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Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein
白色念珠菌中响应抗真菌蛋白Hog1磷酸化水平的检测   

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参见作者原研究论文

本实验方案简略版
Antimicrobial Agents and Chemotherapy
Aug 2013

Abstract

It is becoming increasingly apparent that stress signalling is important for tolerance of fungal species to antifungal chemicals and proteins. The high-osmolarity glycerol (HOG) pathway responds to a number of stressors including osmotic and oxidative stress. This protocol describes a method to detect activation of the Candida albicans (C. albicans) MAPK Hog1 by monitoring its phosphorylation in response to an antifungal protein.

Materials and Reagents

  1. Candida albicans wild-type (WT)
  2. C. albicans hog1Δ/Δ deletion mutant
  3. Half-strength potato dextrose broth (½ PDB) (BD, DifcoTM, catalog number: 254920 )
  4. Protein of interest (e.g. the plant defensin NaD1)
  5. 5 M NaCl
  6. Ultrapure ice-cold water
  7. Dry ice
  8. 20% trichloroacetic acid (TCA) (20% w/v)
  9. 10% trichloroacetic acid (TCA) (10% w/v)
  10. 100% ice-cold acetone
  11. 4x NuPAGE LDS sample buffer (Life Technologies, catalog number: NP0007 )
  12. Bond-Breaker TCEP solution (neutral pH) (Thermo Fisher Scientific, catalog number: 77720 )
  13. 1 M Tris (pH 8.0)
  14. Any kD Mini-PROTEAN TGX precast polyacrylamide gels (15 well) (Bio-Rad Laboratories, catalog number: 456-9036 )
  15. 10x Tris/glycine/SDS running buffer (Bio-Rad Laboratories, catalog number: 161-0072 )
  16. 5% (w/v) skim milk in TBS
  17. Phospho-p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling Technology, catalog number: 9211 )
  18. Hog1 (y-215) antibody (Santa Cruz, catalog number: sc-9079 )
  19. Amersham ECL rabbit IgG (HRP-linked antibody from donkey) (GE, catalog number: NA934 )
  20. Amersham ECL Western blotting detection reagents (GE, catalog number: RPN2106 )
  21. Yeast peptone dextrose broth (YPD) (see Recipes)
  22. Yeast peptone dextrose agar (YPD agar) (see Recipes)
  23. Tris-buffered saline (TBS) (see Recipes)
  24. Tris-buffered saline with Tween 20 (TBST) (see Recipes)

Equipment

  1. 180 μm glass beads (acid-washed) (Sigma-Aldrich, catalog number: G1152 )
  2. Trans-blot turbo mini PVDF transfer pack (Bio-Rad Laboratories, catalog number: 170-4156 )
  3. 50 ml tubes screw-cap tubes
  4. 30 °C shaking incubator
  5. Spectrophotometer
  6. 500 ml baffled flask
  7. Eppendorf 5810 R centrifuge (Eppendorf, catalog number: 5811 000.010 )
  8. 1.5 ml microfuge tubes
  9. Vortex
  10. Bench top centrifuge
  11. Trans-blot turbo transfer system (Bio-Rad Laboratories, catalog number: 170-4155 )
  12. Circular mixer
  13. Bio-Rad ChemiDoc MP imaging system (Bio-Rad Laboratories, catalog number: 170-8280 )

Procedure

  1. Treatment of cells
    1. Plate C. albicans wild-type and hog1Δ strains onto YPD agar and grow overnight at 30 °C.
    2. Take scraping of both C. albicans strains from agar and inoculate 5 ml YPD in a 50 ml tube. Incubate tube overnight at 30 °C (250 rpm).
    3. Dilute both cultures to an OD600 of 0.2 (in 100 ml ½ PDB) and transfer 100 ml to a 500 ml baffled flask.
    4. Incubate cells at 30 °C (250 rpm) for 5 h (or until they reach an OD600 of 1.0).
    5. Dilute cells to an OD600 of 1.0. Transfer 10 ml aliquots of each culture to 50 ml tubes. One tube will be required per treatment (see Table 1 for treatment conditions).
    6. Treat 10 ml aliquots of WT and hog1Δ with NaCl or protein of interest (e.g. 10 μM and 20 μM NaD1 in ultrapure water). Cells will be incubated for 5, 10 or 15 min at 30 °C (250 rpm). For NaCl treatment, add 2.5 ml of 5 M NaCl to the 10 ml of WT or hog1Δ.  
      For protein treatments, add volume to reach desired protein concentration (e.g. volume of protein for a final concentration of 10 μM or 20 μM). For the hog1∆ control treatments, only the 20 µM treatment concentration is required. If no total Hog1 or phosphorylated Hog1 is detected in the hog1∆ cells treated with 20 µM protein, then none will be detectable at a lower protein concentration.

