华润哈丁是由威康信托助学金WT086724支持。

1。 L的制备肺军团菌的感染<ol><li>制备炭酵母提取物（CYE），板（2 g / L的活性炭，10g / L的酵母提取物13克/升琼脂，10g / L的N-（2 - 乙酰胺基）-2 - aminothanesulfonic磺酸（ACES），1 g / L的α-酮戊二酸，0.4 g / L的L-半胱氨酸盐酸和0.25克/升焦磷酸铁，pH值6.9）。 注意:如果需要，添加卡那霉素（25微克/毫升）和/或氯霉素（6微克/毫升）至CYE板。 <ol><li>开展所有L。在生物安全防护水平的2 肺的工作（BSL-2）在微生物安全柜（MSC）的符合当地规则。 </li></ol></li><li>数L。从-80°C甘油股票到CYE平板杆菌 </li><li>在37°C孵育板4天 注:孵化板4天显著增加L的毒力肺军团菌在G蜡螟过温育3天。 </li><li>感染前一天，悬浮一个循环复细菌在1ml预热LL（含有几个菌落）（37℃）ACES酵母提取物（埃）肉汤并用分光光度计测量吸光度在600nm（OD 600）。 </li><li>接种新鲜3毫升埃培养物（以及如果需要抗生素）至最终OD 600为0.1。 <ol><li>包括媒体只能控制，以确保媒体不育。 </li></ol></li><li>在摇床以200rpm 21小时在37°C。 注:细菌应生长到指数后期感染38。生长21小时允许实验进行标准化。 注:如果需要的蛋白的诱导，添加0.5mM的异丙基β-D-1-硫代半乳糖苷（IPTG）诱导过夜。 </li><li>测量外径600（细菌应该是在后期指数生长期，外径600 2.5-3）。 </li><li>稀释细菌培养物，得到1×10 9 CFU / ml的无菌Dulbecco氏磷酸盐缓冲盐水（D-PBS）中。 注:基于先前结果的OD 600为1对应于1×10 9 CFU / ml的，然而，这应该被证实为不同的L。杆菌菌株。 注意:如果需要感应从质粒的蛋白质，加入IPTG 1mM的对接种物。 </li><li>板作为在第9描述作为对照，以确保预期的CFU存在于接种物接种物。 </li></ol> 2。幼虫的制备<ol><li>购买足够的G。蜡螟幼虫从商业供应商。幼虫被运送于5 个或6 个龄期（约之间2-3厘米长），并适合立即使用。 注意:描述如何后方幼虫以前已经39描述方法。 注:幼虫可存放在室温下长达两个星期，不需要食物。立即丢弃任何幼虫化蛹显示的迹象。 </li><li>通过的Plac准备容器幼虫ING为10厘米的滤纸在一个10cm培养皿的底部的圆。 </li><li>用钝头镊子，放置约同样大小十大健康幼虫放入培养皿中。 注:放弃不健康的棕色或斑点寻找昆虫。健康的幼体均匀的奶油色的，没有地区色变暗，并能够迅速向右自己，如果翻过来。 </li></ol> 3。 G的感染蜡螟幼虫<ol><li>通过绑扎的滤纸圆的表面制备的注射平台。 </li><li>牢固水平带的P1000尖端到滤纸来创建一个注入平台。这并不需要是无菌的。 </li><li>通过吸液70％的乙醇和孵育至少10分钟灭菌一个20微升微量注射器。 <ol><li>同时注入戴上防刺穿手套， </li></ol></li><li>通过抽吸和排出无菌水几次去除残留的乙醇。 </li><li> USI纳克注射器，吸液10微升L的肺军团菌 1×10 9 CFU / ml的悬浮液。 </li><li>拿一个幼虫，轻轻地但坚定地把它在它的后面，弯腰的P1000一角。 </li><li>将注射器的针尖对幼虫的前面，右侧前脚。 </li><li>轻轻地，将针尖到前脚，确保它是幼虫在里面，并顺利注入所有的注射器内容。 注意:如果该注射器安装正确时，它应该有可能只用注射器将其放置到腔拿起幼虫。 <ol><li>注射后，观察幼虫几秒钟。幼虫会开始后几秒钟抓取，但不应该排泄液。 </li></ol></li><li>简要地观察幼虫几个小时后感染;感染10 7 CFU L.肺 130b中不引起所述第一5-8小时圆周内的任何症状，因此，如果幼虫都转向灰/黑这一点之前，叔他的实验时应停药。 注意:幼虫用1mM IPTG的接种不影响幼虫存活在实验的过程中。 </li><li>以相同的方式，总共每条件包括10个幼虫注射用D-PBS作为对照，分析幼虫死亡率10幼虫注射。 </li><li>磁带培养皿关闭，发生在二次安全壳。 </li><li>孵育在一个标准的细菌培养箱中，在37℃，在实验的持续时间。 </li></ol> 4。幼虫预处理用化学抑制剂<ol><li>在此之前的感染，如第3.1-3.6的介绍准备幼虫注射。 </li><li>注射10微升的细胞松弛素D的100μM的溶液进入前，左10幼虫前脚。 注:抑制剂注入细菌悬液不同的前脚，以减少伤害幼虫。 </li><li>注射10微升的DMSO中成10个幼虫作为对照。 </li><li>孵化幼虫37°C为4小时。 </li><li>注入预处理幼虫在第3节入前所述，无论是1×10 7右侧前脚WT L的CFU 肺或PBS对照。 </li></ol> 5。幼虫死亡率分析<ol><li>在18小时后感染（PI），检查所有受感染的幼虫的​​死亡率。 </li><li>要检查死亡率，用钝头镊子改过的幼虫，寻找腿部的运动，健康的幼虫应迅速合适自己。色素沉着表示了强烈的免疫反应，感染。