研究在实验室被授予由德意志研究联合会（DFG医管局3125/3-2，DFG医管局3125/4-2）和德国联邦教育与研究部（BMBF）医疗感染基因组学（FKZ 0315828A）到上海的支持。

这里所描述的动物感染实验必须严格按照当地和国际准则和规定的使用脊椎动物（ 如欧洲健康实验动物科学协会（FELASA）的联邦法）进行。实验必须由当地的伦理板和机构动物护理委员会的批准。所有实验与S肺炎在实验室或动物感染的II类生物安全柜进行。 1。生物发光Pneumocococi的传染性股票的制备<ol><li>准备在Todd-Hewitt肉汤补充有1％（重量/体积）酵母提取物和20％甘油进行维护的终浓度在80℃下的库存肺炎球菌培养物</li><li>板块少量速冻股票肺炎球菌文化到血琼脂平板上，并在5％CO 2培养细菌10小时，在37℃。 Ŧ他血液琼脂平板上包含用于luxABCDE基因盒插入基因组的转座子插入适当的抗生素如卡那霉素（150微克/毫升）。 </li><li>子培养出了新的殖民地到一个新的血琼脂平板上最多10小时的。 </li><li>接种THY培养基补充有10％（V / V）热失活（30分钟，56℃）胎牛血清（FBS）与肺炎双球菌，开始培养时的OD 600的0.05-0.07。 </li><li>培养肺炎球菌，不加搅拌，在37℃和5％CO 2，直到培养物达到的OD 600的0.35-0.40。增长将需要3-4小时之间。 </li><li>通过离心收获的肺炎球菌在3750×g离心10分钟，并重新悬浮生物发光性的肺炎球菌的磷酸盐缓冲盐水pH为7.4（PBS）中补充有0.5％FBS中。 </li><li>调整接种物至所需浓度在PBS/10％FBS中。感染剂量取决于所用的肺炎链球菌菌株和麦克风的遗传背景Ë。感染远交CD-1小鼠滴鼻S。肺炎 D39 勒克斯感染剂量应在20微升补充有90单位的透明质酸酶的悬浮液调整到1×10 7 CFU。请勿摇晃或涡流肺炎双球菌，因为这将触发自溶。 </li><li>通过电镀10倍连续稀释到血琼脂和菌落计数在37℃下在5％CO 2培养过夜后验证接种浓度。 </li><li>用光学成像系统测量生物发光验证肺炎球菌接种物的生物发光。 </li></ol> 2。鼻内和小鼠腹腔感染生物发光肺炎球菌<ol><li>使用雌性远交小鼠在7-10周龄时为急性肺炎和败血症模型。这些小鼠的重量应该是20-30克。 </li><li>腹腔注射氯胺酮和甲苯噻嗪麻醉小鼠。配制混合液1.0毫克在100μl无菌0.9％氯胺酮和0.1毫克甲苯噻嗪（重量/体积）的氯化钠对小鼠体重为20g。这与50毫克/公斤体重的氯胺酮和5毫克/公斤体重为甲苯噻嗪的剂量是一致的。要准确，注射了麻醉剂之前权衡小鼠。 </li><li>注入混合物进入腹膜腔，然后将动物放回笼子，直到麻醉作用和动物是narcotized。小鼠的呼吸会变得非常缓慢，很有规律。小鼠必须充分narcotized，从而使鼻内接种细菌悬浮液和吸入不会导致打喷嚏，因此，接种物的损失。这可以通过捏鼠标在他们的尾巴末端进行评估;完全narcotized鼠标将缺乏响应。 </li><li>轻轻拿起narcotized鼠标和保持手指和拇指与鼻子直立（ 图1）之间的鼠标。 </li><li>用移液管与狭长的小技巧（凝胶装载机提示），把多个小液滴生物发光肺炎球菌到鼠标26-29的鼻孔（10微升/鼻孔）。鼠标会不由自主地吸入的细菌。按住鼠标1-2分钟直立，观察小鼠是否打喷嚏或保持接种。 </li><li>感染7-10周龄的CD-1小鼠经鼻内用1×10 7或7.5×10 7个肺炎链球菌菌株D39和TIGR4，一种感染剂量分别监测感染的实时性。检出限为系统而不由宿主组织的任何吸收是约1×10 6个发光细菌。 </li><li>感染小鼠腹腔注射（IP）来评估和肺炎球菌的败血症小鼠感染模型BioImage以毒性。使用5×10 3 CFU在100μl菌株D39和1×10 4株TIGR4的。使用IP路由时鼠标不麻醉。 </li><li>接种对照组用PBS。 <img alt="图1" fo:conteNT-宽度="5英寸"FO:SRC ="/ files/ftp_upload/51174/51174fig1highres.jpg"SRC ="/ files/ftp_upload/51174/51174fig1.jpg"宽度="500px的"/&gt; 图1。鼻内感染CD-1小鼠的与S肺炎 。 <a href="https://www.jove.com/files/ftp_upload/51174/51174fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 </li></ol> 3。