The authors thank Dara Frank and Carrie Harwood for their generous contribution of strains. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. This research was supported by the National Institutes of Health (R00-GM083147). The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

1.制备培养基，抗生素，和2- [2-硝基-4-（三氟甲基）苯甲酰基] -1,3-环己二酮（NTBC） <ol><li>使LB肉汤（1％胰蛋白胨，0.5％酵母提取物，0.25％NaCl的H 2 O）和分装成适当体积。通过高压灭菌消毒。室温保存。 </li><li>达到100ml LB琼脂（1％胰蛋白胨，0.5％酵母提取物，0.25％的NaCl，在H 2 O的1.5％琼脂）的250ml烧瓶中。通过高压釜中，在室温下储存消毒。确保琼脂倒入盘前熔化。 注：含有100ml的LB琼脂将产生4板保温瓶。 LB琼脂的量可以改变以对应于所需的测定板的数量。 </li><li>使PBS（137 mM氯化钠，2.7毫米氯化钾，10毫的 Na 2 HPO 4，2mM的KH 2 PO 4）16。通过高压灭菌器或过滤和储存在室温下消毒。 </li><li>准备抗生素股票方案的庆大霉素，卡那霉素，一种ð妥布霉素。 <ol><li>准备适当的抗生素浓度股票为P.假单胞菌菌株含有100mg / ml的庆大霉素，30毫克/毫升卡那霉素，和10毫克/毫升妥布霉素。溶解抗生素在水中，过滤杀菌（0.2微米），并储存在4℃。改变这取决于所研究的细菌的抗生素和浓度。 </li></ol></li><li>准备NTBC股票的解决方案。溶解10毫克NTBC在400微升DMSO中。这就产生了75.9毫米NTBC的浓度。商店NTBC储备溶液在-20℃。解冻溶液在室温下按需要。 注：不同NTBC的来源有溶解度的差异。确定适当的车辆中，以溶解根据制造商的建议的NTBC和调整步骤1.5相应。 </li></ol> 2.细菌菌株的NTBC滴定<ol><li>菌株的设置过夜培养来进行测试。加入2ml LB培养基为16×150毫米的试管S（每株一个），接种从每一株1孤立的殖民地。孵育过夜，在37℃通气到组织培养旋转器中的空气培养箱中。 </li><li>第二天，准备NTBC的滴定在LB肉汤。使用的初始范围从0到900微米NTBC自不同菌株具有的感光度NTBC差异。 <ol><li>加入1毫升的LB肉汤到每株4到5支试管（16×150毫米）。 </li><li>添加NTBC储液（75.9毫米）的试管（16×150毫米）的范围内的浓度。 见表 1 NTBC浓度和相应的库存量增加至1ml LB肉汤。 </li></ol></li><li>测量过夜培养的OD 600。以OD 600的读数，以消除pyomelanin出现在媒体面前洗的文化。 <ol><li>通过离心1毫升文化的一个离心16,000 XG 2分钟洗净文化。去除上清，任何松散沉淀的细胞与移液器和悬浮固体细胞沉淀在1毫升LB中</li></ol></li><li>接种滴定管，在OD 600 0.05。计算接种管所需的洗涤培养的量。 注：使用清洗培养的接种，因为pyomelanin不应该存在。 </li><li>孵育滴定管约24小时，在37℃使用组织培养旋转器中的空气培养箱曝气。 </li><li>拍摄滴定管和内和菌株之间比较颜料生产，以确定要用于MIC和氧化应激测定NTBC的量。使用的OD 600读数，以确定pyomelanin的量在无细胞培养物上清液，并确定细胞密度。 注：在培养物上清液的OD 600比率pyomelanin至细胞可以计算量化与NTBC处理后的差异在pyomelanin生产。 </li></ol> 3.抗生素最少INHIBI保守党浓度（MIC）测定在96孔板<ol><li>菌株的设置过夜培养要在LB有和没有NTBC测试。 注：此协议使用300微米NTBC代表级别描述。 NTBC的要使用的适当水平是在步骤2.6确定。 <ol><li>添加300μMNTBC至2ml LB。加载体（DMSO）等体积的2毫升LB培养基的无NTBC条件。 </li><li>使用无菌牙签，接种管与细菌的一个孤立的群体。将有一个文化与NTBC和一种文化，而不NTBC每一株。孵育过夜，在37℃，使用的组织培养旋转器中的空气培养箱曝气。 </li></ol></li><li>第二天，让LB + NTBC和LB + DMSO主解决方案用于设置MIC检测。添加NTBC在600μM，因为这一个浓度将稀释两倍时接种物添加，得到300微米的最终浓度。 <ol><li>为了测试一个antibi耳一紧张，加上600μMNTBC〜2毫升LB和组合，使NTBC主的解决方案。加载体（DMSO）的等效体积至2ml LB和混合以使无NTBC母液。使用这些解决方案，用于创建抗生素储备溶液以及用于设置稀释系列，在96孔板中。 注：主溶液制剂将产生额外的解决方案，以弥补移液错误。主溶液可以放大或缩小取决于抗生素和应变测试的数目为必需的。 </li></ol></li><li>准备抗生素解决方案，在LB + NTBC或LB + DMSO主的解决方案。 注意：在这些溶液中的抗生素浓度应双倍的最终所需的浓度。够溶液应使在一个96孔板100微升转移到四口井。 <ol><li>制备庆大霉素+/- NTBC原液在64微克/毫升。为了使这个解决方案，增加0.288微升庆大霉素的股票（100毫克/毫升）450微升LB + NTBC或LB + DMSO主解决方案。 注：庆大霉素为P的最大浓度绿脓杆菌 PAO1是32微克/毫升。 </li><li>使卡那霉素+/- NTBC原液在256微克/毫升。为了使这个解决方案中，添加3.84微升卡那霉素股票（30毫克/毫升），以450微升LB + NTBC或LB + DMSO主的解决方案。 注：卡那霉素的P的最大浓度绿脓杆菌 PAO1为128微克/毫升。 </li><li>制备妥布霉素+/- NTBC原液在8微克/毫升。为了使这个解决方案中，添加0.36微升妥布霉素股票（10毫克/毫升），以450微升LB + NTBC或LB + DMSO主的解决方案。 注：为P.假单胞菌 PAO1，妥布霉素的最大浓度为4微克/毫升。 注：抗生素和浓度可以调整为细菌进行测试。 </li></ol></li><li>加入100微升每2×抗生素溶液的四个井在96孔板中。将这些解决方案中的行A.