如果有任何动静，算不算活着。 </li><li>死者和活着的幼虫的记录数。 </li><li>在选择了其它所有时间点重复此过程。 注意:如果继续培养超过三天，蛹可以看出。删除任何蛹和通过在冷冻安乐死 - 20°C才变态可能发生。 </li></ol> 6。血淋巴的提取<ol><li>随机选择3幼虫并放入14毫升管在选择的时间点，例如5和18小时圆周率， </li><li>将此管在冰上5-10分钟，直到幼虫的腿没有运动可以被观察到。 </li><li>将麻醉的幼虫到培养皿，用解剖刀，使附近的幼虫的尾部两个段之间形成切口。 </li><li>挤幼虫进入无菌1.5毫升离心管收集血淋巴。 <ol><li>池血淋巴从至少三个人。 15-50微升血淋巴中的一个幼虫给人视大小而定。 注意:在血淋巴中提取它是很容易扰乱肠道，造成潜在的样品的污染。通过切割幼虫靠近尾部（远离肠）然而，抗生素的选择将永远被镀出的细菌时，需要减少污染。 注意:为了防止从血淋巴内采集后10分钟变成褐色和凝固，过程中血淋巴。 </li></ol></li><li>丢弃幼虫体内转化为一个新的14毫升猎鹰管，密封和地点 - 20℃过夜，以确保幼虫都死了。 </li><li>高压灭菌死亡的幼虫，并根据当地法规处理。 </li></ol> 7。活力血球的测定<ol><li>提取如上所述的血淋巴。 </li><li>混合20微升提取的血淋巴与20μl的0.02％（体积/体积），台盼蓝的PBS中在96孔板的一个孔中。 </li><li>孵育5分钟，在RT。 </li><li>负载10μL血淋巴到血球计数和可行的（不是蓝色）细胞。 </li><li>计算每个样品一式三份，以减少误差。 </li></ol> 8。提取的血细胞进行免疫荧光显微镜的处理<ol><li>混合血淋巴汇集来自至少三个幼虫和吸管到10-15毫米的玻璃盖玻片的24孔板中提取。 注:盖玻片不需要治疗，血细胞可以附着到玻璃上。 </li><li>加入0.5毫升的D-PBS中，通过上下吹打拌匀。 </li><li>离心平板10分钟，在500×g离心，在室温（RT）用气密离心机板固定器。 </li><li>使用倒置显微镜来检查血细胞都坚持检查每口井。 </li><li>去除上清，并小心地加入0.5ml的D-PBS以井的壁，摇动板2-3倍，并用移液管取出的D-PBS洗涤细胞3次。 </li><li>通过加入0.5ml的4％（体积/体积）的多聚甲醛（PFA）的PBS固定细胞。 </li><li>孵育的细胞在室温20-30分钟之间。 </li><li>如前用D-PBS洗涤细胞3次。 </li><li>加入0.5毫升15 mM的NH 4 Cl等在PBS中以淬灭残余的煤灰和在室温下孵育15分钟。 </li><li>用D-PBS洗涤细胞3次。 注意:在这个阶段，盖玻片可以一夜之间在4℃下保存</li><li>加入0.5毫升的0.1％的Triton X-100的PBS中孵育5分钟，在RT通透细胞。 </li&gt; <li> 1小时以封闭液（2％（重量/体积）BSA的PBS）块。 </li><li>孵育1小时，在室温下在黑暗中与稀释在封闭液在由制造商指定的稀释的第一抗体。 </li><li>用PBS洗3次。 </li><li>孵育1小时，用第二抗体和DAPI对如上述细菌的可视化。 </li><li>用PBS洗3次。 </li><li>用一滴安装试剂到玻片上安装的盖玻片。 </li><li>孵育过夜在黑暗中在RT下充分干燥该安装的解决方案。 </li><li>图片幻灯片上的荧光显微镜。 </li></ol> 9。细菌菌落形成单位的量化<ol><li>添加100微克/毫升大观霉素对CYE板，以避免污染肠道菌群。L.肺军团菌菌株130B自然是耐壮观霉素40。 </li><li>血淋巴提取前，权衡1.5毫升离心管中。 </li><li>抽取血淋巴中第6条所述并放置在称重吨ubes，加入1μl5 mg / ml的毛地黄皂苷，拌匀，静置5分钟，在室温裂解血细胞。 </li><li>再次称重该管与血淋巴和确定血淋巴中提取出的权重。 </li><li>在无菌AYE媒体进行血淋巴的10倍系列稀释。 </li><li>用一支笔，CYE板的底座分为相等的六部门和标签。 </li><li>板3滴25微升每种稀释液（从最稀）在所述板的每个部分。 </li><li>培养板在37℃下至上过夜盖子</li><li>一旦滴干了充分，转板过​​来，在37℃下至少再两天。 </li><li>量化由在每个稀释度计数的菌落中提取的细菌和规范化到血淋巴中提取的重量。 </li></ol> 10。竞争指数的测定（CI） <ol><li>确认这两个菌株生长同样在肉汤培养和CYE琼脂平板PRIOr来尝试的有竞争力的指标。 </li><li>准备WT或卡那霉素抗性突变菌悬液第1条中所述及混合比例为1:1。 <ol><li>接种到CYE壮观霉素（100微克/毫升）和CYE壮观霉素/卡那霉素平板系列稀释</li></ol></li><li>感染幼虫和提取物在血淋巴如上所述合适的时间点。 </li><li>通过提取血淋巴和电镀系列稀释到CYE大观霉素和壮观霉素CYE /卡那霉素确定活菌数。 </li><li>计算具有竞争力指数（CI），如下所示:CI =（突变输出/输出WT）/（突变体接种物/ WT接种物）。 </li></ol>

