Name: fission yeast as a platform for antibacterial drug screens targeting bacterial cytoskeleton proteins
Uploaded: 2024-04-26T14:35:00.000+00:00
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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的大肠杆菌表达载体pREP42中，如前所述14,15。如20,21中所述，从大肠杆菌菌株（DH10β）中维持，扩增和分离质粒。 注：也可以使用其他 大肠杆 菌菌株，如DH5α、XL1Blue、TOP10等或其他常规用于分子克隆的市售感受态细胞。</li>
	<li>质粒pREP42-GFP-EcMreB和pREP42-SaFtsZ-GFP转化为 鲳鱼链球菌
	<ol><li>使用醋酸锂方法22将质粒pCCD3（pREP42-GFP-EcMreB）和pCCD713（pREP42-SaFtsZ-GFP）转化为庞贝链球菌株（h-leu1-32 ura4-D18），如以下步骤所述。</li>
		<li>第 1 天 - 初次培养：将一环新鲜条纹的 S. pombe 培养物接种到 3 mL 高压灭菌酵母提取物和补充剂 （YES） 肉汤中。将其在30°C（O / N）的轨道振荡器中孵育过夜。</li>
		<li>第2天：<ol><li>二次培养：将约 500 μL 原代培养物加入 30 mL 高压灭菌的 YES 肉汤中。在30°C下孵育3-4小时，振荡至OD600 达到0.4-0.6。 注：对于每次转化，使用 30 mL。</li>
			<li>在室温下以 2,500 x g 沉淀 30 mL 培养物 6-8 分钟。弃去上清液并用 50 mL 无菌蒸馏水 （D/W） 洗涤细胞。再次沉淀并丢弃上清液。将细胞重悬于 1 mL 无菌 D/W 中，并将其转移到 2 mL 离心管中。如上离心，弃去上清液。</li>
			<li>加入 1 mL 0.1 M 醋酸锂、Tris-EDTA （LiAc-TE） 溶液并如上所述离心。弃去上清液并重悬于 1 mL 的 0.1 M LiAc-TE 中。离心并弃去上清液，留下 100 μL 溶液。</li>
			<li>加入 10 - 20 μg 载体 DNA（鲑鱼精子 DNA;在冰上变性并快速冷却）和 2 - 3 μg 需要转化的质粒 DNA。轻轻混合。在室温下孵育 10 分钟。</li>
			<li>加入 260 μL 40% PEG/LiAc-TE;轻轻混合。在振荡器或30°C的热混合器中孵育60分钟，轻轻混合。加入 43 μL DMSO;轻轻混合。</li>
			<li>在热混合器中在42°C下热休克10分钟。以2,500× g 沉淀6-8分钟，弃去上清液。用 1 mL 无菌 D/W 洗涤沉淀 1 次。</li>
			<li>沉淀，弃去上清液并将沉淀重悬于200μL无菌D / W中。 板100μL在含有5μg/ mL硫胺素（抑制 nmt41 启动子）和氨基酸补充剂腺嘌呤（0.225mg / mL），组氨酸（0.225mg / mL）和亮氨酸（0.225mg / mL）但缺乏尿嘧啶（pREP42质粒的选择标记）。 注：交替，将 70 μL 和 130 μL 板放在两个不同的板中。接种两种不同体积（70 μL 和 130 μL）以在至少一个板上获得分离的菌落，具体取决于转化效率，转化效率可能因实验而异。</li>
			<li>将板在30°C孵育2-3天，直到菌落出现。将单个菌落与 100 μL 无菌 D/W 混合，并铺在含有硫胺素但缺乏尿嘧啶的新鲜 EMM 板上，如上所述。</li>
			<li>在30°C孵育2-3天，直到出现完整的草坪。使用接种环废弃生长的细胞草坪，并将其重悬于冻存瓶中含有 30% 甘油的 1 mL YES 培养基中。</li>
			<li>在液氮中快速冷冻冷冻冻存瓶并储存在-80°C以保存冷冻酵母储备液。</li>
		</ol></li>
	</ol></li>
	<li>细菌细胞骨架蛋白在裂变酵母中的表达<ol><li>将甘油原液中的一块无菌划线到新鲜的酵母特异性板上，以获得足够的培养物用于进一步的实验。 注意：在 pREP42 的情况下，我们使用含有腺嘌呤、组氨酸和亮氨酸的 EMM 琼脂平板（最小培养基）（使用 S. pombe 菌株 （h-leu1-32 ura4-D18））而不使用尿嘧啶（尿嘧啶存在于 pREP42 中作为选择标记）。我们将 15 - 20 μM 硫胺素添加到琼脂平板中（因为 pREP42 具有硫胺素可抑制的 nmt41 启动子），以在平板上生长时抑制目的基因的表达。</li>
