作者感谢埃利森医学基金会的支持。我们还要感谢艾比Dernburg实验室的成员，使用的wormsorter援助。

1。获得年轻的成年动物的同步文化<ol><li>蠕虫生长在接种OP50-1 大肠杆菌数NGM板大肠杆菌菌（10倍浓缩从饱和的文化），直到许多蠕虫都达到了妊娠阶段。 </li><li>治疗妊娠动物的卵制备的溶液，以获得同步培养（卵）。 </li><li>在几个60毫米NGM板板蛋接种10倍浓缩OP50-1在大约150-200个卵/板的密度。 </li><li>孵育板在25°C为2天（直到蠕虫已经达到了L4 - 青少年阶段）。 </li></ol> 2。准备绿脓杆菌板，感染蠕虫<ol><li>对鸡蛋准备（步骤1.1）的一天接种PA14 :: GFP文化于2ml为100微克/毫升的最终浓度金氏B培养基含有利福平。孵育培养，在37℃下搅拌的O / N。 </li><li>板的PA14 :: GFP的尽头根据需要测定TURE到尽可能多的缓慢杀灭板（SKP）。在每个150毫米SKP培养皿移液器75-100微升饱和培养均匀涂抹用无菌玻璃吊具。培养板在37℃下20-24小时。 </li><li>从孵化器中取出板，让他们冷却至室温（约20分钟）。用M9缓冲液中，来自步骤1.4中的液体的最小量洗蠕虫到PA14 :: GFP板。当使用的150mm板，几百蠕虫可以被放置在一个单一的板。 </li><li>孵育板在25℃，18〜21小时。对于P。铜绿假单胞菌暴露年轻的成虫，这是时间窗口，显示的殖民者的最优分配比noncolonized动物。 </li></ol> 3。设立Wormsorter 在进行样品排序，确保机器正常工作。详情可http://www.unionbio.com/support/documents.aspx?id=43获得，日以下ae基本步骤进行了概述。 <ol><li>设置止回阀的压力约5磅（4.5-5.5范围内）。 </li><li>以验证鞘液的流速是合适的，放置15毫升锥形管中的分配器的下方，打开鞘阀，并关闭分选器阀。保持管内的分配器60秒的下方。在管中的体积应为9-10毫升。如果在此范围之外，相应地调整鞘阀压力。 </li><li>以确保鞘液流动速率（设置为在步骤3.2）将允许精确分选，测试使用40微米的颗粒控制的排序率： </li><li>关闭止回阀。加约25ml的控制粒子溶液的贮存器。 </li><li>打开鞘阀，样品阀。 </li><li>在计算机软件，导航到“控制粒子模式”，然后点击获取。 </li><li>确保颗粒流量为〜5-8粒/秒，用TOF（时间飞行）近似宽度颗粒，40微米。这样的TOF表明只有一个40微米的对象是通过在一个时间流逝。如果值在这个范围之外，调整设置（按照上述方向）相应。 </li></ol> 4。排序殖民者与Noncolonized蠕虫<ol><li>使用M9，洗蠕虫成15毫升锥形管。允许成虫由重力下沉到试管底部并除去上清液。填充管与新鲜的M9。重复这个过程3-4倍。这消除了很大比例的幼虫以及过量的细菌并需要大约10分钟。 </li><li>蠕虫添加到wormsorter水库，轻轻混合小搅拌棒。 </li><li>由绿色光电倍增管的红色和黄色的PMT设置为600和200的初始调整信号增益。 </li><li>开始采集数据调整设置。 </li><li>瞄准25-30事件（蠕虫）每秒一种速度。如果数率太高，淡化蠕虫与M9相应的贮存器。如果利率是o低，在小体积的M9添加更多的蠕虫。 </li><li>如果实验需要的殖民一个非常严格的分离与noncolonized蠕虫，设置“巧合检查”到“纯”。这确保了在事件的两个对象发生太靠近彼此或在相同的压降，该机器将拒绝该压降，而不是收集它。 </li><li>要排序的利益群体，绘制一个围绕轴门表示大小（以获得成虫）和荧光强度（ 即高的殖民统治，低noncolonized）。对于荧光门控的值必须根据经验来确定。 </li><li>将一个多孔板或分配器下方的培养皿收集蠕虫然后打开阀门排序开始整理蠕虫。 </li><li>收集一小群动物的分类人口的利益群体在显微镜下进行验证。如果不是这样，相应地调整门控参数，并继续与样品采集。 </li></ol>

