我们感谢箴。伯纳德香格里拉斯科拉的有益的技术咨询和amoebal共培养和amoebal充实有趣的讨论。我们也感谢博士文森特托马斯，他在实施该技术在我们实验室的帮助。

1。 Amoebal共培养 1.1样品制备<ol><li> 环境样品 <ol><li> 水样 通过0.22μm孔径的膜过滤水样（500毫升至1升）。然后，摇动膜在页面的阿米巴盐水介质首席助理秘书长（120毫克氯化钠，4毫克硫酸镁4•7H 2 O，4毫克氯化钙2•2H 2 O，142毫克的Na 2 HPO 4，和136毫克的KH 2 PO 4在1升的蒸馏水）。 </li><li> 固体样品 悬浮固体样品像土壤或沙子样品和半固体样品，如活性污泥在蒸馏水或PBS中，通过0.22μm孔径的膜过滤。然后，在摇PAS膜。 </li><li> 样品严重污染的内生阿米巴原虫和 首先通过低速离心（180×g离心）FO沉淀样品-R 10分钟或通过5微米孔径的膜过滤。进一步处理上清液（分别滤液），如在1.1.1。 注：其他净化技术可以用于进一步净化环境样品：将样品加热，在50℃下进行30分钟或治疗用酸性或碱性溶液14。 </li></ol></li><li> 临床样本 处理临床样品根据它们的物理 - 化学性质。过滤或离心液去除大杂质。悬浮固体样品。使用的组织，通过用Dounce homogeneiser或玻璃珠研磨它们，例如，和裂解细胞以释放细胞内的细菌。 </li></ol> 1.2变形虫准备<ol><li> 肉汤准备 准备以下介质：含蛋白胨，酵母提取物和葡萄糖（PYG富媒体;100克胨，10克酵母提取物4.9克MgSO 4干燥•7H 2的输出，5克柠檬酸钠•2H 2 O，0.1g的铁（NH 4）2（SO 4）2•6H 2 O，1.7克KH 2 PO 4，1.97克Na 2 HPO 4的•7H 2 O 45克 ​​葡萄糖，和0.295克的CaCl 2在5升蒸馏水）和非营养性介质，如PAS为棘阿米巴物种。 </li><li> Amoebal文化 <ol><li>在装有30ml PYG培养基的细胞培养瓶中培养变形虫（优先氏棘阿米巴 ATCC 30010或A多食领潮-AP1）在25°C。 </li><li>由烧瓶中剧烈摇晃收获变形虫和离心细胞悬液10分钟，1,500×g的。与PAS中等两次洗涤沉淀。计数细胞在KOVA幻灯片，调整音量，以获得每毫升5×10 5细胞悬浮液。 </li><li>转移amoebal悬挂到微孔板。使用每孔1毫升12 - 和24-W埃尔板，500μl的48孔板中，并加入300微升96孔板中。孵育微孔板至少2小时，在25℃下这允许沉积和附着在变形虫的每个孔的底部。 </li></ol></li></ol> 1.3共培养<ol><li> 样品接种 <ol><li>从2.3.3接种板与试样的从1.1或1.2系列稀释液，通常以10倍稀释系列，从用100μl稀释的样品。 </li><li>离心微孔板在1800×g离心10分钟，沉淀在amoebal草坪上的微生物可能存在于样品中。这增加了接触和微生物的吞噬变形虫。 </li><li>孵育板45分钟，在25℃下并且通过与新鲜的PAS更换介质洗涤三次，PAS。添加每孔1毫升PAS添加或不添加抗生素（链霉素，青霉素，庆大霉素和/或万古霉素）的，取决于bacteri被搜索的人的物种。 </li><li>孵育微孔板在32℃，饱和湿度条件下，以避免阿米巴的包囊。每天用20倍物镜观察每孔检测细菌入侵和裂解变形虫的存在。 </li><li>在溶解的情况下，通过接种100μl的共培养物中，以约10 5变形虫/ cm 2的单分子层上执行新鲜变形虫一个传代培养。要明确地隔离特定的细菌种类，接种设计也为这些细菌（ 即 BCYE琼脂军团菌 ）的具体琼脂培养基。 </li><li>在没有溶解，取100微升共同培养的初次接种后4至7天，接种900微升新鲜amoebal文化在24孔板。如果不带细菌的增殖观察变形虫的快速裂解，病毒可能存在。在这种情况下，过滤上清液在0.22微米和使用该过滤悬浮液感染新鲜的变形虫。 </li></醇&gt; </li></ol> 1.4细菌分离和鉴定<ol><li> 细菌染色 直接在24孔微孔板上执行的玻璃盖玻片上共培养物。