对于为例即，庆大霉素应到A4置于A1中，卡那霉素应至A8被放置在A5和妥布霉素应通过A12被放置在A9。 参见图1A，用于一个96孔板设置的示意图。 注意：多个抗生素可以在一个板进行试验，但只有一个应变应该每板进行测试，以消除交叉污染，从其它菌株的潜力。 </li><li>添加50微升LB + NTBC或LB + DMSO主溶液的行B至96孔板的小时。确保一个板块是LB + NTBC，一个板块是LB + DMSO。参见图1A。 <ol><li>使用LB + NTBC在LB + NTBC的抗生素。使用LB + DMSO在LB + DMSO的抗生素。 </li></ol></li><li>用微量转增50微升的解决方案，从行A到B行混合的解决方案，改变枪头执行的抗生素两倍系列稀释液，并转移50微升的解决方案从B行与行C.重复以上步骤，该remaini吴行。稀释G行后，取​​出50微升该行和丢弃的解决方案。使用行H作为一个无抗生素控制细菌的生长。参见图1B。 注：每个井在排A至G现在包含50微升抗生素的LB + NTBC或LB + DMSO以2X的最终所需的浓度。行H中的LB + NTBC或LB + DMSO没有抗生素。 </li><li>测量过夜培养的OD 600。以OD 600的读数，以消除pyomelanin出现在媒体面前清洗所有的文化。 <ol><li>通过离心1毫升文化的一个离心16,000 XG 2分钟洗净文化。用微量去除上清，重悬细胞沉淀在1毫升LB中</li></ol></li><li>稀释过夜培养在LB中2.75x10 5 CFU /毫升 注意：假设一个OD 600单位为1×10 9 CFU /毫升P.相当于铜绿假单胞菌 。 OD以CFU / ml的转换可能是different在其他细菌。 </li><li>加入50微升稀释的细菌培养物，以适当的井。 注：在NTBC生长培养应加入到含有NTBC和培养在DMSO生长应添加到含有的DMSO的孔的孔中。参见图1B。 <ol><li>添加细菌三眼井每一株和抗生素浓度。加入50微升的LB至第四孔充当细菌污染的控制。参见图1B。 </li><li>使用多通道移液器以接种的孔中。保证吸管加入接种物时防止周围井污染提示是井的底部附近。 注意：添加细菌培养到孔会冲淡抗生素和NTBC浓度的两倍。 </li></ol></li><li>覆盖96孔板用封口膜，并培育约24小时，在37℃下。孵育96孔板静态，在空气培养箱中。 </li><li>审查所述板对中的细菌生长的孔中。的MIC是抗生素的最低浓度，其中无细菌生长被认为对于每个菌株的所有三次重复。 <ol><li>用肉眼检验板的增长或读取用酶标仪集到OD 600。 </li></ol></li></ol> 4.现货板检定的氧化应激反应<ol><li>菌株的设置过夜培养将在LB有和没有NTBC按照步骤3.1中所述进行测试。 </li><li>第二天，制备含有的 H 2 O 2作为氧化应激的LB琼脂平板上。 h的范围 2 O 2的浓度从0到1毫米是一个很好的起点。 <ol><li>融化LB琼脂瓶。冷媒介在室温下约50℃。 </li><li>直接添加的 H 2 O 2到冷却介质在所需的浓度。旋流烧瓶混合。 见表 2 h的浓度2 O 2和浓H 2 O 2的体积增加。这些值是基于100 ml的LB琼脂的。 </li><li>加入H 2 O 2和火焰表面，以去除气泡后，立即倒入盘子。产率是每100毫升的LB琼脂4板。标记用H 2 O 2浓度的板。 </li><li>放置揭示在生物流罩与运行30分钟风扇从板除去多余的水分的板。 注意：使用它们制备当天的板。如果不这样做，可能会导致数据不一致。 注：氧化应激，如百草枯可以取代的 H 2 O 2在该试验中。用于其它的氧化应激的浓度可以比那些用于H 2 O 2的不同。 </li></ol></li><li>洗净按步骤3.7说明测量外径600过夜培养的。 </li><li>规范化的所有一夜之间立方米的OD 600ltures到对于该组的菌株的最低值被测试。P.假单胞菌通常具有约2.5时在LB + NTBC或LB + DMSO过夜生长的OD 600。 <ol><li>确定培养的稀释培养物以最低的OD 600为1毫升的总体积所需要的体积。例如，如果一个培养具有的OD 600的3和最低的OD 600对于该组的菌株是2.5，执行以下计算：（2.5）（1毫升）=（3）（x）的。 X =0.833毫升。 0.833毫升培养将被放置在一个离心管。 </li><li>计算使培养体积至1ml所需LB + NTBC（300μM）或LB + DMSO的量。对于在步骤4.4.1的例子中，加入LB + NTBC或LB + DMSO的量在培养。将0.167毫升（1 ml的总体积 - 0.833 ml的培养物）。使储备溶液LB + NTBC和LB + DMSO的用于这些稀释液根据需要稀释所有菌株在卷上。 </li><li>混合的文化和LB + NTBC或LB + DMSO涡旋。 </li></ol></li><li>为了维持培养物在NTBC或DMSO的浓度恒定，在执行PBS + NTBC或PBS + DMSO规格化过夜培养的10倍系列稀释。 <ol><li>使股票的解决方案PBS + NTBC和PBS + DMSO中。对一组稀释液为一种菌株，混合300μMNTBC或DMSO等体积的PBS，得到的720微升的总体积。缩放这些股票上升或下降取决于有多少株进行了测试。 </li><li>标签离心管10 -1至 10 -7系列稀释。添加90微升PBS + NTBC或PBS + DMSO中至适当管中。用PBS + NTBC在LB + NTBC生长的菌株，用PBS + DMSO在LB + DMSO生长的菌株。 </li><li>加入10微升培养物，以适当的10 -1稀释管。通过涡旋混合并转移10微升10 -1稀释到10 -2稀释管。重复，直到所有的稀释已PERFORmed指。更改稀释之间的枪头。 </li></ol></li><li>点5微升10 -3的通过10 -7稀释在LB + H 2 O 2的平板式两份每个菌株。使用一个枪头，如果斑点镀大多数稀释至至少稀（10-7至10-3）。不要翻倒或倾斜板，直到液体干燥成板。 </li><li>孵育所述板为24-48小时，在37℃（空气培养箱），根据不同的应变。 注：P。培养24小时后， 铜绿假单胞菌 PAO1将对LB好户型的殖民地。孵育菌株直到他们有菌落大致相同的尺寸PAO1。 </li><li>拍摄使用上述一个transluminator CCD照相机的板。任选地，编辑照片的对比度和作物为相同大小。算在每个点的菌落数来确定的变化灵敏度氧化应激。 </li></ol>

