We thank our fellow Lee Lab colleagues, who provided valuable insight and expertise that greatly assisted this research. This work was supported by the Young Innovator Award from the National Institute of Health, awarded to S.W. Lee (NIH-1DP2OD008468-01). R.A. Flaherty is supported by the Albertus Magnus Fellowship provided by Dr. Robert C. Boguslaski, and a teaching assistantship through the Eck Institute for Global Health at the University of Notre Dame.

1.细菌培养<ol><li>产生等基因突变株用于感兴趣21的具体分泌因子的研究。 注意:这些实验包括使用的GASM1T1 5448株 WT 气"， 佐贺 Δ 猫 SLS缺陷的突变（简称文本为ΔSAGA）和SAGA补充株（简称文本为ΔSAGA SAGA +）21。 </li><li>制备感兴趣的细菌菌株的过夜液体培养物。生长气体M1T1在-10毫升的Todd-Hewitt肉汤的5448菌株在37℃下16-20小时感染人体细胞之前。 </li><li>隔夜离心细菌培养（2 400 RCF 10分钟），并悬浮在新鲜的细菌培养基。 注意:在相同的体积重悬的过夜培养物在（5-10毫升）生长通常产生期望初始光密度。 </li><li>规范化的细菌培养，以通过在600nm（OD 600）稀释在附加细菌培养基中再悬浮培养物，直至相同的光密度等于起始浓度获得用于所有菌株进行测试（OD的大约1.0 600被推荐为方便起见）。 注意:在这些研究中，固定相细菌培养物用于感染宿主细胞立即正常化以下。然而，如细菌出口固定相的一些菌株nonsynchronously它可能更合适，如果这被发现是对所关注的菌株的情况下，开始与对数期培养物宿主的感染。 </li><li>之前进一步分析，执行一个菌落计数测定以确定在起始培养物存在的每毫升菌落形成单位（CFU），使感染复数（MOI），或每宿主细胞的细菌的比率，可以被确定。 <ol><li>制备1毫升已归一化到所需的光学DENSI细菌培养物的系列稀释液在磷酸盐缓冲盐水（PBS）或适当的细菌生长培养基（的Todd-Hewitt肉汤在这些研究中使用）TY。制备稀释从10 -1至10 -6，以产生至少一组与可数菌落（30-300集落）琼脂平板。一式三份进行所有稀释。 </li><li>转移100μl的各系列稀释的到被分析含有用于细菌种类适当培养基的琼脂平板。 注意:在这些研究中，使用的Todd-休伊特琼脂。 </li><li>使用玻璃或塑料细菌吊具均匀分布的100微升等分试样在整个琼脂板的整个表面上，并且继续传播，直到液体已经被吸收到琼脂。使用无菌技术下进行的火焰。 </li><li>孵育在37℃的琼脂平板过夜，或直到可数的菌落出现。 </li><li>计数生长在各平板上的克隆和由以下formu计算在原始培养物中的CFU / ml的LA:菌落数×镀毫升连续稀释/培养体积成反比。 例如:（200个菌落点¯x1/10 -5稀释）/ 0.1毫升镀= 2.0×108 CFU /毫升</li></ol></li></ol> 2.宿主细胞培养<ol><li>获得用于所需的分析合适的宿主细胞系。 注:角质形成人体上皮角质月29日 ，从五Nizet一种礼物，被用于这些研究。 <ol><li>维持在100mM培养皿HaCaT细胞在Dulbecco改进的Eagle培养基（DMEM）和10％热灭活的胎牛血清（FBS）。孵育在37℃，5％的CO 2。 </li></ol></li></ol> 3.宿主细胞感染渗透膜插入系统<ol><li>板细胞在合适的组织培养皿中，让他们成长，直到他们达到90％汇合。 注意:在这些研究中通常使用2〜3天之内到达汇合的HaCaT细胞电镀后AYS。 <ol><li>对于溶胞产物收集（ 例如信令分析和三磷酸腺苷（ATP）的测定测定），板HaCaT细胞在6孔培养皿中以大约3×10 5个细胞的接种密度/孔。 </li><li>对于免疫荧光成像实验，板的细胞上在6孔培养皿中无菌盖玻片在约3×10 5个细胞的接种密度/孔。 </li><li>为乙锭同型二聚体膜通透性和乳酸脱氢酶（LDH）释放测定法，镀HaCaT细胞在24孔培养皿中以大约5×10 4个细胞的接种密度/孔。 </li></ol></li><li>紧接在治疗之前，洗涤细胞以1×无菌PBS。 </li><li>应用新鲜生长培养基细胞。对于这里所描述的条件下，应用每阱2毫升培养基为6孔板或每孔0.5毫升介质24孔板中。如果药物治疗正在测试中，拌入新鲜培养基，这期间STE申请页。 注意:有些试验可能需要替代的媒体。例如，如酚红和高血清浓度与LDH释放测定干扰，无酚红的DMEM补充有1％牛血清白蛋白（BSA）（重量/体积），2mM的L-谷氨酰胺和1mM丙酮酸钠是这些实验利用。 </li><li>新鲜培养基已经施加之后，小心地在每一个无菌0.4微米透过膜插入井。执行在层流罩此步骤中，并使用无菌镊子对渗透膜插入物转移到每孔中。 </li><li>根据制造商的说明适用新鲜的细胞培养基中的每个孔中的上腔室。对于这里所描述的条件下，应用每孔1ml培养基6孔板或每孔0.1毫升介质24孔板中。 </li><li>申请归细菌培养物（见第1）至渗透膜的插入系统的上部腔室的适当体积。使用细菌培养基对照感染醇治疗。对于这里所描述的结果，添加25微升每室的归一化培养物的24孔板中，并加入100微升每室中培养的6孔板中。 注意:在这些研究中，野生型或突变体气体施加到宿主细胞 在10 MOI的MOI基于最终的真核细胞数，计算（ 例如 2×10 6个细胞/孔），使得均在感染期的开始（ 例如 ，2×每宿主细胞中加入10倍的细菌每孔10 7 CFU）。适当的MOI将与不同的细菌种类，并与所希望的后续分析而变化。 注意:除了使用细菌培养基未感染的对照，对于本技术的某些应用可以包括热灭活的细菌或用于比较花费细菌介质其他有用的控制。 </li><li>孵育感染的细胞在37℃，5％的CO 2。 </li></ol> 4.样品采集 &lt;OL&gt; <li>收集细胞培养基，宿主细胞裂解物，或玻璃盖玻片与完整细胞的后续分析中，开始通过仔细地除去用无菌镊子渗透膜插入。 </li><li>对于分泌主机Protiens的评估: <ol><li>收集来自下部腔室中的介质进入1.5毫升管中。避免收集过程中的干扰单层。 </li><li>在4℃下14000 RCF离心10分钟离心样品以除去细胞碎片。 </li><li>在-20°C删除所有，但50微升上清到一个新的1.5 ml管和储存或立即使用。 </li></ol></li><li>对于宿主细胞Lystates的评估: <ol><li>轻轻吸宿主细胞的单层以上的介质。避免打扰单层，因为这可能会导致样品损失。 </li><li>用1X PBS冲洗细胞一次。 </li><li>轻轻吸PBS。立即应用冰冷的裂解缓冲液的适当体积，以获得所需的蛋白质浓度，并孵育在冰上15分钟的样品。 200微升和350微升每每6孔板的裂解缓冲液之间适用以实现0.5和1.5毫克/毫升之间蛋白浓度。 注意:在这些研究中所用的裂解缓冲液的组成为去离子水，1％（V / V）的Nonidet P40替代，磷酸酶抑制剂混合物，和蛋白酶抑制剂混合物。具体的试剂列在材料和设备部分。 </li><li>使用细胞刮棒从每个的板面分离细胞并在每个的全部内容吸取井到1.5毫升管。 </li><li>离心样品在4℃14000 RCF离心20分钟。 </li><li>为了评估水溶性成分溶解产物，去除上清到一个新的管并储存在-20℃或立即使用。 </li><li>评估核或其他不溶性裂解物组分，保留沉淀并贮存在-20℃或立即使用。 </li></ol></li><li>通过免疫荧光成像技术评价: <ol><li>如海盗中，洗1X冷PBS细胞。 </li><li>过夜在4％低聚甲醛（PFA）溶液固定细胞在PBS（重量/体积）。 注意:在这些研究中，将1ml的PFA每每6孔板的孔中加入。 </li></ol></li></ol> 5.建议应用<ol><li>主机信号转导分析SDS-PAGE和Western印迹<ol><li>收集宿主细胞裂解物如4.3节所述。 </li><li>确定由二辛可宁酸（BCA）测定法或等效使用蛋白质标准30各样品裂解物的蛋白质浓度。 </li><li>标准化样品之间蛋白浓度之前加载和运行在4-15％聚丙烯酰胺凝胶或合适的替代31样本。 <ol><li>样品体积-通过从每个样品（ 例如 20微克）吹打相同蛋白量成新的1.5毫升管并加入裂解缓冲液的可变体积（缓冲量=总体积正常化样品浓度加）到各管中，使总体积（ 例如 50微升）和最终蛋白质浓度相当于整个样品。 </li></ol></li><li>转移样品到一个聚偏二氟乙烯（PVDF）膜（或等效物）。对于这里描述的结果，增加电压至70伏为额外的45分钟，然后在25伏转移样品2小时。使用200mM的Tris碱，1.5M甘氨酸和去离子水的20％甲醇（体积/体积）组成的转移缓冲液。 </li><li>在5％BSA + 0.1％吐温20的Tris块膜缓冲盐水（TBS）。相应地调整根据制造商的建议，将要使用的抗体。 </li><li>孵育初级抗体的膜在4℃下过夜。在使用厂家推荐的稀释。 </li><li>洗膜在TBS + 0.1％吐温20 1.5小时;刷新缓冲每次10-15分钟。 </li><li>与辣根过氧化物酶（HRP）缀合的孵育二级抗体以1:1的稀释度:5000，在室温TEMPERAT 1.5小时URE。 </li><li>洗膜在TBS + 0.1％吐温20 1.5小时;刷新缓存每10 - 15分钟。 </li><li>根据制造商的说明在胶片显影前孵育化学发光检测试剂膜。如需要，可以使用其它的检测方法。 </li></ol></li><li>宿主细胞免疫荧光染色和成像<ol><li>如第4.4节所述的修复含有宿主细胞盖玻片。 </li><li>除去PFA溶液和洗涤含有处理的细胞两次在PBS盖玻片，洗涤之间进行抽吸。 注:PFA是剧毒的，必须适当处置。 </li><li>用1％阻断在PBS中室温下搅拌2小时的盖玻片（重量/体积）正常山羊血清，2％（V / V）的Triton X-100和0.5％（体积/体积）吐温20。 </li><li>用PBS洗涤盖玻片30分钟，刷新缓冲每10分钟。 </li><li>孵育初级抗体的盖玻片在1:50比率（或由制造商推荐的）在阻断溶胶ution过夜，在4℃。 </li><li>洗盖玻片在PBS 1.5小时，刷新缓冲区每隔30分钟。 </li><li>孵育盖玻片在二级抗体室温2小时用1（山羊抗兔IgG AlexaFluor488被用于这些研究）:抗体的阻断溶液200比。 </li><li>洗盖玻片1小时，刷新缓冲区每隔20分钟。 </li><li>应用所需的污渍。 注意:在封闭溶液1000:利用DAPI（核染色）中，在1比罗丹明 - 鬼笔环肽（肌动蛋白染色）这些研究。 </li><li>孵育核和肌动蛋白污渍盖玻片在室温下30分钟。 </li><li>洗盖玻片在PBS，在室温下30分钟，刷新缓冲每10分钟。 </li><li>安装在使用安装介质载玻片盖玻片。 </li><li>允许盖玻片前一夜密封和成像设置幻灯片。对于这里描述的结果，在一个荧光显微镜USI采集图像纳克一个标准的20倍的目标。使用ImageJ的和显微镜的成像软件来处理拍摄的图像。 </li></ol></li><li>乙锭同型二聚体细胞死亡含量<ol><li>从每个孔洗细胞两次用无菌PBS吸介质。 </li><li>在PBS申请4μM的乙锭同型二聚体-1。 </li><li>在室温下孵育细胞，在黑暗中30分钟。 </li><li>确定用酶标仪设定为528纳米激发和617与590纳米的截止值nm发射的荧光的程度。 </li><li>加0.1％（重量/体积）皂苷对每个孔之后的初始读数，并允许读出板的第二时间在相同的设置前板孵育在室温下（与摇动）一个额外的20分钟。 </li><li>由第二荧光读数（后皂素）除以初始荧光读数（后处理）单独确定百分之膜通透为每个孔中。 </li></ol></li><li> ATP含量测定<ol><li&gt;如上述第4.3节收集裂解液。 </li><li>通过BCA测定或类似30正常化的裂解物的蛋白质水平。 </li><li>确定使用基于发光的ATP的测定试剂盒，或根据制造商的说明等效细胞ATP水平。 </li><li>正常化处理值，以相应的未感染的对照样品。 </li></ol></li><li> LDH释放分析<ol><li>感染后，收集上清如4.3节所述。 </li><li>根据制造商的说明使用细胞毒性检测试剂盒为LDH释放评价LDH释放。 </li></ol></li></ol>

