We thank Dr. Antonio Virgilio&#160;Failla for providing the FRET acquisition quantification algorithm and Erwin Bohn for providing the pMK-bla and pMK-ova constructs.

1.峰值发射和串扰测定（参见图2） <ol><li>为了确定该发射峰和施主之间的串扰（香豆素衍生物V450）和受体（荧光素）的染料量，执行标记的细胞都在405nm激发个体荧光团的光谱扫描。 </li><li>确定每种染料的将被用于供体和受体通道（这里455-465纳米和525-535纳米）的峰在10纳米的带宽。积归一化强度在波长，并计算在受体通道，反之亦然（图2）的串扰供体染料的平均百分比。 </li></ol> 2.准备Y的菌菌株用于细胞培养的感染<ol><li>使用下面的毒株:WA-314 PMK-OVA（WA-314转化的质粒PMK-BLA 17和PMK-OVA 17，分别）和WA-314ΔYopEPMK-BLA（WA-314ΔYopE用该质粒转化PMK-BLA 17） </li><li>感染连胜Y.前至少两天菌菌株WA-314 PMK-OVA，WA-314 PMK-BLA和WA-314ΔEPMK-BLA从包含所需的抗生素（卡那霉素WA-314 PMK-BLA和WA-314 PMK-OVA LB琼脂平板冷冻库存;卡那霉素和四环素WA-314ΔEPMK-BLA）和成长O / N在27℃。此后保持板长达一个星期4℃。 </li><li>感染前一天接种含有所需抗生素3 ml的LB培养基的与各自的菌株的一个菌落，并培育O / N在27℃在摇床以180rpm。 </li><li>关于感染当天接种2毫升的O / N培养的成40毫升的新鲜LB培养基无抗生素并在摇床孵育90分钟，在37℃下以180rpm以引起III型分泌系统的表达。 </li><li>转移培养物到合适的管中，保持培养物在冰上或在4℃下今后。离心培养物10分钟，在5000×g离心和4℃。 </li><li>重悬细菌沉淀在2-3毫升冰冷的PBS。通过使用分光光度计稀释细菌悬浮液，以8×10 8 CFU / ml的浓度。分别确定相应的光密度。保持这个悬挂在冰上，直到感染的HeLa细胞。 </li></ol> 3.电镀HeLa细胞<ol><li>一天感染种子前1.5×10 4个细胞的HeLa细胞中含有在96孔板的孔中的10％热灭活胎牛血清（FCS），在37℃下培养在5％CO 2培养箱200μl的DMEM中。种子三眼井每一株（三个不同的培养时间）。 </li></ol> 4.感染HeLa细胞与CCF4加载<ol><li>感染HeLa细胞通过加入3.5微升细菌悬浮于培养基中，并轻轻吹打培养基两次混合。让细菌沉积物上到细胞中，以每细胞约100细菌的MOI（感染部分）。对于时间过程开始感染在-90分钟，-60分钟，-30分钟，并在37℃下在5％CO 2培养箱直到0分钟以实现共同的终点。 </li><li>当孵化完成后，小心吸媒体不去除HeLa细胞，并加入50微升PBS含20微克/毫升氯霉素停止效应和2.5mM丙磺舒的表达，减少CCF4出口。 </li><li>在这一点上96孔板转移到显微镜载物台，并定义合适的斑点在每个以后的分析孔。在10分钟内完成这个步骤（对收购事项之详情参见5.1节）。 </li><li>每孔中，加70微升CCF4 / AM加载溶液（0.24微升溶液A，1.2微升B溶液，18.56微升溶液C（溶液A，B和C提供的范围内CCF4 / AM装载套件）和50微升的PBS（含20微克/ ml氯霉素和2.5mM丙磺舒）使用多pipette孵育5分钟，在室温。从现在开始，工作不中断。 </li><li>吸加载溶液，并添加含有20微克/ ml氯霉素和2.5mM丙磺舒的使用多移液管将100μlPBS中。然后迅速在96孔板转移到显微镜载物台，并从加载溶液的吸液在10分钟内开始采集。 </li></ol> 5.激光扫描显微镜和数据分析<ol><li>图像采集<ol><li>设定成像条件的方式以最大化总光子计数和最小化光毒性，漂白和交叉激发。 <ol><li>在现代激光共聚焦显微镜，使用20X高数值孔径浸泡的目的，打开探测器针孔2.5艾里单位（ 即宽光学切片），选择高灵敏度的GaAsP探测器同时与主动供体和受体通道，8位深度，〜750nm的像素大小，3倍帧平均和EXCITe为405nm的二极管激光器的10％。 </li><li>为了确保正确的量化设置两个通道的检测器增益为相同的值，并避免任何饱和的像素。 注意:在此，增益被设定为150％。 </li></ol></li><li>保证连续浸没用1ml注射器扩频浸没介质到孔的底部，例如，避免任何气泡的俘获。 </li><li>激活的透射光，并找到在每个井的视图的代表字段。使用正确校准自动阶段，标志着该软件的位置。 </li><li>去除板染色后（参见4.3节），重新插入板和顶部增加的重量，以避免焦点漂移。回顾与激光扫描方式保存的位置，并适当调整的重点和侧卧位。 </li><li>设置的时间间隔为2分钟，持续时间为2小时，并开始收购。 </li></ol></li><li>图像量化（例子是位平面软件选装定是诸如了Imaris） <ol><li>导入数据集到软件，使像素矩阵的算术运算。通过拖放含有供体和软件图标受体通道的源文件执行此操作。 </li><li>使用相同的偏移减去在这两个信道的背景。要做到这一点，请单击菜单，然后单击图像&gt;处理&gt;基线扣除。选择每个通道和输入基准值。 </li><li>纠正渗出通过供给方信道（​​CH1）到受体通道（CH2）用的预先确定的校正系数f（参见1.1）的，创建一个新的信道: CH2 = CH 2 - 通道1 *˚F 注:渗出通过受体进入施主信道的则没有检测到过度的噪音水平。 <ol><li>点击菜单&gt;图像处理&gt;通道运算在和进入ABS（CH 2 - （CH1 * 0.33））。随后删除频道2通过单击菜单&gt;编辑&gt;删除频道。确保渗出通过修正受体通道保持作为新的渠道2.可选:通过进入菜单&gt;编辑&gt;图像属性更改从灰色渠道颜色与原来的颜色。选择4频道&gt;映射颜色&gt;基础颜色和改变它，例如，绿色环保。 </li></ol></li><li>创建具有以下操作来确定相对FRET系数的新渠道: CH 3 = CH 2&#39;/（CH1 + CH2"） <ol><li>进入菜单&gt;图像处理&gt;通道运算在和输入CH2 ./（CH1 + CH2） </li></ol></li><li>对于一个8位图像的FRET通道进行适当的可视化，创建第四个通道: CH4 = 255 * CH3 <ol><li>进入菜单&gt;图像处理&gt;通道运算在并输入255 * CH 3。通过进入菜单&gt;编辑&gt;图像属性改变通道的颜色。选择4频道&gt;映射颜色&gt;颜色表文件&gt;喷射。 </li></ol></li><li>应用一个3x3的中值滤波通道CH3和CH4塔吨将减少图像中的噪声。要做到这一点，请单击菜单&gt;图像处理&gt;平滑&gt;中值滤波器。选择这两个渠道，应用过滤器大小"3x3x1"。 </li><li>对于蜂窝信号定量，检测的细胞在通道4通过适当地设置偏移和过滤结构由尺寸排除太小的结构。 <ol><li>选择超越查看并点击图标" 添加新的表面 "上。限定在表面窗口的检测算法参数。为了更快的处理，激活两个复选框"段利息只有一个区域"和"处理整个图像最后"。通过编辑其尺寸限定感兴趣的区域。 </li><li>选择通道4作为信源通道和阈值&gt;背景减法（局部对比度）设置的阈值，并设置直径10微米。设置最小强度阈手动0和最大阈值的最大的IntensiTY。过滤结构由区，并设置最小值例如，187平方微米。完成表面检测。 </li></ol></li><li>提取的平均值为供体荧光强度（CH1）和相对的FRET系数（CH3）和在蜂窝实体它们的标准偏差。要做到这一点，去表面性能。通过选择面风格/质量&gt;表面改变结构的外观。通过单击过滤器选项卡上选择的所有结构。通过将流程选择&gt;统一统一的检测机构。导出通过点击统计标签表面的统计数据为MS Excel&gt;导出所有统计到文件。 </li><li>随着时间的推移绘制这些值。确定的信道供体荧光增加为每个条件的合理参数来表示易位效应物的量最大斜率的10分钟时间间隔。计算斜率为m =Δ供体荧光强度/Δ时间（分钟）。 </li></ol></l我&gt; </ol>

