Name: production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization
Uploaded: 2018-08-11T15:00:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

研究得到了国家过敏和传染性疾病研究所 (NIH-NIAID) 奖 R15AI079685 的支持。

1. 解决方案准备
注: 准备步骤1.1–1.9 和1.8–1.11 中描述的解决方案, 以便分别生产大肠杆菌spheroplasts 和巨大原生质体。
<ol><li>制备1米三氯, pH 7.8 通过溶解10.34 克三盐酸和 4.17 g 的三 OH 在50毫升的 dH 2O 在125毫升瓶。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>20毫米氯化镁2, 0.7 米蔗糖, 10 毫米三氯, pH 7.8), 通过溶解0.10 克氯化镁2 (95.2 克/摩尔) 和11.98 克蔗糖 (342.3 克/摩尔) 25 毫升 dH 在2毫升的瓶中。添加500µL 1 M 三氯 (pH 7.8) 和调整体积为50毫升。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>10毫米氯化镁2, 0.8 米蔗糖, 10 毫米三氯, pH 7.8), 通过溶解0.05 克氯化镁2 (95.2 克/摩尔) 和13.69 毫升的蔗糖 (342.3 克/摩尔) 在 25 ml dH2毫升瓶中。添加500µL 1 M 三氯 (pH 7.8) 和调整体积为50毫升。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>在2毫升的烧瓶中, 将13.69 克蔗糖 (342.3 克/摩尔) 溶解在50 毫升 dH 中, 制备0.8 米蔗糖。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>准备5毫克/毫升脱氧核糖核酸酶 (DNase i) 通过溶解0.015 克 DNase i 3 毫升的 dH2O 在50毫升瓶。通过25毫米注射器过滤器, 用0.2 µm 膜过滤消毒。整除溶液成离心管, 贮存在-20 摄氏度。</li>
	<li>用15毫升的烧瓶将0.698 克 EDTA 二钠脱水 (372.2 克/摩尔) 分解为2毫升 dH, 制备0.125 米乙氧乙酸 (edta), pH 8.0。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>600µg/毫升苄溶解0.03 克的苄水合物 (365.404 克/摩尔) 在50毫升的 dH2O 在125毫升瓶。通过过滤25毫米注射器过滤器与0.2 µm 膜, 并贮存在一个锥形管4°c 消毒。</li>
	<li>5毫克/毫升溶菌酶溶出0.015 克溶菌酶3毫升的 dH2O 在50毫升瓶。通过25毫米注射器过滤器, 用0.2 µm 膜过滤消毒。整除溶液成离心管, 贮存在-20 摄氏度。</li>
	<li>在2升瓶中, 在1升的 dH2O 中溶解30克的胰酪胨, 准备3% 瓦特/v 的大豆肉汤。整除将溶液放入含有25毫升或100毫升的烧瓶中, 分别用于制备大肠杆菌spheroplasts 或巨大原生质体.蒸压瓶消毒液体培养基。无菌的可储存在室温或4摄氏度。</li>
	<li>准备溶液 C (1 米蔗糖, 0.04 米马来酸, 0.04 米氯化镁2, pH 6.5) 溶解34.23 克蔗糖 (342.3 克/摩尔), 0.46 克马来酸 (116.07 克/摩尔), 和0.38 克氯化镁2 (95.21 克/摩尔) 在100毫升的 dH2 毫升瓶。将 pH 值调整为6.5。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>在1升玻璃瓶中混合100毫升3% 瓦特/v 型和100毫升溶液 C, 制备200毫升原生质体培养基。蒸压釜消毒溶液并贮存在室温下。</li>
</ol>2. 隔夜文化的准备
注: 使用适当的无菌技术执行2-4 节。如果需要, 细菌菌株可以含有一种抗抗生素的质粒, 以减少潜在的污染。如果使用耐药性菌株, 在步骤2.1、3.1–3.2 和4.1–4.4 中添加必要的抗生素。
<ol><li>准备一夜文化, 通过采摘一个单一的细菌菌落使用不育的吸管尖端, 并将其放入一个14毫升培养管含有2–3毫升3% 瓦特/v。孵育在37摄氏度, 而颤抖 16–21 h。</li>
</ol>3. 革兰阴性大肠杆菌Spheroplasts 的制备
<ol><li>在一个250毫升的烧瓶中, 稀释隔夜文化1:100 在25毫升容量3% 瓦特/v 和孵化细菌溶液在37°c, 而颤抖大约 2.5 h, 直到解决达到了0.5–0.8 的光学密度在600毫微米。使用分光光度计测量光学密度。</li>
	<li>在一个250毫升瓶, 稀释这个文化1:10 在30毫升3% 瓦特/v 和孵化在37°c, 而颤抖 2.5 h 在存在60µg/毫升苄 (347.4 克/摩尔), 以产生约50–150µm 的单细胞花丝长度, 可在使用光显微镜的1,000X 放大率下观察到 (图 1B)。</li>
	<li>通过离心细菌溶液在 1500 x g, 4 °c 为4分钟醒酒, 并丢弃上清, 保留颗粒, 以收获花丝。</li>
	<li>轻轻地加入1毫升0.8 米蔗糖, 小心不要扰乱颗粒, 以洗涤花丝。孵育1分钟, 然后丢弃上清, 不干扰颗粒。</li>
