Name: automated, high-throughput detection of bacterial adherence to host cells
Uploaded: 2021-09-17T14:00:00
Description: Watch this Scientific Journal Video about Automated, High-Throughput Detection of Bacterial Adherence to Host Cells at JoVE.com

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1. A549细胞培养
<ol><li>将A549细胞系维持在补充有10%胎牛血清（FBS）的F-12K培养基中，并在37°C，5%CO2下孵育。</li>
	<li>每3-4天更换一次培养基，以85%-95%的汇合度通过。</li>
	<li>简而言之，用1x磷酸盐缓冲盐水（PBS，pH 7.4，除非另有说明）冲洗细胞，并在37°C下用1ml 0.25%胰蛋白酶-0.53mM乙二胺四乙酸（EDTA）溶液（浸没细胞层）处理约2分钟。</li>
	<li>加入额外的6mL完全生长培养基（F-12K培养基+ 10%F...

<ol>
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P., Barton, B., Morgenstein, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Imaging+of+bacterial+cells+for+accurate+cellular+representations+and+precise+protein+localization.">Three-dimensional Imaging of bacterial cells for accurate cellular representations and precise protein localization.</a> Journal of Visualized Experiments. (152), e60350 (2019).</li><li>Hoffmann, 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=High-throughput+quantification+of+bacterial-cell+interactions+using+virtual+colony+counts.">High-throughput quantification of bacterial-cell interactions using virtual colony counts.</a> Frontiers in Cellular and Infection Microbiology. 8, 43 (2018).</li><li>Hazan, R., Que, Y. -. A., Maura, D., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high+throughput+determination+of+viable+bacteria+cell+counts+in+96-well+plates.">A method for high throughput determination of viable bacteria cell counts in 96-well plates.</a> BMC Microbiology. 12 (1), 259 (2012).</li><li>Gellatly, S. L., Hancock, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa:+new+insights+into+pathogenesis+and+host+defenses.">Pseudomonas aeruginosa: new insights into pathogenesis and host defenses.</a> Pathogens and Disease. 67, 159-173 (2013).</li><li>Jiang, R. D., Shen, H., Piao, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphometrical+analysis+on+the+ultrastructure+of+A549+cells.">The morphometrical analysis on the ultrastructure of A549 cells.</a> Romanian Journal of Morphology and Embryology. 51 (4), 663-667 (2010).</li><li>Farinha, M. 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=Alteration+of+the+pilin+adhesin+of+Pseudomonas+aeruginosa+PAO+results+in+normal+pilus+biogenesis+but+a+loss+of+adherence+to+human+pneumocyte+cells+and+decreased+virulence+in+mice.">Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice.</a> Infection and Immunity. 62 (10), 4118-4123 (1994).</li><li>R&#233;glier-Poupet, H., Pellegrini, E., Charbit, A., Berche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+LpeA,+a+PsaA-Like+membrane+protein+that+promotes+cell+entry+by+Listeria+monocytogenes.">Identification of LpeA, a PsaA-Like membrane protein that promotes cell entry by Listeria monocytogenes.</a> Infection and immunity. 71 (1), 474-482 (2003).</li><li>Ortega, F. 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=Adhesion+to+the+host+cell+surface+is+sufficient+to+mediate+Listeria+monocytogenes+entry+into+epithelial+cells.">Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.</a> Molecular Biology of the Cell. 28 (22), 2945-2957 (2017).</li></ol>

该协议描述了一种用于枚举细菌附着在宿主细胞上的自动化方法。与传统方法相比，所描述的方法具有几个有吸引力的优点。首先，这种方法能够精确定量附着在单个宿主细胞上的微生物病原体细胞的数量。重要的是，这种定量可以在不需要费力的细菌收获，连续稀释，在固体培养基上电镀以及CFU10，11，12的测定的情况下进行...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62764">Automated, High-Throughput Detection of Bacterial Adherence to Host Cells</a>. The Authors section was updated.
The Authors section was updated from:
Jing Yang1, Qing-Ming Qin1, Paul de Figueiredo1,2 
1Department of Microbial Pathogenesis and Immunology, Texas A&#38;M Health Science Center 
2Department of Veterinary Pathobiology, Texas A&#38;M College of Veterinary Medicine
to:
Jing Yang1, Qing-Ming Qin1, Erin Van Schaik1, James E. Samuel1, Paul de Figueiredo1,2 
1Department of Microbial Pathogenesis and Immunology, Texas A&#38;M Health Science Center 
2Department of Veterinary Pathobiology, Texas A&#38;M College of Veterinary Medicine

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62764">Automated, High-Throughput Detection of Bacterial Adherence to Host Cells</a>. The Authors section was updated.