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The authors declare that they have no competing financial interests.

我们在这里成功地应用于基于TEM-1β-内酰胺酶报告基因分析的效应易位中用 Y定量分析菌 。这种敏感，特异的和相对简单的技术的许多不同的变化，在文献中已经描述。在这项研究中的激光扫描显微镜进行最灵敏和最精确的检测荧光信号的进行。专门校正的供体和受体染料和单独调节检测的带宽之间的串扰允许测量更高的精度相比该方法的其它变型。结果清楚地表明，该方法是能够?...

III型分泌系统，它们由不同的属的革兰氏阴性菌，直接递送细菌编码的效应蛋白到真核靶细胞利用专门蛋白出口的机器。而分泌机制本身是高度保守的，专门集效应蛋白进化的不同细菌种类中，以操纵细胞信号途径和促进特定细菌的毒力的策略1。万一耶尔森氏菌，最多七个效应蛋白，所谓Yops（ 耶尔森氏菌外蛋白），被易位在宿主细胞接触并一起行动颠覆免疫细胞反应，如吞噬作用和细胞因子产生， 即，以允许细菌的胞外生存2-4。易位的处理严格控制在不同阶段5。它被确定，T3SS初级活化通过它的接触到目标策触发LL 6。然而，这种引发的精确机制仍有待阐明。在耶尔森所谓微调易位的第二电平由上调或下调细胞的Rho GTP结合蛋白的Rac1或RhoA的活性实现的。 Rac1的激活例如通过侵袭素或细胞毒性坏死因子Y（CNF-Y）导致增加的易位7-9，而GAP（GTP酶活化蛋白质）易位YopE的功能下调Rac1的活性，因此通过一个负反馈型降低易位机制10,11。 有效和精确的方法是前提，以研究如何易位是在小肠结肠炎耶尔森氏宿主细胞相互作用调控。许多不同的系统已被用于这个目的，每个具有特定的优点和缺点。一些方法依赖于裂解感染的细胞，但不细菌由不同的去污剂随后免疫印迹分析。钍这些方法Ë共同的缺点是次要的，但不可避免的细菌裂解潜在污染的细胞裂解液与细菌相关的效应蛋白。然而，提出了治疗细胞用蛋白酶K以降解细胞外的效应蛋白和后续使用毛地黄皂苷为真核细胞的选择性裂解，以尽量减少此问题12。重要的是，这些检测主要依赖于高品质抗效应的抗体，其中大多是尚未投放市场。企图利用效应蛋白翻译融合和荧光蛋白GFP的像并监控易位没有成功可能是由于该荧光蛋白的球状三级结构和分泌装置的无法分泌13之前展开它们。 百日咳毒素然而，有几个不同的记者的标签，如CYA（钙调蛋白依赖的腺苷酸环化酶）域cyclolysin 14或闪存标签被成功用于分析易位。在前者的测定CYA的酶活性被用于扩增胞内融合蛋白的信号，而闪光标签，在很短的四半胱氨酸（4Cys）基序标签，允许与biarsenical染料的Flash标签，而不会干扰分泌的过程中15。 这里所应用的方法是由夏邦杰等人报道首次并基于对细胞内的荧光共振能量转移（FRET）染料CCF4通过易位效应TEM-1β-内酰胺酶融合体16（图1A）的转换。 CCF4 / AM是其中香豆素衍生物（供体）和荧光素部分（受体）由头孢菌素核连接的细胞穿透物化合物。在被动进入真核细胞，所述非荧光酯化CCF4 / AM化合物由蜂窝酯酶处理以带电和荧光CCF4从而被困内的小区。激发香豆素结构部分在405nm处的结果的FRET的荧光素部分，其在530nm发射的绿色荧光信号。头孢菌素核的β-内酰胺酶的FRET的裂解之后被破坏，香豆素结构部分的激励导致的蓝色荧光发射at460纳米。该方法的不同应用在文献中突出了其通用性已被描述。该方法允许易位体外的分析以及在体内，例如，该技术被用来在小鼠感染模型来识别体内 17-19针对易位的白细胞群。信号读出可使用酶标仪，流式细胞仪或荧光显微镜进行。值得注意的是，该方法还提供了感染过程20,21期间监视易位实时由活细胞显微术的可能性。这里激光扫描荧光显微镜涂敷了用于readou荧光信号吨，因为它提供最高的灵敏度和精确度。特别是，能力调整与纳米精度与高敏感检测器组合的发射窗有助于优化荧光检测和最小化串扰。此外，该显微镜的设置可适于易位实时监测和有可能允许对宿主 - 病原体相互作用的在细胞水平上同时分析。 在这项研究中易位Y.率菌野生型菌株和YopE缺失突变体表现出hypertranslocator型10,11示范性地进行了分析。