取出介质，并进行染色或免疫荧光。 <ol><li>修改Romanowsky染色<ol><li>让盖玻片干。浸入盖玻片在固定液（2毫克/升坚牢绿在甲醇中）5次。 </li><li>沉浸在盖玻片五次染色溶液I（1.22 g / L的曙红的G磷酸盐缓冲液pH值6.6） </li><li>最后，将其浸入盖玻片5倍，在染色溶液II（在磷酸盐缓冲液pH 6.61.1克/ L噻嗪）。 </li><li>冲洗样品用蒸馏水冲洗。让干燥并通过显微镜观察。 </li></ol></li><li>抗酸染色<ol><li>让盖玻片干。盖上施乐品红样品。加热染料具有阻燃，直到蒸气出现。 </li><li>在室温下冷却至少5分钟并用蒸馏水冲洗。覆盖所述样品与在异丙醇中进行2分钟的3％盐酸溶液，并用蒸馏水冲洗。 </li><li>覆盖30秒用亚甲蓝和用蒸馏水冲洗。让干燥并通过显微镜观察22。 </li></ol></li><li>希门尼斯染色<ol><li>通过将100毫升10％碱性品红（10克碱性品红在100毫升95％的乙醇），加入250毫升4％的含水苯酚和650毫升蒸馏水中制备碱性品红贮备液。在37℃，使用前48小时。 </li><li>让盖玻片干燥和修复通过火焰传递给它。盖在2分钟新鲜过滤的碱性品红（4毫升碱性品红储备液在10毫升的M 0.1的磷酸钠缓冲液，pH 7.45）的样品。 </li><li>冲洗样品与水和10秒的孵育中孔雀绿（在蒸馏水中的0.8％）。用清水再次冲洗和重复孔雀石绿染色。用清水再次冲洗。让盖玻片干，M&#39;mount它，并通过显微镜观察23。 </li></ol></li><li>免疫荧光<ol><li>孵化甲醇5分钟，或用多聚甲醛10分钟4％固定盖玻片。 </li><li>用PBS洗涤三次，并用封闭液（5％BSA，0.1％皂苷的PBS中）在室温下孵育2小时。 </li><li>孵育盖玻片1小时阻断含有提出对感兴趣的微生物的抗体溶液。 </li><li>在PBS中再次洗涤三次，并孵育1小时用针对第一抗体和连接到荧光团的第二抗体。用PBS洗三次，安装盖玻片，用荧光显微镜观察。 </li></ol></li></ol></li><li> DNA的PCR检测 从100到200微升amoebal共存的提取DNA。检测微生物与靶向16S rRNA基因或特异性引物对感兴趣的物种，如分枝杆菌24通用引物，军团菌25或Chlamydiales 26的成员。 </li></ol> 2。 Amoebal富集 2.1。样品制备通过涡旋重新悬浮固体和半固体样品中PAS。离心悬浮液以低速（180×g离心）10分钟。这允许在小球自由生活阿米巴富集。上清液可用于amoebal共存，沉淀为amoebal富集21。 2.2。培养基的配制<ol><li>加入1.5克琼脂的至100ml的PAS和高压灭菌该培养基15分钟，在121℃下倒入温暖的介质在培养皿中，让在室温下固化。 </li><li>在LB或巯基乙酸盐肉汤生长的大肠杆菌 （ATCC 25922）中过夜，在37℃下用PBS洗两次的细菌和悬浮在其中的PAS媒体。稀释10倍的PAS和传播2-3毫升这种稀释对的PAS琼脂平板上，并让其干燥。 </li></ol>加一滴样品（或一块滤波器）在板的一侧，并且让它流过培养皿中，形成在盘的中心的直线。 2.4。 Amoebal增长和amoebal亚文化<ol><li>每天观察培养皿中。如果amoebal迁移前被检测到，切出一小块琼脂在迁移前和接种新鲜NNA培养皿盖上E的草坪大肠杆菌 。 </li><li>重复该回接几次取决于样品的纯度，以便具有给定的amoebal菌株的纯培养物。 </li></ol> 2.5。变形虫和细菌鉴定<ol><li>刮细胞，重悬在他们的PAS。 </li><li>提取DNA并确定变形虫和/或细菌共生菌通过PCR和测序（16S rRNA基因的扩增对细菌，呼吸道。18S rRNA基因的变形虫和测序，例如）的身份。 </li> &lt;李&gt;使用在这些PAS刮下细胞接种到新鲜变形虫和执行amoebal共培养，以检测可能存在于样品中的细菌。 </li></ol>

