在我们的实验室研究是由马克斯·冯·Pettenkofer研究所，慕尼黑路德维希 - 马克西米利安大学德国研究基金会（DFG，您好1511/3-1）的Bundesministerium献给教化和Forschung（BMBF）的“医疗感染基因组学”的倡议（ 0315834C）。

1。轻型商用车分离的筹备工作<ol><li>连续出嗜肺军团菌生产甘油股票CYE平板DsRed的，快13,14轻型商用车隔离之前的5微克/毫升氯霉素（CAM）四天。细菌在37°C。 </li><li>种子出1×10 7 D。 discoideum生产GFP-calnexin或75厘米2组织培养的RAW264.7巨噬细胞瓶前一天的感染。使用10毫升20微克/毫升为四 G418的HL5中等discoideum和孵化的变形虫在23°C对于RAW264.7巨噬细胞用10毫升辅以10％FCS（热灭活）和1％谷氨酰胺的RPMI 1640培养基，生长的细胞在37°C和5％的CO 2。使用至少有三个75厘米2％感染和样本瓶（至少6×10 7细胞）。 </li><li>接种过夜培养与研究肺从CYE板。以埃中等3毫升15毫升试管凸轮5微克/毫升。 100的产量为0.1 OD 600nm的细菌悬浮液接种。在37°C孵育架空旋转轮上过夜培养21-22小时。 </li></ol> 2。轻型商用车的分离<ol><li>改变四中等discoideum细胞清除以下感染的抗生素，这会干扰。 </li><li>测量外径过夜培养。细菌已达到其峰值传染性≥3 OD 600nm的 ，这相当于2×10 9菌/毫升。 </li><li>感染细胞，加入约500μL的研究肺过夜培养增长10毫升HL5介质（ 四discoideum）或10毫升的RPMI 1640（巨噬细胞）的细胞。这相当于一个感染复数（MOI），50。随后，500×G同步感染到细菌细胞的离心10分钟。离心后由孵化了D.在25°C和RAW264.7巨噬细胞在37°C和5％CO 2，分别discoideum细胞。感染1小时的时间，包括离心步骤。 </li><li>感染后取出介质和洗一次删除外的细菌细胞。使用D.冰冷的SORC缓冲区discoideum和冰冷的RAW264.7巨噬细胞的PBS。加入3毫升HS缓冲区，辅以蛋白酶抑制剂（罗氏）每个烧瓶，用细胞刮刀收集细胞。相应的样品池在15毫升的试管。 </li><li>样品用3毫升塑料露儿锁注射器和不锈钢球均质同质化。确保您在冰工作。在开始之前，用蒸馏水洗球均质，以避免任何洗涤剂污染和冲洗冰冷协缓冲区得到消除气泡。使用8微米的间隙球。 </li><li>填充到第3毫升注射器UND安装在THé均质。按样品来回通过均质九倍。交换后的注射器，以避免与非均质材料的污染。收集和池均质样品15 mL试管微观分析，并采取了150μL样品。在不同的样本出发之前，拆除和洗球均质。 </li><li>阻止振动筛上的冰30分钟或架空纺车（10-20转）匀浆2％CS或FCS在4°C。 </li><li>阻断后使用对SIDC（1:3000稀释）或反对任何其他细菌指标完全结合轻型商用车膜亲和纯化抗体。涡抗体溶液，然后再将其添加到匀浆。样品在4°C孵育1小时上冰筛上或架空纺车（10-20转） </li><li>在此期间，准备Histodenz梯度。使用每梯度15毫升的试管和3个75厘米2瓶的一个样本。冷杉ST，加入5.75毫升的35％Histodenz管，第二，小心加入5.75毫升10％Histodenz解决方案之上。事后仔细铺设管水平为1小时。 </li><li>阻断试剂和抗体，在600×g下离心15分钟，在4°C至颗粒样品匀浆后孵化。弃上清，悬浮颗粒在1.5毫升HS缓冲区。转移到一个新的15毫升试管样品和微观分析40μL样品后。 </li><li>孵化与二次抗体沉淀 - 磁珠山羊抗兔IgG微珠（1:25稀释） - 振动筛为30分钟，在4°C在此期间，在Mac上的磁持有的​​列和0.5毫升协缓冲区平衡。 </li><li>随后，适用于样品列。通过收集流量为150μL，在随后的微观分析。用0.5毫升HS缓冲液洗列的三倍。 </li><li>从T中删除列他的磁铁和洗脱液LCVs直接喷射0.5毫升协缓冲区。微观分析，以20μL样品。免疫磁分离纯化步骤，预计产量每1×10 7感染D。 discoideum细胞约1×10 6的完整LCVs，其中约七倍在洗脱液浓缩与流过13。 </li><li>提出的Histodenz梯度管，垂直位置，谨慎地装载在上面的洗脱液。 1小时离心管3350×G在4°C。 </li><li>离心后，取1.5毫升的分数，从试管底部开始使用长玻璃巴斯德吸管。共收集8个组分，每个从试管底部。观察不可见的层在连续Histodenz的梯度。产量最高的LCVs通常〜2的比例预计在分数4×10 5个完整的LCVs每1×10 7染病变形虫13。未成年人数量LCVs也存在组分3和5，分别。因此，纯化LCVs的整体产量是1×10 6×10 7 6％不变LCVs感染D。 discoideum细胞13。在室温（RT），可以进行恢复LCVs和的LCVs出现了几个小时的稳定。 </li></ol> 3。采集的微观分析<ol><li>准备与聚-L-赖氨酸涂层盖玻片的24孔平底培养板。 </li><li>吸取轻型商用车净化加150微升到24孔板的每一个密度梯度分数在采取每一个样本。每孔加入0.5毫升HS缓冲区冲淡高Histodenz浓度。此后，离心10分钟600×G板在4°C。小心取出上清液，并修复与4％多聚甲醛在室温为20分钟（PFA）的样品。固定后洗两次样本。我们为四 éSORC discoideum和PBS RAW264.7巨噬细胞。 </li><li><ol class="lalpha"><li> GFP-calnexin装入样品，表示D。 discoideum，在轻型商用车上直接使用如Vectashield玻璃幻灯片隔离，采取不同的步骤。 </li><li>样品孵育RAW264.7巨噬细胞，采取不同的步骤，在轻型商用车隔离，封闭液在室温为10分钟（1％BSA的PBS中）。使用反SIDC抗体，染色LCVs在湿室（亲和纯化兔抗SIDC;封闭液1:1000）在室温孵育1小时。随后，洗样品，用PBS三次。孵化与二次抗体（如抗兔Cy5的;封闭液1:200）为30分钟，在室温下湿暗室。最后，洗样品用PBS三次，并安装上使用如Vectashield玻璃的幻灯片。 </li></ol></li><li>形态，完整性和分离GFP-calnexin LCVs的数量表达D。 DIScoideum或从RAW264.7巨噬细胞分析，荧光显微镜，配备足够的过滤器。 </li></ol> 4。代表结果可通过免疫亲和分离和密度梯度离心的质量和产量的轻型商用车净化后用荧光显微镜（ 图1）。 D.轻型商用车隔离期间收集样品的概述discoideu米或巨噬细胞（ 图2）所示。 L 的匀浆肺感染的巨噬细胞显示完整LCVs，但也有大量细胞碎片和细胞外细菌。制粒样品后，图像的类似的匀浆，有时有点密集。由于完整LCVs要坚持列，流过主要包含外的细菌和细胞碎片。列样品洗脱后，洗脱液中包含大量完整LCVs。在我们的手中，轻型商用车purificati<html>上似乎是更有效地为D。 discoideum比为巨噬细胞。 在 D discoideum的病原体空泡出现全才和的孤立LCVs产量是与巨噬细胞（ 图3）相比，高出10倍以上。在完整的巨噬细胞与不同的抗体染色的低消费量国家还出现更多的不规则形状。 <img alt="图1" src="/files/ftp_upload/4118/4118fig1.jpg" /> 图1。原理概述轻型商用车隔离。一）分离LCVs使用对细菌效应蛋白SIDC亲和纯化抗体，完全本地化的轻型商用车膜免疫磁性分离。用于分离细胞碎片LCVs次要磁珠微珠耦合抗体和磁铁。 b）在免疫磁分离LCVs的是由Histodenz密度梯度离心进一步纯化。 LCVs丰富的分数4。 upload/4118/4118fig2.jpg“/&gt; 图2。样品收集在轻型商用车净化。从匀浆，沉淀，图片流过， 从 D洗脱液discoideum或巨噬细胞被描绘。该样本显示L。肺产生在 D DsRed的表达（红色） discoideum生产绿色荧光，calnexin信号（绿色，上游面板）或抗的SIDC抗体（青色，下部面板）染色的巨噬细胞。酒吧，10微米。 <img alt="图3" src="/files/ftp_upload/4118/4118fig3.jpg" /> 图3。纯化从 D LCVs discoideum或巨噬细胞。 研究 Histodenz密度梯度离心后的样品显示分数4 肺 （一）D生产DsRed的表达（红色） discoideum生产绿色荧光，calnexin信号（绿色）或（B）与抗的SIDC抗体（青色）染色的巨噬细胞。酒吧，10微米。

