Name: 作者聚焦：探索细胞骨架动力学，通过创新的基于细胞的检测揭示新型抗生素
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 和 AKS 感谢从原子能部国家科学教育与研究所获得的奖学金。RS 承认原子能部的校内资金支持，这项工作得到了生物技术部 （DBT） 对 RS 的研究补助金 （BT/PR42977/MED/29/1603/2022） 的支持。作者还感谢 V Badireenath Konkimalla 在协议开发过程中的评论、建议和讨论。

1. GFP标记的细菌细胞骨架蛋白在鲳鱼中的表达
注：有关此处使用的所有质粒和菌株的信息，请参阅 表1 。请参阅 表 2 了解所有介质成分。
<ol><li>使用N端GFP融合（GFP-MreB）和携带C端GFP（SaFtsZ-GFP）的金黄色葡萄球菌FtsZ克隆大肠杆菌MreB到具有中等强度硫胺素抑制启动子nmt4118,19<sup...

鉴定影响细菌细胞骨架蛋白聚合的药物

<ol>
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抗微生物药物耐药性（AMR）是一种严重的全球健康威胁，迫切需要具有新靶点的新型抗生素。细菌细胞骨架已成为开发新抗生素的一个有吸引力的靶点，细胞分裂蛋白FtsZ的小分子抑制剂，如TXA709，已经处于I期临床试验30。已经开发了几种方法来鉴定 FtsZ 聚合抑制剂 7,31。我们最近在基于细胞的测定中使用真核裂变酵母表明，FtsZ 稳定药...

目前用于对抗细菌感染的几乎所有抗生素都具有广泛的耐药性，因此迫切需要新型抗生素。2019 年的一份报告指出，抗生素耐药性感染导致 127 万人丧生，考虑到耐药细菌感染的并发症，总共有 495 万人死亡1。虽然在临床实践中仍然有效，但目前的抗生素库主要针对窄谱细胞过程，主要集中在细胞壁、DNA 和蛋白质合成上。在过去的半个世纪中，只有不到 30 种蛋白质被商业开发为开发新型抗菌剂的靶标 2,3。这种有限的可行靶点范围极大地限制了发现新的抗生素或其衍生物以对抗抗生素耐药细菌。因此，为了克服新出现的抗生素耐药性问题，需要开发具有新靶点和作用机制的新型抗生素。
理想情况下，抗菌靶标应该是细菌细胞生长的重要组成部分，在系统发育多样性的物种中是保守的，表现出最少的真核同源性，并且可以使用抗生素4。自从发现参与细胞分裂和细胞形状维持的细菌细胞骨架蛋白以来，它们已成为开发抗菌化合物的有前途的焦点5。这些蛋白质对细菌活力至关重要，在维持细胞形状（MreB、CreS）、分裂（FtsZ、FtsA）和 DNA 分离（ParM、TubZ、PhuZ、AlfA）方面发挥着关键作用，类似于真核细胞中的细胞骨架。值得注意的是，FtsZ在广泛的原核生物中表现出非常高的保守性水平，而MreB几乎存在于所有棒状细菌中。如此广泛的分布和细胞活力的相关性使这些蛋白质成为抗生素研究中的一个引人入胜的靶标 5,6,7。
采用多管齐下的方法至关重要，该方法将体内观察、体外相互作用和酶学实验相结合，以彻底验证细菌细胞骨架蛋白作为潜在抑制剂的主要靶点7。繁琐的程序或巨大的成本影响阻碍了许多用于此目的的可用检测。这些是它们在筛选可能影响细菌细胞骨架的先导化合物中广泛使用的显着障碍。其中，显微镜作为一种非常有效和快速的方法脱颖而出，它通过直接检查细胞形态的变化来评估化合物的有效性。然而，靶蛋白与其他细胞骨架复合物的异质结合、脱靶结合和膜电位变化导致的间接影响、难以有效穿透细胞以及外排泵的存在，尤其是在革兰氏阴性细菌中，使得确定细菌细胞变形的确切原因变得总体复杂 8,9,10。
裂殖酵母，或俗称的裂变酵母，是一种杆状单细胞真核生物。裂变酵母在细胞和分子生物学中被广泛用作模式生物，因为在细胞过程（如细胞周期和分裂、细胞组织以及与高等真核生物（包括人类）的染色体复制中具有非凡的保守性11,12。此外，Errington及其同事在裂变酵母中表达了极点定位的细菌细胞骨架蛋白DivIVA，以证明DivIVA积累在负曲面上13。同样，Balasubramanian和团队首次将裂变酵母建立为细胞模型系统，以为大肠杆菌肌动蛋白细胞骨架蛋白（如MreB14和微管蛋白同源物FtsZ15）的机制、组装和动力学带来新的见解。他们还证明了 A22 在酵母14 中表达时通过落射荧光显微镜有效阻止 MreB 聚合的能力。在此之后，其他研究小组也成功地利用裂变酵母研究了叶绿体 FtsZ1 和 FtsZ2 蛋白的组装特性16。最近，我们通过全面评估三种已知的 FtsZ 抑制剂（血根碱、小檗碱和 PC190723-on FtsZ 蛋白）的影响，建立了使用裂变酵母作为细胞平台特异性筛选细菌细胞骨架抑制剂的概念验证，这些蛋白来自两种致病细菌，即金黄色葡萄球菌和幽门螺杆菌 17.此外，这种基于细胞的单步法检测被证明有助于最大限度地降低识别可能对真核细胞具有潜在毒性的化合物的风险。
在本报告中，利用裂变酵母系统，我们提出了一种系统的工作流程，使用标准 96 孔板进行半自动筛选和定量小分子抑制剂靶向 金黄色葡萄球 菌 FtsZ 和 大肠杆菌 MreB 的作用。在这里，我们使用已建立的抑制剂 PC190723 和 A22 分别专门针对 FtsZ 和 MreB 来设置和优化半自动工作流程。该工作流程使用配备电动高精度载物台的落射荧光显微镜和标准 96 孔板中的自动图像采集，以改进当前的标准化。因此，它可以应用于合成化学库的中通量和高通量筛选，并规避了上述一些挑战。

