This work has been supported by Elizabeth E. Kennedy Research Award.

1，准备生产菌株的<ol><li>连胜肺炎球菌菌株对细菌生产的5％羊血的胰蛋白酶大豆琼脂从-80°C冰箱个股板块进行测试。 注:该菌株的抑制作用被测试的初期生长，使用血板大大增加了实验的可重复性。此外，其他能产生细菌素的非肺炎球菌菌株可使用虽然生长条件可能需要修改为特定的有机体。我们已经用缓症链球菌和乳酸乳球菌中的覆盖试验成功。 </li><li>在37℃下孵育平板过夜（O / N），用5％的CO 2。 </li><li>该生产菌株的培养后，吹打过氧化氢酶的3000至4000台准备胰酶大豆琼脂（TSA）平板上有25ml的TSA板，并使用无菌玻璃珠流传过氧化氢酶的解决方案。让板干燥下约10分钟生物安全柜。 </li><li>收集细菌的数量明显到一个枪头。刺枪头到TSA的干板。 注:不止一个的生产菌株可捅成一个单一的TSA板。它使得得到的光晕不重叠是很重要的，以空间出来的刺伤。 <ol><li>如果可能的话，包括已知的细菌素生产的，作为阳性对照，并在覆盖作为阴性对照使用的菌株。 注:阳性和阴性对照是此前已公布，并根据要求提供10。 </li></ol></li><li>孵育TSA板包含多个刺，在37℃，5％CO 2中6小时。 </li></ol>叠加应变2制作甘油股票<ol><li>备之前，覆盖测定当天甘油原液（培养物中加入甘油后生长至中期指数期，然后保存于-80℃）。接种肺炎球菌的海峡殖民地5-10AIN为敏感或信息素的分泌托德休伊特中有0.5％的酵母提取物（THY）进行测试。 注:BLPC诱导记者，施工已如前所述2株可根据要求提供。 </li><li>孵育5-10殖民地玻璃管用5毫升THY与盖紧盖子在37℃水浴中，不晃动。 </li><li>孵育培养物在37℃，直到它们达到的OD 0.3 620〜0.5。 注意:这将需要大约4-5小时，具体取决于所使用的菌株。 <ol><li>轻轻翻转试管前测量外径几次。 </li></ol></li><li>如果实验不能在同一天进行，或者在将来的应变会被多次使用，使甘油原液和恢复试验在稍后的日期。甘油股，加甘油文化的20％的最终浓度。分装到培养的1.5 ml离心管中，并储存在-80℃。注:样品可为stored几个月在-80℃。如果不是在甘油的股票，直接利用样本进行叠加分析。 </li></ol> 3，叠加分析<ol><li>之前叠加应用，混合叠加组成部分。在15 mL锥形管，加入5 ml祢，过氧化氢酶3,000-4,000 U和200微升这是不是长大的那一天，或者是在室温下解冻文化的甘油覆盖应变肉汤培养物。如果使用报告菌株，添加50μl的第X-gal的（40毫克/毫升），以覆盖混合物。用一个锥形管，每盘。 </li><li>在室温下平衡，然后加入熔融的TSA的覆盖混合物。 </li><li>从孵化器刺伤应变删除TSA板。熔化的固体TSA微波炉，并保持在55℃水浴中待用。加入熔融TSA 3毫升与1.5％琼脂冷却至55℃以叠加混合​​物。 <ol><li>立即移液之前，请准备叠加混合，混合物会开始凝固很快。注:如果叠加电镀开始之前固化，其结果将是无法解释的。 </li></ol></li><li>移液器向上和向下几次了500毫升吸管小心不要引入气泡到混合物混合叠加混合物。慢慢的吸液管直接覆盖混合到捅TSA板。保持在台式数分钟使琼脂变硬的板。 <ol><li>不要旋转盘来分发叠加混合，这将拉动从被刺的文化发展到覆盖。 </li></ol></li><li>小心地TSA板覆盖到含有5％的CO 2 O 2 / N（约16-18小时）37℃培养箱中培养。 </li><li>过夜生长后观察平板上。该板块应该会出现在覆盖株生长不透明的，除非发生抑制或信息素信号的地方的地方。这些将分别出现在左右应变刺伤清除或蓝色区域。 注:虽然测量电的蓝色或结算的直径对此语句可以用于定量评估来确定，在直径不同也可取决于细菌的被刺到板的量。覆盖法是一种定性分析，任何定量评估应谨慎解释。任何可见的结算相比于阴性对照被视为活性。 </li></ol>

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<li>Wladyka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+biochemical+characterization%2C+and+cloning+of+a+bacteriocin+from+the+poultry-associated+Staphylococcus+aureus+strain+CH-91%5BTitle%5D)+AND+%22Appl+Microbiol+Biotechnol%22%5BJournal%5D)">Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91.</a> Appl Microbiol Biotechnol. 97 (16), 7229-7239 (2013).</li>
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<li>Son, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conserved+mutations+in+the+pneumococcal+bacteriocin+transporter+gene%2C+blpA%2C+result+in+a+complex+population+consisting+of+producers+and+cheaters%5BTitle%5D)+AND+%22MBio%22%5BJournal%5D)">Conserved mutations in the pneumococcal bacteriocin transporter gene, blpA, result in a complex population consisting of producers and cheaters.</a> MBio. 2 (5), (2011).</li>
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</ol>

The authors have nothing to disclose.