		<li>将来自条纹贴片的一小环接种物接种到缺乏硫胺素的5mL酵母特异性EMM培养基中，并在30°C下孵育10-12小时。 注：在 nmt41 启动子下，不同物种的 FtsZ 和 MreB 在 S. pombe 中的表达可能有所不同，通常为 16 至 30 小时。在不带硫胺素的情况下，GFP-EcMreB和SaFtsZ-GFP在30 °C下蛋白表达的最佳时间分别为20 - 24 h和16 - 20 h。</li>
	</ol></li>
</ol>2. 表达 GFP 标记的细菌细胞骨架蛋白 的 S. pombe 培养物的处理
注：在 96 孔板中对过夜培养的酵母培养物测试许多不同的药物分子。
<ol><li>通过使用半自动 96 孔多通道移液仪器将 50 μL 过夜培养物转移到含有 150 μL 新鲜酵母 EMM 培养基的 96 孔板的每个孔中来接种新鲜培养物。</li>
	<li>在使用半自动 96 孔多通道移液器移入和移出以进行适当混合时，缓慢地将不同浓度的药物引入生长培养物中，每个药物一式三份，如图 1 中的示意图所示。</li>
	<li>作为阳性对照，使用已知的药物，PC190723用于（金黄色葡萄球菌 FtsZ）和 A22（用于 大肠杆菌 MreB）一式两份。 注：如前所述，PC190723和A22的浓度分别为56.2μM17和72.6μM 14,17。同时，A22处理SaFtsZ和PC190723处理EcMreB分别为阴性对照。</li>
	<li>根据药物的最低和最高浓度，使用DMSO作为三种不同浓度的溶剂对照。 注：溶解药物的溶剂用作溶剂对照。</li>
	<li>将对照和处理过的培养物在30°C孵育6-10小时（直到细胞显示细菌荧光蛋白的表达），然后使用落射荧光显微镜成像。</li>
</ol>3. 聚合物的可视化
<ol><li>将 20 μL 1 mg/mL 刀豆球蛋白 A 的包被涂在光学透明的底部 96 孔板（用于荧光成像的黑壁）的每个孔中，并在室温下孵育 20 分钟。吸出液体，让它风干 10 分钟。</li>
	<li>将 20 μL 细胞从培养板转移到每个相应的孔中，静置 10 分钟。用无菌 EMM 培养基洗涤细胞 3x-4x。</li>
	<li>在要使用的物镜就位（参见步骤 3.6）的情况下，使用图像采集软件采集面板中的导航器模式进行 96 孔板对准和孔覆盖。在导航器上对齐孔边缘。</li>
	<li>接下来，选择孔覆盖参数，其中包括孔中感兴趣区域的多位置选择，并使用具有按需模式的自适应自动对焦控制，以在成像过程中保持样品对焦。</li>
	<li>使用微分干涉对比 （DIC） 和荧光采集图像。分别设置475/28nm和525/48nm的激发和发射滤光片，以对酵母中表达的GFP标记的细菌蛋白进行成像。通过酵母细胞的厚度（5μm），以0.2μm的步长获得Z-stacks。</li>
	<li>使用配备 100x、1.4-NA 油浸物镜和 2,000 x 2,000 sCMOS 相机（6.5 μm x 6.5 μm 像素尺寸）的倒置落射荧光显微镜捕获图像。使用 LED 照明系统激发荧光团。 注：任何具有用于 96 孔板的电动载物台的落射荧光显微镜系统都应该是合适的，该平台具有精确的多点定位。在1×1的分档和15%-20%的照明下，以相似的曝光时间（通常在0.3-0.5秒的范围内）获取对照和药物处理的所有细胞图像，以最小化实验变量并保持一致性。</li>
</ol>4. 使用 ImageJ 对图像进行量化
<ol><li>处理细胞图像并测量 FtsZ17 的每个细胞的斑点数和密度。在 MreB 的情况下，测量密度和各向异性23 ，并使用 Fiji （v2.0.0-rc-69/1.52p）24 比较对照细胞和处理过的细胞。</li>
	<li>使用 DIC 通道中的图像，首先使用手绘工具勾勒出单个酵母细胞的轮廓，然后在 ROI 管理器中另存为感兴趣区域 （ROI）。此步骤尚未自动化，但可以尝试使用机器学习25,26 的最近开发的工具，并在不久的将来进行整合。</li>
	<li>如27 中所述，遮罩单元格的外部区域并填充黑色。</li>
	<li>使用 OPS 阈值 IJ1 分析宏（斐济的内置功能）来计算点数24。</li>
	<li>使用 otsu 方法进行自动阈值设置并屏蔽分割的粒子。使用宏运行插件分析颗粒。</li>
	<li>为了测量聚合物密度（细胞中每单位面积的细胞骨架量），如所述处理图像，包括掩蔽和骨架化步骤27,28。在 lpx 插件中使用 Lpx 2Dfilter 对图像进行骨架化。</li>
	<li>有关详细信息，请查看最近的出版物17。如前所述23中所述执行EcMreB聚合物定量，如前所述23，使用各向异性使用FibrilTool29量化空间组织。 注：这些分析方法可用于量化经药物处理或未经处理的任何其他细菌细胞骨架蛋白。</li>
</ol>

