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Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species
酵母菌中能量依赖性罗丹明6G流出量的测定   

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Abstract

Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an energy-dependent manner. Incubation of yeast cells in the presence of 2-deoxy-D-glucose (inhibitor of glycolysis) and R6G (mitochondrial ATPase inhibitor) leads to marked depletion of intracellular ATP pool (Kolaczkowski et al., 1996). An active KlPdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer.

Keywords: Rhodamine 6G(罗丹明6G), Fluorescence(荧光), Kluyveromyces lactis(乳酸克鲁维酵母), ABC transporter(ABC转运蛋白), Transport activity assay(转运活性测定)

Background

Multidrug efflux pumps are widely distributed and can be found in all living species. They represent an important mechanism of antimicrobial resistance. The ability to quantify the activity of efflux pumps is necessary for understanding of their contribution to physiological processes and assessment of the validity of potential therapeutics (e.g., efflux inhibitors) (Blair and Piddock, 2016). Methods for efflux activity measurements largely rely on two different mechanisms. Some methods directly measure the substrate efflux, i.e., how much of the substrate is pumped out, and others measure substrate molecule accumulation inside the cell, the levels of which is then used to infer efflux indirectly. However, the latter is less sensitive due to variable membrane permeability that alters dye influx rates (Blair and Piddock, 2016). Accumulation of R6G in growing C. albicans cells inversely correlates with the level of the ABC transporter Candida drug resistance 1 (CDR1) mRNA expression, establishing levels of intracellular R6G accumulation can be therefore used for identification of azole-resistant strains (Maesaki et al., 1999). Historically, this was carried out by measurements of accumulated radiolabelled-substrates. More recently, fluorescence-based methods are being used. Accumulation of fluorescent dye in a single cell can be also measured by flow cytometry. The benefit of this approach lies in the ability to measure variation in efflux activity among individual cells.

The protocol of the above described method involves preloading the cell population with a fluorescent substrate prior to the efflux assay. After the loading step, substrate accumulates within the cells at maximum concentration. Cells are then washed to remove the substrate. Subsequently, glucose is supplemented to the culture as a source of energy, and the fluorescence signal of substrate is monitored. The method is suitable for use with any yeast species (Borecka-Melkusova et al., 2008).

Materials and Reagents

  1. Pipettes tips
  2. Sterile inoculation loop
  3. 50-ml polypropylene centrifuge tubes (TPP Techno Plastic Procucts, catalog number: 91050 )
  4. 1.5-ml microcentrifuge tubes
  5. 96-well flat bottom with lid MicroWell plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167008 )
  6. Yeast strain to be analysed
  7. Yeast extract (Biolife Italiana, catalog number: 4122202 )
  8. Bacto peptone (Biolife Italiana, catalog number: 4122592 )
  9. HEPES, free acid (AMRESCO, catalog number: 0511 )
  10. Sodium hydroxide (NaOH), 1 mol/L (Merck, catalog number: 109137 )
  11. 2-deoxy-D-glucose (Sigma-Aldrich, catalog number: D6134 )
  12. Rhodamine 6G (Sigma-Aldrich, catalog number: R4127 )
  13. D-glucose (Biolife Italiana, catalog number: 4125012 )
  14. YEPD rich growth medium (see Recipes)
  15. 20 mM glucose (see Recipes)
  16. 50 mM HEPES/NaOH assay buffer (see Recipes)
  17. 2-deoxy-D-glucose in HEPES/NaOH buffer (see Recipes)
  18. 10 mM rhodamine 6G (see Recipes)

Equipment

  1. Pipettes
    Xplorer® 15-300 µl (Eppendorf, catalog number: 4861000031 )
    Research® plus 2-0 µl (Eppendorf, catalog number: 3120000038 )
  2. pH meter (Xylem, WTW, model: inoLab® pH 7110 )
  3. Incubation shaker Unitron (Infors, model: Plus AJ252 )
  4. Haemocytometer
  5. Centrifuge 5804R (Eppendorf, model: 5804 R )
  6. Centrifuge Mikro 200R (Hettich Lab Technology, model: MIKRO 200 R )
  7. Spectrofluorometer Varioscan Flash (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5250500 )
  8. Autoclave

