We would like to thank Dr. Hiroshi Miyazaki, Dr. Scott Henderson, Dr. Todd Kitten, Dr. Justin Hutcherson, Dr. Catherine Jauregui, and Collin R. Berry. This work was supported by NIH NIDCR grants R01DE016124, R01DE018039, and R01DE023304 to J.P. Lewis.
Microscopy was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant (5P30NS047463).

以下协议将描述由厌氧种，P.培养和研究入侵方法牙龈 ;然而，也可以用于大量的厌氧病原体这些协议。虽然内皮细胞的使用，该协议可以用于其他的真核细胞两者的免疫和非免疫。 1.厌氧室中使用与维护 注意 : 牙龈是厌氧菌对氧的正常水平在环境空气中遇到敏感。受控的厌氧环境是体育的培养至关重要牙龈 。 <ol><li>这里，维持指定为混合厌氧气体（80％的N 2，10％H 2，10％的CO 2）中的乙烯基厌氧室中（图1A）的人工气氛。从实验室环境转印各色的厌氧室中使用一个气闸（图1B）。气闸手动操作，TW冰吹扫用的 N 2气体引入该混合厌氧气体之前。 </li><li>免维护除去不希望的硫化氢的使用硫化氢去除塔（图1C）。放置室内的除湿机，以除去H 2 O的通过催化剂产生并避免气溶胶促进污染扩散。 注意:硫化氢是许多厌氧细菌的天然代谢副产物和它的积累是有毒的细菌，并可能导致损害电子和降低的催化剂的寿命。 </li><li>使用一个风扇箱通过钯催化剂，从而消除在氢的存在下（图1D）氧气流通腔室的气氛。 注:再循环空气（HEPA）过滤器去除空气中的污染物以0.22微米或更大的尺寸。 </li><li>培养厌氧细菌在37℃培养箱位于厌氧的内室。厌氧室内工作时，使用标准的无菌技术。 </li></ol><img alt="图1" src="/files/ftp_upload/53408/53408fig1.jpg" /> 图1.厌氧乙烯基室及其组件。（A）的乙烯基厌氧室中从大气中的氧完全密封（32英寸×78英寸）提供工作空间的两个人的时间。它包含一个孵化器设置在37℃（背面中间部分）。 （B）中的气闸，用于物品从实验室环境转移至厌氧室中。图为是一个自动气闸通过可以被编程以自动执行以创建一个厌氧环境所需的真空和吹扫步骤的控制器操作。 （ 三）硫化氢去除列提供免维护高容量去除不需要的硫化氢。 （D）两个催化剂风扇框放在整个厌氧室的形式，通过钯催化剂，其中，在氢的存在，除去氧循环室的气氛。厌氧室设置按照厂家的说明。 <a href="https://www.jove.com/files/ftp_upload/53408/53408fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.准备厌氧菌注:P。牙龈是耐氧和可以存储在有氧条件，但在比6％17,18更高水平的氧的存在也不会增加。厌氧培养室是为适当地栽培P的牙龈和其他厌氧物种（ 图1）。适当的培训和教育，厌氧室中使用与microanaerobes 19工作之前需要。 <ol><li>平衡所有的液体介质和板厌氧康迪特至少12小时之前，实验离子以除去残余的氧。 </li><li>转移P.从-80°C冰箱厌氧室牙龈 ，让解冻。 </li><li>条纹P.牙龈上胰蛋白酶大豆血琼脂平板（TSA II含5％绵羊血）。在厌氧培养箱包裹板在封口膜，并储存在37℃下4-7天。 </li><li>接种P.牙龈于3ml脑心脏浸液（BHI）肉汤补充有氯化血红素和甲萘醌，富集非选择性液体培养基中厌氧和苛养微生物的分离和培养，使用无菌环路。 注意:对于长期储存，混合制得的BHI与甘油或DMSO（10〜20％最终浓度），并将其放置在-80℃冷冻的细菌培养。 </li><li>准备P的发酵剂培养物牙龈通过使1:10稀释，并允许细菌生长，直到对数中期。 </li></ol>注意:BAC的光密度terial悬浮液被确定并且该细菌浓度为每个应变要被检查被调整。对于P.牙龈的悬浮液以0.7对应OD 660至对数中期和〜7×10 8细胞/ ml。以上在协议中所述生长条件是特异性的P.牙龈和可能需要适合于其他细菌菌株。 3.内皮细胞培养注意:购买在5％CO 2合并含有血管内皮细胞生长因子（VEGF）的初级内皮细胞和培养在基础培养基在37℃下，根据制造商的说明。 <ol><li>种子的HUVECs在T-75烧瓶以2.5×10 5个细胞/烧瓶在15毫升的VEGF介质。 注:1稀释4.0％台盼蓝:通过1检查可行性。细胞有损害膜将保留台盼蓝，健康的细胞胎膜完整双目光学显​​微镜下观察时，将显示为白色。 COUN牛逼100个细胞，保证细胞超过80％是可行的20。 </li><li>更换介质每2天预热的新鲜VEGF介质，直至细胞达到约80％汇合。 </li><li>用预热的PBS洗一次细胞。通过用2ml胰蛋白酶-EDTA（0.25％）中5分钟，随后2 ml胰蛋白酶中和溶液孵育解放从T75烧瓶的细胞。 </li><li>收集在50毫升离心管中悬浮血管内皮细胞。从T-75瓶用PBS洗出任何额外的细胞和转移到50毫升锥形管。 </li><li>离心细胞，在200×g离心10分钟。 </li><li>除去上清液，悬浮细胞沉淀在10毫升预热VEGF媒体。 </li><li>确定使用血球或相似的细胞计数装置细胞浓度。 </li><li>计算的细胞悬液量添加至任一6孔板（400,000 /孔）或12孔板用盖玻片（50,000 /孔）。内皮细胞将准备试验的第二天。 </li></ol> 4.存活试验入侵/互动（电镀） 注意:当进行这种测定，准备接种在40万个细胞/孔的内皮细胞的两个6孔平板。一个板将被用来评估附着并宿主细胞内在化细菌。其他板块将占到胞内细菌。在6孔板允许在一个实验中要执行的两个样品的一式三份。对于此协议的概要请参考存活测定法流程图（ 图2）。 <ol><li>制备如上所述（参见第1部分），直到他们达到中等对数生长（OD 660 0.5-0.7）厌氧菌。 </li><li>离心机菌在5000×g离心10分钟。 注意:如果离心机是外厌氧室中，携带细菌样品在密封15ml试管，包住盖用封口膜以防止氧气泄漏。 </li><li>将沉淀P.牙龈回到室内，弃上清。用PBS重悬在VEGF我之前再次洗涤，沉淀细菌直径。制备悬浮液的所有细菌菌株以0.7对应于对数中期（〜7×10 8细胞/ ml）OD 660进行测试。细菌现在准备感染。 </li><li>转印6孔板含有内皮细胞从组织培养箱成厌氧室中。取出介质和洗三次厌氧PBS。加2ml厌氧VEGF介质到每个孔中，并将板于37℃在厌氧培养箱20分钟以平衡感染的温度。 注意:血琼脂平板上平板的细菌，以确保用于感染的那些是均匀的和感染时不被污染。 </li><li>感染宿主细胞与细菌于感染复数（MOI菌:主机）100:1。 注意:HUVEC细胞数目由感染之前，在单一井进行锥虫排斥试验测定。细菌细胞数目通过光密度测定（例如，外径为0.5 = 5×10 8个细胞/ ml）。乙acterial浓度基于对HUVECs浓度21调整到适当的MOI。 </li><li>放置6孔板与受感染的HUVEC成厌氧培养箱和使细菌与宿主细胞相互作用30分钟。 </li><li>制备皂苷在BHI（1.0％重量/体积）里面的厌氧室中并通过0.2微米的过滤器进行过滤。 </li><li>生存附加和内在的细菌。 <ol><li>从培养箱，吸介质除去板，洗三次，厌氧PBS中，加2ml滤1.0％皂甙（如在步骤4.8中所述制备）。孵育15分钟，以允许宿主细胞裂解。 </li><li>每刮底以及与细胞刮刀。收集从每个孔的细胞混合物，并以1:1稀释于BHI。 </li><li>继续以使样品的系列稀释液。根据细菌种类和浓度，调整连续稀释。开始1:100和1:1000的稀释液。 </li><li>板200微升血琼脂平板上所需的稀释。裹吨他板在封口膜和发生在厌氧培养箱在37℃。 </li><li>七天的培养在37℃，取出板和计数使用光框手动计数菌落菌落形成单位（CFU的）。 注:CFU的列举。对于较大数量的CFU的，图像可以被获取并且计算机软件可以被用来促进的CFUs的枚举。 </li></ol></li><li>生存内在的细菌。 <ol><li>从培养箱中取出盘子。厌氧PBS洗三次，并加入2毫升的VEGF媒体用补充抗生素（庆大霉素300微克/毫升和甲硝唑的400微克/毫升）。 </li><li>孵育1小时。一定要测试的抗生素，所以他们是100％有效杀灭所需的细菌菌株，并确保它们不会穿透宿主细胞22,23。 </li><li>吸媒体，加2ml过滤1.0％皂甙。孵育15分钟，以允许宿主细胞裂解。 </li><li>刮每个逢底l在细胞刮刀。从每个孔收集细胞混合物，并1:1稀释在BHI。 </li><li>制备样品（1:100，1:1000）的系列稀释液。 </li><li>板200微升血琼脂平板上所需的稀释。