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对大蜡螟的嗜肺军团菌感染的幼虫模型是发病的体内研究的有用工具。这里，示出了一个数字巨噬细胞的感染方面的可以在杠杆被概括蜡螟模型，包括点/ ICM T4BSS的毒力和细菌复制的作用和点/ ICM-效应上实发展的定位。此外，我们证明了肌动蛋白聚合的化学抑制剂显著降低幼虫死亡率，极似在macophages 47得到的结果和支持的证据表明，细菌的内在需要引起幼虫死亡率。先前，这已经证实，L之间的偏差在毒力见于其他感染模型肺军团菌菌株能在G进行验证蜡螟和诱导毒力因子在后期指数生长期所需的细菌的毒力<su对&gt; 36，确认了G。蜡螟是一个合适的模型，L。肺军团菌感染。 确定L的CFU从幼虫杆菌感染它们可以单独或混合感染大大增加了模型的实用性。以前，有几个因素已经发现，在感染29,48-51中的一个或多个模型上细菌复制微妙的影响。虽然幼虫不具备适应性免疫系统，先天免疫反应的存在提供了强的选择相比，单独的巨噬细胞，其可以用于扩增细微的表现型。因此，它是可能的，而这些菌株可能不会显著影响幼虫死亡率，它们可以显示在G.减少细菌复制或健身蜡螟模型。以及L的复制肺中的幼虫，我们已经表明显著血细胞耗竭后期的感染。由于L。肺预计裂解宿主细胞，在其复制周期结束时，测定血细胞耗竭也可作为细菌复制的间接测量。血细胞耗竭之前已与相关感染3,52昆虫死亡，虽然最近的研究结果表明，这张照片是更复杂的比第一belived 37。最近，已经表明，幼虫饥饿通过免疫反应53抑制导致增加对感染的易感性。在这里描述的试验，幼虫不喂了研究的持续时间和它不知道有多好喂养幼虫会到L回应肺军团菌感染。 一个G的优点蜡螟作为模式生物是便于提取感染幼虫血细胞和定量。以前的视频显示各种方法，从昆虫54,55血细胞中提取然而，遇见HOD这里介绍的是简单，适合立即处理。一旦提取，血细胞可轻易量化，用于免疫荧光，透射电子显微镜36或流式细胞仪56或培养和感染的体外 3使细胞对病毒感染的反应进行详细调查。这显著增加了模型的灵活性。有一点需要注意在G免疫荧光蜡螟是抗体的供应有限验证对G。蜡螟的蛋白质。然而，研究证明产生抗体对抗幼虫蛋白57抗体及抗人免疫相关蛋白的发现认识到G。蜡螟蛋白11展示了潜在的免疫荧光测定对G。蜡螟血细胞。 G的方便蜡螟感染可以快速，中等通量筛选，可以用于比较各种军团杆菌物种和菌株的毒力，并可以用于进一步分析先前发现的毒力因子，如粘附分子58或2型分泌系统59，它在其它模型中所需的毒力。此外，使用这种模式将使新型毒力因子包括分泌和转运效应蛋白的鉴定和进一步鉴定。最近，已经表明L的该磷脂酶C活性肺军团菌在G的作用蜡螟的毒力60，而点/ ICM效应蛋白SDHA是必需的毒力37。此外，我们最近表明，有在G中观察到的表型之间的相关性蜡螟和在A /系小鼠品系37。 这表明了这个工具的价值，以配合环境原生动物和单细胞宿主和穆里NE感染模型。在G。蜡螟模型将更加有价值成为今后，一旦幼虫基因组序列将可与多种遗传工具被建立。在这个方向的步骤包括最近出版的详细免疫相关转录组61和主动性，提升基因沉默在鳞翅目属62的形成。 通过使用G。蜡螟幼虫，我们有许多细菌毒力的简单，快速读出，可用于研究L的发病机制肺军团菌 。建立这些分析和L的更广泛的筛查肺军团菌血清型和将增加这个新工具的实用工具，将有助于我们L.的理解肺的发病机制。