这个覆盖测定法是一种快速的方式来确定用于细菌素生产活动的范围，并表明肺炎球菌菌株之间可能出现的竞争性相互作用。覆盖测定法可适于用于筛选多株为细菌素生产上的单个板。我们已通过使用一个48针复制器从一个半到96孔板的接种SBA板成功筛选大应变的集合与该测定。隔夜培养后，成长正在使用的复制和穿孔板与复制的引脚的琼脂表面转移到TSA的板块。 被调节在像羊毛硫抗生素的浓度依赖性其他细菌分泌抗微生物肽也可以使用覆盖技术7,12,13检查。这是当细菌或羊毛硫抗生素的纯化是困难的特别有用的。虽然上清液可用于抗菌测试活性，在培养液的BLP衍生细菌素的表达是非常有限的或不存在（数据未示出）。覆盖测定法允许生长到较高密度未达到时，生物体生长在液体中。在固体培养基上的生长也可能更密切地反映鼻咽的体内生长条件。 覆盖测定法也可用于检查被刺菌株分泌的细菌素的特定信令信息素的能力。这需要结构的报告菌株其表达的信息素受体 ​​蛋白质，并且其中的信息素反应性启动子被放置在lacZ基因的上游。信息素的分泌被刺伤应变升值作为BLP轨迹活性的读出是特别有用的，如果被刺应变不抑制叠加应变。抑制叠加应变无力可能是一个非功能性的BLP轨迹（例如在结果与转运突变株），或者因为分泌的细菌素不积极反对特定的叠加压力测试。记者张力叠加规定，生产者的轨迹有一个全功能的双组分体系和BlpA转运允许上调的信息素分泌更多的信息。这意味着，任何编码细菌素也被分泌出来。 有一些限制该测定。叠加分析是定性的分析中，以便抑制活性或不同品系的信息素的分泌之间的比较，必须小心进行。增长率和条件应比较不同毒株的时候，因为实验的结果在很大程度上依赖于覆盖和刺伤菌株的生长速度加以考虑。在加入过氧化氢 ​​酶以两个板和所述覆盖是重要的，以便消除肺炎球菌H 2 O 2生产上的抑制作用覆盖菌株的生长，特别是当在覆盖使用过氧化氢酶阴性的有机体。定的，在肺炎球菌人口的基因组内容已知的多样性，看到与此测定法的任何抑制活性不能自动归因于BLP轨迹而不缺失分析。

肺炎链球菌 ，鼻咽部的多种微生物社会的共同殖民者，能够通过生产细菌产生抗菌肽（细菌素）与其他肺炎球菌和密切相关的物种竞争。每一个完全测序肺炎球菌菌株检测到包含最新版本的B acteriocin- 升 IKE p eptide轨迹，BLP的。细菌素生产由肺炎球菌已被证明是既在体外的结果，并在体内竞争1-4重要。竞争的动力学是由沿与同源免疫蛋白多样的细菌素的表达响应于一个特定的肽信息素的影响。感应的BLP轨迹是由一个群体感应系统，其中的肽信息素，BLPC，结合到传感器激酶，BlpH控制，并开始导致信号级联转录BLP轨迹5,6。有BLPC四个等位基因变异，每绑定到其同源BlpH 6。对于细胞生存其自身细菌素的影响，其编码同源免疫蛋白的基因被转录于同一操纵子作为各细菌的基因。如果竞争者应变是不能够上调BLP轨迹响应于外源信息素，或如菌株缺少所需的保护的特定免疫基因发生杀伤。该转运体复合物，BlpAB是所必需的肽信息素的分泌，加工和细菌的肽2,4。最近，它已被证明，肺炎球菌菌株的显著一部分是"骗子"，这意味着他们在blpA基因，使它们无法分泌信息素和细菌素1,2保守突变。这些菌株能够外感BLPC 2所分泌的嘶鸣回应无聊株允许生产的免疫蛋白。 虽然细菌素，从上清液粗制剂可以从生产者菌株用生化方法步骤，以达到纯度来制备，这种方法是不实用的筛选分离的大的集合，如果细菌素的表达水平是低的肉汤生长培养2,7- 9。覆盖测定法作为一种快速的方法来调查可能存在的在体外的菌株之间的竞争动态。要做到这一点，肺炎球菌菌株是预先接种到琼脂平板，让它生长，以达到局部细菌浓度足以诱导BLP轨迹。第二应变，然后接种到熔融的软琼脂溶液中，然后施加在预接种菌株来测试灵敏度。以评估为BLP轨迹（独立抑制活性）的活性，菌株可与一系列日来覆盖稀土前面所述BLPC记者株。这些菌株被构造成包含三个不同blpH等位基因是由肺炎链球菌分泌的三种最常见的BLPC信息素响应之一。记者菌株具有一个集成的lacZ基因，该基因是一个BlpH依赖的启动子的控制下，并携带的缺失在BLPC基因，防止自励10,11。

