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A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii
采用坎氏弧菌进行群体感应研究的生物测定法   

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Abstract

Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important role in bacterial biofilm formation and virulence factor production in many pathogenic strains, QS disruptors have become a hot topic in current antimicrobial research. There are several reporter strains exhibiting QS-regulated phenotypes that have been engineered for the identification of QS inhibitors, including, for example, pigment production (González and Keshavan, 2006; Steindler and Venturi, 2007), gfp, lacZ or lux reporter gene fusions (González and Keshavan, 2006; Steindler and Venturi, 2007), or lethal gene fusions downstream QS-controlled promoters (Weiland-Bräuer et al., 2015). With three parallel QS circuits, the bioluminescent marine bacterium Vibrio campbellii (formerly harveyi, Lin et al., 2010) constitutes a complex Gram-negative model for which an extensive body of knowledge exists, including an array of mutant biosensors. In V. campbellii, bioluminescence is regulated by QS. However, bioluminescence is the result of complex biochemical networks that converge with cell respiration and fatty acid metabolism. It is also an energy-demanding reaction that strongly depends on the overall metabolic state of the bacterium, consuming up to 1/5 of the cell resources (Munn, 2011). Thus, disruption of QS-controlled phenotypes might be the result of toxic side effects or interference with the above-mentioned biochemical pathways rather than QS signaling. Therefore, adequate control experiments should be included. The protocol described herein provides a method and workflow for the identification of putative QS-disrupting compounds in Vibrio. It can also be easily adapted for other QS studies (e.g., detection of AI molecules).

Keywords: Quorum Sensing(群体感应), Vibrio(弧菌), Bioluminescence(生物发光), Inhibitors(抑制剂), Screening(筛选)

Materials and Reagents

  1. White, clear-bottom 96-well plates (Sigma-Aldrich, Corning® Costar®, catalog number: 3610 )
  2. Sterile sealing membrane (Sigma-Aldrich, Breathe-Easy®, catalog number: Z380059 )
  3. 15-ml tubes (VWR international, catalog number: 525-0150 )
  4. 96-well plate (Thermo Fisher Scientific, NuncTM MicroWellTM, catalog number: 167008 )
  5. Cryovials (optional) (Cryoinstant) (Deltalab, catalog number: 409113/6 )
  6. Disposable 1 mm path length cuvettes (Sigma-Aldrich, Brand®, catalog number: Z637092 )
  7. Vibrio campbellii strain
  8. Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  9. NaCl (Sigma-Aldrich, catalog number: S7653 )
  10. MgSO4 (Sigma-Aldrich, catalog number: M7506 )
  11. Protein Hydrolysate Amicase (Sigma-Aldrich, catalog number: 82514 )
  12. DI water
  13. NaOH (Sigma-Aldrich, catalog number: S5881 )
  14. Glycerol
  15. Potassium phosphate monobasic (Sigma-Aldrich, catalog number: P5655 )
  16. Potassium phosphate dibasic (Sigma-Aldrich, catalog number: 1551128 )
  17. L-arginine (Sigma-Aldrich, catalog number: A5006 )
  18. Kanamycin (Sigma-Aldrich, catalog number: K1377 )
  19. Cinnamaldehyde (Sigma-Aldrich, catalog number: W228613 )
  20. Marine Agar (MA) (Conda, catalog number: 1217 )
  21. Autoinducer Bioassay (AB) medium (see Recipes)

Equipment

  1. EnSpire® Multimode Plate Reader (PerkinElmer, model: 2300-0000 )
  2. Tube rotator (Grant-bio, model: PTR35 )
  3. Spectrophotometer (Beckman Coulter, model: DU 530 )