      Table 1. Treatment conditions. Treatment conditions of C. albicans WT and hog1∆ cells with the protein of interest. 1 M NaCl is included as a positive control. No-protein and hog1∆ treatments are included as negative controls.
      C. albicans strain
      Protein or NaCl concentration
      Incubation time
      WT
      No treatment
      -
      WT
      1 M NaCl
      10 min
      WT
      10 μM protein
      5 min
      WT
      10 μM protein
      10 min
      WT
      10 μM protein
      15 min
      WT
      20 μM protein
      5 min
      WT
      20 μM protein
      10 min
      WT
      20 μM protein
      15 min
      hog1Δ
      No treatment
      -
      hog1Δ
      1 M NaCl
      10 min
      hog1Δ
      20 μM protein
      5 min
      hog1Δ
      20 μM protein
      10 min
      hog1Δ
      20 μM protein
      15 min

    7. After incubation spin cells down at 1,800 x g for 5 min (at RT). Discard the supernatant.
    8. Resuspend cells in 500 μl ice-cold water. Transfer to a 1.5 ml microfuge tube (Tube A).
    9. Centrifuge cells at 3,000 x g for 5 min (at RT). Discard the supernatant.
    10. Freeze pellets with dry ice.

  2. Protein preparation
    1. Resuspend cell pellets in 100 μl 20% TCA. Also add approximately 100 μl of 180 μm glass beads. Keep pellets on dry ice.
    2. Vortex tubes for 2 min (at highest speed).
    3. Transfer supernatant from Tube A to a new tube (Tube B).
    4. Wash the remaining beads in Tube A with 500 μl 10% TCA. Transfer the 500 μl TCA and add it to Tube B.
    5. Spin Tube B at 18,000 x g for 5 min (at RT). Remove supernatant.
    6. Resuspend pellets in 1 ml ice-cold acetone.
    7. Spin again at 18,000 x g for 5 min (at RT). Remove supernatant.
    8. Resuspend pellets with 50 μl loading buffer (1x NuPAGE LDS sample buffer and 50 mM Bond-Breaker). If the buffer turns yellow, add a few microliters of 1 M Tris (pH 8.0) until the color becomes blue.
    9. Heat samples at 70 °C for 10 min.

  3. SDS-PAGE and Western blots
    1. Load 25 μl of each sample into the wells of a Bio-Rad any kD Mini-PROTEAN TGX precast polyacrylamide gels, 15 well (Gel 1). Repeat this for a second gel (Gel 2).
    2. Run gel for 30 min at 200 V (with 1x Tris/Glycine/SDS running buffer).
    3. Transfer protein to Trans-blot turbo mini PVDF membranes (from trans-blot turbo mini PVDF transfer pack) using the Bio-Rad trans-blot turbo transfer system (1.3 A, 25 V, 7 min).
    4. Block PVDF membranes in 5% skim milk (40 ml TBS, in a 50 ml tube) for 2 h at RT (on a circular mixer). Discard skim milk after incubation.
    5. Add primary antibodies and incubate overnight on a circular mixer at 4 °C (in a 50 ml tube).
      For Gel 1, dilute Hog1 (y-215) antibody to 1 in 200 with 10 ml 5% skim milk in TBST.
      For Gel 2, dilute Phospho-p38 MAPK (Thr180/Tyr182) antibody to 1 in 200 with 10 ml 5% skim milk in TBST.
    6. Remove primary antibodies. Wash membranes with TBST (3x, 10 min per wash).
    7. Dilute secondary antibody (ECL rabbit IgG, HRP-linked) to 1 in 20,000 with 10 ml TBST and incubate with membranes overnight at 4 °C.
    8. Remove secondary antibodies. Wash membranes with TBST (3x, 10 min per wash).
    9. Mix 1 ml ECL detection reagent 1 and 1 ml of ECL detection reagent 2 for each membrane. Add 2 ml of the mixture to each membrane.
    10. Expose membranes using chemiluminescent settings on a Bio-Rad ChemiDoc MP imaging system. Expose membranes for ~ 1 min (or longer if the signal is weak).
      See Figure 1 for an example.