可视化肺炎球菌传播到肺和血液使用成像系统肺炎双球菌的雌性远系繁殖的CD1小鼠中实时使用体内成像系统的鼻内感染后图像的传播。 <ol><li>切换所述成像系统上，并启动位于preconfigurated计算机上的图像分析软件。 </li><li>所述成像系统自动初始化和CCD相机热电冷却至90℃，即活性之前。 </li><li>的设置，分级取决于测量小鼠的数目同时且在生物发光信号的强度。视场（FOV）的字段来监控动物的生物发光是可变的，为22.5厘米FOV之间的放大倍数范围内测量五只小鼠，和3.9厘米FOV测量单个鼠标一样的胸部的动物或零件或头部。 </li><li>使用常规的1分钟，分级的中等程度，和22.5厘米的FOV（组D）的曝光时间。首先，选择发光成像模式，然后设置图像参数。 </li><li>的信号的发射可能依赖于细菌菌株和所用的小鼠品系，以及鼠标的颜料。剃鼠的胸使用例如 C57BL / 6小鼠时，成像肺炎在这些小鼠中的发展时，它可以是有利的。 </li><li>在prechosen时间间隔形象感染小鼠。测量小鼠急性肺炎模型的生物发光在最大8-12小时的时间间隔。通过observi还监测感染小鼠的福利纳克自己的外表，行为和决心重量的损失。 </li><li>在成像设备的腔室中的测量之前的麻醉系统的麻醉室麻醉小鼠。开始吸入异氟醚和氧气的混合物，等到老鼠缓慢地呼吸，并定期。 </li><li>放置麻醉动物进入系统的成像室，并保持小鼠仰卧位。上腔，然后将图像中的小鼠呼吸道顶​​部的CCD相机。 </li><li>将用自己的鼻子动物进入试管，使吸入的麻醉药。 </li><li> 通过气体管道异氟烷进入成像室被允许维持麻醉。 </li><li>要激活在腔顶部的CCD摄像头，并在成像软件启动生物发光光学成像，按"采集"。紧接小鼠在封闭腔室中的照片显示在屏幕上。一分钟后<html>测量生物发光数据的叠加和照片被示出。 </li><li>停止麻醉的图像叠加显示在软件的​​窗口后，把老鼠放回笼子。 </li><li>监测小鼠进行恢复。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51174/51174fig2highres.jpg" src="/files/ftp_upload/51174/51174fig2.jpg" width="500px" /> 图2。麻醉和监测使用IVIS麻醉和IVIS频谱系统，分别为A）的IVIS光谱成像系统B）的IVIS系统麻醉的孵化室。 三）麻醉小鼠在孵化室感染肺炎球菌生物发光小鼠D）小鼠位于IVIS光谱成像室中与自己的鼻子吸入麻醉剂。_blank"&gt;点击这里查看大图。 </li></ol> 4。定量感染小鼠生物发光S的生物发光及评价肺炎 <ol><li>通过使用图像分析软件定量的总光子发射（光子/秒）确定的小鼠的选定区域的小鼠或生物发光强度。 </li><li>使用光子发射在Excel数据表中所提供的数值，制成曲线图。由于数据的变化往往是分组小鼠内高，产生一个盒子晶须图形显示在感染肺炎球菌不同品系的小鼠的差异。 </li><li>提供每小鼠的光子或备选的结果作为辐射率的所选区域的平均值（光子/秒/ cm 2 / SR）。 </li><li> ROI（感兴趣区域的）在光子模式的测量数据可以在不同的定量比较在不同的体内成像系统来RA的设置，因为在四射的单位测量自动考虑相机设置（ 如积分时间，分级，F /停止，和视场）。 </li></ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+pneumococcal+infections+in+the+lungs+of+live+mice+using+bioluminescent+Streptococcus+pneumoniae+transformed+with+a+novel+gram-positive+lux+transposon.">Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.</a> Infect. Immun. 69, 3350-3358 (2001).</li><li>Basavanna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+methionine+acquisition+and+synthesis+on+Streptococcus+pneumoniae+growth+and+virulence.">The effects of methionine acquisition and synthesis on Streptococcus pneumoniae growth and virulence.