感染的动物模型中的细菌毒力因子的确定是无价的。然而，无脊椎动物模型已经受到越来越多的关注，一个可行的替代感染哺乳动物的传统模式。蜡蛾幼虫， 大蜡螟越来越多地被用来研究一些重要的人类病原体，包括革兰氏阳性1和革兰氏阴性菌2,3和几种病原真菌4,5。使用昆虫模型具有许多优于传统的哺乳动物模型的优点，如无脊椎动物，G.蜡螟不受哺乳动物模型的道德限制。此外，幼 ​​虫可容易地维持，感染通过注射无麻醉，经过预处理化学抑制剂6和维持培养，在37℃7。有趣的是，在G中几个微生物的致病性之间有良好的相关性MELlonella和感染的哺乳动物模型已经建立2,8。 G的免疫系统的增加的了解蜡螟还协助在这个模型有机体的特征。虽然昆虫没有一个适应性免疫系统在哺乳动物中发现，他们有成熟的细胞和肱骨防御包括生产抗菌肽9。血细胞是细胞防御系统的主要介质，并在G的血淋巴（或血液）中发现的数量最多的细胞类型蜡螟 10，这些细胞是专业的吞噬细胞和由两个占用，并在吞噬溶酶体隔室10,11降解细菌和周围侵入的细菌形成结节，物理限制细菌复制12执行类似的功能，以人类巨噬细胞和嗜中性粒细胞。 嗜肺军团菌是一种呼吸道病原体引起严重pneumoni一个（军团病）的易感人群，如老人或免疫功能低下13。 军团菌在环境和人为的水源，它是不同种类的淡水变形虫14,15的病原体无处不找到。 军团菌生存和通过利用多蛋白复合物被称为点/ ICM（有缺陷的细胞器贩卖/胞内乘法）型4分泌系统（T4SS）易位超过275个效应蛋白进入宿主细胞16-20这些专业的吞噬细胞内复制。这些蛋白质有助于颠覆了正常的宿主细胞吞噬途径，从而创建包含液泡（LCV）的军团菌 。的LCV避免与溶酶体融合，而是招募质网（ER）衍生的小泡，导致一个专门的隔室，类似于粗糙ER 21,22。L.肺军团菌被认为是一种偶然的人类路径OGEN;相同的策略，允许它在变形虫复制，也让人类肺泡巨噬细胞23复制。 哺乳动物宿主已被定性为模型人类军团菌感染，包括小鼠和豚鼠24,25。然而，大多数的小鼠品系有抗药性的军团菌感染26唯一的例外是近交系白化病的A /系小鼠，其中开发一个温和的，自限性感染24。虽然豚鼠模型更类似于人类疾病25，缺乏突变体和增加的成本阻碍了它们的使用27。此外，一些无脊椎动物模型已经被开发用于嗜肺军团菌感染，包括线虫 28， 果蝇变形虫30-32的几个品种29和。然而，这些模型也有弱点，毒力在C。线虫 </em&gt;系统不点/ ICM-28依赖性，限制了该模型的实用性。 果蝇模型已被证明有效的调查细菌的毒力因子29，似乎不过是有前途，这种模式还没有得到充分的特点。单细胞变形虫是L的主机环境肺军团菌 ，是理想的调查致病因素的作用在分子水平上33但是缺乏哺乳动物宿主细胞对感染的反应，如半胱天冬34的几个重要介质。现有模型的弱点，伴随着高成本和有关哺乳动物的实验伦理问题，导致了寻找其他合适的模式生物29,35。 最近，我们证明了G。蜡螟是一个合适的模型，L。肺发病机制36,37。这个协议的细节用于感染实验技术ING G。蜡螟幼虫，幼虫分析道德，提取血细胞计数和免疫荧光和确定了可行的CFU计数的复制从受感染的幼虫。