	<li>添加150µL 1 M 三氯 (pH 7.8), 120 µL 5 毫克/毫升溶菌酶, 30 µL 5 毫克/毫升 DNase I, 120 米 EDTA µL 在各自的顺序对颗粒和孵化溶液在室温下为0.125 分钟。</li>
	<li>增加1毫升溶液 a 逐渐超过1分钟的解决方案, 在3.5 使用微, 同时轻轻旋转的解决方案手工。在室温下孵化溶液4分钟。</li>
	<li>将7毫升4°c 溶液 B 放入两个15毫升锥形管中。在3.6 中为这两个管中的每一个添加相同数量的溶液。离心溶液在 1500 x g, 4 °c 为4分钟。</li>
	<li>使用血清吸管, 小心去除所有, 但 1-2 毫升的上清, 而不干扰颗粒。用 P1000 微轻轻吹打上下并用重悬颗粒。通过使用光显微镜观察1,000X 放大器上的样品, 目视检查以查看 spheroplasts 的形成情况 (图 1C)。</li>
	<li>储存的 spheroplasts 在-20 摄氏度, 直到一个星期或直到他们已经经历了3冻融循环。</li>
</ol>4. 革兰阳性B. 巨大原生质体的制备
<ol><li>在一个250毫升瓶, 稀释隔夜文化 1:1, 000 在100毫升 3% w/v 和孵化细菌溶液在37°c, 而颤抖约4.5 小时, 直到解决方案已达到0.9 毫微米的光学密度的一.使用分光光度计测量光学密度。</li>
	<li>把液体培养成两个50毫升圆锥管和离心机在 2000 x g, 4 °c 10 分钟。</li>
	<li>使用血清吸管, 从两个圆锥管中去掉上清。并用重悬2.5 毫升原生质体培养基中的每个颗粒, 并将悬浮溶液组合成一个圆锥管。在125毫升的烧瓶中用吸管将悬浮溶液混合。</li>
	<li>加入1毫升5毫克/毫升溶菌酶和孵育1小时在37摄氏度, 而颤抖。</li>
	<li>用光镜观察1,000x 放大后原生质体的生长情况, 注意任何不规则的现象, 如出现肿胀棒而不是球体的细菌 (图 2)。使用血清吸管, 将溶液转移到15毫升圆锥管, 离心机在 2000 x g, 4 °c 10 分钟。</li>
	<li>醒酒5毫升原生质体培养基上清液并重新悬浮颗粒。将原生质体贮存在-20 摄氏度以上, 直到他们经历了3次冻融循环。</li>
</ol>5. 制备多肽溶液和膜染料用于成像
<ol><li>
肽溶液
	<ol>
<li>在用铝箔包裹的离心管中, 溶解2毫克 FITC 标记为 BF2 P11A 800 µL dH2o 使用含有至少一色氨酸残留物的肽, 以进行蛋白质浓度测量。</li>
		<li>Spectrophotometrically, 测量 280 nm 的多肽溶液的吸光度。用摩尔消光系数计算色氨酸 (5700/立方米) 的肽浓度。</li>
		<li>将 dH2O 的肽浓度稀释至最终浓度的100–200µM. 贮存在一个离心管在-20 °c 包裹在铝箔, 以防止光线。</li>
	</ol>
</li>
	<li>
膜染料
	<ol>
<li>在离心管中, 通过将5毫克的 di-8-ANEPPS 溶解在亚砜的843.3 µL 中, 准备10毫米 di-8-ANEPPS (592.9 克/摩尔) 的股票。储存在4°c 包裹在铝箔, 以防止光线。</li>
		<li>在离心管中, 通过增加3µL 10 毫米 di-8-ANEPPS 到997µL 亚砜, 准备1毫升0.03 毫米 di-8-ANEPPS。贮存在一个离心管在4°c 包裹在铝箔, 以防止光线。</li>
	</ol>
</li>
</ol>6. 应用共焦显微术对大肠杆菌Spheroplasts 和巨大原生质体的可视化研究
<ol><li>吸管5µL spheroplasts 或原生质体在聚 l-赖氨酸涂层玻璃滑动。添加2µL FITC 标记安培 (100-200 µM) 到幻灯片和孵化3分钟保护免受光照。</li>
	<li>添加1µL 的膜染料 di-8-ANEPPS (0.03 毫米) 的幻灯片和孵化3分钟保护免受光照。用玻璃盖玻片盖上, 用指甲油封住。</li>
	<li>打开共焦显微镜和氩激光。将氩激光器调整为20% 功率输出和20% 传输。</li>
	<li>分别设置 FITC 标记肽发射通道和 di-8-ANEPPS-labeled 膜染料发射通道的 499–532 nm 和 670–745 nm 的发射波长范围。</li>
	<li>图像原生质体或 spheroplasts (图 1和图 2) 与63X 目标。使用成像软件获得8位, 512 x 512 复合 z 叠加图像由0.5 µm 切片整体的 spheroplast 或原生质体。 注意: 在成像 spheroplasts 和原生质体时, 要仔细考虑减少放射出血。有关详细信息, 请参阅讨论。</li>
</ol>7. 安培定位的表征
<ol><li>在成像软件中打开复合 z 堆栈图像。找到 spheroplast 或原生质体的中心-多数切片, 并将一个循环区域 (roi) (0.3 µm 直径) 放在膜上 (roi 1), 一个位于 spheroplast 或原生质体的中心 (roi 2), 一个远离 spheroplast 或原生质体来测量背景荧光 (ROI 3) (图 5)。避免包含饱和像素。每个 ROI 的荧光强度都可以通过成像软件来计算。 注意: 如果肽不局部化到膜的整体, 在肽被本地化的膜区域上绘制 ROI 1。</li>
	<li>使用以下等式确定细胞内肽荧光强度与膜肽荧光强度的比值: <img alt="Equation" src="/files/ftp_upload/57904/57904eq1.jpg"> 注: roi 3 中的荧光强度从 roi 2 和 roi 1 中的荧光强度中减去, 以说明背景荧光。</li>
</ol>