    Figure 1. Treatment of C. albicans with NaD1 leads to Hog1 phosphorylation.
    Western blots showing C. albicans Hog1 phosphorylation (detected using Phospho-p38 MAPK antibody) after treatment with the plant defensin NaD1. Bands corresponding to total Hog1 levels are indicated with an arrow. Total Hog1 levels are consistent across all WT treatments and are absent from the hog1∆ mutant. No phosphorylated Hog1 is detected in the no treatment or hog1∆ negative controls. Hog1 phosphorylation is detected in the 1 M NaCl positive control and in all NaD1 treated samples. In this example only one time point is shown for the 20 µM NaD1 treated hog1∆ control. Image originally published in Reference 1 (Hayes et al., 2013).

Recipes

  1. YPD or YPD agar
    1% yeast extract
    2% peptone
    2% dextrose
    If making plates also add 2% agar
  2. TBS and TBST
    400 mM Tris (pH 7.5)
    3 M Tris
    For TBST also add 0.1% Tween-20

Acknowledgments

This protocol has been adapted from Hayes et al. (2013), in which a brief description of the protocol was presented.

References

  1. Hayes, B. M., Bleackley, M. R., Wiltshire, J. L., Anderson, M. A., Traven, A. and van der Weerden, N. L. (2013). Identification and mechanism of action of the plant defensin NaD1 as a new member of the antifungal drug arsenal against Candida albicans. Antimicrob Agents Chemother 57(8): 3667-3675.

简介

越来越明显的是,应激信号传导对于真菌物种对抗真菌化学品和蛋白质的耐受性是重要的。 高渗透压甘油(HOG)途径对包括渗透和氧化应激在内的许多应激物起反应。 该方案描述了通过监测其对抗真菌蛋白的磷酸化来检测白色假丝酵母(embiculans)(白色念珠菌)MAPK Hog1的活化的方法。

材料和试剂

  1. 白色念珠菌野生型(WT)
  2. C。 白色念珠菌hog1Δ/Δ缺失突变体
  3. 半强度马铃薯葡萄糖肉汤(1/2 PDB)(BD,Difco TM ,目录号:254920)
  4. 感兴趣的蛋白质(例如植物防御素NaD1)
  5. 5 M NaCl
  6. 超纯冰水
  7. 干冰
  8. 20%三氯乙酸(TCA)(20%w/v)
  9. 10%三氯乙酸(TCA)(10%w/v)
  10. 100%冰冷的丙酮
  11. 4x NuPAGE LDS样品缓冲液(Life Technologies,目录号:NP0007)
  12. Bond-Breaker TCEP溶液(中性pH)(Thermo Fisher Scientific,目录号:77720)
  13. 1 M Tris(pH 8.0)
  14. 任何kD Mini-PROTEAN TGX预制聚丙烯酰胺凝胶(15孔)(Bio-Rad Laboratories,目录号:456-9036)
  15. 10x Tris /甘氨酸/SDS运行缓冲液(Bio-Rad Laboratories,目录号:161-0072)
  16. 5%(w/v)脱脂牛奶在TBS中
  17. 磷酸-p38MAPK(Thr180/Tyr182)抗体(Cell Signaling Technology,目录号:9211)
  18. Hog1(y-215)抗体(Santa Cruz,目录号:sc-9079)
  19. Amersham ECL兔IgG(来自驴的HRP-连接的抗体)(GE,目录号:NA934)
  20. Amersham ECL Western印迹检测试剂(GE,目录号:RPN2106)
  21. 酵母蛋白胨葡萄糖肉汤(YPD)(参见食谱)
  22. 酵母蛋白胨葡萄糖琼脂(YPD琼脂)(见Recipes)
  23. Tris缓冲盐水(TBS)(见Recipes)
  24. 含Tween 20的Tris缓冲盐水(TBST)(见Recipes)

