这项工作是由威康信托基金会（WT 0795880）。

以下协议将介绍由S.研究内皮细胞的侵袭金黄色葡萄球菌 ，但理论上可以用来研究任何可培养的细菌细胞的入侵。 到 S阶段的具体金黄色葡萄球菌和血管内皮细胞的表示。 1。细菌的制备<ol><li>文化S。 4-16 H（上生长阶段所需而定）在10毫升的脑心浸液（BHI）肉汤在37 ° C在空气中晃动在200转金黄色葡萄球菌菌株。这些生长条件具体为S.金黄色葡萄球菌和可能需要对其他细菌的适应。 </li><li>洗净细菌三个贝科的修改鹰的培养基倍（DMEM培养液; Invitrogen公司）离心备用轮在房间温度（5000 x 克 ，10分钟），去除一个的DMEM相当于体积的文化上清和的细菌沉淀重新悬浮。然后可以根据需要调整的细菌产生的悬浮测量光密度。为S.金黄色葡萄球菌，我们准备在外径600 = 1〜10 9 CFU毫升-1，对应暂停。 </li></ol> 2。内皮细胞培养<ol><li>文化内皮细胞株EA.hy926 1的DMEM补充胎牛血清（FBS，10％）在37和L -谷氨酰胺（2毫米）℃，5％的CO 2。另外，汇集小学的人脐静脉（内皮细胞）的内皮细胞可以购买从龙沙（瑞士巴塞尔，）和内皮基底介质与2％FBS，牛脑提取物（包括肝素），人血管内皮生长因子和氢化可的松，在37培养℃，5％CO 2，根据制造商的说明（龙沙） 。这些增长的条件是针对这些细胞和其他类型的宿主细胞，可能需要适应。 </li><li>血管内皮细胞生长在T75烧瓶完成汇合，用肉眼验证。 </li><li>准备插入的盖玻片的24孔板。精细镊子（火焰消毒）需要移动盖玻片，其中有一个不透明的，表面有光泽。在24孔板与不透明表面朝上，让细胞附着井发生盖玻片。 </li><li>从3毫升胰蛋白酶- EDTA（0.25％）与T75烧瓶中解放出来的细胞，并添加到10毫升的有关培养基。 </li><li>添加500μL悬浮细胞Thermanox盖玻片的24孔板。一个T75细胞融合烧瓶两个24孔板提供足够的细胞，造成约5 × 10 5个细胞（500μL介质） 。 </li><li>孵育48小时板块如上所述，并验证100％细胞汇合倒光镜。 </li><li>浸在PBS冲洗盖玻片，并添加新的24孔板中，含有490μLDMEM培养液，每孔含10％胎牛血清。 </li><li>为了研究细胞浸润在特定的代谢过程中的作用，抑制剂可以被添加到培养的细胞1小时之前，除了细菌和浓度保持在检测。例如，要确定的肌动蛋白重排的 S中的作用黄色葡萄球菌的内皮细胞的侵袭，50微米的细胞松弛素D可以添加或作用的小窝，5毫米甲基-β-环糊精可以增加。 </li></ol> 3。侵袭实验<ol><li>加入洗净的细菌10μL（约2 × 10 7 CFU 毫升 -1金黄色葡萄球菌）以及包含在490μLDMEM培养液与内皮细胞的融合层含10％胎牛血清的一个洗涤盖玻片。 </li><li>孵育15-90分钟，在37℃，5％CO 2的彗星。 </li><li>要测量的细胞（贴壁和内在）的细菌总数，浸洗盖玻片在PBS的3倍，并添加到PBS中含有500μL0.5％Triton X - 100的的新鲜井。为了确保细胞充分裂解，并释放所有的内在细菌，吸几次，直接在盖玻片表面的尖端指向移液器。 </li><li>列举细菌悬浮液（或稀释，这在必要时）TSA的板表面镀上。由于TX - 100裂解许多革兰阴性菌，皂甙，可以用来代替 2 。 </li><li>要衡量化的细菌数量，请从每口井含有未绑定的细菌培养上清和替换500μLDMEM/10％FBS的补充200微克毫升-1庆大霉素。我们经常使用庆大霉素而比溶葡球菌酶，因为它是便宜的，让我们实验使用不同类型的细菌（如葡萄球菌和Lactococci）之间进行切换，而无需改变实验的协议。 </li><li>板在37 ° C在5％CO 2为60分钟，杀死所有外细菌。 </li><li>洗盖玻片在PBS的3倍，裂解枚举镀上TSA的描述上面的粘附实验。 </li><li>在某些情况下它可能是最好目视计数使用光学显微镜的细菌数量。在这种情况下，具体到 S 金黄色葡萄球菌细胞的侵袭，使用溶葡球菌酶（-1毫升10微克），而不是庆大霉素，物理破坏外细菌。 </li><li>在37 ° C的CO 2的溶葡球菌酶的解决方案，然后用清水冲洗并修复Cytopath（Cellpath）为20分钟盖玻片。 </li><li>洪水与结晶紫（0.5％，W / V）为5min盖玻片。 </li><li>浸在水，空气干燥冲洗，并安装到玻片上。利用光镜，可以量化融合的内皮细胞每平方毫米2的细菌数量。 </li></ol> 4。代表性的成果： 纤维连接蛋白结合蛋白的表达 S的表面上（FnBPA和FnBPB ） 金黄色葡萄球菌赋予侵入血管内皮细胞的能力。最近的工作重新FnBPA 3纤维连接蛋白结合结构域。野生型（WT）S金黄色葡萄球菌 8325.4侵入内皮细胞具有效率高，而应变，缺乏FnBPA和B（ΔFNB）显示的内部化水平显着降低（图1A） 。与质粒编码FNB的突变体的互补A减去纤维连接蛋白结合结构域（pFnR0）没有促进入侵（图1A ）。相比之下，与质粒编码整个FNB（pFnBA4）入侵恢复到野生型水平（图1A）一个基因突变的互补性。 FnBPA在内皮细胞的侵袭中的作用也可以证明使用的异源表达主机乳酸乳球菌。表达质粒编码的FnBPA 在 L 球菌 （pRM9 9）没有显着增强附着力内皮细胞相比，细菌表达没有FnBPA（CTL）（开放的酒吧，图1B） 。相比之下，FnBPA表达L。比非表达株（封闭式酒吧，图1B）水平显着高于球菌入侵血管内皮细胞。 实验进行了四次重复和均值±标准差是。 *代表WT或CTL值显着性差异（P &lt;0.05）的数据。 <img src="/files/ftp_upload/2693/2693fig1.jpg" alt="图1" /> 图1。EA的入侵。 Hy926内皮细胞由 S 金黄色葡萄球菌 （A）或L。球菌 （二）。