<table border="1">
 <tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material/ Equipment</td>
		 </tr>
		 <tr>
			<td>ACES yeast extract (AYE) broth</td>
			<td></td>
			<td></td>
			<td>4 g ACES, 4 g yeast extract, 0.4 g &alpha;-ketoglutarate, pH 6.9, 400 ml H2O, autoclaved, 4 ml iron solution*, 4 ml cysteine solution* 
		 *add sterile ingredients after autoclaving</td>
		 </tr>
		 <tr>
			<td>Charcoal buffered yeast extract (CYE) plates</td>
			<td></td>
			<td></td>
			<td>As above, with the addition of 0.6 g activated charcoal and 6 g agar</td>
		 </tr>
		 <tr>
			<td>Sterile iron solution (Ferric Pyrophosphate)</td>
			<td>Sigma</td>
			<td>P6526 </td>
			<td>0.6 mM solution, sterile filtered</td>
		 </tr>
		 <tr>
			<td>Sterile cysteine solution</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td>3.3 mM solution, sterile filtered</td>
		 </tr>
		 <tr>
			<td>G. mellonella (waxworms)</td>
			<td>Livefood UK</td>
			<td>W250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-HA-Tetramethyl Rhodamine Isothiocyanate (TRITC)</td>
			<td>Sigma</td>
			<td>H9037</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-Legionella LPS</td>
			<td>Cambridge Biosciences</td>
			<td>PA1-7227 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-Rabbit IgG Alexa488</td>
			<td>Jackson Immunoreach</td>
			<td>711-485-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Sigma</td>
			<td>I6758</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbeccos phosphate buffered saline (D-PBS)</td>
			<td>Sigma</td>
			<td>D8662</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Paraformaldehyde</td>
			<td>Agar Scientific</td>
			<td>R1026</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ammonium chloride (NH4Cl)</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D141</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cytochalasin D</td>
			<td>Biomol International</td>
			<td>BML-T109-0001</td>
			<td></td>
		 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>[header]</td>
 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Microtiter Syringe</td>
			<td>Sigma</td>
			<td>24544</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cell counter, double, Improved Neubauer </td>
			<td>VWR</td>
			<td>631-0926</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For centrifuging plates </td>
		 </tr>
		 <tr>
			<td>Fluorescence microscope</td>
			<td></td>
			<td></td>
			<td>Any microscope with appropriate filters for the required fluorophores</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td></td>
			<td></td>
			<td>For viable cell counting </td>
		 </tr>
		 <tr>
			<td>Puncture-proof glove</td>
			<td>Turtleskin </td>
			<td></td>
			<td></td>
		 </tr>
 </tbody>
 </table>

use of galleria mellonella as a model organism to study legionella pneumophila infection