Software

  1. GraphPad Prism (GraphPad Software, Inc.)
  2. Microsoft Excel (Microsoft Corporation)

Procedure

  1. Streak a MA plate with the cryopreserved V. campbellii strain of interest. Incubate for single colonies at 30 °C for 24-36 h. If required, supplement the plates with suitable antibiotics (e.g., kanamycin, see flow chart in Figure 4 for more details).
    Note: We generally prepare two bacterial stocks: the first in commercial cryovials with beads for storage in our strain repository, and the second in 25-30% glycerol to be used as a working stock. Both are preserved at -80 °C. Even if we have not experienced any issue, we use this copy of the stock since repeated freezing and thawing can damage the cells, particularly if they are used routinely.
    For one streak we just use a 1-μl inoculating loop or any other sterile material. We start the assays from single colonies after 24-36 h incubation. This agar plate can be preserved at 4-5 °C for at least 1 week without loss of reproducibility. Alternatively, cultures can be prepared directly from the stocks; in that case, we recommend using 10 μl for inoculating 3 ml of AB medium (see next step).
  2. Prepare a culture of the V. campbellii strain of interest by inoculating 3 ml of AB medium in a 15-ml tube with a fresh colony. Incubate overnight in a tube rotator (40 rpm) at 30 °C to an OD600 of around 0.5 (1 mm path length).
  3. Distribute 100 µl of a 2x dilution of the test compounds in AB medium into the wells of a white, clear-bottom 96-well plate. Serial dilutions, if required, can be prepared in the plate.
    Note: We usually prepare 80 mM stocks of the test products in DMSO. Using a cut-off dose of 200 µM yields a maximum solvent concentration of 0.25% v/v that has not significant effect on bacterial growth and luminescence.
    No-treatment controls and solvent controls are included in each plate, as well as 100 μM cinnamaldehyde (positive control) and water (negative control) (Figure 1).


    Figure 1. Plate distribution showing sample wells (x80), No-Treatment Control wells (NTC, x3), Solvent Control wells (SC, x3), Positive Control wells (PC, x3), Negative Control wells (NC, x3) and Blank wells (x4). Serial two-fold dilutions of the test compounds, if required, are proposed from rows A to H.

  4. Dilute the overnight V. campbellii culture 1:50 in fresh AB medium. This gives an inoculum of approximately 1-2 x 107 CFU ml-1 that will be further diluted 1:2 (see next step).
    Note: When establishing this protocol in the laboratory, we recommend verifying the cell density of the inoculum as described by Harrison et al., 2010. Briefly, 180 µl of AB medium are distributed into four columns of a sterile 96-well plate, from row A to H. Twenty µl of inoculum are pipetted into the A-row wells and serially diluted (-1 to -8 dilutions). Using a multichannel pipette, 10 µl of the dilutions are spotted onto the surface of a MA plate. Up to four replicates of each dilution can be easily spotted onto a standard 90-100 mm agar plate. The spots are dried inside a biosafety cabinet and the plates incubated for 24 h at 30 °C for CFU count. For an accurate determination of the cell density, take into consideration the lowest concentration in which individual colonies can be counted. The CFU per ml will be = x 10 (d+2), where n represents the colony count in each of the 4 replicates, and d the corresponding dilution. The actual cell density in the assay will be half of this calculation. 
  5. Distribute 100 µl of the diluted bacterial culture into the wells.
  6. Seal the plate with a sterile, breathable and transparent sealing film (Figure 2).
    Note: Use a brayer for carefully sealing the plate. Do not remove the external protective cover until the plate is properly sealed in order to avoid contamination.


    Figure 2. Aspect of a sealed, white, clear-bottom 96-well plate

  7. Incubate the plate for 18 h at 30 °C and internal orbital shaking (150 rpm). Take OD600 and luminescence readings every 15 min.
    Note: Plate dimension, measurement height and Z’ optimizations are advised when using EnSpire® Multimode Plate Reader. Similarly, flatfield and crosstalk corrections optimize the readings. We do not recommend using a distance between plate and detector lower than 0.6 mm (otherwise, contaminations might occur due to damage of the sealing film). Activate the condensation prevention option for sealed plates (upper heater temperature 2 °C warmer than lower heater temperature).