<ol>
	<li>Hilbi, H., Hoffmann, C., Harrison, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+spp.+outdoors:+colonization,+communication+and+persistence.">Legionella spp. outdoors: colonization, communication and persistence.</a> Environ. Microbiol. Rep. 3, 286-296 (2011).</li><li>Zhu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+identification+of+protein+substrates+of+the+Dot/Icm+type+IV+transporter+of+Legionella+pneumophila.">Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.</a> PLoS ONE. 6, e17638 (2011).</li><li>Hilbi, H., Weber, S., Finsel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anchors+for+effectors:+subversion+of+phospho-inositide+lipids+by+Legionella.">Anchors for effectors: subversion of phospho-inositide lipids by Legionella.</a> Front Microbiol. 2, 91 (2011).</li><li>Hubber, A., Roy, C. R. <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+host+cell+function+by+Legionella+pneumophila+type+IV+effectors.">Modulation of host cell function by Legionella pneumophila type IV effectors.</a> Annu. Rev. Cell Dev. Biol. 26, 261-283 (2010).</li><li>Kima, P. E., Dunn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+calnexin+expression+on+phagosomes+to+isolate+Leishmania+parasitophorous+vacuoles.">Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles.</a> Microb. Pathog. 38, 139-145 (2005).</li><li>L&#252;hrmann, A., Haas, A. <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+to+purify+bacteria-containing+phagosomes+from+infected+macrophages.">A method to purify bacteria-containing phagosomes from infected macrophages.</a> Methods Cell Sci. 22, 329-341 (2000).</li><li>Sturgill-Koszycki, S., Haddix, P. L., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+Mycobacterium+and+the+macrophage+analyzed+by+two-dimensional+polyacrylamide+gel+electrophoresis.">The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis.</a> Electrophoresis. 18, 2558-2565 (1997).</li><li>Fernandez-Mora, E., Polidori, M., L&#252;hrmann, A., Schaible, U. E., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+Rhodococcus+equi-containing+vacuoles+is+arrested+after+completion+of+the+early+endosome+stage.">Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage.</a> Traffic. 6, 635-653 (2005).</li><li>Mills, S. D., Finlay, B. B. <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+characterization+of+Salmonella+typhimurium+and+Yersinia+pseudotuberculosis-containing+phagosomes+from+infected+mouse+macrophages:+Y.+pseudotuberculosis+traffics+to+terminal+lysosomes+where+they+are+degraded.">Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded.</a> Eur. J. Cell Biol. 77, 35-47 (1998).</li><li>Shevchuk, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+Legionella-containing+phagosomes+isolated+from+Dictyostelium.">Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium.</a> Int. J. Med. Microbiol. 299, 489-508 (2009).</li><li>Brombacher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab1+guanine+nucleotide+exchange+factor+SidM+is+a+major+phosphatidylinositol+4-phosphate-binding+effector+protein+of+Legionella+pneumophila.">Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.</a> J. Biol. Chem. 284, 4846-4856 (2009).</li><li>Urwyler, S., Finsel, I., Ragaz, C., Hilbi, 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+of+Legionella-containing+vacuoles+by+immuno-magnetic+separation.">Isolation of Legionella-containing vacuoles by immuno-magnetic separation.</a> Curr. Protoc. Cell Biol. Chapter 3, Unit 3 (2010).</li><li>Urwyler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Legionella+vacuoles+purified+by+magnetic+immunoseparation+reveals+secretory+and+endosomal+GTPases.">Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases.</a> Traffic. 10, 76-87 (2009).</li><li>Mampel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planktonic+replication+is+essential+for+biofilm+formation+by+Legionella+pneumophila+in+a+complex+medium+under+static+and+dynamic+flow+conditions.">Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions.</a> Appl. Environ. Microbiol. 72, 2885-2895 (2006).</li></ol>

