Name: imaging ca2+ responses during shigella infection of epithelial cells
Uploaded: 2018-05-24T14:45:00
Description: Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

我们感谢珍妮-李. Thomassin 在编辑手稿方面的帮助。这项工作得到了情报局赠款 MITOPATHO 和 PATHIMMUN 的支持, Labex Memolife 和人民党 IDEX Shigaforce 提供赠款。春晖孙是来自中国奖学金委员会的博士助学金的接受者。劳伦 Combettes 和家伙搬运车 Nhieu 是 WBI-法国交换 Tournesol 计划 N°31268YG (Wallonie 布鲁塞尔国际, 全宗研究所科学研究, 部法国, étrangères et européennes, 部 de 的接受者。l ' Enseignement 高级研究所 dans 乐干部 des Partenariats 休伯特克瑞恩)。

1. 筹备工作
<ol><li>
细菌制剂
	<ol>
<li>板细菌-志贺菌野生型菌株, 表达 AfaE 黏附 (M90T AfaE)-在胰酪胨大豆 (TCS) 琼脂板上含有0.01% 刚果红 (CR) 和孵化他们18小时37摄氏度。 注意: 为了提高其重现性, 在步骤1.1.1 中获得的志贺菌板块储存在4摄氏度, 应在一周内使用, 因为 CR 培养基上的所有菌落最终会随着时间而变红。</li>
		<li>接种 TCS 肉汤前培养, 从条纹板采摘3红殖民地。 ?...

<ol>
	<li>Ashida, H., Ogawa, M., Kim, M., Mimuro, H., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+and+host+interactions+in+the+gut+epithelial+barrier.">Bacteria and host interactions in the gut epithelial barrier.</a> Nature Chemical Biology. 8 (1), 36-45 (2012).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signaling.">The versatility and universality of calcium signaling.</a> Nature Reviews Molecular Cell Biology. 1 (1), 11-21 (2000).</li><li>Strehler, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+calcium+ATPases:+from+generic+Ca(2+)+sump+pumps+to+versatile+systems+for+fine-tuning+cellular+Ca(2).">Plasma membrane calcium ATPases: from generic Ca(2+) sump pumps to versatile systems for fine-tuning cellular Ca(2).</a> Biochemical and Biophysical Research Communications. 460 (1), 26-33 (2015).</li><li>Muallem, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+Ca2++signals:+a+question+of+timing.">Decoding Ca2+ signals: a question of timing.</a> Journal of Cell Biology. 170 (2), 173-175 (2005).</li><li>Uhlen, P., Fritz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+calcium+oscillations.">Biochemistry of calcium oscillations.</a> Biochemical and Biophysical Research Communications. 396 (1), 28-32 (2010).</li><li>Carneiro, L. 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=Shigella+induces+mitochondrial+dysfunction+and+cell+death+in+nonmyleoid+cells.">Shigella induces mitochondrial dysfunction and cell death in nonmyleoid cells.</a> Cell Host &amp; Microbe. 5 (2), 123-136 (2009).</li><li>Horng, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+and+mitochondrial+destabilization+in+the+triggering+of+the+NLRP3+inflammasome.">Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome.</a> Trends in Immunology. 35 (6), 253-261 (2014).</li><li>Galan, J. E., Lara-Tejero, M., Marlovits, T. C., Wagner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+type+III+secretion+systems:+specialized+nanomachines+for+protein+delivery+into+target+cells.">Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells.</a> Annual Review of Microbiology. 68, 415-438 (2014).</li><li>Ashida, H., Mimuro, H., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+manipulates+host+immune+responses+by+delivering+effector+proteins+with+specific+roles.">Shigella manipulates host immune responses by delivering effector proteins with specific roles.</a> Frontiers in Immunology. 6, 219 (2015).</li><li>Tran Van Nhieu, 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=Actin-based+confinement+of+calcium+responses+during+Shigella+invasion.">Actin-based confinement of calcium responses during Shigella invasion.</a> Nature Communications. 4, 1567 (2013).</li><li>Niebuhr, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+PtdIns(4,5)P(2)+into+PtdIns(5)P+by+the+S.+flexneri+effector+IpgD+reorganizes+host+cell+morphology.">Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S. flexneri effector IpgD reorganizes host cell morphology.</a> The EMBO Journal. 21 (19), 5069-5078 (2002).</li><li>Konradt, C., 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=The+Shigella+flexneri+type+three+secretion+system+effector+IpgD+inhibits+T+cell+migration+by+manipulating+host+phosphoinositide+metabolism.">The Shigella flexneri type three secretion system effector IpgD inhibits T cell migration by manipulating host phosphoinositide metabolism.</a> Cell Host &amp; Microbe. 9 (4), 263-272 (2011).</li><li>Friedrich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intriguing+Ca2++requirement+of+calpain+activation.">The intriguing Ca2+ requirement of calpain activation.</a> Biochemical and Biophysical Research Communications. 323 (4), 1131-1133 (2004).</li><li>Thomas, D., 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+comparison+of+fluorescent+Ca2++indicator+properties+and+their+use+in+measuring+elementary+and+global+Ca2++signals.">A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals.</a> Cell Calcium. 28 (4), 213-223 (2000).</li><li>Sun, C. H., 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=The+Shigella+type+III+effector+IpgD+recodes+Ca2++signals+during+invasion+of+epithelial+cells.">The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells.</a> The EMBO Journal. 36 (17), 2567-2580 (2017).</li><li>Allaoui, A., Menard, R., Sansonetti, P. J., Parsot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Shigella+flexneri+IpgD+and+IpgF+genes,+which+are+located+in+the+proximal+part+of+the+mxi+locus.">Characterization of the Shigella flexneri IpgD and IpgF genes, which are located in the proximal part of the mxi locus.</a> Infection and Immunity. 61 (5), 1707-1714 (1993).</li></ol>