<ol>
	<li>Baltzer, S. A., Brown, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+promising+alternatives+to+conventional+antibiotics.">Antimicrobial peptides: promising alternatives to conventional antibiotics.</a> Journal of Molecular Microbiology Biotechnology. 20 (4), 228-235 (2011).</li><li>Hancock, R. E., Sahl, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+and+host-defense+peptides+as+new+anti-infective+therapeutic+strategies.">Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.</a> Nature Biotechnology. 24 (12), 1551-1557 (2006).</li><li>Jenssen, H., Hamill, P., Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+antimicrobial+agents.">Peptide antimicrobial agents.</a> Clinical Microbiology Reviews. 19 (3), 491-511 (2006).</li><li>Toke, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+new+candidates+in+the+fight+against+bacterial+infections.">Antimicrobial peptides: new candidates in the fight against bacterial infections.</a> Biopolymers. 80 (6), 717-735 (2005).</li><li>Wang, G., 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=Antimicrobial+peptides+in+2014.">Antimicrobial peptides in 2014.</a> Pharmaceuticals. 8 (1), 123-150 (2015).</li><li>Epand, R. M., Vogel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+antimicrobial+peptides+and+their+mechanisms+of+action.">Diversity of antimicrobial peptides and their mechanisms of action.</a> Biochim Biophys Acta. 1462, 11-28 (1999).</li><li>Drin, G., Rousselle, C., Scherrmann, J. -. M., Rees, A. R., Temsamani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+Delivery+to+the+Brain+via+Adsorptive-Mediated+Endocytosis:+Advances+With+SynB+Vectors.">Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors.</a> AAPS PharmSciTech. 4 (4), 61-67 (2002).</li><li>Splith, K., Neundorf, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+with+cell-penetrating+peptide+properties+and+vice+versa.">Antimicrobial peptides with cell-penetrating peptide properties and vice versa.</a> European Biophysics Journal. 40 (4), 387-397 (2011).</li><li>Bustillo, M. E., 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=Modular+analysis+of+hipposin,+a+histone-derived+antimicrobial+peptide+consisting+of+membrane+translocating+and+membrane+permeabilizing+fragments.">Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments.</a> Biochim Biophys Acta. 1838 (9), 2228-2233 (2014).</li><li>Koo, Y. 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=Structure-activity+relations+of+parasin+I,+a+histone+H2A-derived+antimicrobial+peptide.">Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide.</a> Peptides. 29 (7), 1102-1108 (2008).</li><li>Libardo, M. D., Cervantes, J. L., Salazar, J. C., Angeles-Boza, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+bioactivity+of+antimicrobial+peptides+by+addition+of+amino-terminal+copper+and+nickel+(ATCUN)+binding+motifs.">Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.</a> ChemMedChem. 9 (8), 1892-1901 (2014).</li><li>Park, C. B., Kim, H. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Action+of+the+Antimicrobial+Peptide+Buforin+II:+Buforin+II+Kills+Microorganisms+by+Penetrating+the+Cell+Membrane+and+Inhibiting+Cellular+Functions.">Mechanism of Action of the Antimicrobial Peptide Buforin II: Buforin II Kills Microorganisms by Penetrating the Cell Membrane and Inhibiting Cellular Functions.</a> Biochemical and Biophysical Research Communications. , 253-257 (1998).</li><li>Park, C. B., Yi, K. -. S., Matsuzaki, K., Kim, M. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+analysis+of+buforin+II,+a+histone+H2A-derived+antimicrobial+peptide:+The+proline+hinge+is+responsible+for+the+cell-penetrating+ability+of+buforin+II.">Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (15), 8245-8250 (2000).</li><li>Pavia, K. E., Spinella, S. A., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+histone-derived+antimicrobial+peptides+use+different+antimicrobial+mechanisms.">Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms.</a> Biochim Biophys Acta. 1818 (3), 869-876 (2012).</li><li>Wei, L., LaBouyer, M. A., Darling, L. E., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Spheroplasts+as+a+Model+for+Visualizing+Membrane+Translocation+of+Antimicrobial+Peptides.">Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.</a> Antimicrobial Agents and Chemotherapy. 