这项工作是支持的，一部分资金从巴斯德研究所和国家法甲抗癌症（补助R05/75-129和RS07/75-75），协会倾LA RECHERCHE SUR LE癌症（赠款ARC A09 /补助1/5031和4867），和国家法新社​​德拉RECHERCHE（MIME 009 02 ANR09的ANR07冕026 01）。 MM收件人的MENRT奖学金。

1。酵母双杂交放映<ol><li>进行以下所需的解决方案： <ol><li>完成合成跌落式（SD）：与葡萄糖在1升水中溶解26.7克的最小SD基地。添加氨基酸混合物制备如下：将2克精氨酸盐酸盐，2克2克腺嘌呤硫酸盐，2克盐酸组氨酸异亮氨酸，2克，4克亮氨酸盐酸赖氨酸，将2克，蛋氨酸2克，苯丙氨酸3克G L 2丝氨酸，2 G L-苏氨酸， 色氨酸 3克，2克酪氨酸，1.2克尿嘧啶，缬氨酸9克。 </li><li>选择性输出SD-L/-W/-H下降：SD每一个人体必需的氨基酸，除了那些指定（-L-亮氨酸-W-色氨酸-H-组氨酸）。 </li><li> YPGLU：对于1升，重10克的酵母提取物，细菌用胰蛋白胨10克，20克葡萄糖。完成25克细菌琼脂固体培养基。 </li><li> YCM：与为YPGLU，但是pH值调整到4.5。 </li></ol></li><li>交配和蔓延<ol><li>使用冷冻等分预变换ED Y187酵母菌株（交配型α）cDNA文库克隆PACT2。 SD-L板电镀系列稀释检查细胞活力。 </li><li>病原体ORF克隆到酵母表达载体pGBKT7，重组质粒转化AH109酵母菌株（交配型）使用的标准程序。 </li><li>差价构建转化AH109酵母SD-W板。在培养箱中孵育至少两天30℃。板可在4℃下储存，​​直到双杂交筛选。 </li><li>使用3 - 氨基三唑（3-AT），HI​​S3酶的竞争性抑制剂，抵消潜在的HIS3报道基因的转录自病原体的蛋白质。执行自动激活试验确定的浓度为3-AT的构建转化酵母的生长，从而防止在SD-W/-H平板。 </li><li> SD-W种子30毫升的构建转化AH109几个殖民地。在30°C孵育30小时旋转。 </li><li>种子的SD-W与30毫升200毫升的AH109文化。在30℃下孵育20小时左右旋转</li><li>孵育，孵育10分钟，在30℃下用旋转20毫升YPGLU的解冻cDNA文库转化Y18​​7。 </li><li>离心构建转化AH109用相当量的5分钟，在3,500 rpm转速的可行Y187酵母细胞的培养相应的体积在20℃下小心翼翼地撤回上清，重悬沉淀在10毫升YPGLU。 </li><li>混合悬浮的AH109酵母与含Y187库。在50毫升管中的传输。离心5分钟，在3,500 rpm转速在20°C，仔细退出上清（脆弱沉淀）。 </li><li>一共拿到在YPGLU悬浮颗粒1.5毫升。 </li><li>传播酵母3 YCM-琼脂150毫米板，0.5毫升/板。孵育4.5小时，在30°C。 </li><li>收集从YCM板，刮耙玻璃，在10毫升SD-L/-W/-H交配酵母。冲洗板两次用5毫升SD-L/-W/-H媒体和游泳池。 </li><li>在3,500 rpm转速离心5分钟，20℃。弃上清，悬浮酵母沉淀在5毫升SD-L/-W/-H介质。 </li><li>差价10板（0.5毫升/板）辅以适当浓度的3 - 氨基三唑（3-AT）在自动激活测试确定的SD-L/-W/-H琼脂酵母交配。在30°C孵育，湿度条件下6-10天。 </li><li>确定二倍体（2n）的速率，以评估通过传播250μl的系列稀释液（10 -4〜10 -6）的交配的酵母悬浮液上SD-L/-W板的配合效率。两天，在30℃下孵育板。与计数菌落生长SD-L/-W，推断出总的二倍体数存在于酵母的交配的初始体积。报告这个二倍体可行库含酵母数计算的二倍体率。它的范围应该10-25％左右。 </li><li> 6-10天孵化后，在30°C，挑交配的酵母菌菌落在新鲜SD-L/-W/-H + 3-AT板。 [ 提示：为了酵母在计划8线再现96孔板中的12个孔，这样它会更容易之后测序]。让酵母菌菌落生长为4-5天，在30℃在加湿的气氛中。 </li></ol></li><li>筛选的HIS3阳性克隆通过PCR和测序 我们的目标是PCR扩增的ORF中所载的质粒pACT2的酵母载体上生长的菌落选择性SD-L/-H/-W板。 <ol><li>将96孔PCR板在冰上。 </li><li>以裂解酵母细胞，每孔50μl的Zymolase 20T溶液（2.5 mg / ml的10mM的HEPES缓冲液pH 7.7）添加。 </li><li>轻轻悬浮每一个菌落。 </li><li>在37℃和15分钟，在95℃下孵育15分钟</li><li>把盘子放在冰上。每孔加入50微升的蒸馏水。上下吹打拌匀。 </li><li>离心5分钟，在3,500 rpm和4℃。 </li><li> PCR扩增10微升来检测插入物，使用引物退火是来自于cDNA的质粒pACT2载体的ORF两侧。该协议是g尔文96 ExTaq聚合酶裂解酵母菌落的扩增。需准备以下组合：525微升10×ExTaq缓冲液，420微升的dNTPs混合（2.5毫摩尔），10.5微升正向引物（1微克/微升）10.5微升反向引物（1微克/微升）31.5微升ExTaq（5 U /μl的）和3202.5微升H 2 O中 。分发40微升的搭配，每孔96孔PCR板。加入10微升裂解酵母每口井。执行放大如下：94℃3分钟，然后35个循环，在94℃下30秒，60℃1分钟，72℃下4分钟，结束与72℃7分钟。运行3微升的PCR产物在1.2％琼脂糖凝胶电泳检测PCR阳性克隆。需要注意的是使用预投了96以及琼脂糖凝胶建议。 [提示：板可以在-20°C存储了几个月，并重新用于新的PCR] </li><li>序列的PCR扩增片段孤立从HIS3的阳性克隆。执行BLAST分析识别蜂窝ORF。低信任路线（E值＆&gt; 10 -10，身份&lt;80％，&lt;20个氨基酸肽长度）被丢弃，以及frameshifted和含有提前终止密码子序列。 </li></ol></li></ol> 2。 HT-GPCA矩阵建设 HT-GPCA是哺乳动物的基于细胞的测定，其中的病原体和宿主蛋白瞬时表达融合到分割Gaussia颤荧光素酶片段互补。当贸易伙伴的相互作用，这种酶被重新允许其酶活性的措施。相互作用强度，从而推导出归一发光比率（见下图1） <ol start="4"><li>选择宿主细胞伙伴的<ol><li>选择确定在哺乳动物细胞中的进一步验证在Y2H放映的三倍以上的蛋白质，以增加的数据集的信心9。 </li><li>添加已知位置的病原体蛋白质相互作用的合作伙伴VE控制。 </li></ol></li><li>转移到HT-GPCA兼容的载体<ol><li>克隆每个病原体蛋白的ORF中的pSPICA-N2向量生成病原体蛋白与氨基酸110至185的萤光素酶融合在其N-末端（GL2-病原体蛋白融合）的人源化Gaussia颤 。 </li><li> PCR扩增的细胞蛋白的ORF直接从酵母双杂交阳性克隆或从其他ORF的资源（人类的ORFeome <a href="http://horfdb.dfci.harvard.edu/" target="_blank">http://horfdb.dfci.harvard.edu/</a> ）的，并将它们传送到pSPICA-N1载体中，以产生融合蛋白在N-末端与氨基酸18至109，人性化Gaussia荧光素酶颤 （GL1主机蛋白融合）。 [提示：对于大规模克隆，网关克隆系统的建议。在这种情况下，进行PCR扩增，使用设计为包含网关兼容的位点的引物]。 </li><li>所有序列的表达载体。 pSPICA-N1和pSPICA的-N2的序列的参考文献8中可以找到。 </li></ol></li></ol>请注意，质粒结构可以反转， 即病原体蛋白在pSPICA-N1和宿主蛋白到pSPICA-N2。 3。高通量Gaussia颤基于荧光素酶蛋白质互补技术（HT-GPCA） <ol start="6"><li>细胞转染<ol><li>种子染293T细胞，在35万个细胞/ ml的DMEM，10％胎牛血清（FBS）的无抗生素。分发100μl/孔，在无菌的白色培养板。在一次实验中，不超过10板。让细胞生长24小时，在37℃5％CO 2。 [ 提示：，由于pSPICA质粒包含SV40原点的，293T细胞中的使用允许其在转染细胞中的复制，从而导致过度的两种荧光素酶片段。] </li><li>次日，使用任何适当的转染协议的转染细胞。需要注意的是，怒江一定的转染试剂MBER种子细胞可能会进行调整。使用100纳克每口井的病原体和宿主ORF质粒结构。共转染10纳克每口井的CMV-萤火虫荧光素酶质粒转染效率的正常化。每个点都一式三份进行测试。 [ 提示：转染前一天，可以进行DNA混合，并储存在-20℃下用粘接剂密封。] </li><li>转染混合物转移至293T细胞培养板。要小心，轻轻地沉积到细胞的DNA混合，293T强粘附性，和容易地从井的底部分离。用5％的CO 2在37℃下孵育24-30小时，在细胞培养孵化器。 </li></ol></li></ol><ol start="7"><li>细胞的裂解和Gaussia荧光素酶用量<ol><li>洗涤细胞，用PBS（100μl/孔）。加入40μl1X 海肾裂解缓冲液。搅拌下，在室温下孵育30分钟。 </li><li> 10微升的细胞裂解液保存在另一个白色平台E随后剂量的萤火虫荧光素酶的活性。储存在4℃下，或者在-20℃下与密封。 </li><li>测量的Gaussia活动的剩余30μL细胞裂解液加入100微升海肾萤光素酶检测试剂和计数光度计发光，持续10秒。 96孔板的剂量需要约半小时。因为冷冻或长期储存可能会影响相互作用介导Gaussia活动的新鲜细胞提取物进行测量。 </li></ol></li></ol><ol start="8"><li>萤火虫活动的测量<ol><li>测量萤火虫活性，对保存的10微升的细胞裂解液，加入50μl的萤火虫荧光素酶测定试剂和数量，持续10秒的发光。 96孔板的剂量需要约半小时。 [ 提示：萤火虫荧光素酶的用量可以进行冷冻裂解后的第二天。 </li></ol></li></ol><ol start="9"><li> NLR计算<ol><li&gt;对于每个交互，计算Gaussia荧光素酶活性和萤火虫荧光素酶活性，以获得归一化Gaussia考虑到账户的转染效率和细胞存活率的发光值之间的比率。计算三次重复的平均值。 </li><li>要监视的相互作用，估计每个病原体宿主对归一发光比率（NLR）。要做到这一点，除归Gaussia发光测量控制井的活动的总和（改编自8参考图1）病原体和宿主蛋白存在活性检测。据估计，大约3.5 8 NLR良性互动的截止值。 </li></ol></li></ol><ol start="10"><li>数据分析<ol><li>使用R统计软件包进行分层聚类。首先，组NLR数据进行分类，然后计算出一个互动树状使用完整的联动方法。这个可视化“树形图10 JavaTreeView。计算互动Pearson相关系数聚类分析和进化树之间的距离。 </li><li>要产生互动网络，选择良性互动从HT-GPCA数据集和使用在Cytoscape的软件1。宿主蛋白的提取度，并绘制其分布访问本地网络的动态。主机病原体网络可以叠加到宿主相互作用组病原体蛋白在宿主细胞网络在全球的影响提供了一个评估。 </li><li>提取与目标细胞因子与大卫的生物信息学数据库12评估数据集的功能富集的GO（Gene Ontology的）条款。 </li></ol></li></ol>

