这项工作是由威康信托基金会资助WT091939MA。阿兰·Filloux是由英国皇家学会的支持。

1。细菌菌株和文化<ol><li>工程师变换（使用标准的氯化钙处理或电）15 E.大肠杆菌受体细胞（猎物，P）与允许的 lacZ基因（ 表1）中的a-互补的质粒的大肠杆菌 DH5α细胞。板的Luria-Bertani琼脂平板上的转化细胞（LBA，1.5％琼脂），含有5 - 溴-4 - 氯 - 吲哚基-β-D-吡喃半乳糖苷（X-gal的），在40毫克/毫升的最终浓度和合适的抗生素。孵育在37°C过夜，第二天早上选择蓝色的转化（见第1.3节）。 </li><li>成长的铜绿假单胞菌 ，T6SS活性的供体细胞（D +）或T6SS的惰性（D-），（表1）上的LBA过夜，在37℃下在给出的例子在这里我们使用的是P.铜绿假单胞菌可再生能源技术的应变拥有一个组成活性H1-T6SS 16和一个等位基因突变的的H1-T6SS的基因簇（ 表1）的删除。 </li><li>第二天，准备通宵充气烧瓶液体培养接种明亮的蓝色克隆之间的E. §1.1中描述的大肠杆菌转化体（P），从板，在5毫升的胰蛋白酶大豆肉汤（TSB）辅以适当的抗生素。在搅拌下成长，在37°C。 §1.2中描述的从板的单菌落（D +）和（D - ），继续进行同样。 </li></ol> 2。竞争法<ol><li> LBA板准备第二天的实验。确保这些板块干燥（附近的本生灯，或在层流柜）。准备一个“检测输入”板（A输入）的菌株（D +），（D - ），和（P </strong&gt;）。准备“法输出”（A-输出）的菌株板（D - + P）和（D + + P）。划分和标记板。 </li><li>过夜后生长（见第1.3节），测量输入细菌培养（D - ，D +和P）的光密度（OD 600nm的），并计算所需要的体积，得到细胞密度相当于1个单位的每个菌株OD 600nm为。每个“输入”细胞培养物（D - ，D +和P）的最初收集在一个无菌1.5毫升Eppendorf管中。 </li><li>以13,000 rpm离心细菌样品在室温下的1分钟期间，并弃去上清液。 </li><li>重悬的粒料的 D - ，（D）+，和 P的文化，在100μl新鲜TSB通过轻轻移液接种10毫升的各对应作为一个单一的点上的“A-输入”板（§2.1）中制备的相应的应变。 </li><li>接种“A-输出板（§2.1编制）。更精确地，轻轻混匀，30微升（D +）与30微升（P）和30微升的（D - ）用30毫升（P）的两个单独的Eppendorf管中（注意，所使用的培养是在§2.4中描述的那些） 。接种20μl的混合物（D）+（/ P）和（D - / P），“输出”的板作为单独的点上。允许斑点附近的一个本生灯干燥并放置一段适当的时间，在此期间，杀死细菌正在发生的板在37℃的培养箱中。在的情况下的 P 铜绿假单胞菌 ，有效的杀死细菌培养5小时后观察比较T6SS积极与T6SS缺陷株。 </li></ol> 3。定性观察的t他杀死细菌<ol><li>准备的LBA板含有40微克/毫升X-gal的用于读出的测定。板将被称为“读数输入”或“R-输入”发现分离出的细菌或“读出输出”或“R-输出”发现混合细菌培养，这样读出的查杀性能。这些板块也需要得到适当的干燥。除以板在它的后面的四个相等的部分，这些部件和注释0，10 -1，10 -2，10 -3，指定的细菌培养物的稀释液的琼脂平板上发现（见第3.3节）。的稀释将允许在同一个地方的蓝/白菌比半定量评价。 </li><li>收集的无菌环的单个细菌斑点从“A-输入板（见§2.4）和”输出“板（见§2.5），重悬在不同的1.5 mL的Eppendorf管中含有1毫升TSB每个点。将在30分钟到离心振动筛块重悬有效地细菌。 </li><li>准备了一系列的5倍的3个Eppendorf管含900微升TSB的，每个重悬点至10倍系列稀释到10 -3继续进行。请一定要更换吸头和涡5秒之间每个稀释步骤。 </li><li>继续LBA板干燥最摊薄未稀释的悬浮液中含40μg/ ml的X-gal的准备§3.3细菌稀释，使得这些“R-输入”车牌为“A检举输入点“的”R-输出“为”A-输出点“（ 图2）的板。涡简要每个管中，然后再进行检举保持均匀的菌悬液。的可重复性的结果，在一个象限中的点20μl，以一式三份（ 图2）。你的盘子的相对干燥，在这​​个阶段是很重要的，因为点需要保持单独分离板，而不是向对方滑行。 的竞争测定甲量化也可以在此阶段的实验。之后的步骤3.3中，100微升稀释10 -3 LBA板含有40微克/毫升X-gal的传播。对于重复性的结果，从“A-输出”板电镀一式三份的每一个点。将稀释板中，在37℃培养箱中培养过夜（相应于孵育16小时）。继续的蓝色菌落计数（P细胞仍然活着）。典型的竞争5小时后，得到的结果如图3所示。 </li><li>发布的无菌区域内板开（无盖），以允许过量的液体的吸收，各点内。 </li><li>板放置在37℃培养箱中过夜。在此温育时间，杀害仍然发生在混合（D + / P）的时间这两个菌株仍处于接触。 </li><li>拍摄一张照片或扫描您的输出分析的板（ 图2）。点仍然在很大程度上蓝色表明，E.菌没有被杀死的P.铜绿假单胞菌 。这是E.大肠杆菌混合与T6SS缺陷P.铜绿假单胞菌菌株（D-）（图2中 ，板在右下角）。 </li></ol>