没有利益冲突的声明。

在此前公布的方法相比，该协议是基于两个步骤，首先通过免疫磁性分离的LCVs，和第二，通过密度梯度离心LCVs净化。轻型商用车的隔离，可以很容易地后，荧光显微镜，要么荧光标记的细菌和细胞产生的细胞或抗体染色时使用。这里描述的协议是一个简单的，直接的方法，该方法在高纯度浓缩铀的LCVs结果。重要的是，在同质化缓冲区的蛋白酶抑制剂是必需的RAW264.7巨噬细胞，但D. discoideum ...

purification of pathogen vacuoles from legionella-infected phagocytes

条件致病菌嗜肺军团菌是一种抗阿米巴的细菌，这也是在肺泡巨噬细胞，从而导致重症肺炎“退伍军人病”1复制。在原生动物和哺乳动物吞噬，L。肺采用了保守的机制，以形成特定的许可，复制，车厢，“含军团菌的空泡”（轻型商用车）。轻型商用车的形成需要ICM / DOT细菌IV型分泌系统（T4SS）“275效应”的蛋白质，其中的translocates进入宿主细胞。效应操纵宿主蛋白以及血脂和分泌，内体和线粒体的细胞器2-4沟通。 的LCVs形成一个复杂的，强大的和冗余的过程，这是很难把握，在还原的方式。一个综合性的做法需要全面了解轻型商用车的形成，包括一个全球性的路径分析ogen宿主因子的相互作用及其时空动态。为实现这一目标的第一步，完整LCVs纯化和蛋白质组学和脂类组学分析。 已被调查的蛋白质组学分析，使用液相色谱或2-D电泳质谱仪耦合的组成和形成的含病原体的空泡。 9， 沙门氏菌 ， 球菌 8，7 分枝杆菌 ， 李斯特菌 6 利什曼 5 军团菌分离的社会土壤变形虫粘菌或哺乳动物吞噬细胞空泡窝藏10。然而，在这些研究中采用的协议是净化费时和繁琐，因为他们需要如电子显微镜分析轻型商用车的形态，完整性和纯度。此外，这些协议不利用EN病原体液泡的具体特点富集。 这里介绍的方法克服了这些限制，由用人四discoideum生产轻型商用车荧光标记和针对细菌效应蛋白SIDC，选择性地结合到磷酸肌醇4 -磷酸（PtdIns（4）P）的3,11轻型商用车膜锚。第一步由SIDC和二级抗体，加上磁珠古典Histodenz密度梯度离心12,13（ 图1），其次是第二步，对主要抗体亲和纯化的免疫磁性分离富集LCVs 。 一个蛋白质孤立LCVs的研究从 D discoideum发现560多宿主细胞包括吞噬囊泡，线粒体，ER和高尔基，以及一些GTP酶，它没有被牵连13日之前在轻型商用车形成相关蛋白的蛋白质，。丰富和纯化LCVs协议概述，这里还可以进一步分析显微镜（免疫荧光法，电子显微镜），生化方法（免疫印迹）和蛋白质或lipidomic办法。

Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

本文介绍了一个完整的隔离和净化的方法<em&gt;军团</em&gt;从变形虫和巨噬细胞含有空泡（LCVs）。两步的协议，包括轻型商用车通过使用对细菌的轻型商用车标记和密度梯度离心进一步纯化抗体的免疫磁性分离富集。

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

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Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K 次观看.  Ludwig-Maximilians-Universität. 本文介绍了一个完整的隔离和净化的方法军团从变形虫和巨噬细胞含有空泡（LCVs）。两步的协议，包括轻型商用车通过使用对细菌的轻型商用车标记和密度梯度离心进一步纯化抗体的免疫磁性分离富集。

视频: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

科研

免疫与感染

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host 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. ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.