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The authors have nothing to disclose.

本文中描述了利用渗透膜基于插入物感染系统研究的在体外系统人类上皮角质化细胞的细菌毒素链球菌溶血素S的影响的方法。这个协议可以适用于其他细菌分泌的毒力因子的研究，以及替代宿主细胞类型。这家新近开发的系统提供了超过，利用纯化毒素或细菌过滤上清液1实验方法几个优点- 3,8,18 - 20。可渗透基于插入膜系统提供有关细菌毒素，因为它产生对宿主细胞，其允许维持最大毒素活性的保持恒定的剂量也实验之间增加的一致性。此外，该系统更紧密地通过允许分泌因子随时间累积的感染的进展和由ELIMI模拟生理条件内廷需要任意地选择具体的毒素浓度以适用于宿主细胞。此外，该系统也将提供装置，用于调查，以评估所关注的细菌因子是否被递送到宿主细胞在接触依赖方式。接触依赖性通常是通过在加入或通过使用该防止附着的试剂的缺陷等基因的细菌的突变体的产生进行测试。这里所描述的系统将提供一个简单的替代来补充或替代这些传统的方法。 除了在该协议的部分5中所述的测试的应用程序，其它应用到这种感染系统可以很容易地适应包括用于细胞因子阵列和ELISA测定的样品的集合。在这两种情况下，一个类似的协议，对于LDH释放测定法描述可以跟随。虽然这里未示出，该系统已被​​用于有效地收集宿主细胞样品是一个通过流式细胞仪nalyzed。在本申请中，渗透膜插入件是继感染期间除去，将细胞洗涤以除去细胞培养基，并胰蛋白酶被施加以使细胞进行分析的汇编。细菌从宿主细胞的物理分离是这种应用是特别有用的，因为它有助于防止由附着的细菌和宿主细胞中，可以以其他方式与精确细胞计数通过流式细胞仪干涉的大的聚集体的形成。 虽然比较与传统的直接感染研究渗透膜基于插入物感染的研究的结果时，很一致宿主反应已经观察到，在这些宿主反应的动力学一些显着的差异已经观察到21。例如，在宿主信令和膜 - 基于细胞毒性的变化在渗透膜基于插入物感染模型比心病发生取30-50％更长响应直接感染模型。因为渗透膜基于嵌件的系统需要介质要施加到每个上部和下部隔室以及，对于此处描述的研究开发的系统所需的总培养基体积增加了30％，每孔相比相应的先前使用的直接感染模型21。这种差异在总培养基体积，加上细菌与宿主细胞之间的距离增加的直接接触被禁止时，有可能会增加花费SLS通过介质扩散到达宿主细胞和引发所观察到的效果的时间。此外，可渗透基于插入膜系统删除许多气体毒力因子有可能向宿主细胞损害;没有在该系统中这些额外的因素也可能有助于在宿主细胞死亡和主机信号事件的开始相比直接感染21的延迟。这些应考虑的因素设计实验时，评估其他分泌的细菌成分。 找出最有意义的条件来测试对宿主细胞的分泌细菌因素的影响，检测的范围内的时间点的渗透膜以插入感染系统的每一个应用建议。在什么条件下感兴趣的特定致病因子最佳地生成（如对数期，静止期，响应于某些环境信号等 ），以便成功地观察其效果考虑是很重要的。在实验中重点是评估在宿主信号蛋白的变化，有必要选择，允许足够的时间用于SLS达到宿主细胞和以足够的量，以产生以诱导信令改变21的时间点。同时，在主机信令所需的更改的评估将之前SLS诱导膜通透性，因为这影响下进行，omplicates宿主细胞裂解物进行分析的集合。由SLS诱导的细胞死亡可在直接和渗透膜基于插入物感染模型被最佳观察到的报告信号事件21的开始后数小时。另外，在选择感兴趣的时间点，这也是重要的，以确保所研究的细菌是无法在研究期间穿透多孔插入物膜。在较长时间内生产某些细菌成分的可能破坏膜的完整性，并允许细菌的通道到下隔室。以确定这是否是在感兴趣的系统中的潜在问题，加以研究的细菌菌株可以在的范围内的时间点施加到可渗透的基于插入膜系统的上腔室。在每个时间点，插入件可以被小心地去除，并从底腔的介质可以被收集，并以标准菌落共同利用unting试验（参见1.5节）。如果没有菌落从在下部隔室中的细胞培养基形成，插入膜可以假定为有效地防止细菌的通道在所测试的时间点和细菌负荷。 另一个实验设计的组件，很可能需要优化的分泌细菌因子的有效的分析是感染（MOI）的多重性。的MOI是指每宿主细胞的细菌细胞的比率，并且由宿主细胞汇合感染的时间，由细菌菌落形成单位的（CFU）的数目，因此影响施加到细胞。在这些研究中，角质细胞细胞生长至90％汇合，这使得这些细胞形成用完整的紧密连接有凝聚力的单层。宿主细胞的生理组织的考虑周到进行研究是必要选择为感染实验的适当汇合。一旦appropr每孔被确定宿主细胞的莱特号码，合适的细菌的CFU可以计算基于该所需的MOI。在此处描述的研究，气体施加在10适当MOI的MOI对宿主细胞将与不同的细菌，并与所希望的后续分析而变化。低开始的MOI是典型地更生理学相关，并允许针对感兴趣的细菌因子积累足够慢在宿主细胞生存力的急剧变化之前捕获在宿主细胞中信号传导的细微变化是显而易见的。更高的MOI在许多研究中评估的细胞毒性使用的，但这种效果也可以通过用低的MOI开始并允许感染的时间更长的时间取得的进展。重要的是要确定一个准确的CFU至光密度推论为要测试的所有菌株，如同基因的突变体可以比野生型的细菌具有改变的生长速率，因此可能需要更高或更低的CFU每加到很好地确保菌株之间的宿主反应的一个合适的比较。在所研究的哺乳动物宿主细胞能够杀死细菌或抑制其生长，或者如果细菌菌株之间非同步生长被怀疑，它也可以是执行的研究在感染期的结束，以评估最终细菌负荷有用的情况。这可以通过收集可渗透插入件的内容，并执行类似于在该过程的第1.5节中所述的菌落计数测定来完成。 选择适当尺寸的孔中所需测定也用于获得最佳的结果很重要。可渗透膜插入在多种尺寸可用，但使用专为24孔和6孔组织培养板的插入，观察这里示出的研究中最一致的结果。这些刀片尺寸相对容易无菌镊子处理和易于操控的是预防的关键ING从上部隔室到井的下部隔室的细菌不需要的传输。对于涉及宿主细胞裂解物的收集实验中，6孔培养皿中提供每种条件对大多数分析适当的细胞数，并足够大以容纳样品收集细胞刮刀。对于细胞毒性试验，其中宿主细胞保持粘附，并贴有可在读板器（ 例如 ，乙锭同型二聚体测定法，台盼蓝排除法）测量荧光或比色染料，使用24孔板与相应的插入件是推荐的。对于其中细胞培养基将被收集和分析实验（ 如 LDH释放，细胞因子的研究）任一的24孔或6孔平板，可以使用，虽然小的孔一般为这些分析提供足够的样品体积，并尽量减少使用所需的试剂。总体而言，该方法是高度灵活的，并根据需要，制备SAMPL可适于ES众多后续的应用程序。