60 (10), 6350-6352 (2016).</li><li>Chassy, B. M., Giuffrida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+Lysis+of+Gram-Positive,+Asporogenous+Bacteria+with+Lysozyme.">Method for the Lysis of Gram-Positive, Asporogenous Bacteria with Lysozyme.</a> Appl. Environ. Microbiol. 39 (1), 153-158 (1980).</li><li>Fitz-James, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+and+Chemical+Studies+of+the+Browth+of+Protoplasts+of+Bacillus+megaterium.">Cytological and Chemical Studies of the Browth of Protoplasts of Bacillus megaterium.</a> J. Biophysic. and Biochem. Cytol. 4 (3), 257-266 (1958).</li><li>Martinac, B., Buechner, M., Delcour, A. H., Adler, J., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-sensitive+ion+channel+in+Escherichia+coli.">Pressure-sensitive ion channel in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 2297-2301 (1986).</li><li>Martinac, B., Rohde, P. R., Cranfield, C. G., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Methods Mol Biol. 966, 367-380 (2013).</li><li>Nadeau, J. 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> Introduction to Experimental Biophysics: Biological Methods for Physical Scientists. , (2016).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+Escherichia+coli+spheroplast+membranes.">Physical properties of Escherichia coli spheroplast membranes.</a> Biophysical Journal. 107 (9), 2082-2090 (2014).</li><li>Branco, P., Viana, T., Albergaria, H., Arneborg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+(AMPs)+produced+by+Saccharomyces+cerevisiae+induce+alterations+in+the+intracellular+pH,+membrane+permeability+and+culturability+of+Hanseniaspora+guilliermondii+cells.">Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.</a> Int J Food Microbiol. 205, 112-118 (2015).</li><li>Kobayashi, 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=Membrane+Translocation+Mechanism+of+the+Antimicrobial+Peptide+Buforin+2.">Membrane Translocation Mechanism of the Antimicrobial Peptide Buforin 2.</a> Biochemistry. 43 (49), 15610-15616 (2004).</li><li>Spinella, S. A., Nelson, R. B., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+peptide+translocation+into+large+unilamellar+vesicles.">Measuring peptide translocation into large unilamellar vesicles.</a> J Vis Exp. (59), e3571 (2012).</li><li>van der Kraan, M. I., 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=Lactoferrampin:+a+novel+antimicrobial+peptide+in+the+N1-domain+of+bovine+lactoferrin.">Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin.</a> Peptides. 25 (2), 177-183 (2004).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Biophys J. 111 (1), 132-139 (2016).</li><li>Xie, Y., Fleming, E., Chen, J. L., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proline+position+on+the+antimicrobial+mechanism+of+buforin+II.">Effect of proline position on the antimicrobial mechanism of buforin II.</a> Peptides. 32 (4), 677-682 (2011).</li><li>Kobayashi, S., Takeshima, K., Park, C. B., Kim, S. C., Matsuzaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+Novel+Antimicrobial+Peptide+Buforin+2+with+Lipid+Bilayers:+Proline+as+a+Translocation+Promoting+Factor.">Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers: Proline as a Translocation Promoting Factor.</a> Biochem. 39 (29), 8648-8654 (2000).</li><li>Decad, G. M., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+Membrane+of+Gram-Negative+Bacteria+XII.+Molecular-Sieving+Function+of+Cell+Wall.">Outer Membrane of Gram-Negative Bacteria XII. Molecular-Sieving Function of Cell Wall.</a> J. Bacteriol. 128 (1), 325-336 (1976).</li><li>Choi, H., Yang, Z., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell,+real-time+detection+of+oxidative+stress+induced+in+Escherichia+coli+by+the+antimicrobial+peptide+CM15.">Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.</a> Proc Natl Acad Sci U S A. 112 (3), E303-E310 (2015).</li><li>Yang, Z., Choi, H., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melittin-Induced+Permeabilization,+Re-sealing,+and+Re-permeabilization+of+E.+coli+Membranes.">Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes.</a> Biophys J. 114 (2), 368-379 (2018).</li><li>Ruthe, H. J., Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+bacterial+spheroplasts+by+electric+fields.">Fusion of bacterial spheroplasts by electric fields.</a> Biochim. Biophys. Acta. 819 (1), (1985).</li></ol>