Erratum: Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

细菌粘附是细菌附着在其他细胞或表面上的过程。细菌病原体感染的成功建立需要粘附于宿主细胞，定植组织，并且在某些情况下，侵入宿主细胞1，2，3。新出现的传染病构成重大的公共卫生威胁，最近的COVID-19大流行4，5，6证明了这一点。重要的是，使用基于基因组的方法可能无法轻易识别新的或正在出现的病原体，特别是在病原体被设计为逃避检测或不包含将其识别为致病性的基因组特征的情况下。因此，使用直接评估致病性特征的方法（如细菌对宿主细胞的粘附性）鉴定潜在病原体可以在病原体鉴定中起关键作用。
细菌对宿主细胞的粘附性已被用于评估细菌发病机制数十年1，7。显微成像8、9和通过感染后电镀枚举细菌集落形成单元（CFU）10、11、12、13是两种成熟的实验室方法，用于测试微生物的粘附和/或宿主细胞的感染14。考虑到细菌细胞的微米级大小，贴壁细菌细胞的枚举通常需要使用先进的高倍率显微镜技术，以及高分辨率成像方法，包括电子显微镜，膨胀显微镜（ExM）15，16和三维成像17.或者，可以通过在固体琼脂上接种收获的细菌的稀释系列并计数所得的CFU10，12，13来计数与宿主细胞结合或内化在宿主细胞内的细菌的枚举。这种方法费力，包括许多手动步骤，这给建立高通量分析所需的标准化或自动化程序带来了困难18，19。因此，开发用于评估宿主细胞附着的新方法将解决该领域当前的局限性。
这里描述了一种这样的方法，它使用自动化高通量显微镜，结合高通量图像处理和统计分析。为了证明这种方法，对几种细菌病原体进行了实验，包括铜绿假单胞菌，一种人类，动物和植物的机会性革兰氏阴性细菌病原体14，20，它经常被发现定植于宿主防御功能受损患者的呼吸道。这种方法优化了先前研究14，20中描述的显微成像过程。通过荧光标记的宿主细胞和细菌简化了成像检测，以快速跟踪它们的接近程度，这大大减少了显微镜工作量，以获得用于区分细菌的高分辨率图像。此外，对计数宿主细胞和细菌中的图像进行自动统计分析取代了细菌CFU接种的动手实验，以估计每个宿主细胞的贴壁细菌计数的比率。为了证实该方法的相容性，还测试了多种细菌菌株和宿主细胞类型，如单核细胞增多性李斯特菌，金黄色葡萄球菌，蜡样芽孢杆菌和肺炎克雷伯菌，以及人脐静脉内皮细胞（HUVECs），结果支持该方法的多样性和有效性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >10x PBS</td>
			<td >VWR</td>
			<td >45001-130</td>
			<td ></td>
		</tr>
		<tr >
			<td >4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td >Thermo Fisher</td>
			<td>62248</td>
			<td>Host cell staining dye</td>
		</tr>
		<tr >
			<td >96 well plate</td>
			<td >Corning</td>
			<td >3882</td>
			<td>Half area well, flat clear bottom</td>
		</tr>
		<tr >
			<td >A549 cells</td>
			<td >ATCC&#160;</td>
			<td >CCL 185</td>
			<td>Mammalian cell line</td>
		</tr>
		<tr >
			<td >BactoView Live Red</td>
			<td >Biotium</td>
			<td >40101</td>
			<td>Bacteria staning dye</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Eppendorf</td>
			<td >5810R</td>
			<td></td>
		</tr>
		<tr >
			<td >CFSE cell division tracker</td>
			<td >BioLegend</td>
			<td >423801</td>
			<td></td>
		</tr>
		<tr >
			<td >Cytation 5&#160;</td>
			<td >BioTek</td>
			<td >Cytation 5&#160;</td>
			<td>Cell imaging multi-mode reader</td>
		</tr>
		<tr >
			<td >E. coli</td>
			<td ></td>
			<td ></td>
			<td>Laboratory stock&#160;</td>
		</tr>
		<tr >
			<td >EGM bulletKit</td>
			<td >Lonza</td>
			<td >CC-3124</td>
			<td>HUVEC cell culture medium</td>
		</tr>
		<tr >
			<td >EHEC</td>
			<td ></td>
			<td ></td>
			<td >NIST collections</td>
		</tr>
		<tr >
			<td >F-12k medium</td>
			<td >ATCC&#160;</td>
			<td >302004</td>
			<td>A549 cell culture medium</td>
		</tr>
		<tr >
			<td >Fetal bovine serum</td>
			<td >Corning</td>
			<td >35-016-CV</td>
			<td></td>
		</tr>
		<tr >
			<td >HUVEC</td>
			<td ></td>
			<td ></td>
			<td>Laboratory stock&#160;</td>
		</tr>
		<tr >
			<td >L. monocytogenes</td>
			<td ></td>
			<td ></td>
			<td >NIST collections</td>
		</tr>
		<tr >
			<td >OD600 DiluPhotometer</td>
			<td >IMPLEN</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >P. aeruginosa</td>
			<td ></td>
			<td ></td>
			<td>Dr. Lori Burrows laboratory stock</td>
		</tr>
		<tr >
			<td >P. aeruginosa &#916;pilA</td>
			<td ></td>
			<td ></td>
			<td>Dr. Lori Burrows laboratory stock</td>
		</tr>
		<tr >
			<td >S. agalactiae</td>
			<td ></td>
			<td ></td>
			<td >NIST collections</td>
		</tr>
		<tr >
			<td >S. aureus</td>
			<td >BEI</td>
			<td >NR-46543</td>
			<td></td>
		</tr>
		<tr >
			<td >S. aureus &#916;saeR</td>
			<td >BEI</td>
			<td >NR-48164</td>
			<td></td>
		</tr>
		<tr >
			<td >S. rubidaea</td>
			<td ></td>
			<td ></td>
			<td >NIST collections</td>
		</tr>
		<tr >
			<td >Typical soy broth</td>
			<td >Growcells</td>
			<td >MBPE-4040</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated, high-throughput detection of bacterial adherence to host cells

