Name: detecting cortex fragments during bacterial spore germination
Uploaded: 2016-06-25T13:00:00
Description: Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

描述该项目由奖号5R01AI116895从过敏和传染病国家研究所的支持。内容完全是作者的责任，并不一定代表国家过敏和传染病研究所和美国国立卫生研究院的官方意见。

1.生成样本<ol><li>热激活C.难辨孢子在65℃下30分钟，并存储在冰上。 注：C.如先前7,9,10所述艰难梭菌芽孢可以产生并纯化。 </li><li>准备在X + 1毫升，其中X是在测定期间要采取的样本数萌发溶液。 注意：萌发溶液是特定细菌的物种孢子正在研究中。用于测定C.难辨孢子萌发，我们使用的10mM Tris（pH值7.5），150毫摩尔NaCl，在去离子水中的100mM甘氨酸和10mM牛...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+that+degrade+bacterial+cell+walls.">Enzymes that degrade bacterial cell walls.</a> Methods Enzymol. 8, 685-699 (1966).</li><li>Park, J. T., Johnson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+submicrodetermination+of+glucose.">A submicrodetermination of glucose.</a> J Biol Chem. 181, 149-151 (1949).</li><li>Thompson, J. S., Shockman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modification+of+the+Park+and+Johnson+reducing+sugar+determination+suitable+for+the+assay+of+insoluble+materials:+its+application+to+bacterial+cell+walls.">A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls.</a> Anal Biochem. 22, 260-268 (1968).</li><li>Paredes-Sabja, D., Setlow, P., Sarker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SleC+is+essential+for+cortex+peptidoglycan+hydrolysis+during+germination+of+spores+of+the+pathogenic+bacterium+Clostridium+perfringens.">SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens.</a> J Bacteriol. 191, 2711-2720 (2009).</li><li>Paredes-Sabja, D., Sarker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+cortex-lytic+enzyme+SleC+from+non-food-borne+Clostridium+perfringens+on+the+germination+properties+of+SleC-lacking+spores+of+a+food+poisoning+isolate.">Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate.</a> Can J Microbiol. 56, 952-958 (2010).</li><li>Hindle, A. A., Hall, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dipicolinic+acid+(DPA)+assay+revisited+and+appraised+for+spore+detection.">Dipicolinic acid (DPA) assay revisited and appraised for spore detection.</a> The Analyst. 124, 1599-1604 (1999).</li><li>Kort, R., 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=Assessment+of+heat+resistance+of+bacterial+spores+from+food+product+isolates+by+fluorescence+monitoring+of+dipicolinic+acid+release.">Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release.</a> Appl Environ Microbiol. 71, 3556-3564 (2005).</li><li>Elson, L. A., Morgan, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+method+for+the+determination+of+glucosamine+and+chondrosamine.">A colorimetric method for the determination of glucosamine and chondrosamine.</a> The Biochemical journal. 27, 1824-1828 (1933).</li><li>Ramirez, N., Abel-Santos, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+germination+of+Clostridium+sordellii+spores+in+vitro.">Requirements for germination of Clostridium sordellii spores in vitro.</a> J. Bacteriol. 192, 418-425 (2010).</li><li>Akoachere, M., 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=Indentification+of+an+in+vivo+inhibitor+of+Bacillus+anthracis+spore+germination.">Indentification of an in vivo inhibitor of Bacillus anthracis spore germination.</a> J. Biol. Chem. 282, 12112-12118 (2007).</li><li>Duncan, C. L., Strong, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+medium+for+sporulation+of+Clostridium+perfringens.">Improved medium for sporulation of Clostridium perfringens.</a> Appl Microbiol. 16, 82-89 (1968).</li><li>Heffron, J. D., Lambert, E. A., Sherry, N., Popham, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+four+cortex+lytic+enzymes+to+germination+of+Bacillus+anthracis+spores.">Contributions of four cortex lytic enzymes to germination of Bacillus anthracis spores.</a> J Bacteriol. 192, 763-770 (2010).</li><li>Meador-Parton, J., Popham, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+Bacillus+subtilis+spore+peptidoglycan+during+sporulation.">Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation.</a> J Bacteriol. 182, 4491-4499 (2000).</li><li>Lambert, E. A., Popham, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacillus+anthracis+SleL+(YaaH)+protein+is+an+N-acetylglucosaminidase+involved+in+spore+cortex+depolymerization.">The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization.</a> J Bacteriol. 190, 7601-7607 (2008).</li><li>Wang, S., Shen, A., Setlow, P., Li, Y. Q. <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+the+Dynamic+Germination+of+Individual+Clostridium+difficile+Spores+Using+Raman+Spectroscopy+and+Differential+Interference+Contrast+Microscopy.">Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy.</a> J Bacteriol. 197, 2361-2373 (2015).</li></ol>