<ol>
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Microbiol. 49, 2637-2642 (2011).</li><li>Wang, Y., Ogawa, M., Fukuda, K., Miyamoto, H., Taniguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+mycobacteria+from+soils+at+an+illegal+dumping+site+and+landfills+in+Japan.">Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan.</a> Microbiol. Immunol. 50, 513-524 (2006).</li><li>Corsaro, D., Pages, G. S., Catalan, V., Loret, J. F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiversity+of+amoebae+and+amoeba-associated+bacteria+in+water+treatment+plants.">Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants.</a> Int. J. Hygiene Environ. Health. 213, 158-166 (2010).</li><li>La Scola, B., 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=Legionella+drancourtii+sp.+nov.,+a+strictly+intracellular+amoebal+pathogen.">Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen.</a> Int. J. Syst. Evol. Microbiol. 54, 699-703 (2004).</li><li>Thomas, V., Casson, N., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Afipia+and+Bosea+strains+isolated+from+various+water+sources+by+amoebal+co-culture.">New Afipia and Bosea strains isolated from various water sources by amoebal co-culture.</a> Syst. Appl. Microbiol. 30, 572-579 (2007).</li><li>La Scola, B., 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=Amoeba-resisting+bacteria+and+ventilator-associated+pneumonia.">Amoeba-resisting bacteria and ventilator-associated pneumonia.</a> Emerging Infect. Dis. 9, 815-821 (2003).</li><li>Collingro, 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=Recovery+of+an+environmental+Chlamydia+strain+from+activated+sludge+by+co-cultivation+with+Acanthamoeba+sp.">Recovery of an environmental Chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp.</a> Microbiology. 151, 301-309 (2005).</li><li>Lienard, J., Croxatto, A., Prod'hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrella+lausannensis,+a+new+star+in+the+Chlamydiales+order.">Estrella lausannensis, a new star in the Chlamydiales order.</a> Microbes Infect. 13, 1232-1241 (2011).</li><li>La Scola, B., 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+giant+virus+in+amoebae.">A giant virus in amoebae.</a> Science. 299, 2033 (2003).</li><li>Boyer, M., 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=Giant+Marseillevirus+highlights+the+role+of+amoebae+as+a+melting+pot+in+emergence+of+chimeric+microorganisms.">Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 21848-21853 (2009).</li><li>Thomas, V., 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=Lausannevirus,+a+giant+amoebal+virus+encoding+histone+doublets.">Lausannevirus, a giant amoebal virus encoding histone doublets.</a> Environ. Microbiol. 13, 1454-1466 (2011).</li><li>Raoult, D., Renesto, P., Brouqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+infection+of+a+technician+by+mimivirus.">Laboratory infection of a technician by mimivirus.</a> Ann. Internal Med. 144, 702-703 (2006).</li><li>Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parachlamydia+acanthamoebae,+an+emerging+agent+of+pneumonia.">Parachlamydia acanthamoebae, an emerging agent of pneumonia.</a> Clin. Microbiol. Infect. 15, 18-28 (2009).</li><li>Lamoth, F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoebal+pathogens+as+emerging+causal+agents+of+pneumonia.">Amoebal pathogens as emerging causal agents of pneumonia.</a> FEMS Microbiol. Rev. 34, 260-280 (2010).</li><li>Lienard, J. G., Ashbolt, K., Sen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+6.">Ch. 6.</a> Environmental microbiology, current technology and water applications. , 143-162 (2011).</li><li>Boughalmi, M., 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+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ. Microbiol. , (2012).</li><li>Ovrutsky, A. R., 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=Cooccurrence+of+Free-Living+Amoebae+and+Nontuberculous+Mycobacteria+in+Hospital+Water+Networks,+and+Preferential+Growth+of+Mycobacterium+avium+in+Acanthamoeba+lenticulata.">Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata.</a> Appl. Environ. Microbiol. 79, 3185-3192 (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。