Procedure

  1. Transfer a yeast colony from an agar plate with a sterile inoculation loop, and inoculate into 10 ml YEPD medium (see Recipes). Incubate the culture in an incubation shaker at 150 rpm, at 28 °C for 20 h. (see Note 1)
  2. Determine the cell number using haemocytometer. Add 1 ml (total amount of 5 x 108 cells) from overnight culture into 100 ml fresh YEPD medium.
  3. Incubate the cells in an incubation shaker at 150 rpm for 1.5-2 h at 28 °C until they reach the exponential phase (1 x 107 cells/ml). Monitor the cell density by counting the cell number in a haemocytometer.
  4. Harvest the cells by centrifugation at 3,000 x g for 5 min at laboratory temperature and discard the supernatant.
  5. Wash the cell pellet twice with 50 ml of sterile distilled water (room temperature, RT), then with 50 ml HEPES/NaOH (RT, see Recipes) assay buffer, vortex. Collect the cells by centrifugation (3000 x g, 5 min, RT).
  6. Resuspend the cell pellet (total cell number 109) in 50 ml HEPES/NaOH assay buffer (RT) containing 2-deoxy-D-glucose (2 mM) and R6G (10 µM) (see Recipes and Note 2).
  7. Incubate the cell suspension in an incubation shaker at 150 rpm for 2 h at 28 °C.
  8. Harvest the cells by centrifugation (3,000 x g; 5 min; 4 °C) and wash the pellet with 50 ml sterile distilled water (4 °C) first, then with 50 ml HEPES/NaOH (4 °C) assay buffer.
  9. Resuspend the pellet in 50 ml HEPES/NaOH (4 °C) assay buffer to 108 cells/ml.
  10. Transfer 5 ml of the cell suspension into a new 50 ml centrifuge tube and add 500 µl of glucose from a 20 mM stock solution (see Recipes) to a final concentration of 2 mM. For negative control experiment, glucose is not supplemented in the culture medium (see Note 3).
  11. At specified time intervals (0, 5, 10, 15, 20, 30 min, etc.), transfer 400 µl of cell suspension to a microcentrifuge tube and harvest cells by centrifugation at 10,000 x g, 1 min, 4 °C.
  12. Transfer 100 µl of supernatant into microtiter plate well. Supernatant is sufficient for three technical replicates. Store the samples on ice until the end of experiment.
  13. Measure the R6G fluorescence of the samples at laboratory temperature using the spectrofluorometer (the excitation wavelength of 515 nm; emission wavelength of 555 nm).

Data analysis

  1. Calculate the R6G concentrations using a standard curve. The following concentrations of R6G were used to generate the standard curve: 0, 50, 100, 250, 500 and 1,000 nM.
  2. Plot the obtained R6G concentrations. The results are expressed as the mean ± SD for three independent experiments. A representative curve is shown in Figure 1.
  3. These data were fit and display a linear model y = ax, where the coefficient ‘a’ represents the rate constant of the reaction. Control experiments were done without the added glucose.


    Figure 1. Energy-dependent ATP-binding cassette transporter-mediated efflux of R6G from yeast cells. De-energized cells were preloaded with R6G as described in the protocol. The efflux of R6G was followed by direct measurement of the fluorescence following the addition of glucose (2 mM) to suspension of K. lactis cells. The variation indicated for each time point is the standard error of the mean. The numbers on the x-axis represent time in minutes as independent variable, numbers on y-axis represent concentration of R6G (nM) in the supernatant. Glucose was added at time zero. (Konecna et al., 2016).

Notes

  1. Start the culture with fresh (up to 20 h old) inoculum. Starting concentration should be in the range 0.5-1 x 106 cells/ml.
  2. The Kluyveromyces lactis is usually grown at a temperature of 25-28 °C. The upper limit is approximately 40 °C.
  3. Freshly prepared rhodamine 6G solution is recommended.
  4. Control experiment without added glucose is recommended.
  5. To generate the calibration curve use three independent standard dilution sets of R6G weight.
  6. PBS buffer can be used instead of HEPES.
  7. Some spectrofluorometers require the use of Nunc FluoroNunc/LumiNunc plates (Nalge Nunc International, Rochester, NY).