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作者什么都没有透露。

我们所描述的方法利用实体的荧光标记的蜗杆以外，遵循蠕虫和它的环境之间的相互作用。在我们目前的情况下，分离是基于病原体的标记和被雇用，以单独的蠕虫与那些没有（或轻）负荷沉重的病原体负荷。随后的基因表达分析中发现的免疫反应，两组表明它们是独立的病原体负载之间没有差异。启动该响应信号别处被证明在物理上与细菌相关的，而不是被分泌的，这表明这种信号可以是PAMP。...

蠕虫自动分拣很像流式细胞仪，通过在蠕虫（通常由记者蛋白的转基因表达提供）测量荧光信号，因为它通过拉直在管经营，允许根据重定向到任何一个收集管/孔或废物容器到门的研究员1设置参数。该wormsorter可以在许多方面促进研究，采用它作为分析工具的一个例子是一项研究，随后启动子活性的时空模式对近1000个基因2。 然而，wormsorter的主要用途是在遗传筛选，下列靶基因表达水平或荧光蛋白的沿蜗杆3-5的轴线定位。 在这里，我们描述了wormsorter一个新的应用程序，在由荧光标记的病原体以下蠕虫的殖民化。用此作为一种工具，我们专注于pathog之间的关系恩定植/负载和免疫反应，从而获得新的见解负责的蠕虫启动免疫反应的机制。 几乎在研究迄今所有的生物，先天免疫反应，病原微生物开始依赖于识别病原体相关分子模式（PAMP），和/或危险/损伤相关分子模式（抑）6,7。第一是保守的微生物结构，其包括微生物细胞壁，其鞭毛，或它的脂质双分子层6的部件，第二个，包括释放分子（ 如 ATP 8），改变的蛋白或改变的细胞过程9,10的其他标记。这两种类型的信号被指定为模式识别受体（模式识别受体），其在特定的图案分子的结合激活链导致的保护性反应事件的蛋白C。确认。 线虫一直作为tracta非常有用的BLE模型解剖的宿主 - 病原体相互作用的各个方面，但有一点还不是很清楚的是免疫应答是如何在蠕虫发起的。无推定的受体是同源模式识别受体（的PRRs）在其他生物体中已显示出结合的PAMP，许多的PRR是关键对于在其他生物中的免疫应答的直向同源物的展现给蠕虫病原体响应一个令人惊讶的贡献有限和电阻。例如，在果蝇 Toll受体，它是一种抗革兰氏阳性病原体必不可少的，表示以C由一个唯一同源物，TOL-1，这有助于保护从革兰氏阴性病原体沙门氏菌 11，但不从其他检测革兰氏阴性或阳性病原体11,12。这些观察，结合数据线虫表明免疫应答可以通过破坏细胞蛋白质翻译h内诱导为使一些人认为，C。线虫主要检测DAMPS 9,13,14。然而，描述的死病原体诱导的免疫应答能力的报告表明，PAMP结合可能有病原体识别在C的重要作用线虫 15,16。以前的工作重点是与年龄同步的基因完全相同C.免疫反应线虫种群，由细菌革兰氏阴性致病菌绿脓杆菌表现在肠道定植大的个体差异。 然而，转录谱研究处理这些可变定植人群作为一个实体17,18。利用这种变化，我们开发了一套集中的自动化wormsorter分离暴露在表达GFP的体育秀丽隐杆线虫的差异殖民地人口铜绿假单胞菌 。研究基因表达的不同定植人群FACI病原体负载（和相关联的损伤）和免疫应答和有关C.病原体识别提供了新的见解之间的关系litated评估线虫 19。下面我们介绍的协议，它可以应用到排序感染任何荧光标记的病原虫。 给潜在的用户应当注意的是，需要进行排序的蠕虫数目依赖于随后的分析和协议中使用的性质。例如，在微阵列基因表达分析的情况下，&gt; 1,000蠕虫将需要获得足够的RNA，如果使用标准的协议，但〜100蠕虫就足够了，如果扩增采用，允许快速采集材料，因而应力最小化，以该蠕虫。