嗜肺军团菌 ，命名为退伍军人病重症肺炎，病原体是一种重要的人类病原菌，感染和肺泡巨噬细胞内复制。其毒性取决于点/ ICM IV型分泌系统（T4SS），这是必要建立一个复制许可的液泡称为含液泡（LCV）的军团上。L.肺感染可以在小鼠中进行建模然而大多数小鼠品系是不许可的，从而导致了寻找新的感染模型。我们最近表明，蜡蛾蜡螟的幼虫都适合L的调查肺军团菌感染。G。蜡螟被越来越多地用作感染模型的人类病原体和几个菌种毒力之间存在于昆虫和哺乳动物模型很好的相关性。幼虫的免疫防御系统的一个关键组成部分是血细胞，界人巨噬细胞，它占用和破坏的入侵者。L.肺是能够感染，形成一个LCV和这些细胞内复制。在这里，我们展示了用于分析L.协议在G.杆菌毒力蜡螟的模型，其中包括如何成长传染病L。肺 ，预处理与抑制剂的幼虫，感染的幼虫以及如何提取感染细胞进行定量和免疫荧光显微镜。我们还描述如何量化细菌复制和健身竞争分析。这些方法允许突变体的快速筛选，以确定因素L.重要菌毒力，描述一个新的工具来帮助我们这个复杂的病原体的理解。