</a> PLoS One. 8, (2013).</li><li>Hartel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+central+carbon+metabolism+of+Streptococcus+pneumoniae+by+isotopologue+profiling.">Characterization of central carbon metabolism of Streptococcus pneumoniae by isotopologue profiling.</a> J. Biol. Chem. 287, 4260-4274 (2012).</li><li>Hammerschmidt, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+immune+regulator+factor+H+interacts+via+two+contact+sites+with+the+PspC+protein+of+Streptococcus+pneumoniae+and+mediates+adhesion+to+host+epithelial+cells.">The host immune regulator factor H interacts via two contact sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion to host epithelial cells.</a> J. Immunol. 178, 5848-5858 (2007).</li><li>Voss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choline-binding+protein+PspC+of+Streptococcus+pneumoniae+interacts+with+the+C-terminal+heparin-binding+domain+of+vitronectin.">The choline-binding protein PspC of Streptococcus pneumoniae interacts with the C-terminal heparin-binding domain of vitronectin.</a> J. Biol. Chem. , (2013).</li><li>Cartwright, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumococcal+disease+in+western+Europe:+burden+of+disease,+antibiotic+resistance+and+management.">Pneumococcal disease in western Europe: burden of disease, antibiotic resistance and management.</a> Eur. J. Pediatr. 161, 188-195 (2002).</li><li>vander Linden, M., Al-Lahham, A., Nicklas, W., Reinert, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+pneumococcal+isolates+from+pets+and+laboratory+animals.">Molecular characterization of pneumococcal isolates from pets and laboratory animals.</a> PLoS One. 4, (2009).</li><li>Brehm, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+of+the+adenine+methylase+gene+of+the+Streptococcus+faecalis+plasmid+pAM+beta+1.">Sequence of the adenine methylase gene of the Streptococcus faecalis plasmid pAM beta 1.</a> Nucleic Acids Res. 15, 3177 (1987).</li></ol>