Data analysis

  1. Subtract the background values for OD600 and luminescence (Microsoft Excel)
    Note: In luminescence readings, cross-talk differs slightly in function of the position of the wells. Thus, central wells are more susceptible to well-to-well light cross-talk. A tentative plate distribution that takes into account this effect is shown in Figure 1. Optimization of measurement parameters diminishes this phenomenon, although it takes place even with proper optimization. However, luminescence readings in blank wells are always at least two orders of magnitude lower than those recorded for sample wells and consequently, cross-talking exerts a negligible effect with our experimental settings.
  2. Using a scientific graphing software (e.g., GraphPad Prism), represent the time-course curves for bacterial growth and bioluminescence (Figure 3). Determine the area below the curves and calculate the relative inhibition of the treatments with respect to the controls for each variable.
    Note: It could be useful to normalize the luminescence data to the OD600 for each dose or treatment, i.e., NL= where NL = Normalized Luminescence, AULC = Area Under Luminescence Curve and AUGC = Area Under Growth Curve.


    Figure 3. Chart displaying a typical NTC growth curve (black squares) and, overlapped, the corresponding luminescence curve (blue squares) over an assay period of 18 h. Data represent the mean and standard deviation of three replicates.

  3. With these data, calculate the corresponding IC50 values using non-linear regression (GraphPad Prism).
  4. Proposed workflow for the discovery of Quorum Sensing Inhibitors (Figure 4).


    Figure 4. Flowchart for testing and characterizing the activity of putative Quorum Sensing Inhibitors using Vibrio campbellii QS mutants

Recipes

  1. AB Medium
    17.5 g NaCl
    12.3 g MgSO4
    2.0 g casamino acids
    970 ml DI water
    Dissolve the ingredients and bring the pH of the solution to 7.5 with 3 N NaOH
    Autoclave at 121 °C and after completely cooling down, add the following ingredients from separate filter-sterilized stocks:
    10 ml 1 M potassium phosphate (pH 7.0)
    10 ml 0.1 M L-arginine
    10 ml glycerol

Acknowledgments

This protocol was developed with financial support provided by the Spanish Ministry of Economy and Competitiveness, grant CTQ2014-55888-C03-01. A.J.M-R. holds a fellowship from PLOCAN. This protocol is an adaptation from that published in Martín-Rodríguez et al. 2015a and 2015b.

References

  1. González, J. E., Keshavan, N. D. (2006). Messing with bacterial Quorum Sensing. Microbiol Mol Biol Rev 70(4): 859-875.
  2. Harrison, J. J., Stremick, C. A., Turner, R. J., Allan, N. D., Olson, M. E. and Ceri, H. (2010). Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening. Nat Protoc 5(7): 1236-1254.
  3. Lin, B., Wang, Z., Malanoski, A. P., O'Grady, E. A., Wimpee, C. F., Vuddhakul, V., Alves Jr, N., Thompson, F. L., Gomez-Gil, B. and Vora, G. J. (2010). Comparative genomic analyses identify the Vibrio harveyi genome sequenced strains BAA-1116 and HY01 as Vibrio campbellii. Environ Microbiol Rep 2(1): 81-89.
  4. Munn, C. (2011). Marine Microbiology. 2nd ed. New York: Garland Science: 105.
  5. Martín-Rodríguez A. J., Babarro J. M. F., Lahoz F., Sansón M., Martín V. S., Norte M., Fernández J. J. (2015). From broad-spectrum biocides to Quorum Sensing disruptors and mussel repellents: antifouling profile of alkyl triphenylphosphonium salts. PLoS One 10(4): e0123652.
  6. Martín-Rodríguez A. J., Ticona J. C., Jiménez I. A., Flores N., Fernández J. J., Bazzocchi I. (2015). Flavonoids from Piper delineatum modulate Quorum-Sensing regulated phenotypes in Vibrio harveyi. Phytochemistry 117: 98-106.
  7. Steindler, L., Venturi, V. (2007). Detection of Quorum-Sensing N-acyl homoserine lactone signal molecules by bacterial biosensors. FEMS Microbiol Lett 266(1): 1-9.
  8. Weiland-Bräuer N., Pinnow, N., Schmitz R. A. (2015). Novel reporter for identification of interference with acyl homoserine lactone and autoinducer-2 Quorum Sensing. J Bacteriol 81(4): 1477-1489.