设备

  1. 180μm玻璃珠(酸洗)(Sigma-Aldrich,目录号:G1152)
  2. 转印涡轮迷你PVDF转移包(Bio-Rad Laboratories,目录号:170-4156)
  3. 50ml管螺旋盖管
  4. 30℃摇动培养箱
  5. 分光光度计
  6. 500ml带挡板的烧瓶中
  7. Eppendorf 5810R离心机(Eppendorf,目录号:5811000.010)
  8. 1.5 ml微量离心管
  9. 涡流
  10. 台式离心机
  11. Trans-blot涡流转移系统(Bio-Rad Laboratories,目录号:170-4155)
  12. 圆形混合器
  13. Bio-Rad ChemiDoc MP成像系统(Bio-Rad Laboratories,目录号:170-8280)

程序

  1. 细胞治疗
    1. 平板C. 白色念珠菌野生型和emg1Δ菌株在YPD琼脂上并在30℃下生长过夜。
    2. 刮擦两者。 来自琼脂的白色念珠菌菌株,并在50ml试管中接种5ml YPD。 在30℃(250rpm)下孵育试管过夜
    3. 将两种培养物稀释至0.2的OD 600(在100ml1/2PDB中),并转移100ml至500ml带挡板的烧瓶中。
    4. 在30℃(250rpm)孵育细胞5小时(或直到它们达到OD的OD 600为1.0)。
    5. 将细胞稀释至OD 600的1.0。将每份培养物的10ml等分试样转移到50ml试管中。每次治疗需要一个管(见表1的治疗条件)。
    6. 用NaCl或感兴趣的蛋白质(例如,10μM和20μMNaD1在超纯水中)处理10ml等分的WT和hog1Δ。将细胞在30℃(250rpm)温育5,10或15分钟。对于NaCl处理,将2.5ml的5M NaCl加入到10ml WT或hog1Δ中。  
      对于蛋白质处理,加入体积以达到所需的蛋白质浓度(例如,最终浓度为10μM或20μM的蛋白质体积)。对于hog1Δ对照处理,仅需要20μM处理浓度。如果在用20μM蛋白质处理的hog1Δ细胞中未检测到总的Hog1或磷酸化的Hog1,则在较低的蛋白质浓度下没有检测到。
      表1.治疗条件。治疗条件。白色念珠菌WT和感兴趣的蛋白质hog1Δ细胞。包括1M NaCl作为阳性对照。无蛋白质和hog1Δ治疗作为阴性对照
      C。 阿尔巴尼亚人紧张
      蛋白质或NaCl浓度
      孵化时间
      WT
      没有治疗
      -
      WT
      1 M NaCl
      10分钟
      WT
      10μM蛋白质
      5分钟
      WT
      10μM蛋白质
      10分钟
      WT
      10μM蛋白质
      15分钟
      WT
      20μM蛋白质
      5分钟
      WT
      20μM蛋白质
      10分钟
      WT
      20μM蛋白质
      15分钟
      hog1Δ
      没有治疗
      -
      hog1Δ
      1 M NaCl
      10分钟
      hog1Δ
      20μM蛋白质
      5分钟
      hog1Δ
      20μM蛋白质
      10分钟
      hog1Δ
      20μM蛋白质
      15分钟

    7. 温育后,以1,800×g离心细胞5分钟(室温)。 弃去上清液。
    8. 重悬细胞在500μl冰冷的水中。 转移到1.5ml微量离心管(Tube A)
    9. 离心细胞在3,000×g离心5分钟(在室温)。 弃去上清液。
    10. 用干冰冻结颗粒。