在刺激时，进行发芽的过程孢子丧失其抗损伤性，而无需大分子合成。当孢子萌发被触发时，孢子释放核心DPA，以换取水2。由于休眠孢子的高DPA含量，孢子核心是在强烈渗透压和专门肽，皮质，有助于防止芯从通过作用，据推测，作为屏障，以扩张2肿胀。 上述的方法使用艾氏试剂（黄色溶液），以检测还原糖作为可测量的移位到紫色色调的存在。期间皮质?...

芽孢是细菌代谢，使细菌在不利的环境中坚持的休眠形式。在许多孢子形成细菌，孢子形成是通过营养剥夺诱导但此过程可通过改变pH值，暴露于氧或其它应力1进行控制。而在其代谢休眠孢子形式，细菌抵抗紫外线辐射，干燥，更高的温度，和冷冻2。最上的孢子形成过程的知识来自在模式生物的研究， 枯草芽孢杆菌 。在孢子形成过程开始于DNA复制和轴向灯丝3,4的形成。非对称隔膜然后把细胞分成两个大小不等车厢。较大的隔间里，母细胞，吞噬较小的隔间里，forespore。无论是母细胞和forespore协调基因表达成熟的孢子母细胞裂解最终，释放多尔曼吨孢子到周围环境中1。 孢子的结构和组合物在许多细菌物种保守的。孢子芯具有比营养细胞低水含量和有丰富的吡啶二羧酸（DPA）1,2。期间形成孢子，DPA被泵入孢子芯换取水。围绕核心的是一种内在的孢子膜的地方，在多数孢子形成的细菌，其中认识到刺激发芽（germinants）的小分子的受体位于2。刚好位于孢子的内膜外是细胞壁肽聚糖的层。一个专门的肽聚糖层（皮层）围绕细胞壁肽聚糖，并且由许多相同的部件，细胞壁肽聚糖[交替的N-乙酰葡糖胺酶（NAG）和N-乙酰胞壁酸（NAM）]的。但是，在皮质的NAM残基的约50％已被转化为胞壁-δ内酰胺5,6- </s了&gt;。期间孢子萌发，这些胞壁-δ内酰胺残基被孢子皮质裂解酶（SCLEs）的认可，因此允许皮质被降解但不细胞壁（必需完成发芽过程）。周围皮质的外膜和外壳蛋白2几层。 控制发芽的方法对于形成孢子的细菌至关重要。当开端受体各自germinants 2交互萌发开始。在许多孢子形成的细菌，这些germinants是氨基酸，糖，核苷酸，离子或它们的2组合。C.难辨孢子萌发是由某些胆汁酸，[ 例如 ，牛磺胆酸（TA）]和氨基酸（ 例如 ，甘氨酸）7-10的组合引发。虽然有在C之间的差异难辨发芽途径和在所研究的途径其他孢子形成细菌，如B.枯草芽孢杆菌，共同所有的降解孢子皮质以允许营养细胞以从发芽孢子2,8成长的绝对要求。皮质降解可由SCLEs CwlJ / SleB来实现或SLEC（如在枯草芽孢杆菌中发现）（如在许多梭菌找到）。皮质水解降低了细胞壁和孢子的约束。这允许充分芯补液，在重新激活许多必需的细胞代谢2蛋白质的一个必要步骤。 当孢子发芽，从相亮状态到相暗状态，这个过程的休眠孢子的变化可以通过在600光密度（OD）的变化来测量 纳米11。先前的一份报告表明，在很多OD这一变化是由于DPA从12孢子释放。在我们最近的研究中，我们试图比较C的定时难辨孢子萌发和监测DPA和皮质碎片9的释放。在这项研究中是至关重要的监测皮质片段的释放，他们开始由萌芽孢子被释放。 此处所使用的比色测 ​​定法是基于与还原末端检测糖的方法开发Ghuysen 等 13。因为其他人描述协议检测还原糖14或修改这些协议15，关于这一问题的文献可能会造成混淆。在这里，我们详细地一步一步方法还原糖的比色检测从发芽C.解放艰难梭菌芽孢。虽然本研究采用C.艰难梭菌芽孢，从其他孢子形成的细菌孢子的发芽过程中释放的还原糖能够与该协议9,16,17进行检测。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 mL screw cap tube</td>
			<td>USA scientific</td>
			<td>1420-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p20 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-1250uL pipet tips</td>
			<td>VWR</td>
			<td>89079-486</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200uL pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-D-glucosamine</td>
			<td>Sigma</td>
			<td>A3286-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid - 10N</td>
			<td>BDH-Aristar</td>
			<td>BDH3032-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Alfa Aesar</td>
			<td>L04295</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>BDH</td>
			<td>BDH0280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Tetraborate tetrahydrate</td>
			<td>Alfa Aesar</td>
			<td>39435</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>BDH8019-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma</td>
			<td>156477-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturated Phenol</td>
			<td>Fisher</td>
			<td>BP1750-400</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M3</td>
			<td colspan="2">Molecular Devices</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>BDH</td>
			<td>BDH3098-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated, Circulating Water Bath</td>
			<td>VWR Scientific</td>
			<td>Model 1136</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtest 96</td>
			<td>Falcon</td>
			<td>353072</td>
			<td>96 well clear tissue culture plates</td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>VWR</td>
			<td>89000-506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Blocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting cortex fragments during bacterial spore germination