在这里，它被证明G。蜡螟是适当的，易于使用的模型来研究L。肺军团菌感染。先前已经表明，L。在巨噬细胞，变形虫和哺乳动物模型的肺毒性是依赖于点/ ICM分泌系统41-43。G的存在蜡螟幼虫感染如上文所述和野生型（WT）和一个点/ ICM-缺陷型菌株的毒力比较。感染与L的10 7 CFU 肺军团菌菌株130B导致100％的死亡率在24小时内感染后（PI）。然而，L。肺ΔDOTA菌株，其不具有官能点/ ICM T4SS分泌系统，是无毒力在该试验中（ 图1）。这表明L。在G杆菌毒力蜡螟取决于点/ ICM效应的易位，使得这种模式适合于表征这些蛋白质的功能。 最近，它表明，通过细胞松弛素治疗抑制吞噬作用通过酵母白色念珠菌 6增加了幼虫的susceptibly感染。由于L。菌是一种细胞内的病原体，它被决定，以确定细菌的摄取是关键在其发病机制中对该模型。幼虫在37℃下预处理10微升100μM的细胞松弛素D（何秀兰）4小时，然后感染了野生型L的10 7 CFU 肺 130b和死亡率在24小时PI治疗与单纯抑制剂监控并没有影响幼体成活率。然而，相对于DMSO处理的，被感染的昆虫（ 图2）进行预处理，被感染的幼虫显示显著更大的存活（P = 0.0066，非配对t-检验）。何秀兰的治疗作用可被48小时圆周率（结果未显示），这可能是由于药物在G的半衰期蜡螟。这表明L的那个摄取肺为G。蜡螟血细胞是细菌毒力的一个重要方面。 为了验证表达，并确定在杠杆效应器蛋白的亚细胞定位蜡螟，血细胞中提取并处理用于免疫荧光显微术。幼虫感染了野生型和ΔDOTA L.表达良好定义T4SS效应，SIDC 41-918，稠合的4个N-末端HA标签的片段肺 130b中。这个效应被证明的LCV经由磷酸肌醇-4 -磷酸结合域44结合。使用抗-HA（红色）和抗军团菌 （绿色）抗体，4HA-SIDC 41-918定位于LCV在受感染的血细胞（ 图3）。这种本地化曾在变形虫盘基网柄菌和哺乳动物巨噬细胞44,45证实了C被证实omparability这种模式的。 蛋白质对于致病力的重要性通常是通过比较野生型和突变型细菌的生长动力学测定。为了跟随在感染过程中的细菌的复制动力学，3幼虫在每个时间点处死（0，5，18，和24小时圆周率）时，血淋巴收集并汇集并提取血淋巴的CFU/0.1g确定。后以5小时圆周率，对WT细菌的菌落形成单位的初始倾角增大到24小时圆周然而，ΔDOTA应变不发生复制和清除在18小时PI（ 图4）。 L的能力肺军团菌引起的巨噬细胞的裂解在T4SS依赖性早有记载46，但没有类似的研究在体内被执行。循环血细胞浓度为5，18被确定，并且24小时PI幼虫感染了WT或Δ <eM&gt; DOTA嗜肺军团菌130B，血细胞从感染虫和活细胞用台盼蓝排除法计算提取。以5小时圆周率在血球计数的菌株之间没有差别可以看出（ 图5）。然而，在18小时PI有在血细胞浓度显著下降，野生，但不ΔDOTA，受感染的幼虫。这种差异在持续24小时PI在血细胞数量的下降，再加上细胞内的细菌所看到的免疫荧光的存在，表明L。血细胞内的肺 ，然后复制他们裂解，从而使细菌经过几轮复制。 <img alt="图1" fo:content-width="3.5in" src="/files/ftp_upload/50964/50964fig1.jpg" /> 图1。感染L.肺军团菌诱导点/ ICM依赖幼虫死亡率10幼虫感染了单独的PBS或10 <s达&gt; 107 CFU野生型（WT）或ΔDOTA嗜肺军团菌 130b的，在37℃下72小时，记录幼虫的死亡时间。感染与WT所有幼虫死于感染24小时内感染后（PI），但无死亡病例主要出现在幼虫接种用PBS单独或ΔDOTA应变。结果是三个独立的实验中，数±标准差的平均值。 <img alt="图2" fo:content-width="3.5in" src="/files/ftp_upload/50964/50964fig2.jpg" /> 图2。死亡率是依赖于细菌的内化。10 L。杆菌的幼虫进行预处理10微升100μM的细胞松弛素D（何秀兰）4小时，在37℃再感染10 7 WT和死亡率在24小时圆周率预处理幼虫监测显著证明（p = 0.0066，配对t-检验）降低死亡率。结果代表的MEA在四个独立的实验±每个条件10幼虫标准偏差的n。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50964/50964fig3.jpg" /> 图3。在抽取血细胞效应蛋白的免疫荧光成像。血细胞从幼虫感染L.提取肺 130b的WT或ΔDOTA表达4HA-SIDC 41-918以5小时圆周细胞用抗-HA（红色）和抗军团菌 （绿色）的抗体和DAPI染色的DNA染色（蓝色）以可视化的细胞核染色。 4HA-SIDC 41-918观察周围的野生，但不ΔDOTA，细菌。比例尺为5μm。 <img alt="图4" fo:content-width="3.5in" src="/files/ftp_upload/50964/50964fig4.jpg" /> 图4。L.在G.杆菌复制梅洛内拉在一个点/ ICM依赖性。幼虫感染了野生型或ΔDOTA嗜肺军团菌 ，在0，5，18，和24小时PI从汇集3感染昆虫血淋巴，镀上CYE板和CFU决定，归一化到接种物和血淋巴中提取的重量。 WT L。菌复制在实验的过程中，而ΔDOTA菌株于18小时圆周率结果清零是三次独立实验的数±标准差的平均值。 <img alt="图5" fo:content-width="3.5in" src="/files/ftp_upload/50964/50964fig5.jpg" /> 图5。感染与野生型L。菌产生显著血细胞的破坏。血细胞中提取在5，18，和从幼虫24小时圆周率感染WT或ΔDOTA嗜肺军团菌 ，并用血细胞计数器计数活细胞。在无差异细胞数在株间5小时圆周率被看作但是在18小时圆周率只有约15％的血细胞仍然幼虫感染了野生型菌株相比，ΔDOTA应变。结果是三个独立的实验中，数±标准差的平均值。

Watch this Scientific Journal Video about Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection at JoVE.com

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

蜡蛾蜡螟的幼虫最近被确立为一种体内模型来研究嗜肺军团菌感染。在这里，我们展示了基本技术， 军团菌的发病特点的幼虫，包括接种，细菌毒力和复制的测量和提取被感染的血细胞和分析。

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 ...

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JoVE Journal

Immunology and Infection

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

40.1K 次观看.  Imperial College London. 蜡蛾蜡螟的幼虫最近被确立为一种体内模型来研究嗜肺军团菌感染。在这里，我们展示了基本技术， 军团菌的发病特点的幼虫，包括接种，细菌毒力和复制的测量和提取被感染的血细胞和分析。 

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

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 ...

科研

免疫与感染

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

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

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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 ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

嗜肺军团菌外膜囊泡:隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

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