鉴定影响细菌细胞骨架蛋白聚合的药物

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M., Huecas, S., Ara&#250;jo-Baz&#225;n, L., V&#225;zquez-Villa, H., Mart&#237;n-Fontecha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+search+for+antibacterial+inhibitors+targeting+cell+division+protein+ftsz+at+its+nucleotide+and+allosteric+binding+sites.">The search for antibacterial inhibitors targeting cell division protein ftsz at its nucleotide and allosteric binding sites.</a> Biomedicines. 10 (8), 1825 (2022).</li><li>Vje&#353;tica, A., 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+toolbox+of+stable+integration+vectors+in+the+fission+yeast+Schizosaccharomyces+pombe.">A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe.</a> J Cell Sci. 133 (1), jcs240754 (2020).</li><li>Berg, S., 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=ilastik:+interactive+machine+learning+for+(bio)image+analysis.">ilastik: interactive machine learning for (bio)image analysis.</a> Nat Meth. 16 (12), 1226-1232 (2019).</li><li>Shaevitz, J. 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所有作者均声明没有利益冲突。

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><tbody><tr><td>96 Well CC2 Optical CVG Sterile, w/Lid. Black</td><td>Thermo Scientific&amp;trade;</td><td>160376</td><td></td></tr><tr><td>96-well plate</td><td>Corning&amp;nbsp;</td><td>&amp;nbsp;CLS3370</td><td></td></tr><tr><td>A22 Hydrochloride</td><td>Sigma&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Concanavalin A&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>C5275-5MG</td><td></td></tr><tr><td>DMSO</td><td>Sigma&amp;nbsp;</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&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>EDS-500G</td><td></td></tr><tr><td>epMotion&amp;reg; 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&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Lithium acetate&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>517992-100G</td><td></td></tr><tr><td>PC190723</td><td>Merck&amp;nbsp;</td><td>344580</td><td>Dissolved in DMSO</td></tr><tr><td>Polyethylene glycol (PEG)</td><td>Sigma&amp;nbsp;</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&amp;deg;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>&lt;strong&gt;Yeast (&lt;em&gt;S. pombe&lt;/em&gt;) media&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Yeast extract + supplements (YES)&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td></td><td>&lt;strong&gt;Composition&lt;/strong&gt;</td><td>&lt;strong&gt;g/L&lt;/strong&gt;</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>&lt;em&gt;&lt;strong&gt;Edinburg minimal medium (EMM)&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td></td><td>&lt;strong&gt;Composition&lt;/strong&gt;</td><td>&lt;strong&gt;g/L&lt;/strong&gt;</td><td>&lt;strong&gt;concentration&lt;/strong&gt;</td></tr><tr><td></td><td>potassium hydrogen phthallate&amp;nbsp;</td><td>3</td><td>14.7mM</td></tr><tr><td></td><td>Na2HPO4&amp;nbsp;</td><td>2.2</td><td>15.5 mM</td></tr><tr><td></td><td>NH4Cl&amp;nbsp;</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>&amp;nbsp;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>&lt;strong&gt;Salts x 50&amp;nbsp;&lt;/strong&gt;</td><td>52.5 g/l MgCl2.6H20 (0.26 M)&amp;nbsp;</td><td>52.5</td><td>0.26 M</td></tr><tr><td></td><td>0.735 mg/l CaCl2.2H20 (4.99 mM)&amp;nbsp;</td><td>0.000735</td><td>4.99 