Amoebal共培养和amoebal富集是使许多新的细菌和amoebal物种的隔离有效的方法。用这些方法得到的结果证实了这两种阿米巴变形虫和抗细菌在环境中无处不在，最有趣的是在被认为通过化学处理，如氯化，臭氧控制人为供水网络。 Amoebal共培养和amoebal富集是必不可少的工具来隔离和培养这些潜在的病原微生物，并获得菌株纯培养，才能再进一步研究其生物学和致病性。最近，这种方法被改编为巨病毒...

分子诊断来临之前，目前在环境壁龛或临床样品中的微生物经培养他们在不同的选择性培养基，主要是在琼脂培养皿中经常发现。细菌菌落和其代谢活性的表现型，让细菌分类在物种水平。肉汤也可以被用于增加检测的灵敏度。然而，这两种技术不允许该生长缓慢或根本没有在这些介质上的细菌的恢复。这就是为什么分子生物学方法被如此广泛地采用了时下的原因。然而，检测DNA提供了对细菌的生存能力没有线索。另外，相反地，以培养，分子生物学方法不会导致菌株，可以进一步表征。 研究病原体生长不良固体培养基上，或需要细胞生长是复杂的。大多数这些“难以生长”的细菌都讲究不及物动词脱细胞的细菌，经常发现其特征如下，因为它是为嗜肺军团菌的情况下大规模爆发。这种细菌的特点如下，一位美国退伍军人大会期间发生的爆发。多达182人被感染，29死于严重肺炎1,2。后来证明，变形虫是这种细菌的自然宿主，而他们在酒店空调系统和供水网络的存在是在所谓的退伍军人症3爆发的起源。 变形虫是目前全球范围内的土壤，空气，水和人类志愿者（4审查）的鼻腔粘膜分离。这些“自由生活”变形虫一般都划分在自主的环境，但有时可能会侵入许可主机5。通过吞噬作用和随后的溶酶体消化HY上的各种微生物饲料阿米巴drolases 6。许多兼性或专性细胞内细菌能够抵抗消化，从而感染和阿米巴划分为例如军团菌 ， 衣原体相关的细菌或分枝杆菌（7审阅和8）。自由生活的阿米巴可能是一个重要的潜在水库已尚未被发现的细胞内细菌。这导致本集团在洛桑实现两个主要的技术，称为amoebal共培养和amoebal富集，这使得不同的群体从不同环境样品9-15隔离了几个新的专性细胞内微生物。 由于变形虫是专业放牧吞噬细胞对细菌，能抵抗吞噬和这些原生生物生长里面的细菌也可能拓殖人的吞噬细胞和致病走向人类。这是部分地显示出对某些衣原体相关的细菌，如Waddlia町ndrophila。 W. chondrophila不仅可以在变形虫而且在一些细胞类型中，如哺乳动物上皮细胞，巨噬细胞，和鱼细胞系16-18成长。该amoebal共培养也出现相关检测细胞内细菌的临床标本19,20，包括大便这是严重污染，不同的细菌种类21。 在这里，我们描述amoebal共培养和amoebal富集的主要步骤，包括：（一）治疗环境或临床标本;变形虫（二）在无菌介质中生长，对大肠杆菌的细菌草坪及（c）选择和表征胞内细菌。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m pore size membrane</td>
			<td>Merck Millipore, Darmstadt, Germany</td>
			<td>SCVPU11RE</td>
			<td></td>
		</tr>
		<tr>
			<td>proteose peptone</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Kova slide</td>
			<td>Hycor, Indianapolis, IN</td>
			<td>87144</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture microplates</td>
			<td>Corning Inc, Corning, NY</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>Diff-Quik staining kit</td>
			<td>Siemens Healthcare diagn., Munich, Germany</td>
			<td>130832</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziehl fuchsin</td>
			<td>Fluka, St-Louis, MI</td>
			<td>21820</td>
			<td></td>
		</tr>
		<tr>
			<td>basic fuchsin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>857843</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma, St-Louis, MI</td>
			<td>P1037</td>
			<td>Corrosive and mutagenic</td>
		</tr>
		<tr>
			<td>malachite green oxalate</td>
			<td>Fluka, St-Louis, MI</td>
			<td>63160</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>84510</td>
			<td></td>
		</tr>
	</tbody>
</table>

discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches

细胞内病原体如军团菌，分枝杆菌和衣原体样生物是难以分离，因为它们往往较差或根本不生长在所有上，通常是用于培养细菌的选择性培养基。由于这个原因，许多这些病原体被发现仅在最近或以下重要的爆发。这些病原体往往与变形虫，它作为宿主细胞，并允许细菌的存活和生长有关。我们打​​算在这里提供了两种技术，使孤立的细胞内病原体和表征目前临床或环境样品中的示范：在amoebal共培养和amoebal富集。 Amoebal共培养使细胞内细菌的恢复由所研究的样品接种到一个amoebal草坪，可感染并裂解由存在于样品中的胞内细菌。 Amoebal富集使得目前临床或环境样品中变形虫的恢复。钍是可能导致发现新的amoebal物种，但也是新的细胞内细菌在这些变形虫专门生长。总之，这两个技术有助于发现能够在变形虫长出新的细胞内细菌。因为他们的感染变形虫和抵制吞噬能力，​​这些细胞内的细菌也可能会被巨噬细胞吞噬逃脱，因此，是致病性高等真核生物。

使用amoebal共培养和amoebal富集，环境和/或致病菌的整个范围被发现（ 表1）。 Amoebal共培养采​​用由本集团及其他分析环境样品，水处理厂和配水系统。范围广泛的微生物可能是孤立使用这种技术。分离amoebal共培养中最常见的细菌是分枝杆菌属的成员，可以从污水处理厂，并从水网13,14,24,27,28回收。 军团菌和α-变形细菌物种也可从污水处?...

Watch this Scientific Journal Video about Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches at JoVE.com

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Amoebal共存是使用贴壁变形虫以选择性地生长细胞内的病原体能够抵抗吞噬细胞如变形虫和巨噬细胞的细胞培养系统。因此，它代表发现新的传染性病原体的关键工具。 Amoebal富集允许发现其特定的细胞内细菌的新amoebal品种和。

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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Research

JoVE Journal

Immunology and Infection

14.4K 次观看.  University Hospital Center and University of Lausanne.  Amoebal共存是使用贴壁变形虫以选择性地生长细胞内的病原体能够抵抗吞噬细胞如变形虫和巨噬细胞的细胞培养系统。因此，它代表发现新的传染性病原体的关键工具。 Amoebal富集允许发现其特定的细胞内细菌的新amoebal品种和。 

视频: Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

科研

免疫与感染

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

通过胞内细菌，研究内溶酶体系统的改造荧光纳米粒子中的应用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale 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 combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...