<table><tbody><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>Fisher</td><td>B21261X</td><td>For growth from freezer cultures</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C100-500mg</td><td>4,741 Units per plate</td></tr><tr><td>Todd Hewitt Broth + Yeast Extract (THY)</td><td>Fisher</td><td>DF0492-17-6&amp;nbsp;</td><td>30 g of THB, 5 g yeast extract, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>Trypticase Soy Broth + Agar plates (TSA)</td><td>Fisher</td><td>B11768&amp;nbsp;</td><td>30 g of TS, 15 g agar, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside (X-gal)</td><td>Sigma</td><td>B4252-50MG</td><td>Dissolve in dimethylformamide</td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Spectrophometer</td><td></td><td></td><td>Measures the OD&lt;sub&gt;620&lt;/sub&gt; of cultures</td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO2</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td></td><td></td><td></td></tr></tbody></table>

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.

中有重叠测定两种可能的结果。它应当能够看到被刺菌株生长过夜孵育后。在图1A中，覆盖层的应变是敏感的正在生产的细菌素。间隙的区域可以显现周边刺伤应变。此外，被刺应变到覆盖混合物的旋涡是可能的，并且可以在图1A中可以看出。在图1B中，覆盖应变都难被分泌所指出的不存在包围短截卤代的细菌素。它也有可能是在被刺应变不分泌细菌素。对于信令测定中，两个不同的结果是可能的。该刺伤菌株能够分泌素，与覆盖菌株的组氨酸激酶相互作用，通过第X-gal的击穿看去， 如图2A所示 。如果信息素未激活的转录在报告菌株BLP轨迹，或者如果被刺应变不分泌素，不会发生第X-gal的击穿， 如图2B所示 。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51876/51876fig1highres.jpg" width="500" /> 图1，外观BLP介导的抑制与免疫的叠加分析。 （一）应变P133，细菌素生产中掺入到TSA板并使其生长6小时。菌株P250中，细菌素阴性菌株，接种到覆盖。 O 2 / N孵化后板拍照。间隙的区域箭头指示（B）应变P133中掺入到TSA板和保温6小时。菌株130，包含在blpA移码突变，接种到覆盖。 O 2 / N培养后，将板拍照。 图2:信息素介导的激活BLP轨迹的信号检测。 （一）应变P133中，BLPC型R6分泌，被掺入到TSA板和保温6小时。株P981中，BLPC型R6记者与缺失BLPC，接种于覆盖。 O 2 / N孵化后的照片是（B）P133中掺入到TSA的板。培养6小时后，P845中，BLPC型P164记者与缺失BLPC，接种到覆盖。 O 2 / N的增长后的照片拍摄。

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

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 ...

using-overlay-assay-to-qualitatively-measure-bacterial-production

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.5K 次观看.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  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. 

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

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

定量细菌中蛋白质-RNA结合的测定

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

科研

遗传学

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

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 ...

免疫与感染

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 ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

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 ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

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

摘要

探索更多视频

定量细菌中蛋白质-RNA结合的测定

的视觉检测，以监测T6SS介导的细菌竞争

单细胞测量液泡破裂引起细胞内病原体

肺炎链球菌在鼻内殖民期间炎症反应的特征

监测改变膜极性，膜完整性和细胞内离子浓度

使用一个调查益生素对肺炎球菌定植的影响

仿生材料表征细菌宿主相互作用

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

一种高通量志贺氏菌特异性杀菌试验

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

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

摘要

探索更多视频

定量细菌中蛋白质-RNA结合的测定

的视觉检测，以监测T6SS介导的细菌竞争

单细胞测量液泡破裂引起细胞内病原体

肺炎链球菌在鼻内殖民期间炎症反应的特征

监测改变膜极性，膜完整性和细胞内离子浓度

使用一个调查益生素对肺炎球菌定植的影响

仿生材料表征细菌宿主相互作用

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

一种高通量志贺氏菌特异性杀菌试验

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估