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E. <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+melanin-overproducing+transposon+mutants+of+Pseudomonas+putida+F6.">Characterization of melanin-overproducing transposon mutants of Pseudomonas putida F6.</a> FEMS Microbiol Lett. 298, 174-183 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Roy-Burman, A., 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=Type+III+protein+secretion+is+associated+with+death+in+lower+respiratory+and+systemic+Pseudomonas+aeruginosa+infections.">Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections.</a> J Infect Dis. 183, 1767-1774 (2001).</li><li>Jacobs, M. A., 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=Comprehensive+transposon+mutant+library+of+Pseudomonas+aeruginosa.">Comprehensive transposon mutant library of Pseudomonas aeruginosa.</a> Proc Natl Acad Sci USA. 100, 14339-14344 (2003).</li><li>Youngchim, S., Pornsuwan, S., Nosanchuk, J. D., Dankai, W., Vanittanakom, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanogenesis+in+dermatophyte+species+in+vitro++during+infection.">Melanogenesis in dermatophyte species in vitro during infection.</a> Microbiology. 157, 2348-2356 (2011).</li><li>Khajo, A., 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=Protection+of+Melanized+Cryptococcus+neoformans+Lethal+Dose+Gamma+Irradiation+Involves+Changes+in+Melanin's+Chemical+Structure+and+Paramagnetism.">Protection of Melanized Cryptococcus neoformans Lethal Dose Gamma Irradiation Involves Changes in Melanin's Chemical Structure and Paramagnetism.</a> PLoS ONE. 6, e25092 (2011).</li><li>Hancock, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hancock Laboratory Methods. , (2015).</li></ol>