识别新出现的细菌病原体对人类健康和安全至关重要。细菌对宿主细胞的粘附是细菌感染的重要步骤，并构成潜在威胁的标志。因此，检查细菌对宿主细胞的粘附性可以用作细菌威胁评估的一个组成部分。枚举细菌对宿主细胞的粘附的标准方法是将细菌与宿主细胞共同孵育，收获贴壁细菌，将收获的细胞接种在固体培养基上，然后计算所得的菌落形成单位（CFU）。或者，可以使用基于免疫荧光显微镜的方法评估细菌对宿主细胞的依从性。然而，实施这些方法的传统策略既耗时又低效。在这里，描述了最近开发的基于自动荧光显微镜的成像方法。当与高通量图像处理和统计分析相结合时，该方法可以快速定量粘附在宿主细胞上的细菌。测试了两种细菌物种，铜绿革兰氏阴 性假单胞菌 和革兰氏阳性 单核细胞增多性李斯特菌 以及相应的阴性对照，以证明方案。结果表明，这种方法可以快速准确地枚举贴附细菌，并显着减少实验工作量和时间表。

为了开发基于荧光成像的细菌粘附测定法，使用铜绿假单胞菌菌株PAO1及其阴性粘附对应物大肠杆菌来测试方案有效性，因为这些细菌对A549细胞的粘附已有报道14，20，22。首先，分别在各种MOI下将GFP标记的铜绿假单胞菌（PAO1）和GFP标记的大肠杆菌与人类永生化上皮细胞系A549共同孵育。结果表明，PAO1?...