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<html>我们所描述的实验是基于对庆大霉素保护法，已广泛用于研究细菌的宿主细胞的入侵。在早期的研究中利用宿主细胞的入侵细菌 4-8庆大霉素等抗生素杀死细胞内的细菌无法观测。 24，48甚至96孔板的使用，可以生成大量定量，在很短的时间内，在相对较低的成本的重复性数据。该检测也有吸引力，因为它不需要专门设备，它可以根据工作与各种细菌和细胞，可用于研究9入侵细菌和宿主细胞的过程中的作用。事实上，自从最早的实验，庆大霉素保护法已被广泛采用的措施，通过一系列不同的细菌或细菌 10,11甚至组合的宿主细胞浸润。虽然核心原则的方法相同，许多微妙的变化已有报道。在这篇文章中，我们已经描述了我们的版本，并表示，修改可能对其他细菌或宿主细胞所必需的。当设计一个实验来测量使用这种方法也有一些重要的点要考虑的宿主细胞的入侵： 细菌对庆大霉素的敏感性，这可能似乎是显而易见的的，但重要的是要确保正在研究细菌容易对庆大霉素的浓度，温度，并在要使用的持续时间。在不敏感的情况下，它可能会使用其他抗生素 5 。另一种方法，可以用裂解酶（溶葡球菌酶，溶菌酶，mutanolysin）12。 培育和接种时首次执行这个实验中，重要的是要建立的孵育时间和要使用的细菌接种。因此，最初的实验研究随着时间的推移都粘附和侵袭（5分钟到最多6小时）。同样重要的是，以评估如何入侵是感染的多重影响（教学语言;每个细胞中的细菌数）。一般情况下，最好使用最少数量的细菌可能，因为这将减少培养细胞的损害。如果延长的潜伏期或大菌剂使用9,13株之间的重要区别，可能会错过。 细胞损伤。重要的是洗的细菌，在使用前。然而，细胞损伤并不局限于产毒。细菌的侵袭，诱导细胞凋亡和破坏正常的细胞功能都可以引起细胞损伤。这可能会导致庆大霉素渗透进入细胞，造成内在的细菌和给人的印象，入侵并没有发生 14 。 孵化条件。培养基的温度和组成可以有一个入侵的重大影响。例如，由化脓性链球菌的侵袭HeLa细胞依赖的可溶性纤维连接蛋白的存在，这通常是在血清15。另一个要考虑的是在培养液中的细菌生长，在实验的过程中。 文化和细胞内细菌的定量 。虽然TX - 100可能不会杀死细胞内的细菌，它可能会抑制其生长。因此，重要的是要检查固体介质中存在的洗涤剂的细菌复制。凡影响，它可能会使用低浓度的洗涤剂，或使用替代，如皂甙。在结晶紫染色和显微镜检测庆大霉素保护法的一个好处是，它是劳动力密集型少，并允许内在细菌亚群的检测和分化。例如，第金黄色葡萄球菌能产生正常殖民地类型（NCT）或小殖民地变种（SCV）16，这将不个别细菌细胞的显微镜不同。结晶紫染色和显微镜检测对庆大霉素保护法的一个好处是可以和细胞内的细菌进入一个“可行的，但非可培养”的状态将被检测的17个，占的细胞聚集。 研究细菌的入侵宿主细胞的过程中的作用。许多研究都采用主机细胞的功能，如细胞松弛素D，这与肌动蛋白重排干扰抑制剂。这种研究也可以包括抗体，18个特定的基因敲除的靶细胞表面的受体和siRNA。这是非常重要的，以确保这些治疗不影响培养细胞的生存能力或附件或对细菌的毒性作用。 未来的方向： ontent“&gt;由于这种检测通常是在执行多孔细胞培养板，理论上是可能的，以使其适应高通量筛选操作，这可能涉及审查潜在的细胞功能抑制剂库，抗感染药或的或者设计目标细胞内细菌的抗生素。，这种做法可以用来筛选突变体库，以确定参与黏附，侵袭或内生存的基因，在某些情况下它可能是可取的，包括剪切力的模型系统（流动），例如建模时内皮，更加紧密地模拟体内环境中的电流在流动细胞和微流体细胞培养系统的设计和制造的进步提供在宿主病原体模型纳入剪切力的用人庆大霉素保护法的可能性。 总之，这里所描述的协议是基于多年来使用的类似的方法。它广泛适用于多种类型的培养细胞和细菌种类，并能产生大量数据的快速和相对较少的成本。

