています。したがって、合成化学ライブラリーのミディアムスループットおよびハイスループットスクリーニングに適用でき、上記の課題のいくつかを回避できます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well CC2 Optical CVG Sterile, w/Lid. Black</td>
			<td>Thermo Scientific&#8482;</td>
			<td>160376</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning&#160;</td>
			<td>&#160;CLS3370</td>
			<td></td>
		</tr>
		<tr>
			<td>A22 Hydrochloride</td>
			<td>Sigma&#160;</td>
			<td>SML0471</td>
			<td>Dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>FormediumTM</td>
			<td>DOC0229</td>
			<td>225 mg/L of media&#160;</td>
		</tr>
		<tr>
			<td>Concanavalin A&#160;</td>
			<td>Sigma&#160;</td>
			<td>C5275-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma&#160;</td>
			<td>317275</td>
			<td></td>
		</tr>
		<tr>
			<td>Edinburg minimal medium (EMM Agar or EMM Broth)</td>
			<td>FormediumTM</td>
			<td>PMD0210</td>
			<td>See below for composition</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Sigma&#160;</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>epMotion&#174; 96 with 2-position slider</td>
			<td>Eppendorf</td>
			<td>5069000101</td>
			<td></td>
		</tr>
		<tr>
			<td>Histidine</td>
			<td>FormediumTM</td>
			<td>DOC0144</td>
			<td>225 mg/L of media&#160;</td>
		</tr>
		<tr>
			<td>Leica DMi8 inverted fluorescence microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>German company</td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>FormediumTM</td>
			<td>DOC0157</td>
			<td>225 mg/L of media&#160;</td>
		</tr>
		<tr>
			<td>Lithium acetate&#160;</td>
			<td>Sigma&#160;</td>
			<td>517992-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>PC190723</td>
			<td>Merck&#160;</td>
			<td>344580</td>
			<td>Dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Polyethylene glycol (PEG)</td>
			<td>Sigma&#160;</td>
			<td>202398</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Sigma</td>
			<td>T4625</td>
			<td>Filter sterilised</td>
		</tr>
		<tr>
			<td>Tris-Hydrochloride</td>
			<td>MP</td>
			<td>194855</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>FormediumTM</td>
			<td>DOC0214</td>
			<td>225 mg/L of media, Store solution at 36&#176;C</td>
		</tr>
		<tr>
			<td>YES (Yeast extract + supplements) Agar</td>
			<td>FormediumTM</td>
			<td>PCM0410</td>
			<td>See below for composition</td>
		</tr>
		<tr>
			<td>YES (Yeast extract + supplements) Broth</td>
			<td>FormediumTM</td>
			<td>PCM0310</td>
			<td>See below for composition</td>
		</tr>
		<tr>
			<td>Yeast (S. pombe) media&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract + supplements (YES)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Composition</td>
			<td>g/L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Yeast extract</td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Dextrose</td>
			<td>30</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Agar</td>
			<td>17</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Adenine</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>L-Histidine</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>L-Leucine</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>L-Lysine HCl</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Uracil</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td>Edinburg minimal medium (EMM)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Composition</td>
			<td>g/L</td>
			<td>concentration</td>
		</tr>
		<tr>
			<td></td>
			<td>potassium hydrogen phthallate&#160;</td>
			<td>3</td>
			<td>14.7mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Na2HPO4&#160;</td>
			<td>2.2</td>
			<td>15.5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>NH4Cl&#160;</td>
			<td>5</td>
			<td>93.5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>glucose</td>
			<td>2% (w/v) or 20 g/L</td>
			<td>&#160;111 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Salts (stock x 50)</td>
			<td>20 mL/L (v/v)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Vitamins (stock x 1000)</td>
			<td>1 mL/L (v/v)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Minerals (Stock x 10,000)</td>
			<td>0.1 mL/L (v/v)</td>
			<td></td>
		</tr>
		<tr>
			<td>Salts x 50&#160;</td>
			<td>52.5 g/l MgCl2.6H20 (0.26 M)&#160;</td>
			<td>52.5</td>
			<td>0.26 M</td>
		</tr>
		<tr>
			<td></td>
			<td>0.735 mg/l CaCl2.2H20 (4.99 mM)&#160;</td>
			<td>0.000735</td>
			<td>4.99 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>50 g/l KCl (0.67 M)&#160;</td>
			<td>50</td>
			<td>0.67 M</td>
		</tr>
		<tr>
			<td></td>
			<td>2 g/l Na2SO4 (14.l mM)</td>
			<td>2</td>
			<td>14.1 mM</td>
		</tr>
		<tr>
			<td>Vitamins x 1000&#160;</td>
			<td>1 g/l pantothenic acid&#160;</td>
			<td>1</td>
			<td>4.20 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>10 g/l nicotinic acid&#160;</td>
			<td>10</td>
			<td>81.2 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>10 g/l inositol&#160;</td>
			<td>10</td>
			<td>55.5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>10 mg/l biotin&#160;</td>
			<td>0.01</td>
			<td>40.8 &#181;M</td>
		</tr>
		<tr>
			<td>Minerals x 10,000&#160;</td>
			<td>boric acid</td>
			<td>5</td>
			<td>80.9 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>MnSO4&#160;&#160;</td>
			<td>4</td>
			<td>23.7 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>ZnSO4.7H2O</td>
			<td>4</td>
			<td>13.9 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>FeCl2.6H2O&#160;&#160;</td>
			<td>2</td>
			<td>7.40 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>molybdic acid&#160;</td>
			<td>0.4</td>
			<td>2.47 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>KI&#160;</td>
			<td>1</td>
			<td>6.02 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>CuSO4.5H2O&#160;</td>
			<td>0.4</td>
			<td>1.60 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>citric acid&#160;</td>
			<td>10</td>
			<td>47.6 mM</td>
		</tr>
		<tr>
			<td>Strains/ Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strains</td>
			<td>Description</td>
			<td>Reference</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD190</td>
			<td>Escherichia coli DH10&#946;&#160;</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>CCDY4&#160;</td>
			<td>MBY3532; CCDY346/pREP42- GFP-EcMreB</td>
			<td>Srinivasan et al., 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>CCDY340</td>
			<td>CCDY346/pREP42- SaFtsZ-GFP</td>
			<td>Sharma et al., 2023</td>
			<td></td>
		</tr>
		<tr>
			<td>CCDY346</td>
			<td>MBY192; Schizosaccharomyces pombe [ura4-D18, leu1-32, h-]</td>
			<td>Dr. Mithilesh Mishra (DBS, TIFR)</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCCD3</td>
			<td>pREP42-GFP-EcMreB</td>
			<td>Srinivasan et al., 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>pCCD713</td>
			<td>pREP42-SaFtsZ-GFP</td>
			<td>Sharma et al., 2023</td>
			<td></td>
		</tr>
	</tbody>
</table>