<ol>
	<li>Davey, N. E., Trave, G., Gibson, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+viruses+hijack+cell+regulation.">How viruses hijack cell regulation.</a> Trends in Biochemical Sciences. 36, 159-169 (2011).</li><li>Calderwood, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+and+virus+human+protein+interaction+maps.">Epstein-Barr virus and virus human protein interaction maps.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 7606-7611 (2007).</li><li>de Chassey, 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=Hepatitis+C+virus+infection+protein+network.">Hepatitis C virus infection protein network.</a> Mol. Syst. Biol. 4, 230 (2008).</li><li>Simonis, 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=Host-pathogen+interactome+mapping+for+HTLV-1+and+-2+retroviruses.">Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses.</a> Retrovirology. 9, 26 (2012).</li><li>Dyer, M. D., Murali, T. M., Sobral, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+human+proteins+interacting+with+viruses+and+other+pathogens.">The landscape of human proteins interacting with viruses and other pathogens.</a> PLoS Pathog. 4, e32 (2008).</li><li>Rual, J. F., 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=Towards+a+proteome-scale+map+of+the+human+protein-protein+interaction+network.">Towards a proteome-scale map of the human protein-protein interaction network.</a> Nature. 437, 1173-1178 (2005).</li><li>Venkatesan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+empirical+framework+for+binary+interactome+mapping.">An empirical framework for binary interactome mapping.</a> Nat. Methods. 6, 83-90 (2009).</li><li>Cassonnet, P., 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=Benchmarking+a+luciferase+complementation+assay+for+detecting+protein+complexes.">Benchmarking a luciferase complementation assay for detecting protein complexes.</a> Nat. Methods. 8, 990-992 (2011).</li><li>Ito, 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=A+comprehensive+two-hybrid+analysis+to+explore+the+yeast+protein+interactome.">A comprehensive two-hybrid analysis to explore the yeast protein interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 4569-4574 (2001).</li><li>Saldanha, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Java+Treeview--extensible+visualization+of+microarray+data.">Java Treeview--extensible visualization of microarray data.</a> Bioinformatics. 20, 3246-3248 (2004).</li><li>Smoot, M. E., Ono, K., Ruscheinski, J., Wang, P. L., Ideker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoscape+2.8:+new+features+for+data+integration+and+network+visualization.">Cytoscape 2.8: new features for data integration and network visualization.</a> Bioinformatics. 27, 431-432 (2011).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat. Protoc. 4, 44-57 (2009).</li><li>Muller, 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=Large+scale+genotype+comparison+of+human+papillomavirus+E2-host+interaction+networks+provides+new+insights+for+e2+molecular+functions.">Large scale genotype comparison of human papillomavirus E2-host interaction networks provides new insights for e2 molecular functions.</a> PLoS Pathog. 8, e1002761 (2012).</li><li>Neveu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+virus-host+interactomes+with+a+mammalian+high-throughput+protein+complementation+assay+based+on+Gaussia+princeps+luciferase.">Comparative analysis of virus-host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase.</a> Methods. , (2012).</li><li>Braun, P., 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=An+experimentally+derived+confidence+score+for+binary+protein-protein+interactions.">An experimentally derived confidence score for binary protein-protein interactions.</a> Nat. Methods. 6, 91-97 (2009).</li></ol>