简介

Quorum Sensing(QS)或细菌细胞与细胞间的通信是一种微调的机制,通过称为自体诱导物(AI)的细胞外信号分子的产生,分泌和接受,以群体密度依赖性方式调节基因表达。鉴于QS在许多致病菌株中的细菌生物膜形成和毒力因子产生中起重要作用,QS干扰物已经成为当前抗微生物研究的热门话题。存在几种报道菌株,其表现出QS-调节的表型,其已被工程化用于鉴定QS抑制剂,包括例如颜料产生(González和Keshavan,2006; Steindler和Venturi,2007),gfp < (González和Keshavan,2006; Steindler和Venturi,2007)或致死性基因融合下游QS控制的启动子(Weiland-Bräuer > et al 。,2015)。使用三个平行的QS电路,生物发光海洋细菌(Vibrio campbellii)(以前称为harveyi,Lin等人,2010)构成复杂的革兰氏阴性模型,存在广泛的知识,包括一系列突变生物传感器。在中。 campbellii ,生物发光由QS调节。然而,生物发光是与细胞呼吸和脂肪酸代谢汇合的复杂生物化学网络的结果。它也是一种能量需求的反应,强烈依赖于细菌的总体代谢状态,消耗高达细胞资源的1/5(Munn,2011)。因此,QS控制的表型的破坏可能是毒性副作用或干扰上述生物化学途径而不是QS信号的结果。因此,应包括适当的控制实验。本文所述的方案提供了用于鉴定Vibrio中假定的QS破坏化合物的方法和工作流程。它还可以容易地适用于其他QS研究(例如,AI分子的检测)。

关键字:群体感应, 弧菌, 生物发光, 抑制剂, 筛选

材料和试剂

  1. 白色透明底96孔板(Sigma-Aldrich,Corning),目录号:3610)
  2. 无菌密封膜(Sigma-Aldrich,Breathe-Easy ,目录号:Z380059)
  3. 15-ml试管(VWR international,目录号:525-0150)
  4. 96孔板(Thermo Fisher Scientific,目录号:167008)的孔中,
  5. 冷冻瓶(可选)(Cryoinstant)(Deltalab,目录号:409113/6)
  6. 一次性1mm路径长度比色杯(Sigma-Aldrich,Brand ,目录号:Z637092)
  7. Vibrio campbellii 菌株
  8. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  9. NaCl(Sigma-Aldrich,目录号:S7653)
  10. MgSO 4(Sigma-Aldrich,目录号:M7506)
  11. 蛋白水解酶Amicase(Sigma-Aldrich,目录号:82514)
  12. 去离子水
  13. NaOH(Sigma-Aldrich,目录号:S5881)
  14. 甘油
  15. 磷酸二氢钾(Sigma-Aldrich,目录号:P5655)
  16. 磷酸氢二钾(Sigma-Aldrich,目录号:1551128)
  17. L-精氨酸(Sigma-Aldrich,目录号:A5006)
  18. 卡那霉素(Sigma-Aldrich,目录号:K1377)
  19. 肉桂醛(Sigma-Aldrich,目录号:W228613)
  20. 海洋琼脂(MA)(Conda,目录号:1217)
  21. 自动诱导剂生物测定(AB)培养基(参见配方)