Recipes

  1. YEPD rich growth medium
    20 g/L 2% D-glucose
    10 g/L 1% yeast extract
    20 g/L 2% Bacto peptone
    Sterilize using the autoclave (120 °C; 120 kPa for 20 min)
  2. 20 mM glucose
    36 g/100 ml
    Sterilize using the autoclave (120 °C; 120 kPa for 20 min)
  3. 50 mM HEPES/NaOH assay buffer
    Dissolve 5.96 g HEPES in 500 ml distilled water, adjust pH to 7.0 with 1 N NaOH
    Sterilize using the autoclave (120 °C; 120 kPa for 20 min)
  4. 2-deoxy-D-glucose in HEPES/NaOH buffer
    Dissolve 164.2 mg of 2-deoxy-D-glucose in 500 ml HEPES/NaOH pH 7
  5. 10 mM rhodamine 6G
    Dissolve 4.8 mg R6G in 1 ml of sterile distilled water

Acknowledgments

This protocol was adapted from our previous studies (Goffa et al., 2014; Konecna et al., 2016). The work was supported by the Slovak Research and Development Agency grants APVV-0282-10 and VEGA 2/0111/15.

References

  1. Blair, J. M. and Piddock L. J. (2016). How to measure export via bacterial multidrug resistance efflux pumps. mBio 7: e00840-16.
  2. Borecka-Melkusova, S., Kozovska, Z., Hikkel, I., Dzugasova, V. and Subik, J. (2008). RPD3 and ROM2 are required for multidrug resistance in Saccharomyces cerevisiae. FEMS Yeast Res 8(3): 414-424.
  3. Goffa, E., Balazfyova, Z., Toth Hervay, N., Simova, Z., Balazova, M., Griac, P. and Gbelska, Y. (2014). Isolation and functional analysis of the KlPDR16 gene. FEMS Yeast Res 14(2): 337-345.
  4. Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., Soumillion, J. P., Konings, W. N. and Goffeau, A. (1996). Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p. J Biol Chem 271(49): 31543-31548.
  5. Konecna, A., Toth Hervay, N, Valachovic, M. and Gbelska, Y. (2016). ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors. Yeast 33(12):621-632.
  6. Maesaki, S., Marichal, P., Vanden Bossche, H., Sanglard, D. and Kohno, S. (1999). Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candida albicans strains. J Antimicrob Chemother 44(1): 27-31.

简介

罗丹明6G是一种高荧光染料,常用于测定酵母膜外排泵的运输活性。 ATP结合盒转运蛋白Klr5p赋予乳酸克鲁维酵酵菌中几种不相关药物的抗性。 Pdr5p还以能量依赖的方式从完整的酵母细胞中挤出罗丹明6G(R6G)。 在2-脱氧-D-葡萄糖(糖酵解抑制剂)和R6G(线粒体ATP酶抑制剂)存在下酵母细胞的孵育导致细胞内ATP池的显着消耗(Kolaczkowski等人,1996)。 从完整的酵母细胞中可以直接测量R6G的荧光的直接测量PdF5p介导的来自完整酵母细胞的R6G的挤出。
【背景】多药物外排泵广泛分布,可在所有生物种类中找到。它们代表抗菌素耐药性的重要机制。量化外排泵活性的能力对于理解其对生理过程的贡献和评估潜在治疗药物(例如,外排抑制剂)的有效性(Blair和Piddock,2016)是必要的。外排活动测量的方法主要依赖于两种不同的机制。一些方法直接测量底物流出,即,多少基质被泵出,其他方法测量细胞内的底物分子积累,其水平然后用于间接推断流出。然而,后者由于可改变膜渗透性而改变了染料流入速率(Blair和Piddock,2016)的敏感性较差。 R6G在生长中的积累。白念珠菌细胞与ABC转运蛋白念珠菌药物抗性1(CDR1 )mRNA表达的水平反向相关,建立细胞内R6G积累的水平可因此用于鉴定唑类抗性菌株Maesaki等人,1999)。历史上,这是通过测量积累的放射性标记底物进行的。最近,正在使用基于荧光的方法。荧光染料在单细胞中的积累也可以通过流式细胞仪测量。这种方法的好处在于测量各个细胞之间外排活动的变化的能力。
上述方法的方案涉及在流出测定之前用荧光底物预加载细胞群。加载步骤后,底物以最大浓度积聚在细胞内。然后洗涤细胞以除去底物。随后,将葡萄糖作为能量来补充培养物,并监测底物的荧光信号。该方法适用于任何酵母菌种(Borecka-Melkusova et al。,2008)。