了解细菌毒力因子的作用在宿主细胞感染的背景是细菌发病机制研究的主要焦点。许多细菌病原体积极分泌毒素和其它可溶性因子在宿主组织的破坏，以帮助，以引发在宿主细胞中的信号的变化或感染1的过程中，操纵免疫系统反应- 10。虽然方法已经发展成功纯化和产生许多这些重要的致病因素的研究，某些细菌产物具有独特的结构或广泛的翻译后修饰，使对纯化方法他们顽抗候选人，因而不能孤立使用体外系统来研究。例如，A群链球菌，细菌病原体负责的感染范围从咽炎坏死性筋膜炎和中毒性休克综合征无数，产生分泌细菌毒素称为链球菌溶血素S（SLS）11 - 17。此核糖体产生的肽是由链球菌溶血素S相关基因（SAG）簇编码，且成熟的产品，估计为2.7 kDa的大小14 - 17。前毒素，通过SAGA编码，是由几个酶（SAGB，SagC和SAGD）翻译后修饰产生成熟的，功能性的形式15 - 17。在加上毒素的不寻常的氨基酸序列，这些翻译后修饰的复杂性已使该毒素与已尝试迄今15,17所有纯化和结构鉴定的努力不兼容。这些挑战复杂的努力，以确定这种毒素在宿主发病机制中的具体作用。 在的情况下的纯化的毒素或其他分泌的因子的制备或者是复杂的或不可能的，机制，以阐明这些产品的功能传统上一直通过过滤细菌的上清液，然后将其施加到宿主细胞18的制备研究- 20。有与该技术相关联的一些挑战。首先，存储和应用在稍后的时间对宿主细胞时许多这些分泌的因子，包括SLS，不保持最大或一致的活性。此外，当上清液在单一时间点被收集，然后应用到宿主细胞，它是难以确定的生理相关性，得出关于自然感染过程，其中分泌因子允许感染的过程中一个过程中积累的直接结论生理浓度。这第二个挑战不仅适用于使用细菌上清液的，而且要在宿主细胞中的研究1使用的纯化的毒素- 3,8。为了解决这些问题，可透膜樱雪基于RT-感染系统已经开发，以评估在维持最佳毒素活性和也消除细菌和宿主细胞之间的直接接触的可变的方式SLS对宿主细胞的影响。在这个系统中，人上皮细胞生长在一个单层中的两腔室的底部室中井，和细菌被引入到同一井的上部腔室。的多孔膜（0.4微米孔）分隔的上，下腔室，从而使两个室之间交换分泌因子但是防止细菌的通道。该系统允许的宿主反应是仅仅是由于持续分泌的细菌成分，同时消除了通过A组链球菌感染过程中直接接触任何可能发生的反应有效的评价。虽然除了SLS其他分泌细菌因素也可以通过多孔膜，使用的等基因突变型面板，包括野生型（WT）中，SLS-千牛ockout（ΔsagA）和SAGA补充株（ΔsagA+ SAGA）允许对宿主反应是严格SLS-21依赖性精确的评估。 虽然类似渗透膜插入系统已被用于参与病毒感染分泌的因子，肿瘤生物学和免疫细胞迁移22的研究- 26，一些研究涉及与宿主细胞的细菌的相互作用已经利用这种方法6,27,28。即使已采用这种系统，以探索细菌与宿主细胞之间的相互作用的研究主要集中于炎症细胞或细菌迁移通过镀上渗透膜插入上皮或内皮细胞单层。本文所述的可渗透基于插入物膜感染系统是通过一个多孔膜，以评估EFFE依赖于细菌和宿主细胞的分离的简单而有效的方法分泌的毒素，SLS，对宿主细胞膜的完整性，细胞活力，细胞信号转导和分泌宿主细胞因子的克拉。这种技术可以适用于其他分泌的毒力因子在多种哺乳动物宿主细胞类型的研究，调查特定细菌因子的过度的感染过程中的作用。