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

表1示出了使用NAG标准的典型结果。数据被用于产生标准曲线。 表2示出从发芽缓冲器萌发测定补充有100mM的甘氨酸和10mM TA（发芽促进条件）或100mM的甘氨酸只（非发芽条件）的典型结果。在不存在TA，C的艰难梭菌芽孢不发芽并且在皮质片段在溶液中存在的小的变化。然而，在这两个TA和甘氨酸，C的存在艰难梭菌芽孢发芽...

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

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

检测皮质片段在细菌孢子萌发

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

The overall goal of this step-by-step protocol is to quantify the abundance of cortex fragments or reducing sugars during bacterial spore germination. This method will detail the steps required to detect the presence of cortex fragments or reducing sugars during spore germination. The main advantage of this technique is most laboratories have the reagents and equipment necessary to successfully complete the protocol.Begin this procedure by placing a tube of Clostridium difficile spores in a heating block and incubating it 65 degrees Celsius for 30 minutes. After the incubation, place the tube on ice. The germination solution is specific to the species of bacterial spore being studied.For assaying C.difficile spore germination, we use a solution of 10 millimolar Tris at pH 7.5 with 150 millimolar of sodium chloride, 100 millimolar glycine, and 10 millimolar taurocholic acid in deionized water. Prepare one milliliter of germination solution per sample plus an additional one milliliter. Before beginning the assay, transfer one milliliter of the germination solution to a microcentrifuge tube and set it aside.This will serve as a blank for cortex fragment detection. Next, to the germination solution, add spores to an OD600 of about 3.0 and briefly vortex. This amount of spores works well for moderating cortex fragment release during C.difficile spore germination.Immediately after adding the spores, remove a 1.2 milliliter sample to a new microcentrifuge tube and put it into the centrifuge. Then centrifuge at room temperature for one minute at 14, 000 times G.This is the zero time point. After the spin, transfer one milliliter of the supernatant to a fresh two-milliliter screw-cap tube and freeze it at negative 80 degrees Celsius.Repeat the process of taking aliquots, spinning down, and freezing at regular intervals until the sample has been collected for each time point. Because of time required for centrifugation and sampling, time points of less than two minutes are not possible during this procedure, so these samples are taken once every two minutes for 14 minutes. While preparing the cortex samples, should dipicolinic acid need to be monitored, remove a separate 100 microliter sample and dispense in a microcentrifuge tube on ice.Also prepare samples for generating a standard curve with the following amounts of N-acetylglucosamine. Zero, 12.5, 25, 50, 100, 250, 500, and 5, 000 nanomoles in germination solution. After a sample has been collected for each time point, remove the samples from the freezer and lyophilize them along with the blank and the standard curve samples.Then store all of the samples at negative 80 degrees Celsius until use. On the day of the assay, resuspend the lyophilized samples, including the controls and standard curve samples, in 120 microliters of hydrochloric acid with phenol and beta-mercaptoethanol. Next, transfer all of the resuspended samples to fresh two-milliliter screw cap tubes.Then incubate them in a recirculating water bath at 95 degrees Celsius for four hours. After the incubation, place the samples on ice until they are cool. Then add 120 microliters of three molar sodium hydroxide to neutralize them.Next, add 80 microliters of a saturated sodium bicarbonate solution and 80 microliters of a 5%acetic anhydride solution to each sample and vortex it. Then incubate the samples at room temperature for 10 minutes. Following the incubation, transfer the samples back to the 95 degree Celsius water bath for exactly three minutes.Note that this