关键字:罗丹明6G, 荧光, 乳酸克鲁维酵母, ABC转运蛋白, 转运活性测定

材料和试剂

  1. 移液器提示
  2. 无菌接种环
  3. 50毫升聚丙烯离心管(TPP Techno Plastic Procucts,目录号:91050)
  4. 1.5 ml微量离心管
  5. 96孔平底,带盖MicroWell板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:167008)
  6. 待分析的酵母菌株
  7. 酵母提取物(Biolife Italiana,目录号:4122202)
  8. Bacto蛋白胨(Biolife Italiana,目录号:4122592)
  9. HEPES,游离酸(AMRESCO,目录号:0511)
  10. 氢氧化钠(NaOH),1mol / L(Merck,目录号:109137)
  11. 2-脱氧-D-葡萄糖(Sigma-Aldrich,目录号:D6134)
  12. 罗丹明6G(Sigma-Aldrich,目录号:R4127)
  13. D-葡萄糖(Biolife Italiana,目录号:4125012)
  14. YEPD丰富的生长培养基(见食谱)
  15. 20 mM葡萄糖(见食谱)
  16. 50 mM HEPES / NaOH测定缓冲液(见配方)
  17. HEPES / NaOH缓冲液中的2-脱氧-D-葡萄糖(参见食谱)
  18. 10 mM罗丹明6G(见食谱)

设备

  1. 移液器
    Xplorer ®15-300μl(Eppendorf,目录号:4861000031)
    研究®加2-0μl(Eppendorf,目录号:3120000038)
  2. pH计(Xylem,WTW,型号:inoLab pH 7110)
  3. 孵化器Unitron(Infors,型号:Plus AJ252)
  4. 血细胞计数器
  5. 离心机5804R(Eppendorf,型号:5804 R)
  6. 离心机Mikro 200R(Hettich Lab Technology,型号:MIKRO 200 R)
  7. Spectrofluorometer Varioscan Flash(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:5250500)
  8. 高压灭菌器

程序

  1. 用无菌接种环从琼脂平板转移酵母菌落,并接种到10ml YEPD培养基中(参见食谱)。在培养摇床中以150rpm,28℃温育培养20小时。 (见注1)
  2. 使用血细胞计数器确定细胞数。将1ml(总量为5×10 8个细胞)从过夜培养物中加入到100ml新鲜YEPD培养基中。
  3. 在孵育振荡器中以28℃将细胞孵育1.5-2小时,直到达到指数期(1×10 7个细胞/ ml)。通过计数血细胞计数器中的细胞数来监测细胞密度。
  4. 在实验室温度下以3,000×g离心收获细胞5分钟,弃去上清液。
  5. 用50ml无菌蒸馏水(室温,RT),然后用50ml HEPES / NaOH(RT,参见Recipes)测定缓冲液,涡旋洗涤细胞沉淀两次。通过离心(3000×g,5分钟,RT)收集细胞。
  6. 在含有2-脱氧-D-葡萄糖(2mM)和R6G(10μM)的50ml HEPES / NaOH测定缓冲液(RT)中重悬细胞沉淀(总细胞数10次/ sup),参见Recipes和注2)
  7. 在孵育振荡器中,在28℃下以150rpm孵育细胞悬浮液2小时。
  8. 通过离心收集细胞(3,000 xg; 5分钟; 4℃),先用50ml无菌蒸馏水(4℃)洗涤沉淀,然后用50ml HEPES / NaOH(4℃) C)测定缓冲液
  9. 将沉淀重悬于50ml HEPES / NaOH(4℃)测定缓冲液中至10μg/ ml细胞/ ml。
  10. 将5ml细胞悬浮液转移到新的50ml离心管中,并从20mM储备溶液(参见食谱)中加入500μl葡萄糖至终浓度为2mM。对于阴性对照实验,培养基中不补充葡萄糖(见注3)
  11. 以规定的时间间隔(0,5,10,15,20,30分钟,等等),将400μl细胞悬浮液转移到微量离心管中,并以10000xg离心收获细胞,1分钟,4°C。
  12. 将100μl上清液转移到微量滴定板中。上清液足以进行三次技术重复。将样品储存在冰上直至实验结束。
  13. 使用分光荧光计(激发波长515nm;发射波长555nm)在实验室温度下测量样品的R6G荧光。

数据分析

  1. 使用标准曲线计算R6G浓度。使用以下浓度的R6G产生标准曲线:0,50,100,250,500和1,000nM。
  2. 绘制获得的R6G浓度。结果表示为三次独立实验的平均值±SD。代表性的曲线如图1所示
  3. 这些数据拟合并显示线性模型y = ax,其中系数'a'表示反应的速率常数。没有添加葡萄糖进行对照实验