<table><tbody><tr><td>Todd-Hewitt broth</td><td>Acumedia</td><td>7161A</td><td>Use appropriate broth for bacterial species of interest.</td></tr><tr><td>BioSpectrometer</td><td>Eppendorf</td><td>basic model</td><td>Any UV-Vis spectrophotometer is suitable.&amp;nbsp;</td></tr><tr><td>Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713</td><td>Other brands are suitable.</td></tr><tr><td>Agar</td><td>Sigma</td><td>A1296-1kg</td><td>Other brands are suitable.</td></tr><tr><td>HaCaT human epithelial keratinocytes</td><td></td><td></td><td>Gift from lab of Victor Nizet.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Life Technologies</td><td>11995-073</td><td>Use appropriate media for cell line of interest.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biowest</td><td>S162H</td><td>Use appropriate media additives for cell line of interest.</td></tr><tr><td>10 cm tissue culture dishes</td><td>Nunc</td><td>150350</td><td>Other brands are suitable.</td></tr><tr><td>6 well tissue culture dishes</td><td>CytoOne</td><td>cc7682-7506</td><td>Other brands are suitable but need to be compatible with the Corning insert.</td></tr><tr><td>24 well tissue culture dishes</td><td>CytoOne</td><td>cc7682-7524</td><td>Other brands are suitable but need to be compatible with the Corning insert.</td></tr><tr><td>Glass coverslips</td><td>Fisherbrand</td><td>12-541-B</td><td>These must be autoclaved prior to use for cell plating.</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Phenol Red Free DMEM</td><td>Life Technologies</td><td>31053028</td><td>Phenol Red Free media is only required for the LDH release assay.</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030149</td><td>Only required to supplement cell culture media for LDH release assay.</td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>BP356100</td><td>Only required to supplement cell culture media for LDH release assay.</td></tr><tr><td>Collagen-coated 0.4 &amp;mu;m Transwell inserts (6 well)</td><td>Corning</td><td>3540</td><td></td></tr><tr><td>Collagen-coated 0.4 &amp;mu;m Transwell inserts (24 well)</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Forceps</td><td>Fisher</td><td>3120018</td><td>These must be sterilized with ethanol prior to handeling Transwell inserts.</td></tr><tr><td>Cell scrapers</td><td>Fisherbrand</td><td>08-100-241</td><td>These are only required for the collection of cell lysates.</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>04042-500</td><td>TOXIC.&amp;nbsp; This is only required for immunofluorescence imaging.</td></tr><tr><td>Bicinchoninic acid (BCA) assay kit</td><td>Pierce</td><td>23227</td><td>Other protein quantification assays may be used.</td></tr><tr><td>Tris-glycine 4 - 15% polyacrylamide gels</td><td>BioRad</td><td>4561083</td><td>Buffer system and percent polyacrylamide should be selected based on proteins of interest.</td></tr><tr><td>Electrophoresis cassette supplies</td><td>BioRad</td><td>1658063</td><td>Only required for Western Blotting.</td></tr><tr><td>Electrophoresis power supply</td><td>BioRad</td><td>1645050</td><td>Only required for Western Blotting.</td></tr><tr><td>Western blot transfer cassette</td><td>BioRad</td><td></td><td>Only required for Western Blotting.</td></tr><tr><td>Polyvinylidene (PVDF) Membrane</td><td>EMD Millipore</td><td>IPVH00010</td><td>Only required for Western Blotting.</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-500ml</td><td></td></tr><tr><td>Rabbit IgG-HRP secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc2030</td><td>The secondary antibody should be determined based on the selected method of detection.&amp;nbsp;</td></tr><tr><td>Mouse IgG-HRP secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc2031</td><td>The secondary antibody should be determined based on the selected method of detection.&amp;nbsp;</td></tr><tr><td>Phospho-p38 (T180 + T182) MAPK antibody</td><td>Cell Signaling</td><td>4511</td><td>Select appropriate primary antibodies based on proteins of interest.</td></tr><tr><td>Total p38 MAPK antibody</td><td>Cell Signaling</td><td>8690</td><td>Select appropriate primary antibodies based on proteins of interest.</td></tr><tr><td>Goat anti-rabbit IgG Alexafluor 488</td><td>Molecular Probes</td><td>A-11034</td><td>Other secondary antibodies may be used for immunofluoresence imaging detection.</td></tr><tr><td>LumiGLO Detection Reagent</td><td>KPL</td><td>54-61-00</td><td>Only required for Western Blotting with detection on film.</td></tr><tr><td>Detection film</td><td>Biodot</td><td>BDB57</td><td>Only required for Western Blotting with detection on film.</td></tr><tr><td>DeltaVision Nikon 90i fluoresence microscope</td><td>Nikon</td><td></td><td>Other fluorescence microscopes are suitable for these analyses.</td></tr><tr><td>Normal goat serum</td><td>Thermo Scientific</td><td>31873</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284-500mL</td><td></td></tr><tr><td>DAPI nuclear stain</td><td>Cell Signaling</td><td>4083</td><td>Select stains based on specific immunofluorescence applications of interest.</td></tr><tr><td>Rhodamine-phalloidin actin stain</td><td>Molecular Probes</td><td>R415</td><td>Select stains based on specific immunofluorescence applications of interest.</td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td>Only required for immunofluorescence imaging.</td></tr><tr><td>Ethidium homodimer 1</td><td>Molecular Probes</td><td>E1169</td><td>Only required for membrane permeabilization cytotoxicity assay.</td></tr><tr><td>Spectramax M5 Microplate Reader</td><td>Molecular Devices</td><td></td><td>Other microplate readers capable of detecting UV-vis, fluorescence and luminescence may be suitable.</td></tr><tr><td>Saponin</td><td>Sigma</td><td>47036-50G-F</td><td>Only required for membrane permeabilization cytotoxicity assay.</td></tr><tr><td>Molecular Probes ATP Determination Kit</td><td>Life Technologies</td><td>A22066</td><td>Other kits are likely to be suitable.</td></tr><tr><td>Cytotoxicity Detection Kit for LDH release</td><td>Roche</td><td>11644793001</td><td>Other kits are likely to be suitable.</td></tr><tr><td>Nonidet P40 Substitute</td><td>Sigma</td><td>74385-1L</td><td>Other suppliers are suitable.</td></tr><tr><td>HALT Phosphatase Inhibitor Cocktail</td><td>Thermo Scientific</td><td>78420B</td><td>Other cocktails are likely to be suitable.</td></tr><tr><td>SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-free</td><td>Sigma</td><td>S8830-2TAB</td><td>Other cocktails are likely to be suitable.</td></tr></tbody></table>

implementation of a permeable membrane insert-based infection system to study the effects of secreted bacterial toxins on mammalian host cells