The authors declare that they have no competing financial interests.

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates12 (Figure 2). The...

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress1-4 and binds a variety of compounds, including aminoglycoside antibiotics5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)1,9 or through imbalances in the various enzymes in the pathway10. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin11. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)12, which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway13. Hpd is required for the formation of homogentisate, and therefore pyomelanin11. 
We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.
The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa12. Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction. 
The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent12. Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.
Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions14. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.
Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time1, or oxidative stress disc diffusion assays15. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H2O2 and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td>
			<td>Sigma-Aldrich</td>
			<td>SML0269-50mg</td>
			<td>Also called nitisinone.&#160; Soluble in DMSO.</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td>30 wt. % in H2O.&#160; Stabilized.</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gold Bio</td>
			<td>G-400-100</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014-100MG</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
	</tbody>
</table>

methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

NTBC滴定所述NTBC滴定被用来确定是否NTBC能够减少在巴斯德pyomelanin生产假单胞菌，并且还确定NTBC的消除或减少pyomelanin生产供额外分析使用的浓度。有可能的变化在不同的重复产生pyomelanin的水平，但总的趋势仍不变。所述NTBC滴定试验也可以修改，以测试可能影响颜料生产中的其他细菌的其它化合物。这将只工作，但是，如果有一个表型改变，可以在视觉上确定或...

Watch this Scientific Journal Video about Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress at JoVE.com

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

methods-to-inhibit-bacterial-pyomelanin-production-determine

Research

JoVE Journal

Immunology and Infection

8.1K 次观看.  University of Wisconsin Milwaukee. Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

视频: Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

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

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

代谢图谱: 定量酶化学和组织化学法测定脱氢酶在细胞和器官中的活性

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...