<html>在动物身上进行的所有实验必须由地方当局和伦理委员会的批准。在体内实验感染的细菌负荷在感染动物的各种宿主龛，必须在不同时间点在感染后确定。在这些实验条件的动物具有从血液，鼻咽，bronchoalvelar灌洗或器官如肺，脾和脑细菌的隔离之前被牺牲掉。计算每个主机小生细菌的数量，并评估细菌因素对毒力的影响时，血液或器官必须由细菌分离随之恢复和电镀在固体培养基上?...

由病毒或细菌呼吸道感染仍然是全球最常见的社区获得性或临床问题引起全球所有死亡的大约三分之一之一。关键细菌物种是流感嗜血杆菌和肺炎链球菌 2。然而，这些细菌种类是通常的自然呼吸道菌群的常见成分。因此，细菌马车是一定风险的侵入性疾病，并根据免疫状况或个人的倾向也。无症状定植被触发侵袭性感染。 肺炎链球菌是社区获得性肺炎（CAP）和菌血症人类最常见的原因之一，主要病原体。在健康个体S。肺炎链球菌 （肺炎球菌）往往是上呼吸道，在那里他们都面临着非致病性细菌无症状和无害殖民者常住菌也与病原体如嗜血杆菌属的。或金黄色葡萄球菌和人的免疫防御系统的第一道防线。携带率最高的是年幼的孩子（37％），甚至在拥挤的日间护理中心（58％）高3-5。最年轻的人群和老人，通过空气传播的载体和鼻咽部分泌物6接受肺炎球菌，属于使用的肺炎球菌结合疫苗（PCV10或PCV13在儿童和23价多糖PPSV23成人）一个高危人群和疫苗接种建议在美国（美国）和许多欧洲国家4。在PPSV23涵盖负责〜90％的菌血症性肺炎球菌疾病在美国和欧洲，预防成人从而有效地侵入性肺炎球菌疾病（IPD）的血清型，而PCVS涵盖儿童中最常见的血清型。因此，瞳距由于疫苗的种类（VT）是热镀CED但非疫苗血清型显示高毒力潜力及耐药性已经出现4,7-12。鼻咽部的水库是起点肺炎蔓延到鼻窦或中耳引发有害的局部感染。更重要的是，肺炎球菌直接通过气道蔓延至支气管和肺部造成威胁生命的第4,13。肺部感染常常伴随着组织和屏障的破坏，从而使病原体传播到血液中，并造成IPD。 CAP和IPD的发病率最高，免疫功能低下者或在4,13年代的极端。负责从共生与高致病病原体转换的情况仍在争论。然而，除了改变宿主的易感性和进化适应伴随着较高的毒力和增加的抗生素抗性已建议对PNE至关重要的影响umococcal感染14-16。 病原体被赋予了黏附素介导的亲密接触粘膜上皮细胞的多样性。克服气道黏液后，肺炎球菌粘附于宿主细胞是通过表面暴露粘附素与细胞受体相互作用的直接并利用细胞外基质成分或血清蛋白的桥联分子4,17,18便利。多才多艺的病原体肺炎球菌还配备了参与宿主免疫防御机制回避的因素。此外，它们具有适应各种宿主milieus如肺，血液和脑脊液（CSF），分别为5,17,19,20的能力。 细菌因素对发病机制和炎性宿主反应的影响进行了研究中的肺炎，菌血症实验动物模型中，或脑膜炎21-25。尽管是人类的病原体，这些模型是我们LL成立的破译肺炎球菌组织嗜性，致病机制，或肺炎球菌疫苗候选保护性。近交系小鼠品系的遗传背景决定了易患肺炎。鼻内感染了肺炎球菌免疫BALB / c小鼠，结果为:耐腐蚀，而CBA / Ca和SJL小鼠抗肺炎球菌感染22更容易。这意味着，类似于人类的遗传背景和宿主防御机制确定感染的结果。因此，有必要进一步努力解开抗性基因座的小鼠不容易受到肺炎球菌感染的基因组中。该发现导致了变化的体内毒性的协议。而不是过去常常使用的近交系BALB / c小鼠后，极易受到CD-1/MF1远交小鼠品系人现在通常用来研究的功能丧失的肺炎球菌的致病性或适用性的因素26-28的效果。此外，可用生物发光性肺炎和光学成像技术的允许感染的实时生物发光生物成像。在肺炎球菌的优化luxABCDE基因盒（质粒保罗的TN 4001 luxABCDE公里R）已经插入到染色体中通过转座子诱变的单一整合位点。生物发光肺炎链球菌已被用来评估短少在致病性或适用性的因素及其易位从一个解剖部位到另一个26,28-31肺炎球菌突变体的衰减。 在这里，我们提供了一个协议，用于在小鼠肺炎或败血症模型肺炎球菌感染的生物成像。放大和传播生物发光肺炎球菌在鼻内或腹膜内感染的小鼠可以很容易地随着时间的推移，使用光学成像系统和相同的动物在不同时间点监测。