fission yeast as a platform for antibacterial drug screens targeting bacterial cytoskeleton proteins

FtsZやMreBなどの細菌の細胞骨格タンパク質は、細胞分裂や細胞形状の維持など、本質的な機能を果たしています。さらに、FtsZとMreBは、新規抗菌薬発見の重要な標的として浮上しています。これらの細胞骨格タンパク質のヌクレオチド結合と重合を標的とする化合物を同定するために、主にFtsZに焦点を当てたいくつかのアッセイが開発されています。さらに、アッセイの多くは手間がかかるか、費用がかかるため、これらのタンパク質が薬物の細胞標的であるかどうかを確認するには、多くの場合、複数の方法が必要です。最後に、真核細胞に対する薬物の毒性も問題を引き起こします。ここでは、細菌の細胞骨格を標的とする新しい分子を発見し、真核細胞に潜在的に毒性がある可能性のあるヒットを最小限に抑えるためのシングルステップの細胞ベースのアッセイについて説明します。分裂酵母は、顕微鏡法に基づくハイスループットスクリーニングに適しており、視覚的スクリーニングにより、FtsZまたはMreBの重合を変化させる分子を容易に同定できます。私たちのアッセイは、標準的な96ウェルプレートを利用し、分裂酵母などの真核細胞で重合する細菌の細胞骨格タンパク質の能力に依存しています。ここに記載されているプロトコルは分裂酵母用であり、 黄色ブドウ球菌 由来のFtsZおよび 大腸菌由来のMreBを利用していますが、真核生物の発現宿主でポリマーに容易に集合する他の細菌細胞骨格タンパク質に容易に適応できます。ここで説明する方法は、細菌の細胞骨格タンパク質を標的とする新規抗菌薬のさらなる発見を促進するのに役立つはずです。

