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><tbody><tr><td>Vinyl Anaerobic Chamber-Type B</td><td>Coy Laboratory Products</td><td></td><td>Model 2000 incubator&amp;nbsp;</td></tr><tr><td>TSA II Trypticase Soy Agar with 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&amp;nbsp;</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

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

科研

生物化学

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

免疫与感染

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

用于口腔中性粒细胞病评价的鼠期牙周炎的强结合诱导模型

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

厌氧菌小鼠阴道定植模型

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

用于研究体外宿主-病原体相互作用的器官型组织模型系统

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

建立稳定的双生糖核糖核关系从口腔

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

生物学

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Anti-Infective Efficacy in Cystic Fibrosis with Aggregate Biofilm Models

Cystic Fibrosis Aggregate Biofilm Model to Study Infection-relevant Gene Expression

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not reflect the environmental conditions of the hostile lung niche. Current reductionist testing conditions can lead to the progression of compounds along a preclinical pipeline without evidence of their activity under cystic fibrosis lung niche-appropriate conditions. Several approaches used to study traditional antimicrobials may not be suitable for antibiotic alternatives, including anti-virulence therapeutics like anti-quorum sensing agents and siderophore inhibitors. This protocol documents an aggregate biofilm model of Pseudomonas aeruginosa to compare resistance and infection-relevant gene expression in single-species and multi-species cultures (Staphylococcus aureus and Candida albicans), examining colony-forming unit (CFU) reductions and changes in gene expression, using algD as an exemplar. The model was optimized for small, static volumes of bacterial cultures to allow the study of novel compounds in the discovery phase of the drug development pipeline, where compound quantities may be limited. Single-species P. aeruginosa biofilms were formed in Synthetic Cystic Fibrosis Medium 2 (SCFM2) for 24 h before treatment with meropenem at different concentrations (1, 16, and 256 &#181;g/mL) for a further 24 h. Polymicrobial biofilms were established by growing Staphylococcus aureus and Candida albicans together in SCFM2, then inoculating with P. aeruginosa for an additional 24 h and treating with meropenem. The lack of a direct connection between compound efficacy measures in pre-clinical testing and clinical trial results has cast doubt on the applicability of current laboratory screening tools. This model allows us to understand the impact of relevant factors on P. aeruginosa gene expression, including genes contributing to resistance and virulence, thereby bridging this gap.

This study outlines a framework for developing an aggregate polymicrobial biofilm model and optimizing an RNA extraction protocol to assess Pseudomonas aeruginosa gene expression reflective of the cystic fibrosis lung environment. Applications include evaluating antimicrobial effects and studying antibiotic alternatives under conditions relevant to cystic fibrosis.

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not ...

Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells

Source: Wunsch, C. M. et. al., <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate a procedure to visualize internalized Porphyromonas gingivalis, an anaerobic pathogenic bacterium, within human endothelial cells using confocal microscopy. The bacteria are stained with a fluorescent dye while the host endothelial cell actin cytoskeleton is labeled with a different fluorescent dye. The internalized Porphyromonas gingivalis inside endothelial cells are visualized using confocal microscopy.

宿主内皮细胞内化厌氧菌的可视化

1. Internalization of Bacteria into Host Cells (Fluorescent Microscopy)
Note: P. gingivalis is labeled with ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

用于研究厌氧菌-宿主细胞相互作用的细菌感染测定

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...