设备

  1. EnSPire ?多模式读板器(PerkinElmer,型号:2300-0000)
  2. 管旋转器(Grant-bio,型号:PTR35)
  3. 分光光度计(Beckman Coulter,型号:DU 530)

软件

  1. GraphPad Prism(GraphPad Software,Inc。)
  2. Microsoft Excel(Microsoft Corporation)

程序

  1. 用冷冻保存的V板条纹MA板。 Campbellii 菌株。孵育单菌落在30℃下24-36小时。如果需要,用合适的抗生素(例如,卡那霉素,见图4的流程图更多细节)补充板。
    注意:我们通常制备两种细菌菌种:第一种在商业冷冻小瓶中用珠子储存在我们的菌株仓库中,第二种在25-30%甘油中用作工作储备液。两者都保存在-80℃。即使我们没有遇到任何问题,我们使用这个股票的副本,因为反复冻结和解冻可以损坏细胞,特别是如果他们常规使用。
    对于一个条纹,我们只使用1-μl接种环或任何其他无菌材料。我们在孵育24-36小时后从单菌落开始测定。该琼脂平板可以在4-5℃下保存至少1周而不丧失重现性。或者,培养物可以直接从原种制备;在这种情况下,我们建议使用10μl接种3 ml AB培养基(见下一步)。
  2. 准备一个文化的。 Campbellii感兴趣的菌株,通过在15ml管中接种新鲜菌落的3ml AB培养基。在管式旋转器(40rpm)中在30℃下孵育过夜至约0.5(OD路径长度)的OD 600.
  3. 将100μl的测试化合物的2×稀释液在AB培养基中分配到白色透明底96孔板的孔中。如果需要,可以在板中制备系列稀释液。
    注意:我们通常在DMSO中制备80mM的测试产品储备液。使用200μM的截止剂量产生0.25%v/v的最大溶剂浓度,其对细菌生长和发光没有显着影响。
    每个板中包括无处理对照和溶剂对照,以及100μM肉桂醛(阳性对照)和水(阴性对照)(图1)。


    图1.板分布示出样品孔(x80),未处理对照孔(NTC,x3),溶剂对照孔(SC,x3),阳性对照孔(PC,x3),阴性对照孔x3)和空白孔(x4)。如果需要,从A行到H行建议连续两倍稀释的测试化合物。

  4. 稀释隔夜。 campbellii 培养物1:50在新鲜的AB培养基中。这得到将进一步以1:2稀释的约1-2×10 7/mL CFU ml -1 的接种物(参见下一步骤)。
    注意:当在实验室中建立该方案时,我们建议验证接种物的细胞密度,如Harrison等人,2010所述。简言之,将180μl的AB培养基分配到无菌96孔的四个柱板。从A行到H行。将20μl接种物移液到A行孔内并连续稀释(-1至-8稀释)。使用多通道移液管,将10μl的稀释液点在MA板的表面上。每个稀释物的多达四个重复可容易地点在标准的90-100mm琼脂平板上。将斑点在生物安全柜内干燥,将板在30℃下孵育24小时以进行CFU计数。为了精确测定细胞密度,考虑可以计数单个菌落的最低浓度。每ml的CFU为= x 10 (d + 2)/sup>,其中n表示4次重复的每一次中的菌落计数,d表示相应的稀释度。测定中的实际细胞密度将是该计算的一半。
  5. 将100μl稀释的细菌培养物分配到孔中。
  6. 用无菌,透气和透明的密封膜密封板(图2) 注意:使用涂层仔细密封板。在板被正确密封之前,不要取下外部保护盖,以免污染。


    图2.密封,白色,透明底部96孔板的外观

  7. 孵育板在30℃下18小时和内轨道摇动(150 rpm)。取OD <600>和每15分钟的发光读数 注意:使用EnSpire ?多模式板读取器时,建议使用板尺寸,测量高度和Z'优化。类似地,平场和串扰校正优化读数。我们不建议使用板和检测器之间的距离低于0.6 mm(否则,由于密封膜的损坏可能会发生污染)。激活密封板的冷凝防止选项(加热器上部温度比加热器温度低2°C)。