The widespread resistance to nearly all antibiotics presently employed to combat bacterial infections has created an immediate necessity for novel categories of antibiotics. A 2019 report indicated that antibiotic-resistant infections resulted in the loss of 1.27 million lives, contributing to an overall tally of 4.95 million deaths when considering complications from resistant bacterial infections1. While still effective in clinical practice, the current arsenal of antibiotics predominantly targets a narrow spectrum of cellular processes, primarily focusing on cell wall, DNA, and protein synthesis. Over the past half-century, fewer than 30 proteins have been commercially exploited as targets for the development of new anti-bacterials2,3. This limited range of viable targets significantly creates constraints to discovering new antibiotics or their derivatives for combating antibiotic-resistant bacteria. Thus, to overcome the emerging antibiotic resistance problem, there is a need for the development of new antibiotics with novel targets and mechanisms of action.
An antibacterial target should ideally be an essential component of bacterial cell growth, be conserved throughout the phylogenetically diverse species, show least eukaryotic homology, and be accessible to antibiotics4. Since the discovery of bacterial cytoskeletal proteins involved in cell division and cell shape maintenance, they have emerged as a promising focal point for developing antibacterial compounds5. These proteins are essential for bacterial viability and play a pivotal role in maintaining cell shape (MreB, CreS), division (FtsZ, FtsA), and DNA segregation (ParM, TubZ, PhuZ, AlfA), akin to the cytoskeleton in eukaryotic cells. Notably, FtsZ exhibits a remarkably high level of conservation across a wide range of prokaryotic organisms, while MreB is found in nearly all rod-shaped bacteria. Such wide distribution and relevance in cell viability make these proteins a fascinating target in antibiotic research5,6,7.
It is crucial to adopt a multi-pronged approach that combines in vivo observations, in vitro interactions, and enzymatic experiments to thoroughly validate bacterial cytoskeleton proteins as the primary target of a potential inhibitor7. Laborious procedures or substantial cost implications encumber many available assays for this purpose. These are notable obstacles to their widespread utilization in screening lead compounds that could impact the bacterial cytoskeleton. Among these, microscopy stands out as an exceptionally efficient and rapid method for assessing the effectiveness of compounds by directly examining changes in cell morphology. Yet, the hetero-association of target protein with other cytoskeleton complexes, indirect effects due to off-target binding and changes in membrane potential, difficulty in penetrating the cell efficiently, and presence of efflux pumps, especially in Gram-negative bacteria, make it collectively complex to pinpoint the precise cause of bacterial cell deformation8,9,10.
Schizosaccharomyces pombe, or fission yeast as it&#39;s commonly known, is a rod-shaped unicellular eukaryotic organism. Fission yeast is widely used as a model organism in cellular and molecular biology due to the extraordinary conservation in cellular processes such as the cell cycle and division, cellular organization, and chromosome replication with higher eukaryotes, including humans11,12. Furthermore, Errington and colleagues expressed a pole-localized bacterial cytoskeletal protein DivIVA in fission yeast to demonstrate that DivIVA accumulated on negatively curved surfaces13. Again, Balasubramanian and group had first established fission yeast as a cellular model system to bring new insights into the mechanism, assembly, and dynamicity of the E. coli actin cytoskeleton protein like MreB14 and tubulin homolog FtsZ15. They also demonstrate the ability of A22 to efficiently impede the polymerization of MreB through epifluorescence microscopy when expressed in yeast14. Following this, other groups have also successfully employed fission yeast to study the assembly properties of chloroplast FtsZ1 and FtsZ2 proteins16. More recently, we have established a proof-of-concept of the viability of the use of fission yeast as a cellular platform to specifically screen for bacterial cytoskeleton inhibitors by conducting a comprehensive assessment of the impact of three known FtsZ inhibitors-sanguinarine, berberine, and PC190723-on FtsZ proteins derived from two pathogenic bacteria, namely Staphylococcus aureus and Helicobacter pylori17. Additionally, this single-step cell-based assay proves instrumental in minimizing the risk of identifying compounds that may be potentially toxic to eukaryotic cells.
In this report, utilizing the fission yeast system, we propose a systematic workflow using the standard 96-well plate for semi-automated screening and quantification of the effect of small molecule inhibitors targeting FtsZ from Staphylococcus aureus and MreB from Escherichia coli. Here, we set up and optimize the semi-automated workflow using the established inhibitors PC190723 and A22 that specifically target FtsZ and MreB, respectively. This workflow uses an epifluorescence microscope equipped with a motorized high-precision stage and automated image acquisition in a standard 96-well plate to improve upon current standardization. Hence, it can be applied to medium- as well as high-throughput screens of synthetic chemical libraries and circumvents some of the challenges listed above.