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我们什么都没有透露。

在这篇文章中介绍的方法，可以用肉眼观察的T6SS介导的杀菌/抑菌活性。的琼脂板的表面上进行测定。先前已表明，T6SS依赖杀灭试验进行混合细菌液体培养的效率不高，可能是因为缺乏稳定的两种细菌之间的联系8。相信该T6SS是一种机制类似于使用噬菌体注入DNA导入靶细胞的17中的一项操作。在液体培养中，管样结构的T6SS可能更容易地打破，细菌间的接触可以被丢失，和毒素不能有效地传递。 孵育时间，最初5个小时的接触，我们描述的供体菌和猎物之间有足够的观察杀死细菌之间P.绿脓杆菌和大肠杆菌大肠杆菌 ，如在图3中示出</strong&gt;。然而，这是可取的调整通过执行中的动力学为了优化的实验条件下的培养时间。 由于这种方法是基于色彩技术，输出的结果可能会受到影响的供体菌的色素沉着。例如，在的情况下的 P 铜绿假单胞菌 ，某些菌株产生高水平的着色颜料，如绿脓素和pyoverdine，这可能会干扰化验读数，使得从猎物的区别相对困难。其他的显色的β-半乳糖苷酶的底物，如-半乳糖苷酶的品红或红色-半乳糖苷酶，可用于代替的X-gal的（ 表1）。 竞争法可以利用的其他报告基因的读出。例如，也进行了类似的试验，使用绿色荧光蛋白标记的猎物12。 我们的分析，而不能定量，提供了一个很好的指示的T6SS活动，因为它是基于生存或杀害记者的猎物。该技术存在的优点是容易和方便地评价从任何细菌物种的杀菌/抑菌活性T6SSs。到目前为止，的T6SS活性已经显示出对革兰氏阴性菌和革兰氏阳性细菌的T6SS敏感没有明确的例子已报道12。这是交不亲和的文化，以测试不同的细菌种类也很明显， 如生长温度，氧，特定的媒体是要考虑的。 我们的分析也可用于评估的T6SS组件是绝对必要的，因为即使痕迹的分泌毒素足以杀死的猎物。即使是活动能力弱的T6SS就可以清楚地检测到我们的试验相比，标准的程序测试T6SS依赖分泌培养上清液和免疫印迹分析。然而，仍然需要一个适当的集落形成单位（CFU）计数准确定量本T6SS活动。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 铜绿假单胞菌 PAKΔ 可再生能源技术 （D +） （积极T6SS） </td><td>实验室菌株</td><td></td><td>文献16中描述的</td></tr><tr><td> 铜绿假单胞菌 PAKΔ 可再生能源技术ΔH1-T6SS（D - ）（未激活T6SS） </td><td>本研究</td><td></td><td>文献18中描述的步骤后，通过等位基因交换的的H1-T6SS群集（包围基因PA0070 PA0095）已被删除。与下面的一组引物为：向上片段的引物产生的mutator片段： 5&#39;-ATGGTCAACGACATGGAGCTGGAG-3&#39;，和 5&#39;CGAGGCCGATCAGGCCTTCAGAACTGA-3&#39;。向下片段引物： 5&#39;-TCAGGCCTTCAGAACTGAAGCGGCGCA-3&#39;， <pre> 5&#39;-GGTGGCGTTCAACAGTTCCATGTC-3&#39; </pre></td></tr><tr><td> 大肠杆菌 DH5α </td><td> Invitrogen公司</td><td> 18258-012 </td><td> F-φ80， 紫胶 ZΔM15Δ（ 拉丁美洲和加勒比ZYA-ARG F）U169 REC A1 年底 A1 HSD R17（R K - M K +）PHO一个支持 E44λ 氏 -1 GYR A96 相对 A1 </td></tr><tr><td>的pBluescript II SK（+） </td><td>安捷伦</td><td> 212205 </td><td>该载体表达的α肽的β-半乳糖苷酶的用于α-互补。 </td></tr><tr><td> X-gal的</td><td> Invitrogen公司</td><td> 15520-018 </td><td>在40微克/毫升</td></tr><tr><td>卢里亚Bertani琼脂</td><td>默克化学品</td><td> 1.10283.0500 </td><td></td></tr><tr><td>酪蛋白大豆肉汤（TSB） </td><td> Oxoid公司</td><td> CM109 </td><td></td&gt; </tr><tr><td>涡流振动筛天才3 </td><td> IKA </td><td> 3340000 </td><td></td></tr><tr><td>扫描器</td><td>爱普生</td><td> V700 </td><td></td></tr><tr><td>分光光度计</td><td> WPA Biowave </td><td> CO8000密度计</td><td></td></tr><tr><td>洋红色 - 半乳糖苷酶</td><td> Bioworld </td><td> 30350001-1（715241） </td><td></td></tr><tr><td>红加仑</td><td>研究有机物</td><td> 1364C </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1菌株，质粒，材料和使用的试剂。 </td></tr></tbody></table>