裹在板封口膜和发生在厌氧培养箱。 </li><li>七天后的潜伏期在37℃除去板和计数的CFU。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/53408/53408fig2.jpg" /> 图2.用于厌氧菌与真核细胞生存的协议的示意图。这两个试验共细菌的生存和内细菌的生存可以在同一时间进行。 <a href="https://www.jove.com/files/ftp_upload/53408/53408fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.内在细菌进入主策LLS（荧光显微镜） 注:P。牙龈被标记2&#39;，7&#39;-双- （2-羧乙基）-5-（和-6） -羧基，乙酰氧基甲基酯（BCECF-AM）。 BCECF-AM是一个非荧光膜可渗透染料;其通过细胞内酯酶的作用转化为荧光素BCECF可以指示细胞活力。P.牙龈标有BCECF-AM染料，然后用于感染的真核细胞。以下感染，将细胞固定并用DAPI标记和TRITC鬼笔环肽。用于染色的真核细胞核的DAPI染色也将标签细菌细胞核，它提供了应对措施，以确定非活细菌不能代谢裂开的BCECF-AM。宿主细胞突出了TRITC - 鬼笔，一个红色的肌动蛋白染料。 <ol><li>高压灭菌盖玻片。以5×10 4个细胞接种内皮细胞/孔之前无菌添加到盖玻片的12孔板中。 （实验前准备天） </li><lI&gt;有如上所述制备18毫米（1.5＃厚度）圆形盖玻片在12孔板内皮细胞。 </li><li>制备如在第1节中所述生长至对数中期厌氧菌（OD 660 = 0.5-0.7）。 </li><li>洗涤细菌通过离心分离在5000 xg离心并在5-7×10 8个细胞/ ml悬浮沉淀在PBS中2倍厌氧PBS中。 </li><li>加入20微升0.2mM的BCECF-AM至2ml细菌悬浮液（5-7×10 8个细胞/ ml）与BCECF-AM为2μm的最终浓度。 </li><li>在37℃下在黑暗中30分钟。 </li><li>转移板与接种18毫米（1.5＃厚度）圆形盖玻片从组织培养箱进入厌氧室中的内皮细胞。用PBS和厌氧VEGF媒体交换洗涤。 注:验证内皮细胞是在光学显微镜下健康。脐静脉内皮细胞应该〜80％融合，形态应该是媲美的厂商。 </li><li>离心机标记的细菌在5000×g离心10分钟以去除残留的BCECF-AM染料。暂停在2ml厌氧VEGF介质。 </li><li>感染宿主细胞在100 MOI标记的细菌:1（细菌:主机）。 </li><li>孵育在厌氧室中在37℃下30分钟。 </li><li>感染后洗涤细胞，用PBS三次并固定在新鲜制备的4.0％多聚甲醛10分钟。 注意:固定细胞后，实验可以在厌氧室外面进行。 </li><li>洗涤盖玻片用PBS三次。 </li><li>加入1毫升0.2％的Triton X-100的10分钟。 </li><li>洗涤盖玻片用PBS三次。 </li><li>添加50微升TRITC鬼笔环肽（50微克/毫升），以盖玻片45分钟。 </li><li>洗涤盖玻片三次，从12孔板和地方去除一个用含有DAPI的软组安装介质滑动。密封边用指甲油。 注:幻灯片可以储存一两个月在黑暗中。避免光线照射，防止光漂白。 </li><li>查看幻灯片使用共焦显微镜。 <ol><li>在这里，使用围绕AxioObserver（倒）配置了一个34通道光谱系统（32通道阵列检测器和两个侧PMT探测器，再加上透射光检测器）站在电动XY阶段。该系统具有五个激光器:蓝色二极管（405纳米），多线氩（458，488，514纳米），绿色二极管（561纳米），红色氦氖（633纳米）和一个440nm的脉冲激光。装备一个荧光寿命成像系统2混合的GaAsP检测器（用于FRET - FLIM）。 </li><li>使用的双频带滤波器，340-380纳米和540-560纳米，和435-485纳米和570-590纳米的分别发射滤光片的激发波长检测来自DAPI和TRITC荧光一个通道。使用具有440-500纳米的激发波长和510-590纳米的发射滤波器的滤波器检测从BCECF-AM荧光。 注意:控制BCECF-AM应在每个菌株做正在研究，以确保活菌适当的标签。首先验证nonviab勒细菌是DAPI阳性和BCECF阴性。其次，确保活细菌能代谢BCECF-AM为荧光BCECF。可变浓度的细菌或BCECF-AM染料可能需要对最佳标记进行测试。 </li></ol></li></ol>