mM</td></tr><tr><td></td><td>50 g/l KCl (0.67 M)&amp;nbsp;</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>&lt;strong&gt;Vitamins x 1000&amp;nbsp;&lt;/strong&gt;</td><td>1 g/l pantothenic acid&amp;nbsp;</td><td>1</td><td>4.20 mM</td></tr><tr><td></td><td>10 g/l nicotinic acid&amp;nbsp;</td><td>10</td><td>81.2 mM</td></tr><tr><td></td><td>10 g/l inositol&amp;nbsp;</td><td>10</td><td>55.5 mM</td></tr><tr><td></td><td>10 mg/l biotin&amp;nbsp;</td><td>0.01</td><td>40.8 &amp;micro;M</td></tr><tr><td>&lt;strong&gt;Minerals x 10,000&amp;nbsp;&lt;/strong&gt;</td><td>boric acid</td><td>5</td><td>80.9 mM</td></tr><tr><td></td><td>MnSO4&amp;nbsp;&amp;nbsp;</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&amp;nbsp;&amp;nbsp;</td><td>2</td><td>7.40 mM</td></tr><tr><td></td><td>molybdic acid&amp;nbsp;</td><td>0.4</td><td>2.47 mM</td></tr><tr><td></td><td>KI&amp;nbsp;</td><td>1</td><td>6.02 mM</td></tr><tr><td></td><td>CuSO4.5H2O&amp;nbsp;</td><td>0.4</td><td>1.60 mM</td></tr><tr><td></td><td>citric acid&amp;nbsp;</td><td>10</td><td>47.6 mM</td></tr><tr><td>&lt;strong&gt;Strains/ Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td>&lt;strong&gt;Description&lt;/strong&gt;</td><td>&lt;strong&gt;Reference&lt;/strong&gt;</td><td></td></tr><tr><td>CCD190</td><td>&lt;em&gt;Escherichia coli&lt;/em&gt; DH10&amp;beta;&amp;nbsp;</td><td>Invitrogen</td><td></td></tr><tr><td>CCDY4&amp;nbsp;</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; &lt;em&gt;Schizosaccharomyces pombe&lt;/em&gt; [ura4-D18, leu1-32, h-]</td><td>Dr. Mithilesh Mishra (DBS, TIFR)</td><td></td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</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。

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

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。

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

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

745 次观看.  National Institute of Science Education and Research. 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?...

视频: 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

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

科研

遗传学

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

免疫与感染

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

生物学

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

An Introduction to Saccharomyces cerevisiae

Saccharomyces cerevisiae (commonly known as baker&rsquo;s yeast) is a single-celled eukaryote that is frequently used in scientific research. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Because many yeast proteins are similar in sequence and function to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans).
This video provides an introduction to the biology of this model organism, how it was discovered, and why labs all over the world have selected it as their model of choice. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death are also discussed. Finally, the video describes some of the many ways in which yeast cells are put to work in modern scientific research, including protein purification and the study of DNA repair mechanisms and other cellular processes related to Alzheimer&rsquo;s and Parkinson&rsquo;s diseases.

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

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