许多细菌病原体分泌强效的毒素在宿主组织的破坏援，以引发在宿主细胞中的信号的变化或感染的过程中操纵免疫系统的反应。虽然方法已经发展成功纯化和产生许多这些重要的毒力因子，但仍有许多细菌毒素，其独特的结构或广泛的翻译后修饰使它们难以纯化并在体外系统学习。此外，即使当能够得到纯的毒素，存在与学习毒素相关的生理条件下的具体影响相关的许多挑战。大多数在旨在评估分泌细菌毒素对宿主细胞的作用的体外细胞培养模型涉及孵育宿主细胞毒素的一次性剂量。感染，其中的毒素不断所产生过程中的这种方法不好大致与宿主细胞的实际经验细菌细胞，并允许感染的过程中逐渐累积。这个协议描述了基于插入渗透膜细菌感染系统的设计研究链球菌溶血素S，由A群链球菌产生的强效的毒素，对人上皮角质细胞的影响。这个系统的感染比，其中纯毒素或细菌上清液直接施加到宿主细胞的方法中更紧密地模仿自然的生理环境。重要的是，这种方法也消除了宿主反应是由于直接在细菌和宿主细胞之间的接触的偏压。该系统已被​​用来有效地评估链球菌溶血素S（SLS）的对宿主膜完整性，细胞活力和细胞信号的响应的影响。这种技术可以容易地应用于其他分泌的毒力因子的研究在多种哺乳动物宿主细胞的类型，以调查分泌细菌系数d的具体作用uring感染过程。

对于分泌的细菌因子的研究开发的渗透膜基于插入物感染系统协议是图1该系统通过一个多孔膜依赖于细菌和宿主细胞的分离，以评估分泌细菌因素的影响的详细，在这种情况链球菌溶血素S（SLS），对宿主反应如主机膜的完整性，细胞生存力，细胞信号转导，和分泌的宿主细胞因子。 图2提供代表蛋白质印迹数据表明，该系统可被用于评估在接触的活化变化 - 独立主机信号蛋白。具体地，该代表数据显示显著增强p38蛋白的活化在SLS产气菌的存在。该系统还可以应用于显现对宿主蛋白质定位分泌细菌因素通过免疫​​荧光显微镜的影响（&lt;STRONG&gt;图3）。数据显示的关键炎症介质核因子κB（NFκB），其从细胞质在激活21转移到细胞核的依赖性SLS-活化。在图2和3的结果表明，这两种SLS依赖性炎性信号传导的响应不需要的细菌与宿主细胞之间的直接接触。如前面的实验已经表明SLS依赖性p38和NFκB活化的直接感染模型21，在渗透膜基于镶嵌系统三种气体株中的p38或NFκB活化的类似水平将已表明所需的响应之间的直接接触细菌和宿主细胞。 图4表明，该感染系统可以应用于评估通过乙锭同型二聚体和LDH释放测定中在宿主细胞毒性毒素依赖性变化。这是由显著增加我证明Ñ​​既膜通透性和乳酸脱氢酶的从暴露于含有SLS相比暴露于SLS缺失菌株未感染的细胞或细胞GAS菌株的宿主细胞的释放。这些变化在细胞毒性不直接的，如显著效果并不明显，直到12小时后感染膜透的情况下和在LDH释放的情况下，16小时。代表性数据示出选择合适的时间点和感染的条件对这些宿主反应的评价的重要性。除了 ​​允许对宿主信令变化和细胞毒性的评估中，渗透膜基于插入感染系统，适用于代谢变化如准确确定主机ATP水平的研究通过防止宿主细胞裂解物的细菌污染（ 图5） 。这些数据表明响应于GAS感染16小时角化细胞的ATP的显著损失感染后，具有增强的ATP损耗我ñSLS的存在。这些结果与在宿主应激响应信令和细胞毒性所观察到的毒素依赖性增加是一致的。 <img alt="图1" src="/files/ftp_upload/54406/54406fig1.jpg" /> 图1:基于插入渗透的膜示意图感染系统协议，以评估对宿主细胞分泌的细菌因素的影响人角质形成细胞铺在底层舱和成长至90％汇合。用0.4微米孔的胶原涂覆的隔膜分隔的上，下腔室，和细菌被添加到所需感染期间的渗透膜插入系统的上腔室。细胞培养上清和宿主细胞可以被收集后的感染期和各种分析利用。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54406/54406fig1large.jpg" target="_blank">请点击此处查看大图</a>这一数字。 <img alt="图2" src="/files/ftp_upload/54406/54406fig2.jpg" /> 图2:在基于插入渗透膜感染系统可以通过SDS-PAGE和Western印迹被用于宿主细胞裂解物分析中的代表性数据表明SLS增强在感染角化细胞的p38蛋白激酶途径的活化。 （A）中HaCaTs分别经由可渗透膜插入物感染系统感染气体为7小时（MOI = 10）和裂解物进行了评估的p38的活化。 （B）的来自三个独立印迹光密度进行定量的p38的相对激活响应于GAS&#39;infection。从三个生物学重复的平均值被示出，与表示标准偏差误差线。对p38的相对活化被表示为磷酸化/总蛋白水平。相比于被确定统计学意义未受感染的细胞。整体p值是通过ANOVA（p值= 0.0063）测定。邓尼特的测试进行事后比较每个条件对应的未感染控制的意思。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54406/54406fig2large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54406/54406fig3.jpg" /> 图 3: 基于插入渗透膜感染系统允许通过免疫荧光显微镜主机信号的变化的可视化的具有代表性的数据表明，链球菌溶血素s至NFκB的活化增强了促炎症信号。将HaCaT人角质形成细胞感染了气体的M<html>使用可渗透基于插入物膜感染系统8小时10 OI。 NFκB的（A和B）核定位免疫荧光成像评估。核局部化的细胞的百分数通过计数在NFκB已经从细胞质易位到细胞核对于给定的字段细胞的数量除以该号码由细胞同场的总数。刻度条表示100μm左右。 （A）的三次生物学重复的平均值被表示为与表示标准偏差误差棒各条件。整体p值是通过ANOVA确定; P &lt;0.0001。进行Dunnett的检验以各条件比较相应未感染的对照条件。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。/ftp_upload/54406/54406fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54406/54406fig4.jpg" /> 图 4: 基于插入渗透膜感染系统允许细菌介导膜通透性和细胞毒性的细菌和宿主细胞之间的直接接触缺位的决心代表性的数据表明，角质形成细胞的活力活跃SLS毒素的存在减少。 GAS -诱导的细胞死亡是在角质形成细胞WT存在评估，SLS缺陷或补充佐贺角质形成气用渗透膜基于插入感染系统暴露GAS 8-16小时，在10的MOI（。 A）存活率，乙啶同型二聚体检测或（B）LDH释放法检测。在两个面板，3重plicates被平均及误差棒表示标准偏差。对于每个时间点显着性通过ANOVA（A）的8小时，p值= 0.0241确定; 12小时，P &lt;0.0001（B）8小时，P = 0.0287; 12小时，P = 0.1977; 16小时，P = 0.0031。进行Dunnett的检验从每个状态到野生型感染的相应的时间点进行比较的装置。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54406/54406fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54406/54406fig5.jpg" /> 图5: 通过防止BACT对主机ATP水平的准确测定基于插入渗透膜感染系统允许宿主细胞的erial污染裂解液，代表数据表明A组链球菌感染时的ATP依赖的SLS损失。 HaCaT细胞感染了气体使用可渗透基于插入物膜感染系统8-16小时。技术重复（N = 3）从一个代表性生物复制（每个样品2×10 6个细胞）的平均值为每个条件，与表示标准偏差误差线。整体p值分别通过ANOVA确定（12小时，P &lt;0.0001; 16小时，P &lt;0.0001）。进行Dunnett的检验以比较野生型感染每个条件为相应的时间点。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54406/54406fig5large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