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">M9 Buffer</td>
			<td style="width:139px;">Prepared in house</td>
			<td style="width:527px;">Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin&#160;</td>
			<td>Sigma</td>
			<td>R3501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg prep solution&#160;</td>
			<td>Prepared in house</td>
			<td>50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGM plates&#160;</td>
			<td>Prepared in house</td>
			<td>Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKP plates</td>
			<td>Prepared in house</td>
			<td>Recipe same as NGM only 0.35% peptone instead of 0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control test particles&#160;</td>
			<td>Union Biometrica&#160;</td>
			<td>310-5071-001</td>
		</tr>
	</tbody>
</table>

automated separation of c. elegans variably colonized by a bacterial pathogen

该wormsorter是一种手段类似于用在秀丽隐杆线虫的研究，在FACS机器，通常进行排序基于荧光报道的表达蠕虫。在这里，我们重点介绍该仪器的另一种用法，根据由GFP表达他们的病原菌定植程度的排序蠕虫。这个新的使用使我们能够解决肠蠕虫和诱导的免疫应答的定植之间的关系。虽然C.线虫的免疫反应，以不同的病原体已被记录在案，仍是一个未知数，什么因素促使他们。两个主要的可能性（这并不相互排斥）是识别病原体相关的分子模式，并检测感染所引起的损伤。要在两个可能性区分，接触到的病原体必须从它所造成的损害进行分离。蠕虫的wormsorter启用分离的进行了广泛定植由革兰氏-Negative致病菌绿脓杆菌 ，有可能是由病原体负荷造成的损害，从蠕虫，亦放置，而不是，或轻微，定植。这些不同的群体被用来评估病原体负载和转录的免疫应答的诱导之间的关系。该结果表明，这两个解离，配套的病原体识别的可能性。

当年龄相匹配的，遗传上相同的C。线虫暴露于P。假单胞菌 ，广泛分布在定殖（ 图1A）的水平变化。与此处描述的noncolonized从殖民蠕虫有效的分离可以实现（ 图1B）协议的帮助。不像noncolonized蠕虫，蠕虫殖民显示损坏的迹象，如呆滞，减少排便19。后者可能是一个原因，蠕虫殖民地P。绿脓杆菌有困难清除感染。这对描述的协议表明蠕虫排序，n...

Watch this Scientific Journal Video about Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen at JoVE.com

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

该wormsorter通过根据荧光记者表达分拣蠕虫有利于遗传筛选在秀丽隐杆线虫 。在这里，我们描述了一个新的使用：根据定植通过GFP表达病原体排序，我们用它来检查病原体识别在启动免疫反应的了解甚少的作用。

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

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

automated-separation-c-elegans-variably-colonized-bacterial

Research

JoVE Journal

Immunology and Infection

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

8.3K 次观看.  University of California, Berkeley. 该wormsorter通过根据荧光记者表达分拣蠕虫有利于遗传筛选在秀丽隐杆线虫 。在这里，我们描述了一个新的使用：根据定植通过GFP表达病原体排序，我们用它来检查病原体识别在启动免疫反应的了解甚少的作用。 

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

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

科研

免疫与感染

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

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

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

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

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

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

一种高通量、高含量、液态的 Pathosystem线虫

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...