Watch this Scientific Journal Video about Automated, High-Throughput Detection of Bacterial Adherence to Host Cells at JoVE.com

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

使用高通量荧光标记成像以及自动统计分析方法，基于表型粘附性检测宿主 - 细菌病原体相互作用，可以快速评估细菌与宿主细胞的潜在相互作用。

自动、高通量检测细菌对宿主细胞的粘附

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

该方法可快速、高通量地检测细菌对宿主细胞的依从性。与传统的电镀方法相比，它减少了定量粘附细菌所需的手动操作时间。这种技术是自动化的，节省时间的，并且非常可重复。它也广泛地应用于大多数宿主细胞。这种方法，有非常简单。对于第一次试验，必须优化感染的多重性以及宿主细菌共培养时间等条件。首先通过在室温下以13， 000倍G离心两分钟，在指数阶段收获细菌培养物。用一毫升PBS洗涤细胞托盘后，将细菌调色板重悬于一毫升PBS中。通过测量600纳米处的光密度来确定细菌悬浮液的浓度。然后，为了染色细菌悬浮液，将两微升的500倍浓缩原液加入绿色或红色染色染料到一毫升细菌悬浮液中，以稀释染料一倍。将细胞在室温下孵育30分钟，在黑暗中轻轻旋转。30分钟后，将染色的细菌以13， 000倍G离心两分钟，并将沉淀重悬于一毫升PBS中。通过以13， 000倍G离心两分钟收集染色的细菌细胞。将沉淀重悬于一毫升新鲜F12 K培养基中，并在600纳米处测量每种培养物的光密度。随后根据感染的多样性和宿主细胞浓度将培养物稀释至所需浓度。对于细菌粘附性测定，通过向每个孔中加入100微升温PBS并轻轻地上下移液，将先前培养的A549细胞单层洗涤三次。然后丢弃 PBS。或者，添加 PBS 后，等待 10 秒钟，然后使用真空吸尘器取出 PBS。为了确定细菌关联的动力学，用具有不同感染多样性的100微升所需浓度的细菌悬浮液覆盖细胞。将细菌旋转下来，并将受感染的A549细胞在37摄氏度和5%二氧化碳下再孵育一小时。如图所示，用温暖的PBS清洗单层五次，去除未结合的细菌。接下来，通过在冰上96孔板的所有孔中加入100微升4%甲醛来固定细胞。15分钟后，用PBS清洗板三次以除去固定溶液。在室温下用每毫升50纳克DAPI染色剂在细胞核中染色10分钟。用PBS洗涤细胞后，用100微升PBS覆盖受感染的A549细胞以避免干燥，然后将板在四摄氏度下在黑暗中储存长达两天或进行成像。为了保持数据完整性，随机和手动选择每个孔的五个位置，以20倍放大倍率捕获图像。在GFP通道下捕获细菌的荧光图像，在DAPI下捕获A549细胞的荧光图像，并且对于由红色荧光染料染色的细菌使用PE Si五通道。设置滚动球的参数进行平滑处理，并根据目标自动测量图像反卷积的点扩散功能，以实现更好的分辨率。测量宿主细胞和细菌的荧光强度。将宿主细胞和细菌的最弱荧光强度设置为细胞计数阈值，并将15微米距离内靠近宿主细胞的所有细菌计数为粘附细菌。从所有自动图像中对细胞进行计数后，分析关键读数，例如宿主细胞计数，细胞大小和形状，总细菌计数以及每个宿主细胞的平均细菌帐户，这是确定细菌依从性的最重要指标。经过一小时的co孵育后，铜绿假单胞菌菌株PAO1以剂量依赖性方式粘附在A549细胞上。当革兰氏阳性细菌单核细胞增多性李斯特菌及其阴性对照枯草芽孢杆菌时，也观察到类似的结果。宿主贴壁铜绿假单胞菌分割的代表性图像表明，使用该协议对细菌细胞进行计数变得容易。自动分析的结果包括细菌和宿主细胞计数，每个宿主细胞的平均细菌计数和宿主细胞大小以及代表宿主细胞健康状态，细菌粘附水平和细菌细胞毒性的区域。除A549细胞外，HUVECs还进行了粘附测定测试。红沙雷氏菌和无乳链球菌粘附于HUVEC，但不粘附于A549细胞，这表明多个宿主使用该方法研究宿主细菌相互作用的有效检测和适用性。宿主细胞应达到约90%至100%的汇合度，以尽量减少不希望的细菌粘附在孔的底部。这项技术将使研究人员能够探索新的机制，即宿主与害虫的相互作用。

Research

JoVE Journal

Immunology and Infection

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

科研

免疫与感染

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

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 ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

高通量化学化合物筛选，以阐明其对细菌持久性的影响

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...