Watch this Scientific Journal Video about Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells at JoVE.com

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

这里，说明使用可透膜基于插入物感染系统研究链球菌溶血素S，由A群链球菌产生的分泌毒素的效果的方法中，在角质形成细胞。这个系统可以很容易地应用到各种宿主细胞类型的其他分泌的细菌蛋白的感染过程中的研究。

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

implementation-permeable-membrane-insert-based-infection-system-to

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.9K 次观看.  University of Notre Dame. 这里，说明使用可透膜基于插入物感染系统研究链球菌溶血素S，由A群链球菌产生的分泌毒素的效果的方法中，在角质形成细胞。这个系统可以很容易地应用到各种宿主细胞类型的其他分泌的细菌蛋白的感染过程中的研究。 

视频: Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Invasion of Human Cells by a Bacterial Pathogen

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

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

由细菌病原体入侵人体细胞

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

科研

免疫与感染

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

人体血液，脑脊液屏障的脉络丛上皮基于Cell的产品型号从基底外侧面研究细菌感染

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

医学

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

注射脂多糖对小鼠肠道屏障破坏后微生物源产物的模拟进入

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

微生物通过微血管内皮细胞对血脑屏障渗透性分析

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

Source: Sontag, R. L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52296">Electroporation of Functional Bacterial Effectors into Mammalian Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the introduction of bacterial effector proteins into mammalian cells by electroporation. The intracellular localization of the bacterial effector proteins is detected by confocal microscopy following staining with specific fluorophore-conjugated antibodies.

通过电穿孔将细菌效应蛋白引入哺乳动物细胞

Source: Sontag, R. L. et al., Electroporation of Functional Bacterial Effectors into Mammalian Cells. J. Vis. Exp. (2015)
In this video, we demonstrate ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins

Source: Flaherty, R. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54406">Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.</a> J. Vis. Exp. (2016).
This video demonstrates a permeable membrane insert-based infection system to assess the impact of secreted bacterial toxins. By eliminating direct contact between the bacterial pathogen and the host cells using a permeable membrane, this technique allows for studying the effect of toxins secreted by pathogenic bacteria on mammalian host cells.

一种基于渗透膜插入物的感染系统，用于研究细菌毒素的影响

1. Bacterial Culture

	Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest.
	Note: The GAS (Group A ...