a visual assay to monitor t6ss-mediated bacterial competition

Ⅵ型分泌的系统（T6SSs）是分子纳米机器允许革兰阴性菌，运输和注入的蛋白质，用于广泛的各种靶细胞1,2。该T6SS是组成13核心部件，并显示与尾管的噬菌体3的结构上的相似之处。噬菌体使用的球管和删余的移动设备以穿透的目标细菌细胞信封并注入DNA。它提出，T6SS创建一个特定的路径中的细菌细胞信封驱动效应器和毒素的表面是一个倒置的噬菌体的移动设备。这一过程可以采取进一步的T6SS装置可以穿孔与其他细胞的细菌接触，从而注入到这些目标的效应。尾管和穿刺装置部分的T6SS作出与赛和VgrG蛋白中，分别为4,5。 通过s已被证明的多功能性的T6SS各种细菌病原体tudies使用。的的霍乱弧菌 T6SS可以重塑真核宿主细胞骨架的注入“进化”VgrG的一个C-末端的肌动蛋白交联域6,7。另一个明显的例子是最近使用绿脓杆菌能够瞄准并杀死细菌在一个T6SS依赖性，从 ​​而推动建立细菌微生物在特定的细分市场，有竞争力的环境8,9,10记录。 在后者的情况下，三个T6SS分泌蛋白，即TSE1，TSE2及TSE3，已被确定为在目标细菌（ 图1）喷射的毒素。的供体细胞是从这些效应的有害影响的保护，通过抗毒素机制，介导由Tsi1 Tsi2和Tsi3免疫蛋白8,9,10。这种抗菌活性可以被监控当T6SS，精通细菌的共培养在S固体表面，在与其他细菌物种或与T6SS惰性细菌属同一物种的8,11,12,13的竞争。 现有的数据强调了数值方法的细菌竞争法，包括费时CFU计数，它在很大程度上依赖于抗生素制造商。在的情况下的抗生素的耐药菌株，如P.铜绿假单胞菌 ，这些方法可以是不合适的。此外，约200种不同的超过100个细菌基因组的14 T6SS位点的识别，方便的筛选工具，是非常可取的。我们开发了一种检测是容易使用的，并要求标准实验室材料和试剂。该方法提供了一种快速和定性的技术来监测T6SS依赖性杀菌/抑菌活性通过使用记者菌株作为食饵（在这种情况下， 大肠杆菌 DH5α）允许一个互补的 lacZ基因。总体而言，此方法的grap嗝的T6SS相关的表型琼脂平板上，并允许快速识别。这个试验性的协议可适于考虑到特定的条件，例如生长培养基，温度或时间的接触的其他菌株或细菌物种。