    图1.能量依赖的ATP结合盒转运蛋白介导的R6G从酵母细胞的流出。 如方案中所述,断电的电池用R6G预加载。随后,在加入葡萄糖(2mM)至K的K的悬浮液后,直接测量荧光。乳酸细胞。每个时间点指示的变化是平均值的标准误差。 x轴上的数字表示以分钟为单位的时间为自变量,y轴上的数字表示上清液中的R6G(nM)浓度。葡萄糖在零时加入。 (Konecna等人,2016)。

笔记

  1. 用新鲜(高达20小时)的接种物开始培养。起始浓度应在0.5-1×10 6细胞/ ml范围内。
  2. 克氏乳酸克鲁维酵母通常在25-28℃的温度下生长。上限约为40°C。
  3. 推荐新制罗丹明6G溶液。
  4. 建议不添加葡萄糖的对照实验。
  5. 要产生校准曲线,请使用三个独立的R6G重量的标准稀释组。
  6. 可以使用PBS缓冲液代替HEPES。
  7. 一些分光荧光计需要使用Nunc FluoroNunc / LumiNunc板(Nalge Nunc International,Rochester,NY)。

食谱

  1. YEPD丰富的生长培养基
    20g / L 2%D-葡萄糖
    10克/升1%酵母提取物 20克/升2%细菌蛋白胨
    使用高压釜灭菌(120℃; 120kPa 20分钟)
  2. 20 mM葡萄糖
    36 g / 100 ml
    使用高压釜灭菌(120℃; 120kPa 20分钟)
  3. 50mM HEPES / NaOH测定缓冲液
    将5.96 g HEPES溶于500 ml蒸馏水中,用1N NaOH调节pH至7.0 使用高压釜灭菌(120℃; 120kPa 20分钟)
  4. HEPES / NaOH缓冲液中的2-脱氧-D-葡萄糖 将164.2mg 2-脱氧-D-葡萄糖溶解于500ml HEPES / NaOH pH7中
  5. 10 mM罗丹明6G
    将4.8mg R6G溶于1ml无菌蒸馏水中

致谢

该协议是从我们之前的研究(Goffa等人,2014; Konecna等人,2016)中改编而来的。这项工作得到了斯洛伐克研究与发展局的赞助,授权APVV-0282-10和VEGA 2/0111/15。

参考

  1. Blair,JM和Piddock LJ(2016)。  如何通过细菌多药耐药外排泵测量出口。 7:e00840-16。
  2. Borecka-Melkusova,S.,Kozovska,Z.,Hikkel,I.,Dzugasova,V.and Subik,J。(2008)。< a class =“ke-insertfile”href =“http: ncbi.nlm.nih.gov/pubmed/18205807“target =”_ blank“> RPD3 和 ROM2 是酿酒酵母中多重耐药性所必需的。 FEMS酵母研究所 8(3):414-424。
  3. Goffa,E.,Balazfyova,Z.,Toth Hervay,N.,Simova,Z.,Balazova,M.,Griac,P。和Gbelska,Y。(2014)。< a class =“ke-insertfile” href =“http://www.ncbi.nlm.nih.gov/pubmed/24119036”target =“_ blank”> KlPDR16基因的分离和功能分析 FEMS Yeast Res 14(2):337-345。
  4. Kolaczkowski,M.,van der Rest,M.,Cybularz-Kolaczkowska,A.,Soumillion,JP,Konings,WN和Goffeau,A(1996)。抗癌药物,离子型肽和类固醇作为酵母多药转运蛋白Pdr5p的底物。 271(49):31543-31548。
  5. Konecna,A.,Toth Hervay,N,Valachovic,M。和Gbelska,Y。(2016)。 基因缺失修饰乳酸克鲁维酵母对各种生长抑制剂的易感性 酵母 33 (12):621-632。
  6. Maesaki,S.,Marichal,P.,Vanden Bossche,H.,Sanglard,D.and Kohno,S。(1999)。罗丹明6G外排用于检测CDR1 - 去除抗唑抗性的白色念珠菌菌株。 a> J Antimicrob Chemother 44(1):27-31。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gbelska, Y., Toth Hervay, N., Dzugasova, V. and Konecna, A. (2017). Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species. Bio-protocol 7(15): e2428. DOI: 10.21769/BioProtoc.2428.
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