不声明利益冲突。

这里提出的协议使研究人员能够更快地获得更大的细菌图像样本量, 因为扩大的球形细菌更容易定位、定位和图像。这种提高收集数据的能力在几个方面是有价值的。首先, 它能够对多肽定位模式进行更系统的定量分析。虽然从较小的图像集可以证明质量趋势, 但只有大量高质量图像的样本集显示了本地化模式中更微妙的趋势, 如肽 translocates 与本地化的细胞百分比膜。此外, 能够更好地解决内部荧光的膜定位, 使它更容易执行时间课程研究, 以评估如何快速肽与细菌细胞的互动, 以及如何本地化模式变化随着时间的推移。改进后的分辨率还允许研究人员考虑在小细菌中不可能出现的肽定位的趋势。例如, 在我们观察到的一些 spheroplasts 和原生质体中, 肽似乎定位到膜的特定部分,即,肽信号并不形成一个完整的, 在细菌周围的凝聚力环, 而是显示一些点标签。在其他情况下, 多肽可能不会完全均匀分布在细菌膜内,即,肽信号不能完全填充细菌的内部空间。虽然我们没有在目前的分析中追求这些趋势, 但考虑到当成像 spheroplasts 和原生质体而不是标准的细菌细胞时, 这种定位趋势变得可行。这些优势也适用于确定除安培以外的多肽的定位, 如细胞穿透肽。用这些方法产生的 Spheroplasts 和原生质体也可以用于非成像实验, 从增加的细胞大小获益, 如膜片钳电生理学测量18,19。
利用激光扫描共聚焦显微镜对成像协议进行了开发, 但对各特定成像系统的参数进行优化是至关重要的。这包括选择一组参数, 最大限度地检测肽和膜染料荧光, 同时尽量减少出血通过从膜染料到肽的发射通道, 并从 FITC 标记肽进入膜的发射通道。通过选择膜发射通道的波长范围, 不包括 FITC 的任何部分的发射光谱, 避免了从肽进入膜发射通道的出血。从膜染料, di-8-ANEPPS, 进入肽的发射通道的出血, 更难以避免, 因为 di-8-ANEPPS 的发射光谱与大多数 FITC 的发射光谱重叠。特定于本协议, 从膜染料流出到肽的发射通道, 可导致安培作为膜局部化的虚假表征。你可以确定这种性质的出血是由成像 spheroplasts 或原生质体, 完全标记为膜染料和检查是否荧光在肽的发射通道可见。各种成像参数, 包括用于定义肽的发射通道和增益和偏移值的波长范围, 都可以系统地测试, 以确定一组参数, 最大限度地减少出血。一旦建立了一组参数, 就必须评估成像是否能在使用多肽和膜染料标记的 spheroplasts 或原生质体时对荧光信号进行强检测。通过这种方法, 我们成功地建立了成像参数, 最大限度地减少了出血的效果, 同时最大化荧光检测的 FITC 标记安培和膜染料 di-8-ANEPPS。具体地说, 我们发现, 通过 499–532 nm (肽) 和670–745纳米 (膜) 的发射波长范围, 从膜染料到多肽通道的排放流出量最小化, 当增益调整为≤800伏特 (V) (肽) 和≤900 V (膜) 和偏移量被调整为≤-10.0% (肽, 膜)。
虽然我们专注于大肠杆菌和巨大, 但所提出的协议可以用来生产 spheroplasts 或原生质体从许多不同的细菌菌株。例如, 我们已经能够成功地生产出枯草芽孢杆菌原生质体, 这是µm 的直径, 使用4条所述的协议, 并可对该议定书进行修改, 以进一步优化大小。在革兰氏阴性和革兰氏阳性菌中观察肽定位模式的能力对安培的研究特别有用, 因为这两类细菌之间的细胞壁结构差异可能影响安培与细胞膜。然而, 迄今为止, 许多对安培的成像研究完全集中在大肠杆菌菌株上, 因此, 可能无法反映出革兰阳性菌的肽的行为。
Spheroplasts 和原生质体与正常细菌不同, 最明显的是它们缺乏外细胞壁, 因此可能不是所有实验问题的好模型。例如, 革兰阴性菌的外膜已被证明为更大分子29的分子筛。因此, 较大的肽可以在 spheroplasts 中植物常常将, 当它们在正常的细菌中不会这样做。此外, 对 AMP 与细菌细胞壁、外膜和细胞质膜相互作用的详细研究不能用 spheroplasts 和原生质体进行。例如, Weisshaar 实验室最近的工作利用影像学来注意到肽在 CM15 和蜂毒行动30、31的机制中更精确的位置。然而, 正如这里所描述的, 我们的方法并不要求将微流体应用到显微仪器中, 以实现所需的31号决议。虽然以前的工作已经表明, spheroplasts 是可行的21,32, 但他们可能有一些代谢和生理上的差异, 从 "正常" 的细菌, 可能会改变在一些膜相互作用例。此外, 由于我们还没有评估 spheroplast 种群的生存能力, 人们无法区分一个能轻易植物常常将在活细胞的细胞膜上的肽与只能植物常常将在死亡或垂死的细胞。尽管有这些差异, 我们已经看到了许多安培与已知的作用机制15的大肠杆菌spheroplasts 的定位模式, 我们的结果 BF2 P11A 与B. 巨大原生质体在这里提出似乎一致与其他观察为那肽13,27,28。spheroplasts 和原生质体的膜组成也肯定比其他模型系统 (如脂囊) 中使用的典型混合物更具生理学意义。总之, 鉴于 spheroplasts 和原生质体增强的分辨率和成像的易用性, 我们认为它们是在一系列细菌菌株中可视化膜活性剂如安培的有用模型。