本手稿描述了在志贺菌入侵的相对较短的动力学过程中, 我们设计为遵循本地 ca2 +信号的协议, 以及志贺菌扩展动力学过程中的全局 ca2 +响应。下面, 需要解决的关键问题是优化 Ca2 +信号的检测, 同时尽量减少对生物过程的任何干扰。
化学vs基因编码的 Ca2 +探头:
为了图像本地 Ca2 +的变化, 我们使用?...

Ca2 +调节所有已知的单元进程, 包括骨架重组、炎症反应和与宿主-病原体相互作用有关的细胞死亡通路1,2,3。在生理条件下, 基底胞浆 Ca2 +浓度低, 在数以百计的 nM 范围内, 但可能会受到瞬变增加后激动剂刺激。这些变化经常表现出振荡行为, 通过泵和渠道在血浆和内质网膜的作用。这些振荡的模式以 ca2 +的周期、持续时间和振幅为特征, 并由单元格进行解密, 然后在被称为 Ca2 +代码4,5 中触发特定响应..在病理条件下, 胞浆 Ca2 +浓度的持续增加可能导致与线粒体膜通透相关的细胞死亡, 并释放亲凋亡或坏死因子6, 7。
志贺菌是细菌性痢疾的致病剂, 通过使用 III. 型分泌系统 (T3SS)8、9, 将效应器注入宿主细胞, 侵入上皮细胞。志贺菌入侵宿主单元格与 T3SS 引发的本地和全局 Ca2 +信号相关联。至于成孔毒素, T3SS translocon 插入宿主细胞膜, 并需要注入 T3SS 效应剂可能负责激活 PLC 和肌醇 (1, 4, 5) trisphosphate (InsP3) 依赖 Ca2 +释放。局部 PLC 刺激和聚合肌动蛋白在志贺菌入侵部位的聚集, 导致超持久的 InsP3-dependent Ca2 +发行10。III. 型效应器 IpgD, 磷脂 45 bisphosphate (PIP2)-4-磷酸酶, 限制了局部 PIP2 量, 从而控制了可编程序控制器生成 InsP3 的可用基板的数量, 这有助于限制本地 Ca2 +细菌入侵站点的响应11,12。这些本地 Ca2 +响应可能会导致在志贺菌入侵站点10的肌动蛋白聚合。但是, 由志贺氏引起的全局 Ca2 +响应是细菌入侵过程不可缺少的, 但会触发连接蛋白 hemichannels 在等离子膜上的打开和胞外 ATP 的释放。车厢。以分泌方式释放的 ATP 将反过来刺激受感染细胞旁边的单元格中的 Ca2 +振荡响应。IpgD 还负责将全局 Ca2 +响应塑造为不稳定的孤立响应, 并具有缓慢的动态。最后, 在长时间的细菌感染后, IpgD 导致 InsP3-mediated Ca2 +信号的抑制。通过对 ca2 +信号的干扰, IpgD 延迟了与 ca2 +相关的钙蛋白酶激活, 导致了焦点黏附结构的拆卸和感染细胞的过早脱离 13.
虽然 Ca2 +信号涉及病机的关键方面, 但微生物的使用引发了一些在经典激动剂研究中没有遇到的技术挑战。此处描述的协议使用常用的荧光 Ca2 +化学指标 Fluo-4, 我们在志贺病毒感染期间设计了用于表征本地 Ca2 +信号的特性。讨论了对这些信号进行检测的关键步骤, 以及为进行定量分析所需的程序, 以确定细菌效应在 Ca2 +信号中的作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:693px;" width="694">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fluo-4 AM</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:136px;">F14201</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Metamorph version 7.7</td>
			<td style="width:120px;">Universal Imaging</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CoolLED illumination system pE-2</td>
			<td style="width:120px;">Roper Scientific</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">micro-dish 35 mm, high&#160;</td>