著者スポットライト:革新的な細胞ベースのアッセイを通じて新規抗生物質を明らかにするための細胞骨格ダイナミクスの探索

細菌の細胞骨格タンパク質を標的とする抗菌薬スクリーニングのプラットフォームとしての分裂酵母

Our laboratory is interested in understanding how cytoskeletal proteins assemble dynamic structures to regulate functions such as DNA partitioning and cell division in bacteria. We are also interested in discovering compounds that target these cytoskeletal proteins to develop novel antibiotics. Our protocol is a cell-based assay in which the bacterial cytoskeleton protein polymerizes in a molecularly crowded environment in the eukaryotic yeast, Schizosaccharomyces pombe.With this approach, simultaneously, we can identify molecules that directly affects the bacterial cytoskeleton proteins, polymerization, and are potentially toxic. Using this fission yeast platform, we plan to screen libraries of macrocyclic compounds, specifically targeting FtsZ of pathogenic bacteria like Helicobacter pylori and Pseudomonas aeruginosa. We also plan to expand the screens to target actin-like proteins from Clostridium perfringens involved in plasmid segregation.

薬物スクリーニング用の96ウェルプレートのセットアップ 中強度チアミン抑制性プロモーターnmt41を含有するベクター(pREP42)からC末端GFPタグ付き黄色ブドウ球菌FtsZを発現させるS.pombeの使用は以前に確立されており17、同様に、N末端GFPでタグ付けされた大腸菌MreBもS.pombeで発現していた14。また、SaFtsZの特異的...

Read this Scientific Article Protocol about Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays at JoVE.com

ここでは、分裂酵母を異種宿主として利用し、FtsZやMreBなどの細菌細胞骨格タンパク質をGFPとの翻訳融合タンパク質として発現させ、それらの重合を可視化します。また、重合に影響を与える化合物は、蛍光顕微鏡を用いたイメージングによって同定されます。

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...

私たちの研究室では、細胞骨格タンパク質がどのように動的な構造を組み立てて、細菌のDNA分配や細胞分裂などの機能を調節しているかを理解することに興味を持っています。また、これらの細胞骨格タンパク質を標的とする化合物を発見して、新しい抗生物質を開発することにも興味を持っています。私たちのプロトコルは、細菌の細胞骨格タンパク質が真核酵母の分子的に混雑した環境で重合する細胞ベースのアッセイです。このアプローチにより、同時に、細菌の細胞骨格タンパク質、重合に直接影響し、潜在的に毒性のある分子を特定できます。この分裂酵母プラットフォームを用いて、ヘリコバクター・ピロリ菌や緑膿菌などの病原菌のFtsZを特異的に標的とする大環状化合物のライブラリーをスクリーニングする予定です。また、プラスミド分離に関与するClostridium perfringens由来のアクチン様タンパク質を標的とするスクリーニングも拡大する予定です。

fission-yeast-as-platform-for-antibacterial-drug-screens-targeting

Research

JoVE Journal

Biology

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.