Authors have nothing to disclose.

所有上述方法可用于设计特异性试验，以评估厌氧菌与真核细胞的相互作用。然而，有几个方面的考虑，以成功地执行该实验。第一是在一个研究中所用的微生物菌株。 它是在两个菌株均存活测定法的比较以及由显微镜分析至关重要的是，它们是在类似的生长阶段和达到相似的细胞浓度如在上述的任何差异可以影响入侵效率13。当生长曲线两株细菌对之间的不同，调?...

厌氧细菌定殖于人的身体的几乎所有表面。虽然主要在肠道和泌尿生殖道，其中氧气浓度是低的植物，它们也存在着在高水平上的皮肤，口，鼻，喉和1。厌氧细菌是内源性感染的常见原因，并且经常从患病部位分离。由于它们的性质挑剔然而，厌氧菌可能很难分离和培养。研究涉及厌氧菌一定限制条件下进行。现代厌氧培养技术使研究人员模仿学习许多厌氧菌实验室菌株，甚至临床分离2,3所需的厌氧设置。 致病性厌氧菌已经开发出一种动态关系和共同进化与它们所在的宿主细胞。最厌氧菌易受到达infecti之前杀死被宿主的免疫反应OU的水平。然而，一些病原菌已经开发机制从逃脱或破坏宿主的免疫反应。他们实现这一目标，通过机制，如免疫识别，免疫介导的中和，改变细胞介导的免疫，侵入宿主细胞，并改变免疫信号4的逃避。 牙龈卟啉 ，革兰氏阴性厌氧菌参与了口头和口外疾病， 是一个高度适于细菌病原体能够引起在宿主5-7致病变化的一个例子。 生物膜斑块口袋累积在牙齿和牙龈粘膜组织间形成能够携带受保护的大气中的氧气8厌氧菌深的裂缝。这些牙周袋作为利基关于各种厌氧病原体， 诸如铜绿牙龈 9 P。牙龈是一个梯形的病原体，它能够改造的荷兰国际集团的口腔微生物群落的促进发展的牙周病10和发展方式。它产生了大量的有效对抗宿主蛋白广谱并提供机制宿主防御11的逃避毒力因子。它也能够侵入上皮细胞，内皮细胞，成纤维细胞，和牙周膜细胞体外 12-14中 和体内15。通过有效地侵入宿主细胞中，P。牙龈可以逃避宿主免疫。有效侵入宿主细胞不仅允许细菌逃离宿主防御但也作为贮存以备将来再感染以及改变宿主细胞。需要由宿主细胞参与粘附和细菌的内化的分子机制的研究进展。研究几个实验室的重点是了解与体育的内在关联的分子事件牙龈由宿主细胞以及用于抑制并劫持免疫反应和生存的敌对宿主防御机制的机制。 有能够识别和表征的病原体，其能够侵入宿主细胞的许多测定法。 但是 ，在体外研究与厌氧病原体提出了许多实验问题，为研究者主要是因为它是难以执行依赖于在不存在氧的笨重仪器研究。这是由真核细胞所需要的氧气，以成长，因此必须在组织培养孵化器分别制备的事实复杂。避免这种障碍之一的方法是将大气中的氧气下进行研究，但是这将使厌氧细菌的生长是不可能的。另一种方法是使用热灭活的细菌感染和研究宿主 - 细胞相互作用。然而，不同的削弱宿主 - 病原体interacti的相关性热灭活和可行的细菌之间存在16。这是中央对学习活菌与不变的表达与宿主细胞相互作用的;因此，方法培养P.在厌氧环境牙龈给出。此外，两个简单的成本效益的协议被证明为评估P的能力牙龈由人脐静脉内皮细胞（HUVECs）被内化。所述第一协议是基于流行的抗生素保护测定。虽然实验非常简单，利用厌氧微生物时的注意事项给出。第二协议要求使用荧光扫描显微镜的可视化和交互内在P.牙龈 。每个测定有其局限性和将要讨论，以提供研究者的轮廓为研究厌氧菌的侵袭优点。虽然目前的手稿研究P.牙龈和内皮细胞，这些协议可以用于许多其它厌氧细菌以及作为其他类型的宿主细胞。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vinyl Anaerobic Chamber-Type B</td>
			<td>Coy Laboratory Products</td>
			<td></td>
			<td>Model 2000 incubator&#160;</td>
		</tr>
		<tr>
			<td>TSA II Trypticase Soy Agar w/5% Sheep Blood</td>
			<td>BBL</td>
			<td>221261</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells 10-donor Pool</td>
			<td>LifeLine Technology</td>
			<td>FC-0044</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF Medium Complete Kit</td>
			<td>LifeLine Technology</td>
			<td>LL-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypKit</td>
			<td>LifeLine</td>
			<td>LL-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Riedel-de Haen</td>
			<td>16109</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Sulfate Salt</td>
			<td>Sigma-Aldrich</td>
			<td>G-1264</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole</td>
			<td>Sigma-Aldrich</td>
			<td>M-3761</td>
			<td></td>
		</tr>
		<tr>
			<td>BCECF-AM</td>
			<td>LifeTechnologies</td>
			<td>B1150</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC Phalloidin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm Circular Coverslips</td>
			<td>Electron Microscopy Sciences</td>
			<td>72222-01</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