抗菌肽 (安培) 得到了重视, 因为他们的潜在用途作为替代传统抗生素1,2,3,4,5。安培杀死细菌的转运通过细胞膜, 并与细胞内的成分, 如核酸, 或通过 permeabilizing 膜导致细胞含量的泄漏6。除了用作抗生素外, 转运安培还可用于药物传递应用, 因为它们可以无中断地穿过不透水细胞膜7、8。因此, 我们寻求了解基本的 AMP 作用机制, 为其在药物设计中的应用打下基础。
共焦显微镜为评估细菌细胞中荧光标记安培的定位模式提供了一种方法, 提供了对其作用机制9、10、11、12的洞察力,13,14. 通过对细菌膜进行标记, 可以确定是否有荧光标记肽本地化到细胞膜或细菌细胞的细胞内空间。然而, 这种技术受细菌的小尺寸和杆型的限制, 由于常规光学显微镜的分辨率极限和15张幻灯片中细菌的变异方向, 使得成像具有挑战性。
所提出的方法的目的是通过共聚焦显微镜增强荧光标记肽定位模式的可视化。通过将小的、薄的、棒状的革兰阴性大肠杆菌 ( 大肠杆菌) 和革兰阳性芽孢杆菌巨大(B. 巨大) 细菌转化为扩大的球形形式, 将其增强, 称为spheroplasts (为革兰阴性菌株) 和原生质体 (为革兰阳性菌株)16,17,18,19,20,21。Spheroplasts 和原生质体更容易图像, 因为它们的大小和对称的形状, 这使得在幻灯片上的细菌的方向与其成像无关。此外, 我们提出了一个系统的方法来定量分析共焦显微数据, 以表征安培作为一种膜定位或转运。应用这些方法可以更容易地区分荧光标记的肽定位模式。此处提供的协议可用于评估除安培以外的各种膜活性剂的定位, 包括细胞穿透肽。
这种技术的一个明显的优点是, 它提供了对安培在单个细胞水平上的作用机制的洞察, 这可能揭示细胞对细胞的异质性15, 而不是其他常用的荧光化验方法来识别安培的作用机制, 仅提供容量估计9,22,23,24,25。使用 spheroplasts 和原生质体, 以评估 AMP 细胞进入是特别有用的26 , 因为它们比其他模型用于评估细胞进入, 如脂囊泡24的生理相关15 。