<table><tbody><tr><td>LB-Agar</td><td>Roth</td><td>X969.2</td><td></td></tr><tr><td>LB-Medium</td><td>Roth</td><td>X968.2</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8662-500ML</td><td></td></tr><tr><td>96-well plate (black, clear bottom)</td><td>Greiner Bio One</td><td>655087</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td><td>Gibco/Life Technologies</td><td>31966-047</td><td></td></tr><tr><td>Probenecid</td><td>Sigma-Aldrich</td><td>P8761-25G</td><td></td></tr><tr><td>LiveBLAzer FRET-B/G Loading Kit with CCF4-AM</td><td>Life Technologies</td><td>K1095</td><td></td></tr><tr><td>Lectin from Triticum vulgaris (wheat) FITC conjugate</td><td>Sigma-Aldrich</td><td>L4895</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>V450 Rat anti-Mouse CD8a</td><td>BD Bioscience</td><td>560469</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>Immersol W immersion oil&amp;nbsp;</td><td>Zeiss</td><td>444969-0000-000</td><td>Refractive index = 1.3339 @ 23 &amp;deg;C</td></tr><tr><td>TCS SP5 II confocal laser scanning microscope</td><td>Leica microsystems</td><td></td><td>2x GaAsP-Hybrid detectors, 4 channel spectrometer, acusto optical beam spliter, motorized XY stage, adjustable pinhole, objective 20X HC PL APO CS IMM/CORR, 405&amp;#160;nm diode laser 50&amp;#160;mW</td></tr><tr><td>Imaris 7.6 software</td><td>Bitplane</td><td></td><td>Plugins included ImarisXT and MeasurementPro</td></tr><tr><td>MatLab compiler runtime</td><td>MathWorks</td><td></td><td></td></tr><tr><td>Prism 5</td><td>GraphPad software</td><td></td><td></td></tr></tbody></table>

analysis of yersinia enterocolitica effector translocation into host cells using beta-lactamase effector fusions

许多革兰氏阴性细菌包括致病耶尔森菌属使用III型分泌系统，转运的效应蛋白到真核靶细胞。在宿主细胞内的效应蛋白操纵细胞功能的细菌的益处。为了更好地理解III型分泌宿主细胞相互作用，灵敏，准确的检测过程中控制测量易位是必需的。我们在这里描述了基于一小肠结肠炎耶尔森效应蛋白片段的融合的测定法（耶尔森外蛋白; YopE）的应用程序。用TEM-1β-内酰胺酶的易位的定量分析的测定法，依赖于细胞裂解渗透剂FRET染料（CCF4 / AM）通过易位β-内酰胺酶的融合。 CCF4的由β-内酰胺酶的头孢菌素核的裂解之后，从香豆素FRET荧光素被破坏，香豆素结构部分的激励导致的蓝色荧光发射。该方法的不同应用在文献中突出了其通用性已被描述。该方法允许易位体外的分析以及在体内，例如，在小鼠模型。可以用酶标仪，流式细胞仪或荧光显微镜进行检测荧光信号。在由不同耶尔森突变这里描述的，在效应子融合的体外易位至HeLa细胞中设置由激光扫描显微镜监测。通过实时β-内酰胺酶的效应融合录制FRET记者细胞内转化提供了可靠的定量结果。我们在这里展示示范性数据，由Y.展示增加易位菌 YopE突变体比野生型菌株。

为了证明所描述的方法的能力进行定量分析的效应易位至靶细胞，二耶尔森株不同易位动力学进行了研究:Y。菌野生型菌株WA-314（血清型O8，窝藏的毒力质粒pYVO8 22）及其衍生物WA-314ΔYopE（WA-314窝藏pYVO8ΔYopE23）。此前的研究显示，Y.菌突变体缺乏功能YopE表现出显著高于烨转率10。两个菌株用为YopE和TEM-1β-内酰胺酶（PMK-BLA）17的 n-末端分泌信号的?...

Watch this Scientific Journal Video about Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions at JoVE.com

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

analysis-yersinia-enterocolitica-effector-translocation-into-host

Research

JoVE Journal

Immunology and Infection

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

8.5K 次观看.  University Medical Center Hamburg-Eppendorf. Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

视频: Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

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 ...

科研

免疫与感染

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

遗传学

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

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

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

电功能性细菌效应器到哺乳动物细胞

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

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

生物学

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

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

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

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

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

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

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

的产生<em&gt;肠杆菌</em&gt; SP。 YSU营养缺陷型使用转座子突变

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

使用遗传密码扩增对细菌分泌蛋白进行超分辨率成像

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

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