step is time sensitive and longer times can affect downstream signal development. Remove the samples from the water bath and cool them on ice. Add 400 microliters of 6.54%potassium tetraborate tetrahydrate to each tube and vortex.Incubate the samples in a recirculating water bath at 95 degrees Celsius for exactly seven minutes. This step is also time sensitive. Remove the samples and place them on ice for five minutes.While the samples are on ice, prepare the color reagent. Dissolve 0.320 grams of DMAB in 1.9 milliliters of glacial acetic acid. Note that this reagent is time, temperature, and light sensitive and should not be made in advance.After the DMAB completely dissolves, add 100 microliters of 10 normal hydrochloric acid and vortex the sample. Then add five milliliters of glacial acetic acid and vortex again. For the colorimetric reaction, transfer 100 microliters of each cooled cortex or control sample to a new 1.5 milliliter microcentrifuge tube.Add 700 microliters of the freshly prepared color solution to each sample. Immediately transfer the samples to a 37 degree Celsius water bath and incubate the samples for exactly 20 minutes. After the incubation, transfer 200 microliters of each sample to a clear 96-well plate.The samples should be yellow in color to begin with but will shift to purple if reducing sugar, which indicates the presence of cortex, is present. Here, many of the samples do not show a visible purple color. However, the 250, 500, and 5, 000 nanomole samples appear visually different from the negative control zero nanomole.Using a plate reader, determine the absorbance at 585 nanometers. When measured in an OD of 585 nanometers, the signal generated from the standard samples best fits a linear regression. This table shows typical results using N-acetylglucosamine standards.These data were then used to generate the standard curve shown here. A germination assay was performed under germination promoting conditions with germination buffer supplemented with 100 millimolar glycine and 10 millimolar taurocholic acid. In non-germinating conditions, 100 millimolar glycine only.In the absence of taurocholic acid, C.Difficile spores do not germinate, and there is little change in the presence of cortex fragments in the solution. However, in the presence of both taurocholic acid and glycine, C.difficile spores germinate and release cortex fragments into the solution. This is absorbed as an increase in the OD585 over time of the reacted samples.The quantity of cortex fragments released during this assay was calculated using a standard curve. As can be seen here in this assay, most of the cortex was hydrolyzed or released by 15 minutes post-germinant exposure. This suggests that most of the spores have germinated in this time frame.After watching this video, you should have a good understanding of how to detect cortex fragments or reducing sugars in a germination solution during bacterial spore germination. While attempting this procedure, it is important to pay close attention to reaction times so that the assay will develop correctly.

detecting-cortex-fragments-during-bacterial-spore-germination

Research

JoVE Journal

Immunology and Infection

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 ...

科研

免疫与感染

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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. ...

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 ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

作为酶和抗原的非重组显示系统的孢子吸附

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

A High-throughput Shigella-specific Bactericidal Assay

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

A High-throughput Shigella-specific Bactericidal Assay

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

一种高通量志贺氏菌特异性杀菌试验

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

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...