<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

细菌细胞骨架蛋白（如 FtsZ 和 MreB）执行细胞分裂和细胞形状维持等基本功能。此外，FtsZ和MreB已成为新型抗菌药物发现的重要靶点。已经开发了几种检测方法来鉴定靶向这些细胞骨架蛋白的核苷酸结合和聚合的化合物，主要集中在 FtsZ 上。此外，许多检测要么是费力的，要么是成本高昂的，确定这些蛋白质是否是药物的细胞靶标通常需要多种方法。最后，药物对真核细胞的毒性也是一个问题。在这里，我们描述了一种基于细胞的单步测定法，以发现靶向细菌细胞骨架的新分子，并最大限度地减少可能对真核细胞具有潜在毒性的打击。裂变酵母适用于基于显微镜的高通量筛选，视觉筛选可以轻松识别任何改变 FtsZ 或 MreB 聚合的分子。我们的检测使用标准的 96 孔板，并依赖于细菌细胞骨架蛋白在真核细胞（如裂变酵母）中聚合的能力。虽然这里描述的方案是针对裂变酵母的，并利用来自 金黄色葡萄球 菌的FtsZ和来自 大肠杆菌的MreB，但它们很容易适应其他细菌细胞骨架蛋白，这些细菌细胞骨架蛋白很容易在任何真核表达宿主中组装成聚合物。本文所述的方法应有助于进一步发现靶向细菌细胞骨架蛋白的新型抗菌剂。

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.

作者聚焦：探索细胞骨架动力学，通过创新的基于细胞的检测揭示新型抗生素

裂变酵母作为靶向细菌细胞骨架蛋白的抗菌药物筛选平台

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.

设置用于药物筛选的 96 孔板 先前已经确定使用S. pombe从含有中等强度硫胺素可抑制启动子nmt41的载体（pREP42）中表达C-末端GFP标记的金黄色葡萄球菌FtsZ17，同样，用N-末端GFP标记的大肠杆菌MreB也在S. pombe14中表达。我们还表明，PC190723（SaFtsZ 的特异性抑制剂）和 A22（MreB 抑制剂）在酵母14,17</su...

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

裂变酵母在这里用作异源宿主，将细菌细胞骨架蛋白（如 FtsZ 和 MreB）表达为具有 GFP 的翻译融合蛋白，以可视化其聚合。此外，通过使用荧光显微镜进行成像来识别影响聚合的化合物。

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

我们的实验室有兴趣了解细胞骨架蛋白如何组装动态结构以调节细菌中的DNA分配和细胞分裂等功能。我们还对发现靶向这些细胞骨架蛋白的化合物感兴趣，以开发新型抗生素。我们的方案是一种基于细胞的测定，其中细菌细胞骨架蛋白在分子拥挤的环境中在真核酵母 Schizosaccharomyces pombe 中聚合。通过这种方法，同时，我们可以识别出直接影响细菌细胞骨架蛋白、聚合化并具有潜在毒性的分子。利用这种裂变酵母平台，我们计划筛选大环化合物库，专门针对幽门螺杆菌和铜绿假单胞菌等致病菌的FtsZ。我们还计划扩大筛选范围，以靶向参与质粒分离的产气荚膜梭菌中的肌动蛋白样蛋白。

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.