数据分析

  1. 减去OD <600>和发光(Microsoft Excel)的背景值
    注意:在发光读数中,串扰与孔位置的函数略有不同。因此,中心孔更易受井 - 井光串扰的影响。考虑这种效应的暂时板分布示于图1中。测量参数的优化减少了这种现象,尽管它甚至在适当的优化下发生。然而,空白孔中的发光读数总是比对于样品孔记录的读数至少低两个数量级,因此,串扰对我们的实验设置产生可忽略的影响。
  2. 使用科学绘图软件(例如GraphPad Prism)表示细菌生长和生物发光的时间 - 过程曲线(图3)。确定曲线下面的面积,并计算治疗相对于每个变量的对照的相对抑制 注意:对于每个剂量或治疗,将发光数据归一化到OD 600是有用的,即NL = src =其中NL =归一化发光,AULC =发光曲线下面积,AUGC =生长曲线下面积。

    图3.在18小时的测定期间显示典型的NTC生长曲线(黑色方块)和重叠的相应的发光曲线(蓝色方块)的图表。 strong> 数据表示三次重复的平均值和标准偏差
  3. 使用这些数据,使用非线性回归(GraphPad Prism)计算相应的IC <50>值。
  4. 发现Quorum Sensing Inhibitors的建议工作流程(图4)。


    图4.使用vibrio campbellii QS突变体 测试和表征推定的Quorum Sensing Inhibitors的活性的流程图。

食谱

  1. AB中等
    17.5g NaCl
    12.3g MgSO 4 2.0克酪蛋白氨基酸
    970毫升去离子水
    溶解成分,用3N NaOH使溶液的pH值达到7.5 在121℃下高压灭菌,并在完全冷却后,从单独的过滤灭菌的原液中加入以下成分:
    10ml 1M磷酸钾(pH 7.0)
    10ml 0.1M L-精氨酸 10ml甘油

致谢

该协议是由西班牙经济和竞争力部提供的资金支持开发的,授予CTQ2014-55888-C03-01。 A.J.M-R。从PLOCAN获得奖学金。该协议是从Martín-Rodríguez等人发表的改编。 2015a和2015b。

参考文献

  1. González,JE,Keshavan,ND(2006)。  Messing with bacterial Quorum Sensing。 Microbiol Mol Biol Rev 70(4):859-875。
  2. Harrison,JJ,Stremick,CA,Turner,RJ,Allan,ND,Olson,ME和Ceri,H。(2010)。  生长在栓钉上的微生物的微量滴定敏感性测试:用于高通量筛选的小型化生物膜模型 Nat Protoc 5 (7):1236-1254。
  3. Lin,B.,Wang,Z.,Malanoski,AP,O'Grady,EA,Wimpee,CF,Vuddhakul,V.,Alves Jr,N.,Thompson,FL,Gomez-Gil,B.and Vora,GJ 2010)。  比较基因组分析鉴定了Vibrio harveyi基因组测序菌株BAA-1116和HY01作为Campbellii Vibrio。环境微生物学报2(1):81-89。
  4. Munn,C。(2011)。  海洋微生物学。第二版。 :
  5. Martín-RodríguezAJ,Babarro JMF,Lahoz F.,SansónM.,MartínVS,Norte M.,FernándezJJ(2015)。  来自 Piper delineatum的黄酮类化合物在 Vibrio harveyi中调节Quorum-Sensing调节的表型/em> 117:98-106。
  6. Steindler,L.,Venturi,V。(2007)。  通过细菌生物传感器检测群体感应的Nε - 酰基高丝氨酸内酯信号分子。


    FEMS Microbiol Lett 266(1):1-9。 >
  7. Weiland-Br?uerN.,Pinnow,N.,Schmitz RA(2015)。  用于鉴定对酰基高丝氨酸内酯和自诱导物-2干扰的干扰的新型报道基因。细菌 81(4):1477-1489。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Martín-Rodríguez, A. J. and Fernández, J. J. (2016). A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii. Bio-protocol 6(14): e1866. DOI: 10.21769/BioProtoc.1866.
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