<table><tbody><tr><td>Trizma hydrocloride (Tris HCl)</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>Trizma base (Tris OH)</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma</td><td>L6876</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Sigma</td><td>D4527</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma</td><td>106361</td><td>Used Sigma 106361 in original protocol development; 106361 discontinued with ED2SS as replacement</td></tr><tr><td>Cephalexin hydrate</td><td>Sigma</td><td>C4895</td><td></td></tr><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>BP1760</td><td></td></tr><tr><td>BBL Trypticase soy broth</td><td>Fisher Scientific</td><td>B11768</td><td></td></tr><tr><td>BF2 P11A FITC</td><td>NeoScientific</td><td>Custom ordered</td><td></td></tr><tr><td>di-8-ANEPPS</td><td>Biotium</td><td>61012</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>34869</td><td>Used Sigma D8779 in original protocol development; D8779 discontinued with 34869 as replacement</td></tr><tr><td>Maleic acid</td><td>Sigma</td><td>M0375</td><td></td></tr><tr><td>Acrodisc 25 mm Syringe Filter w/ 0.2 &amp;mu;m HT Tuffryn Membrane</td><td>Pall Corporation</td><td>4192</td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td>Leica Microsystems</td><td>TCS SP5 II</td><td>For image acquisition</td></tr><tr><td>Leica Application Suite, Advanced Fluorescence</td><td>Leica Microsystems</td><td></td><td>For image processing</td></tr></tbody></table>

production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization

用共焦显微镜作为一种评估细菌内肽定位模式的方法, 通常被常规光学显微镜的分辨率限制所抑制。由于给定显微镜的分辨率不能很容易地增强, 我们提出了改变小棒状革兰氏阴性大肠杆菌 (大肠杆菌) 和革兰阳性芽孢杆菌巨大(巨大) 的协议 .成更大, 容易成像的球形形式称为 spheroplasts 或原生质体。这种转变使观察者能够迅速、清楚地确定肽是否进入细菌膜 (即膜定位) 或穿过细胞膜进入细胞 (即转运)。利用这种方法, 我们还提出了一种系统的方法来表征多肽作为膜定位或转运。这种方法可用于各种膜活性肽和细菌菌株, 我们通过观察 Buforin II P11A (BF2 P11A)、抗菌肽 (AMP) 与大肠杆菌的相互作用, 证明了本协议的实用性.spheroplasts 和B. 巨大原生质体。