			<td style="width:120px;">IBIDI</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypticase Soy (TCS) broth</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11768</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">TCS agar</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Congo red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:136px;"></td>
			<td>75768</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">M90T-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">Shigella flexneri serotype V. expressing the AfaE adhesin</td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">ipgD-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">isogenic ipgD mutant strain expressing the AfaE adhesin</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging ca2+ responses during shigella infection of epithelial cells

Ca2 +是所有已知的蜂窝进程中普遍存在的一个离子。尽管全局 Ca2 +响应可能会影响细胞的命运, 但在自由 Ca2 +胞浆浓度的局部变化中, 与内部存储区的释放有关, 或者通过等离子膜通道流入, 调节皮层细胞的过程。附着或侵入宿主细胞的病原体触发了宿主血浆膜肌动蛋白细胞骨架的重组, 这可能会影响全局和局部 Ca2 +信号。由于这些事件可能以伪随机方式出现在扩展动力学上, 因此, 分析由病原体引起的 Ca2 +信号会引发需要解决的重大技术挑战。
在这里, 我们报告的协议, 以检测全球和本地 Ca2 +信号后,志贺菌感染上皮细胞。在这些协议中, 与 Ca2 +荧光探针的激发相关的长时间暴露和光损伤关联的手工制品诊断在志贺菌入侵。在使用化学探针 Fluo-4 对扩展感染动力学过程中的全局胞浆 Ca2 +信号的振幅和频率进行严格分析的过程被实施。

志贺菌入侵与非典型的持久性本地 Ca2 +响应关联:
根据上述协议, Fluo-4-loaded HeLa 细胞受到了志贺菌的挑战, 并进行了流采集以分析 Ca2 +信号。一个具有代表性的实验显示在图 1中, 其中的时间推移图像系列的荧光强度的 Fluo-4 探针平均在一个区域感兴趣的一个单元, 和相应的相位?...

Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

在这里, 我们提出的协议, 可视化钙 (Ca2 +) 的反应引起的 HeLa 细胞感染, 由志贺菌。通过对 ca2 +荧光探针的细菌感染和成像参数进行优化, 提出了由细菌引起的非典型全局和局部 Ca2 +信号在大范围的感染动力学中的特征。

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Research

JoVE Journal

Immunology and Infection

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

科研

免疫与感染

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...