作者宣称，他们有没有竞争经济利益。

独立，酵母双杂交和哺乳动物的相互作用分析，，如GST拉下来，LUMIER或MAPPIT，已被证明是有效的工具来检测蛋白质 - 蛋白质相互作用，但假阳性和假阴性率高的限制与这些技术15的相互作用。此外，越来越多的证据正交的方法相结合，提高了可靠性，得到的交互数据集7。这里描述的的HT-GPCA技术的最新发展，不仅提高了检测的灵敏度二进制在哺乳动物细胞中蛋白质相互作用，但也?...

越来越多的数据收集产生的蛋白质 - 蛋白质相互作用图谱打开的角度来进一步了解病原体的感染。随着全球病原体感染的认识开始出现，它提供了访问连接时，病原体蛋白诱导人类蛋白质组1范围内的扰动。因此，它提供了一种方式来理解，病原体如何操纵宿主细胞的机械。特别是，一些病毒宿主相互作用网络的映射显示，病毒蛋白的优先目标主机蛋白高度蜂窝网络中的连接（集线器），或中央许多在一个网络中的路径（瓶颈蛋白）2-4。这些互动让病毒操纵重要的蜂窝流程，这是有助于复制并产生感染性子代。最近进行相关病毒与目标提取信息相对比较相互作用映射发病机制4。此外，研究宿主病原体相互作用景观一直延伸到许多病原体5。针对性的细胞因子识别提供洞察战略病原体感染细胞，并允许潜在的致病性标志物的检测。 酵母双杂交系统是目前最流行的方法来识别二进制交互作用，因为遗传筛查是一种高效，敏感的蛋白质 - 蛋白质相互作用的高通量映射工具。这里提出的方法，进一步提高个别Y2H放映通过评估多种病原体的蛋白质应变变种，从而提供比较概述病原宿主蛋白 - 蛋白相互作用。此外，酵母双杂交筛选的一个主要限制在于较高的假阴性率，因为它恢复约20％的总相互作用6。这意味着，只有一个子集的病理检测的相互作用一族变种可能逃脱检测与他人。因此，从所有双杂交放映个别合作伙伴，新兴的互动与研究菌株齐全的进一步挑战，提供比较相互作用数据库。因为它表明，结合不同的方法强烈地增加了鲁棒性的蛋白-蛋白相互作用的数据集7，此验证在哺乳动物细胞中进行了新开发的蛋白质片段互补法称为HT-GPCA 8。此细胞为基础的系统允许，Gaussia颤通过互补性分裂的蛋白质的相互作用的检测荧光素酶和已被设计为具有高通量的格式兼容。基于其发光技术及其背景噪音低，与其他基于荧光检测相比，HT-GPCA显示了惊人的高灵敏度。 总体而言，这种做法构成了一个高效工具来生成一个全面的映射宿主 - 病原体interplays的，它代表一个全球性的理解宿主细胞劫持的第一步。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Yeast strains</td>
			<td>Clontech</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Minimal SD base</td>
			<td>US biological</td>
			<td>D9500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amino acids</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3-amino-1,2,4-Triazole (3-AT)</td>
			<td>Acros organics</td>
			<td>264571000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zymolase</td>
			<td>Seikagaku</td>
			<td>120491</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>31966</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>BioWest</td>
			<td>S1810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffer Saline (PBS)</td>
			<td>Gibco-Life Technologies</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco-Life Technologies</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Renilla luciferase assay</td>
			<td>Promega</td>
			<td>E2820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>White culture plate</td>
			<td>Greiner Bio-One</td>
			<td>655083</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-wellPCR plates</td>
			<td>4titude</td>
			<td>4t-i0730/C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator (30 &deg;C)</td>
			<td>Memmert</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator (37 &deg;C)</td>
			<td>Heraeus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Berthold</td>
			<td>Centro XS-LB 960</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a comparative approach to characterize the landscape of host-pathogen protein-protein interactions