通过扩大细菌和使它们球状, 我们可以很容易地区分是否多肽定位到细菌膜或容易植物常常将跨细菌膜。常规光学显微镜的分辨率限制使它具有挑战性, 以区分是否在正常细菌的细胞膜或细胞内空间出现多肽信号, 因为在膜上定位的信号似乎会与细胞内空间 (图 3A)。相比之下, spheroplasts (2–5µm) 和原生质体 (2–3µm) 的扩大大小与正常的细菌相比, 通常只有1µm 的直径, 结果在膜标记和细胞内空间之间的清晰分辨率, 使其更容易区分肽的定位 (图 3B)。
在这里, spheroplasts 和原生质体用于显示 N 终端 FITC 标记为安培 BF2 P11A 的本地化模式。我们观察 FITC 标记的 BF2 P11A 的两个局部的膜和植物常常将跨细胞膜的大肠杆菌spheroplasts 和B. 巨大原生质体 (图 4)。虽然野生型 BF2 已观察到植物常常将跨大肠杆菌和脂质囊泡膜, P11A 突变是已知减少此易位13,27,28。我们使用了非抗生素耐药性B. 巨大和氨苄西林耐药 10大肠杆菌(pET45B) 的数据提出。氨苄西林耐药性大肠杆菌生长在氨苄西林 (349.4 克/摩尔) 的存在, 最终浓度在溶液中的25µg/毫升。对于成像, 按照7节中概述的系统方法, ROIs 是在膜、细胞内空间和背景上绘制的 (图 5B)。利用7.2 节所描述的方程, 计算了每个 spheroplast 或原生质体与 BF2 P11A 相互作用的胞内肽荧光强度与肽荧光强度的比值。图 5A显示了这一比例的分布, 所有 spheroplasts 和原生质体得分。spheroplasts 和原生质体得分主要分为两组, spheroplasts 或原生质体的比例小于0.3 或大于1。
考虑到比值的不同组, 我们将肽的定位定义为转运, 当7.2 节计算的比值大于或等于1时。反之, 当7.2 节所描述的比值小于1时, 肽的定位被定义为膜本地化。通过这种评分方法, 我们观察了 BF2 P11A 在大肠杆菌spheroplasts 和巨大原生质体中的相似定位模式, BF2 P11A 在71% 和70% 的情况下对膜进行本地化, 用于大肠杆菌spheroplasts 和B. 巨大原生质体, 分别 (表 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57904/57904fig1.jpg"> 图 1:大肠杆菌的代表性图像。大肠杆菌(B) 大肠杆菌蛇和 (C) 大肠杆菌spheroplast. 图像是以100X 放大倍数拍摄的。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57904/57904fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57904/57904fig2.jpg"> 图 2: B. 巨大的代表性图像。 b. 巨大细菌和 (b) b. 巨大原生质体。图像是以100X 放大倍数拍摄的。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57904/57904fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57904/57904fig3.jpg"> 图 3: B. 巨大的代表性图像。(A)巨大细菌细胞和 (b)巨大原生质体, 用膜标记 di-8-ANEPPS 标记。中间 z 堆栈中的图像显示在 100X (A) 和 63X (B) 放大倍数。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57904/57904fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57904/57904fig4.jpg"> 图 4: 代表大肠杆菌spheroplasts 和B. 巨大原生质体与 FITC BF2 P11A 相互作用.(A)大肠杆菌spheroplast 显示 BF2 P11A 对膜进行定位。(B)大肠杆菌spheroplast 显示 BF2 P11A 转运在细胞膜上。(C) B. 巨大原生质体, 显示 BF2 P11A 在膜上定位。(D)巨大原生质体, 显示 BF2 P11A 转运在细胞膜上。大肠杆菌spheroplasts 和巨大原生质体均用膜标记 di-8-ANEPPS (红色) 和 FITC 标记 BF2 P11A (绿色) 标记。中间 z 堆栈中的图像以63X 放大显示。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57904/57904fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57904/57904fig5.jpg"> 图 5: 肽的定位模式分析.(A) 在背景减法 (roi 2-roi 3)/(roi 1-roi 3)) 后, 细胞内荧光强度 (roi 2) 与膜荧光强度 (roi 1) 的比值分布, 用于大肠杆菌spheroplasts 和B. 巨大原生质体标记与 BF2 P11A (B)大肠杆菌spheroplast 相互作用 FITC 标记 BF2 P11A。感兴趣的循环区域 (roi) 0.3 µm 在膜上绘制 (roi 1) 胞内空间 (roi 2) 和背景 (roi 3)。在每个 roi 中量化了肽的荧光强度, 并通过从 roi 1 和 roi 2 中的荧光强度减去 roi 3 中的荧光强度来解释背景荧光。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57904/57904fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>菌株</td>
			<td>大声 笑spheroplast 或原生质体</td>
			<td>% 膜本地化</td>
			<td>% 转运</td>
		</tr>
<tr>
<td>大肠杆菌</td>
			<td>84</td>
			<td>71</td>
			<td>29</td>
		</tr>
<tr>
<td>B. 巨大</td>
			<td>70</td>
			<td>70</td>
			<td>30</td>
		</tr>
</tbody></table>表 1: BF2 P11A 与之互动的本土化模式大肠杆菌 spheroplasts 和巨大原生质体.

Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

在这里, 我们提出了一个生产革兰氏阴性大肠杆菌 (大肠杆菌) spheroplasts 和革兰氏阳性芽孢杆菌巨大(巨大) 原生质体的协议, 以清楚地形象化和快速表征多肽-细菌的相互作用。这为定义膜定位和转运肽提供了系统的方法。

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

production-visualization-bacterial-spheroplasts-protoplasts-to

Universidad San Sebastian

Research

JoVE Journal

Biology

11.7K 次观看.  Wellesley College. 在这里, 我们提出了一个生产革兰氏阴性大肠杆菌 (大肠杆菌) spheroplasts 和革兰氏阳性芽孢杆菌巨大(巨大) 原生质体的协议, 以清楚地形象化和快速表征多肽-细菌的相互作用。这为定义膜定位和转运肽提供了系统的方法。

视频: Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

科研

生物化学

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

免疫与感染

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

生物工程

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

通过荧光报告基因定量进行全器官病原体追踪

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

作者聚焦:揭示细菌对组织中抗生素和免疫系统的反应

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染的高分辨率三维全器官断层扫描

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

摘要

探索更多视频

此视频中的章节

细菌内膜显示筛查抗体片段的图书馆

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

非规范氨基酸选择性压力合成抗菌肽的研究

枯草芽孢杆菌中细菌体和内膜的可视化

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

使用荧光抗生素探针对细菌耐药性的可视化

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描