Name: assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes
Uploaded: 2015-05-27T14:00:00
Description: Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

这项工作得到了公共卫生服务机构AI41816和AI099339（N.E.F.）和F31AI094886-01（P.D..M）的支持。 内容完全由作者负责，不一定代表资金来源的官方观点。

1. L. 单细胞菌株的 储存和文化条件
<ol><li>通过自动切割脑心输液（BHI）和将熔融介质倒入培养板来准备固体介质。让盘子在室温下干燥过夜，然后在37°C处再通宵干燥。</li>
	<li>使用无菌循环，从以前制造的冰柜储存（BHI液体介质中20%甘油中悬浮的细菌，储存在-80°C）或含有以前被隔离的菌落的藻类板中获取少量 L.单细胞基因 样本。</li>
	<li>使用不化技术，对样本进行单个菌落隔离...

<ol>
	<li>Freitag, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+hot+dogs+to+host+cells:+how+the+bacterial+pathogen+Listeria+monocytogenes+regulates+virulence+gene+expression.">From hot dogs to host cells: how the bacterial pathogen Listeria monocytogenes regulates virulence gene expression.</a> Future Microbiol. 1, 89-101 (2006).</li><li>Drevets, D. A., Bronze, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes:+epidemiology,+human+disease,+and+mechanisms+of+brain+invasion.">Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion.</a> FEMS Immunol Med Microbiol. 53, 151-165 (2008).</li><li>Farber, J. M., Peterkin, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes.+a+food-borne+pathogen.">Listeria monocytogenes. a food-borne pathogen.</a> Microbiol. Rev. 55, 476-511 (1991).</li><li>Lecuit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+how+Listeria+monocytogenes+targets+and+crosses+host+barriers.">Understanding how Listeria monocytogenes targets and crosses host barriers.</a> Clin Microbiol Infect. 11, 430-436 (2005).</li><li>Lecuit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+listeriosis+and+animal+models.">Human listeriosis and animal models.</a> Microbes Infect. 9, 1216-1225 (2007).</li><li>Bou Ghanem, E. N., 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=InlA+promotes+dissemination+of+Listeria+monocytogenes+to+the+mesenteric+lymph+nodes+during+food+borne+infection+of+mice.">InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice.</a> PLoS Pathog. 8, e1003015 (2012).</li><li>Melton-Witt, J. A., Rafelski, S. M., Portnoy, D. A., Bakardjiev, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+infection+with+signature-tagged+Listeria+monocytogenes+reveals+organ-specific+growth+and+dissemination+routes+in+guinea+pigs.">Oral infection with signature-tagged Listeria monocytogenes reveals organ-specific growth and dissemination routes in guinea pigs.</a> Infect Immun. 80, 720-732 (2012).</li><li>Alonzo, F., Bobo, L. D., Skiest, D. J., Freitag, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+subpopulations+of+Listeria+monocytogenes+with+enhanced+invasion+of+cardiac+cells.">Evidence for subpopulations of Listeria monocytogenes with enhanced invasion of cardiac cells.</a> J Med Microbiol. , (2011).</li><li>Adler, 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=Inflammatory+pseudotumor+of+the+heart+caused+by+Listeria+monocytogenes+infection.">Inflammatory pseudotumor of the heart caused by Listeria monocytogenes infection.</a> J Infect. 58, 161-163 (2009).</li><li>Antolin, J., Gutierrez, A., Segoviano, R., Lopez, R., Ciguenza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocarditis+due+to+Listeria:+description+of+two+cases+and+review+of+the+literature.">Endocarditis due to Listeria: description of two cases and review of the literature.</a> Eur J Intern Med. 19, 295-296 (2008).</li><li>Brouqui, P., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocarditis+due+to+rare+and+fastidious+bacteria.">Endocarditis due to rare and fastidious bacteria.</a> Clin Microbiol Rev. 14, 177-207 (2001).</li><li>Brusch, 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=Cardiac+infections+in+the+immunosuppressed+patient.">Cardiac infections in the immunosuppressed patient.</a> Infect Dis Clin North Am. 15, 613-638 (2001).</li><li>McCue, M. J., Moore, 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=Myocarditis+with+microabscess+formation+caused+by+Listeria+monocytogenes+associated+with+myocardial+infarct.">Myocarditis with microabscess formation caused by Listeria monocytogenes associated with myocardial infarct.</a> Hum Pathol. 10, 469-472 (1979).</li><li>Menard, 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=Modulation+of+L-type+calcium+channel+expression+during+retinoic+acid-induced+differentiation+of+H9C2+cardiac+cells.">Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells.</a> J Biol Chem. 274, 29063-29070 (1999).</li><li>Czuprynski, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes:+silage,+sandwiches+and+science.">Listeria monocytogenes: silage, sandwiches and science.</a> Anim Health Res Rev. 6, 211-217 (2005).</li><li>Grundler, T., 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+surface+proteins+InlA+and+InlB+are+interdependently+required+for+polar+basolateral+invasion+by+Listeria+monocytogenes+in+a+human+model+of+the+blood-cerebrospinal+fluid+barrier.">The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.</a> Microbes Infect. 15, 291-301 (2013).</li><li>Zeldovich, V. B., Robbins, J. R., Kapidzic, M., Lauer, P., Bakardjiev, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+extravillous+trophoblasts+restrict+intracellular+growth+and+spread+of+Listeria+monocytogenes.">Invasive extravillous trophoblasts restrict intracellular growth and spread of Listeria monocytogenes.</a> PLoS Pathog. 7, e1002005 (2011).</li><li>Kimes, B. W., Brandt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+a+clonal+muscle+cell+line+from+rat+heart.">Properties of a clonal muscle cell line from rat heart.</a> Experimental cell research. 98, 367-381 (1976).</li><li>McMullen, P. 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=Genome+sequence+of+Listeria+monocytogenes+07PF0776,+a+cardiotropic+serovar+4b+strain.">Genome sequence of Listeria monocytogenes 07PF0776, a cardiotropic serovar 4b strain.</a> J Bacteriol. 194, 3552 (2012).</li></ol>