聚集产生重大努力大型综合性蛋白质 - 蛋白质相互作用网络图。这是有助于理解病原体的宿主关系，基本上是由遗传放映酵母双杂交系统。由Gaussia基于荧光素酶片段互补法检测蛋白质相互作用最近有所改善，现在提供了发展的机会，中西医结合的比较interactomic的方法必须严格比较不同的病原体菌株变种对一组共同的细胞因子的蛋白质的互动型材。 本文特别着重于实用程序相结合的两个正交的方法生成的蛋白质-蛋白质相互作用的数据集：酵母双杂交（Y2H）和一个新的测定法，高通量Gaussia颤蛋白互补法（HT-GPCA）在哺乳动物细胞中进行。 NT“&gt;大型识别病原体蛋白细胞伙伴进行通过交配的酵母双杂交放映cDNA文库使用多个病原体应变变种。是进一步的交互合作伙伴上选择了高可信度的统计评分的一个子集验证整套的病原体变体的蛋白质，使用HT-GPCA成对的相互作用，在哺乳动物细胞中的两个互补的方法，此组合提高了鲁棒性的交互数据集，并允许严格的比较相互作用分析的性能，这样的比较interactomics构成一个可靠和强大战略破译任何病原主机interplays的的。

HT-GPCA的主要优势在于它的高灵敏度，假阳性和假阴性率HPV E2蛋白在图2（改编自参考文献13）所示的评估。要确定假阴性率，已知的相互作用进行了评估，从HPV16 E2 HT-GPCA（ 图2A）。满分18相互作用没有恢复（对应于22％的假阴性率）。假阳性相互作用测定为5.8％，使用12种HPV E2蛋白对细胞的蛋白质（ 图2B）一组随机的。此外，特异性强的方法是突出图2C</s...

Watch this Scientific Journal Video about A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions at JoVE.com

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

本文重点介绍高信心之间的相互作用数据库宿主与病原体的蛋白质结合使用两个正交的方法识别：酵母双杂交，然后通过高通量相互作用的检测在哺乳动物细胞中名为HT-GPCA。

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

a-comparative-approach-to-characterize-landscape-host-pathogen

Research

JoVE Journal

Immunology and Infection

11.1K 次观看.  Institut Pasteur. 本文重点介绍高信心之间的相互作用数据库宿主与病原体的蛋白质结合使用两个正交的方法识别：酵母双杂交，然后通过高通量相互作用的检测在哺乳动物细胞中名为HT-GPCA。

视频: A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to facilitate cross-presentation of chaperoned antigens and elicit innate and adaptive T cell responses. Xenopus skin graft rejection provides an excellent platform to study the ability of gp96 to elicit classical MHC class Ia (class Ia) restricted T cell responses. Additionally, the Xenopus model system also provides an attractive alternative to mice for exploring the ability of gp96 to generate responses against tumors that have down-regulated their class Ia molecules thereby escaping immune surveillance. Recently, we have developed an adoptive cell transfer assay in Xenopus clones using peritoneal leukocytes as antigen presenting cells (APCs), and shown that gp96 can prime CD8 T cell responses in vivo against minor histocompatibility skin antigens as well as against the Xenopus thymic tumor 15/0 that does not express class Ia molecules. We describe here the methodology involved to perform these assays including the elicitation, pulsing and adoptive transfer of peritoneal leukocytes, as well as the skin graft and tumor transplantation assays. Additionally we are also describing the harvesting and separation of peripheral blood leukocytes used for flow cytometry and proliferation assays which allow for further characterization of the effector populations involved in skin rejection and anti-tumor responses.

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.

比较在体内 GP96佐剂研究中的青蛙非洲爪蟾</em

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to ...

科研

免疫与感染

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学：生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

表征形态变化的议定书<em&gt;艰难梭菌</em&gt;应对抗生素治疗

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

无标签定量蛋白经济学工作流程，用于发现驱动的宿主-病原体交互

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...