porphyromonas gingivalis as a model organism for assessing interaction of anaerobic bacteria with host cells

厌氧菌的数量远远超过许多人的壁龛，如肠道，口腔和阴道需氧菌。此外，厌氧菌感染是常见的，经常土著血统的。的一些厌氧病原体侵入人体细胞的能力使它们适应措施逃避先天免疫以及调节宿主细胞中的行为。然而，确保厌氧细菌是试验研究的事件可能会带来的挑战过程中活。 牙龈卟啉 ，革兰氏阴性厌氧菌，能够侵入的各种真核非吞噬细胞的。本文概述了如何成功培养和评估P的能力牙龈侵入人脐静脉内皮细胞（HUVEC）。两个协议被开发:一个用于测量细菌，可以成功地侵入和主机内生存，而其他的可视化的细菌与宿主细胞相互作用。这些技术需要使用一个ANAE的罗比克室提供P.牙龈与最佳生长厌氧环境。 所述第一协议是基于抗生素保护分析，这在很大程度上是用于研究的宿主细胞的入侵的细菌。然而，抗生素保护测定是有限的;仅胞内细菌是培养的下列抗生素治疗和宿主细胞裂解计量。为了评估与宿主细胞，无论是活的和死的相互作用所有的细菌，我们开发了使用荧光显微镜检查宿主 - 病原体相互作用的协议。细菌荧光标记与2&#39;，7&#39;-二（2-羧乙基）-5-（和-6） - 羧基乙酰甲酯（BCECF-AM）和用于感染的真核细胞在厌氧条件下。以下定影用多聚甲醛和透用0.2％的Triton X-100，宿主细胞被标以TRITC鬼笔环肽和DAPI分别以标记细胞骨架和细胞核。多IMA在不同的联络点（Z堆栈）采取GES是为细菌的时空可视化获得。在这项研究中使用的方法可以适用于任何可耕厌氧菌和任何真核细胞类型。

上述协议在研究P之间宿主-病原体相互作用中使用牙龈和内皮细胞。P.牙龈 W83和P.牙龈 V3150携带缺失PG0228的是在研究中使用。的PG0228被预测可能改变的RNA和蛋白质的水平的蛋白质，其可最终影响P的相互作用编码牙龈与宿主细胞。研究PG0228对体育的影响牙龈的与宿主细胞相互作用的能力，亲和突变株与内皮细胞相互作用的能力进行比较。生存协?...

Watch this Scientific Journal Video about Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells at JoVE.com

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

porphyromonas-gingivalis-as-model-organism-for-assessing-interaction

Research

JoVE Journal

Immunology and Infection

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

26.2K 次观看.  Virginia Commonwealth University. This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type. 

视频: Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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 ...

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

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

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

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

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

摘要

探索更多视频

此视频中的章节