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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<li>Nikodinovic-Runic, J., Martin, L. B., Babu, R., Blau, W., O'Connor, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+melanin-overproducing+transposon+mutants+of+Pseudomonas+putida+F6%5BTitle%5D)+AND+%22FEMS+Microbiol+Lett%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSambrook%2C+J+%22Molecular+cloning%3A+a+laboratory+manual%22">Molecular cloning: a laboratory manual</a>. , 3 edn, Cold Spring Harbor Laboratory Press. (2001).</li>
<li>Roy-Burman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+III+protein+secretion+is+associated+with+death+in+lower+respiratory+and+systemic+Pseudomonas+aeruginosa+infections%5BTitle%5D)+AND+%22J+Infect+Dis%22%5BJournal%5D)">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/pubmed?term=((Comprehensive+transposon+mutant+library+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">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/pubmed?term=((Melanogenesis+in+dermatophyte+species+in+vitro++during+infection%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">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/pubmed?term=((Protection+of+Melanized+Cryptococcus+neoformans+Lethal+Dose+Gamma+Irradiation+Involves+Changes+in+Melanin%27s+Chemical+Structure+and+Paramagnetism%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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. Hancock Laboratory Methods. , University of British Columbia, Department of Microbiology and Immunology. British Columbia, Canada. Available from:
	 
 <a target="_blank" href="http://www.cmdr.ubc.ca/bobh/methods.htm">http://www.cmdr.ubc.ca/bobh/methods.htm</a> (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><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.&amp;nbsp; Soluble in DMSO.</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>216763-100ML</td><td>30 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Stabilized.</td></tr><tr><td>Gentamicin</td><td>Gold Bio</td><td>G-400-100</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014-100MG</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; 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

8.2K 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

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

C2C12 Myotubes 测量反式等离子体膜电子传输

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

生物化学

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

医学

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

<meta charset="utf8"></head><body>吸附生物工艺提高了 Pseudomonas stutzeri 黑色素的产量

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

吸附生物工艺提高了 Pseudomonas stutzeri 黑色素的产量

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

工程学

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

生物学

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

铜 绿假单胞 菌分离株铁载体产量的评估

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.
The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

作者聚焦：定量铁载体和绿茶素用于感染控制 

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

铜绿假单胞菌产生铁载体的定性和定量分析

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

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

摘要

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用基因编码荧光传感器评估毒理学氧化应激的成像方法

C2C12 Myotubes 测量反式等离子体膜电子传输

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表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

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吸附生物工艺提高了 Pseudomonas stutzeri 黑色素的产量

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