  2. 蛋白质制备
    1. 重悬细胞沉淀在100微升20%TCA。 还要加入大约100微升180微米的玻璃珠。 将颗粒保留在干冰上。
    2. 涡旋管2分钟(最高速度)。
    3. 将管A的上清液转移到新管(管B)
    4. 用500μl10%TCA洗涤管A中的剩余珠子。 转移500μlTCA,并将其加入试管B.
    5. 旋转管B以18,000×g离心5分钟(室温)。 除去上清液。
    6. 将沉淀重悬在1ml冰冷的丙酮中
    7. 再次以18,000×g离心5分钟(室温)。 除去上清液。
    8. 用50μl上样缓冲液(1x NuPAGE LDS样品缓冲液和50mM Bond-Breaker)重悬沉淀。 如果缓冲液变黄,加入几微升1M Tris(pH 8.0)直至颜色变蓝。
    9. 在70℃加热样品10分钟
  3. SDS-PAGE和Western印迹
    1. 加载25微升的每个样品到Bio-Rad的任何kD Mini-PROTEAN TGX预制聚丙烯酰胺凝胶,15孔(凝胶1)的孔中。对第二块凝胶(凝胶2)重复此操作。
    2. 在200V(使用1X Tris /甘氨酸/SDS运行缓冲液)下运行凝胶30分钟
    3. 使用Bio-Rad trans-blot涡轮转移系统(1.3A,25V,7分钟)将转移蛋白转移到Trans-blot涡轮迷你PVDF膜(来自转印涡轮迷你PVDF转移组件)。
    4. 在室温下(在圆形混合器上)在5%脱脂乳(40ml TBS,在50ml管中)封闭PVDF膜2小时。孵育后丢弃脱脂牛奶。
    5. 加入一抗,并在4℃下在圆形混合器中孵育过夜(在50ml管中) 对于凝胶1,将稀释的Hog1(y-215)抗体在TBST中用10ml 5%脱脂乳稀释200倍。
      对于凝胶2,用TBST中的10ml 5%脱脂牛奶稀释磷酸-p38MAPK(Thr180/Tyr182)抗体至200μl。
    6. 删除一抗。 用TBST洗涤膜(3x,每次洗涤10分钟)。
    7. 用10ml TBST稀释第二抗体(ECL兔IgG,HRP-连接的)至20,000,并与膜在4℃下孵育过夜。
    8. 删除二级抗体。 用TBST洗涤膜(3x,每次洗涤10分钟)。
    9. 每个膜混合1毫升ECL检测试剂1和1毫升ECL检测试剂2。 向每个膜中加入2ml混合物
    10. 在Bio-Rad ChemiDoc MP成像系统上使用化学发光设置来暴露膜。 暴露膜约1分钟(如果信号较弱,则更长时间)。
      参见图1的示例。


    图1.C的治疗。白蛋白与NaD1的结合导致Hog1磷酸化。 Western印迹显示C。白色念珠菌 Hog1磷酸化(用Phospho-p38 MAPK抗体检测)。对应于总Hog1水平的带用箭头指示。总Hog1水平在所有WT处理中一致,并且不存在于hog1Δ突变体中。在未处理或hog1Δ阴性对照中未检测到磷酸化的Hog1。在1M NaCl阳性对照和所有NaD1处理的样品中检测到Hog1磷酸化。在该实施例中,对于20μMNaD1处理的hog1Δ对照,仅显示一个时间点。图片最初发表于参考文献1(Hayes等人,2013年)。

食谱

  1. YPD或YPD琼脂 1%酵母提取物
    2%蛋白胨
    2%葡萄糖 如果制作板也添加2%琼脂
  2. TBS和TBST
    400mM Tris(pH7.5) 3 M Tris
    对于TBST还添加0.1%Tween-20

致谢

该协议已经由Hayes等人(2013)修改,其中提出了协议的简要描述。

参考文献

  1. Hayes,B.M.,Bleackley,M.R.,Wiltshire,J.L.,Anderson,M.A.,Traven,A.and van der Weerden,N.L。(2013)。 识别和作用机制 的植物防御素NaD1作为针对白假丝酵母的抗真菌药物砷的新成员。抗微生物剂化学 57(8):3667-3675。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hayes, B. M. and Weerden, N. L. (2014). Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein. Bio-protocol 4(18): e1244. DOI: 10.21769/BioProtoc.1244.
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