L. 单细胞基因 是一种广泛且特征良好的人类病原体，能够引起多种不同的疾病表现。此前，该细菌曾被描述为能够跨越障碍物（如血脑屏障和胎盘-胎儿屏障）进行迁移，以便分别到达和殖民中枢神经系统并发育胎儿。生物体 的体内 殖民能力通常辅之以 体外 入侵构成目标器官的培养中具有代表性的细胞的能力。例如，胆囊丛中上皮细胞的入侵与生物体殖民CNS16</su...

李斯特菌单细胞基因是一种革兰阳性细胞内病原体，可在易感人群中引起严重疾病，包括老年人、孕妇、艾滋病毒/艾滋病感染者和接受化疗1的人。这些人群的感染往往是摄入受污染的食品造成的，大多数感染都与2，3的大规模食源性疫情有关。在人类和其他哺乳动物中，L.单细胞基因能够跨越小肠的上皮边界，然后被输送到肝脏4，5。动物模型表明，摄入的细菌在肠道内复制，并通过门户静脉传播到肝脏，或通过间质淋巴结扩散到血液中，导致血液向肝脏和脾脏传播6，7。在肝脏和脾脏中，细菌能够介入专业噬菌体和常驻帕丘奇细胞，并迅速在这些器官内建立感染。随着细菌负荷的增加，许多细菌被分散回血液中，在那里它们能够进一步殖民易感组织，包括中枢神经系统和胎盘（在目前）。这些部位的殖民化排除了人类李斯特菌病的最常见表现，包括脑膜炎、脑炎和胎儿流产2。
选定的L.单细胞基因亚种最近被证明具有增强的能力，入侵和复制心脏组织8。心脏介入的表现是多种多样的，范围从心内膜炎和心包炎，到完全由传导异常9-13完成的足部心肌炎。每年L.单细胞基因心脏病例总数较低，但估计可能不足，因为这种感染方面没有得到普遍认可。通过病原体殖民化心脏通常需要宿主倾向，如预先存在的血管损伤或人工心脏瓣膜。然而，已经发现的L.单细胞基因的分离物，是值得注意的能力殖民感染动物的心脏，没有任何心脏损伤和/或异常8。
本文介绍了 体外 和 体内 方法，用于评估受感染动物心脏组织中的细菌殖民化，使用组织培养和活体动物感染的入侵检测。这些方法不仅有助于识别有可能在受感染动物中殖民心脏组织的菌株，而且应该有助于识别细菌基因产品，这些基因产品有助于增强心脏细胞入侵和/或导致心脏病理学的变化。这些方法有助于比较多种菌株之间的心肌梗塞。对于此处描述的方法 ，L. 单细胞基因 10403S 用作非心电应变的经过充分研究的代表，临床隔离 07PF0776 用作心电应变的代表性示例。这两种菌株的选择为细菌入侵心脏细胞 体外 和殖民化受感染的小鼠的心脏 在体内提供了比较。分离07PF0776是一种临床隔离物，从导致艾滋病毒患者8致命心律失常的心室间脓肿中恢复过来。 L. 单细胞基因 分离物的毒性潜力可能有所不同，鉴于 李斯特菌 感染免疫抑制者和孕妇的倾向，这些人群中的人在评估不同的临床隔离物时应谨慎行事。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Difco</td>
			<td>BHI Broth Base</td>
			<td>237200</td>
			<td>2KG of Brain Heart Infusion powder (without agar)</td>
		</tr>
		<tr>
			<td>Difco</td>
			<td>Agar</td>
			<td>214510</td>
			<td>2KG of Granulated Agar</td>
		</tr>
		<tr>
			<td>Difco</td>
			<td>BHI Agar</td>
			<td>241810</td>
			<td>2KG of Brain Heart Infusion Powder with 7.5% Agar</td>
		</tr>
		<tr>
			<td>in vitrogen</td>
			<td>LB Broth Base</td>
			<td>12975-084</td>
			<td>2KG of Luria Broth Base Powder (without agar)</td>
		</tr>
		<tr>
			<td>Fisher</td>
			<td>Glass Coverslips</td>
			<td>12-545-80</td>
			<td>12mm glass coverslips</td>
		</tr>
		<tr>
			<td>Falcon</td>
			<td>24-well Tissue Culture Plate</td>
			<td>35-3047</td>
			<td>24 Well, Plasma-treated Tissue Culture Plate</td>
		</tr>
		<tr>
			<td>Falcon</td>
			<td>14mL Polystyrene tube</td>
			<td>352051</td>
			<td>14mL Polystyrene tubes</td>
		</tr>
		<tr>
			<td>Falcon</td>
			<td>96-well U-bottom plate</td>
			<td>35-3077</td>
			<td>Non-tissue culture treated 96-well plate</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>0.05% Trypsin, 0.53 mM EDTA (1X)</td>
			<td>25-052-CI</td>
			<td>Stock solution of Trypsin EDTA for tissue culture</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>DMEM (High glucose, high pyruvate)</td>
			<td>15-013-CU</td>
			<td>DMEM media without FBS, glutamine, or antibiotics</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>Pen/Strep/Glut Solution</td>
			<td>30-009-CI</td>
			<td>Stock mixture of penicillin, streptomycin, and L-glutamine for tissue culture</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>Gentamicin</td>
			<td>30-005-CR</td>
			<td>50mg/mL Stock solution of Gentamicin for tissue culture</td>
		</tr>
		<tr>
			<td>Cellgro (Corning)</td>
			<td>L-Glutamine</td>
			<td>25005197</td>
			<td>Stock L-glutamine solution without antibiotics</td>
		</tr>
		<tr>
			<td>Denville</td>
			<td>75cm^2 Flask</td>
			<td>T1225</td>
			<td>75cm^2 tissue culture treated flask</td>
		</tr>
		<tr>
			<td>Denville</td>
			<td>1.5mL microcentrifuge tubes</td>
			<td>2013004</td>
			<td>1.5mL microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>ATCC</td>
			<td>H9c2 Cardiac Myoblasts</td>
			<td>CRL-1446</td>
			<td>Rat cardiac myoblasts</td>
		</tr>
		<tr>
			<td>Hyclone</td>
			<td>Fetal Bovine Serum</td>
			<td>SH30070.63</td>
			<td>Fetal Bovine Serum</td>
		</tr>
	</tbody>
</table>

assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes

李斯特菌单细胞基因 是一种革兰氏阳性的细胞内病原体，能够引起免疫功能低下患者、老年人和孕妇的严重侵入性感染。 人类李斯特菌病最常见的表现包括脑膜炎、脑炎和胎儿流产。 侵入性 L.单细胞基因 感染的重要但记录较少的后遗症涉及心脏。 尽管治疗，心脏病的死亡率可能高达35%，但是对于心脏组织的 L.单细胞基因 殖民化及其由此产生的病理，人们所知甚少。 此外，最近很明显 ，L.单细胞基因 的亚群具有增强的能力，入侵和生长在心脏组织内。 该协议详细描述了 体外 和 体内 可用于评估 L.单细胞基因 分离心肌梗塞的方法。 介绍了在组织培养中感染H9c2大鼠心脏肌细胞的方法，以及确定受感染小鼠心脏的细菌殖民化。 这些方法不仅可用于识别有可能在受感染动物中殖民心脏组织的菌株，而且还可能促进细菌基因产品的识别，这些基因产品有助于增强心脏细胞入侵和/或推动心脏病理学的变化。 这些方法还规定了多个 L.单细胞基因 菌株之间的心电图谱的直接比较。

选定的 单细胞基因 分离物在细胞培养和小鼠感染模型中表现出对心脏细胞的增强入侵。 图1 显示了在阿加介质上当场电镀悬浮后细菌菌落可能如何出现的示例。此方法允许在样品中准确评估 CF，而无需使用大量 agar 介质板。 图2 显示了一个基于组织培养的检测示例，该分析将10403S菌株入侵心脏细胞的能力与菌株07PF0776的能力进行了比较。与感染10403S的细胞相比，?...

Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

李斯特菌单细胞基因 导致孕妇胎儿感染和易感人群脑膜炎。 细菌的亚种群可以殖民心脏组织，导致患者和实验室动物心肌炎。 在这里，我们提出了一个协议，描述了如何评估 L.单细胞基因 心脏细胞 入侵体外 和心脏殖民化感染动物。

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

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The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...