典型结果示于图1，表1中描述的菌株和试剂。在该图中所示的板的被扫描后，孵育过夜。 “读数输入”板示出了在该测定中使用的菌株的连续稀释图案。正如预期的那样， 大肠杆菌大肠杆菌猎物点（P）过度表达的 lacZ基因，用X-gal培养基上出现蓝色的，而捐助者P.铜绿假单胞菌菌株（D +，T6SS有源）和（D - ，T6SS无效）保持白色。 “读出输出板之间的搭配的猎物和T6SS积极应变（D / P）已被发现，显示的蓝色消失的猎物，从而表明它已被杀害。这表明捐助，outcompete的猎物的能力。持久性的蓝色的（D - / P）板演示，一个无效T6SS供体不能杀蓝色的猎物。 <img src="/files/ftp_upload/50103/50103fig1.jpg" alt="图1"/> 图1。杀害E.大肠杆菌的T6SS精通P.铜绿假单胞菌。P. 铜绿假单胞菌注射到E.毒素大肠杆菌在T6SS依赖性靶细胞（白色箭头所示）。两种毒素TSE1和TSE3（橙色和红色圆圈）被注入到E.大肠杆菌周质和降解肽聚糖9。 （黄圈）TSE2毒素注射到E.大肠杆菌细胞质，并有抑菌活性8,10。共同作用的毒素杀死目标细胞（闪光的闪电和头骨）。可以通过监测所产生的β-半乳糖苷酶的活性（也可参见图2）检测到的靶细胞的生存。P.一eruginosa是受保护的的免疫力的蛋白质Tsi1，Tsi2和Tsi3（橙色，黄色和红色的正方形），分别8,9,10拮抗活性的毒素。 <img src="/files/ftp_upload/50103/50103fig2.jpg" alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50103/50103fig2highres.jpg"/> 图2。琼脂平板测定监视T6SS依赖杀死细菌，在该图中所示的上半部分的“读出输入”板组成的系列稀释的 D - ，D +和 P输入单元格。 P输入细胞是蓝色由于到α-互补的 lacZ基因，并由此产生了β-半乳糖苷酶酶切的X-gal。中下部所示的“读出输出”的板组成的系列稀释的细菌混合之间的活性（D + / P） </strONG&gt;或无效（D - / P），T6SS捐助P.铜绿假单胞菌菌株与E.大肠杆菌的猎物。 <a href="https://www.jove.com/files/ftp_upload/50103/50103fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50103/50103fig3.jpg" alt="图3"/> 图3。 E.杀害的量化大肠杆菌的P.铜绿假单胞菌 5小时后孵化。图介绍了CFU计数E.大肠杆菌中描述的步骤3.4。这里给出的结果显示的T6SS +和该T6SS菌株的3倍的差异，这表明大部分的杀灭发生在最初的5个小时的接触。

Watch this Scientific Journal Video about A Visual Assay to Monitor T6SS-mediated Bacterial Competition at JoVE.com

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

我们描述了定性检测，监测细菌介导的竞争<em&gt;铜绿假单胞菌</em&gt;类型的VI分泌系统（T6SS）的。分析依赖的生存/杀死大肠杆菌靶细胞背着<em&gt; lacZ的</em&gt;记者。这种技术是可调的，以评估杀菌/抑菌活性精通T6SS的微生物。

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

a-visual-assay-to-monitor-t6ss-mediated-bacterial-competition

Research

JoVE Journal

Immunology and Infection

15.5K 次观看.  Imperial College London. 我们描述了定性检测，监测细菌介导的竞争铜绿假单胞菌类型的VI分泌系统（T6SS）的。分析依赖的生存/杀死大肠杆菌靶细胞背着 lacZ的记者。这种技术是可调的，以评估杀菌/抑菌活性精通T6SS的微生物。

视频: A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

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 ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

检测皮质片段在细菌孢子萌发

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

链霉菌发育突变体的视觉和显微评价

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...