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		<th>Comment</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31885-023</td>
<td>&nbsp;</td>
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<td>Brain heart infusion</td>
<td>&nbsp;</td>
<td>BioChemika</td>
<td>53286</td>
<td>&nbsp;</td>
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<td>Foetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>HUVECs</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-2519</td>
<td>&nbsp;</td>
<tr>
<td>Endothelial basal medium</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3121</td>
<td>&nbsp;</td>
<tr>
<td>Bovine brain extract (including heparin)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092</td>
<td>&nbsp;</td>
<tr>
<td>Human endothelial growth factor</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4017C</td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4035C</td>
<td>&nbsp;</td>
<tr>
<td>GA-1000</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092C</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Lysostaphin</td>
<td>&nbsp;</td>
<td>AMBI</td>
<td>LSPN-50</td>
<td>&nbsp;</td>
<tr>
<td>Thermanox coverslips</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>TKT-210-330P</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Cytopath</td>
<td>&nbsp;</td>
<td>TCS Biosciences</td>
<td>HC8595</td>
<td>&nbsp;</td>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3886</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4049</td>
<td>&nbsp;</td>
<tr>
<td>T75 flasks</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>430641</td>
<td>&nbsp;</td>
<tr>
<td>Cytochalasin D</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2618</td>
<td>&nbsp;</td>
<tr>
<td>Methyl-B-cyclodextrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>332615</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

invasion of human cells by a bacterial pathogen

在这里，我们将描述我们如何研究细菌病原体的金黄色葡萄球菌的人类内皮细胞的入侵。一般的协议，可以适用于几乎任何可培养细菌的细胞侵袭的研究。可以研究在哪些具体方面的入侵阶段，如对肌动蛋白重排或小窝的作用，将突出显示。宿主细胞是生长在烧瓶中，并准备使用时，种子Thermanox盖玻片的24孔板。使用盖玻片允许随后从井的细胞去除，以减少血清蛋白沉积到井（ 金黄色葡萄球菌会附上）双方的干扰。细菌生长所需的密度和清洗，以消除任何分泌蛋白（如毒素）。与融合的内皮细胞层的盖玻片被转移到新的24孔板中，含有新鲜培养液中的细菌除了前。细菌和细胞培养在5％的CO 2一起，所需的时间，金额在37 ° C 。为S.这个金黄色葡萄球菌通常是15-90分钟之间。 Thermanox盖玻片从每口井，并浸在PBS洗去独立的细菌。如果相关的细菌总数（壁和内在）予以量化，盖玻片，然后放置在一个新鲜的以及含有0.5％的Triton X - 100的PBS中。轻柔吹打，导致完整的细胞裂解和细菌连续稀释和电镀列举到琼脂。如果细菌侵入细胞的数量是必要的，盖玻片添加到井含500μL组织培养基辅以庆大霉素和孵化，持续1小时，这将杀死所有外部的细菌。然后可以洗盖玻片，细胞裂解和细菌列举到琼脂电镀如上所述。如果实验需要直接可视化，盖玻片，可以固定和染色光，荧光或共聚焦显微镜或电子显微镜的准备。

Watch this Scientific Journal Video about Invasion of Human Cells by a Bacterial Pathogen at JoVE.com

Invasion of Human Cells by a Bacterial Pathogen

一个协议，由细菌病原体入侵宿主细胞的研究一般，注重<em&gt;金黄色葡萄球菌</em&gt;和人类内皮细胞。

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 ...

invasion-of-human-cells-by-a-bacterial-pathogen

Research

JoVE Journal

Immunology and Infection

34.6K 次观看.  University of Bath. 一个协议，由细菌病原体入侵宿主细胞的研究一般，注重金黄色葡萄球菌和人类内皮细胞。

视频: Invasion of Human Cells by a Bacterial Pathogen

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

肠道细菌侵入肠上皮细胞<em&gt;体外</em&gt;使用垂直扩散室模型，显着增强

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.