<table border="0" cellpadding="0" cellspacing="0" width="985">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Todd Hewitt broth</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">X936.1</td>
			<td style="width:280px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">2363.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Blood agar plates</td>
			<td style="width:280px;">Oxoid, Wesel, Germany</td>
			<td style="width:129px;">PB5039A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">T832.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erythromycin</td>
			<td style="width:280px;">Sigma-Aldrich,Taufkirchen, Germany</td>
			<td style="width:129px;">E6376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">fetal bovine serum (FBS)</td>
			<td style="width:280px;">PAA Laboratories, Coelbe, Germany</td>
			<td style="width:129px;">A11-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CD-1 mice, female</td>
			<td style="width:280px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:129px;">CD1SIFE06W08W</td>
			<td>female CD-1 mice, six to eight weeks old</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamin 500mg, Curamed injection solution</td>
			<td>Schwabe-Curamed, Karlsruhe, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Rompun 2%, injection solution</td>
			<td>Bayer Animal Health, Monheim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Plastipak 1 ml syringes</td>
			<td>Becton Dickinson, Heidelberg, Germany</td>
			<td style="width:129px;">300015</td>
			<td>sterile Luer-Lok&#8482; syringes with needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Gel Loader Tips</td>
			<td style="width:280px;">peqlab</td>
			<td style="width:129px;">81-13790</td>
			<td>M&#181;ltiFlex&#8482; Tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hyaluronidase</td>
			<td style="width:280px;">Sigma-Aldrich</td>
			<td style="width:129px;">H3884-100mg</td>
			<td>Hyaluronidase Type IV-S from Bovine test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Oxygen</td>
			<td style="width:280px;">Air Liquide, D&#252;sseldorf, Germany</td>
			<td style="width:129px;">M1001L50R2A001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Isofluoran</td>
			<td style="width:280px;">Baxter, Unterschlei&#223;heim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pGEM-T Easy</td>
			<td style="width:280px;">Promega, Mannheim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Oligonucleotides </td>
			<td style="width:280px;">Eurofins MWG, Ebersberg, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Qiaprep Spin Midiprep Kit </td>
			<td style="width:280px;">Qiagen, Hilden, Germany</td>
			<td style="width:129px;">27104</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:296px;">PCR DNA purification kit</td>
			<td style="width:280px;">Qiagen, Hilden, Germany</td>
			<td style="width:129px;">28106</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:296px;">Equipment</td>
			<td style="width:280px;"> </td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Living Image 4.1 software</td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">XGI-8 Gas Anesthesia System</td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">IVIS Spectrum Imaging System </td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Biophotometer</td>
			<td style="width:280px;">Eppendorf AG, Hamburg, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
	</tbody>
</table>

following in real time the impact of pneumococcal virulence factors in an acute mouse pneumonia model using bioluminescent bacteria

肺炎是发展中国家的主要医疗保健问题和工业化国家之一，具有相当的发病率和死亡率。尽管在知识这个疾病，重症监护病房（ICU）的可用性，并使用有效的抗菌药物和有效的疫苗的进步，死亡率仍然很高1。 肺炎链球菌是社区获得性肺炎的病原体领先（CAP）和菌血症的人类最常见的原因之一。这种病菌配备的表面暴露的黏附和毒力因素导致肺炎和侵入性肺炎球菌疾病（IPD）的医疗设备。细菌健身或毒力因子在体内的作用进行评估是最重要的解开S。肺炎链球菌致病机制。肺炎，菌血症和脑膜炎的小鼠模型已被用于确定肺炎球菌因素在昼夜温差的影响ferent感染的阶段。在这里，我们描述了一个协议来监控实时传播肺炎球菌在小鼠鼻腔或腹腔内感染发光细菌之后。该结果表明，繁殖和传播的肺炎球菌的下呼吸道和血液，从而可以可视化并进行评价使用成像系统和所附的分析软件。

蛋氨酸收购和吸收是对肺炎球菌，保持健康在他们的主机利基32,33至关重要。蛋氨酸ABC转运脂蛋白是由SPD _ 0151基因（TIGR4:sp_0149）编码在D39和命名MetQ 32。肺炎球菌进一步产生蛋氨酸合成酶（D39:Spd_0510 - Spd_0511; TIGR4 Sp_0585 - Sp_0586，丈量和metF的）。蛋氨酸在一个化学成分确定的培养基中缺乏影响肺炎和类似，缺乏蛋氨酸32,33的蛋氨酸结合脂蛋白MetQ受损摄...

Watch this Scientific Journal Video about Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria at JoVE.com

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

肺炎链球菌是导致病原体引起严重的社区获得性肺炎，负责超过200万世界各地的死亡。的牵连健身或致病细菌因素的影响，可以在实时中采用发光细菌急性小鼠肺炎或菌血症模型来监测。

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

following-real-time-impact-pneumococcal-virulence-factors-an-acute

Research

JoVE Journal

Immunology and Infection

15.0K 次观看.  University of Greifswald.  肺炎链球菌是导致病原体引起严重的社区获得性肺炎，负责超过200万世界各地的死亡。的牵连健身或致病细菌因素的影响，可以在实时中采用发光细菌急性小鼠肺炎或菌血症模型来监测。 

视频: Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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

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

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

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

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

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

线虫模型中气单胞菌感染的毒力及发病机制评价

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

李斯特菌单细胞基因感染后小鼠脾脏原位干扰素伽马生产的成像

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

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