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Activity Assays for Bacteriophage Endolysin PlyPy
噬菌体细胞内溶素PlyPy的活性分析   

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Abstract

Bacterial viruses (bacteriophages) escape and kill their host by degrading the bacterial peptidoglycan layer through the mechanism of enzymes called endolysins: peptidoglycan degrading enzymes. The method included here is useful for the initial characterization of any endolysin, regardless of the specific catalytic domain (as long as the activity results in a reduction in the optical density), in order to determine its optimal enzymatic (lytic) activity. This protocol is specific for the Streptococcus pyogenes phage endolysin PlyPy, but can be adapted for any peptidoglycan degrading enzyme.

Keywords: Phage lysins(噬菌体裂解酶), Lysis(裂解), Lytic activity(溶解活性), Cell wall hydrolase(细胞壁水解酶), Peptidoglycan cleavage(肽聚糖裂解)

Materials and Reagents

  1. Streptococcus pyogenes (S. pyogenes) strain MGAS 3 15
  2. Purified phage lysin PlyPy (or other lysins of interest)
  3. Brain heart infusion (BHI) broth (BD, catalog number: 237500 )
  4. Agar (BD, catalog number: 214010 )
  5. Tris (Thermo Fisher Scientific, catalog number: BP152-1 )
  6. HCl (Thermo Fisher Scientific, catalog number: A144S-500 )
  7. CaCl2.2H2O (Thermo Fisher Scientific, catalog number: C70-500 )
  8. MgCl2.7H2O (AMRESCO, catalog number: 0288-100G )
  9. ZnCl2 (Fluka, catalog number: 96468-50G )
  10. DTT (Sigma-Aldrich, catalog number: D0632-10G )
  11. Sodium acetate (Thermo Fisher Scientific, catalog number: S210-500 )
  12. NaCl (Thermo Fisher Scientific, catalog number: S271-3 )
  13. PlyPy activity buffer (see Recipes)

Equipment

  1. 37 °C incubator
  2. Spectrophotometer (Bio-Rad Laboratories, SmartSpecTM, model: 3000 )
  3. Table centrifuge (Eppendorf, model: 5810 R )
  4. pH meter (Thermo Fisher Scientific, AccumetTM, model: 15)
  5. 15 ml tubes (Falcon®, catalog number: 352097 )
  6. 50 ml tubes (Falcon®, catalog number: 352098 )
  7. Cuvettes (Sarstedt AG, catalog number: 67.742 )
  8. 96-well flat bottom plates (Falcon®, catalog number: 35-1172 )
  9. 96-well plate reader (Molecular Devices, SpectraMax Plus Reader)

Procedure

Note: Streak S. pyogenes strain MGAS 315 on a BHI agar plate (or other plates supporting the growth of your bacteria, including blood agar and THY agar plates) and allow to incubate overnight at 37 °C until single colonies are visible.

  1. Inoculate one colony of MGAS 315 in 5 ml BHI in a 15 ml tube. Incubate overnight at 37 °C.
  2. Preheat BHI by adding 45 ml BHI to a 50 ml tube, and incubate overnight at 37 °C.
  3. Transfer the 5 ml overnight culture of MGAS 315 into the preheated BHI (1:10 dilution), and incubate until OD600 ∼ 0.3. The time for the culture to reach this OD600 varies depending on what strain is used, but will in general take 3-4 h for S. pyogenes.
    Note: Cells can be taken at other OD600 values, but bacteria are generally more susceptible to the effect of endogenously added lysins when in mid log phase as compared to stationary phase cells. Therefore it is recommended to do a growth curve of your bacteria to determine the mid log phase before conducting any further experiments.
  4. Harvest cells by centrifugation (4,000 x g; 15 min; 4 °C).
  5. Discard supernatant, and wash cells three times in 20 mM Tris-HCl (pH 6.8) (4,000 x g; 15 min; 4 °C).
    Note: Other buffers might prove more efficient for other peptidoglycan degrading enzymes, but buffers in the pH-range 6-8 are usually a good starting point since bacteria generally tolerate this pH well, and most lysins are highly active within this pH range.
  6. Resuspend the washed cells in 20 mM Tris-HCl (pH 6.8) to an OD600 ∼ 1.4.
    Note: For best experimental resolution, a final OD600 of 0.5-1.0 is recommended. Washed cells will be diluted 1:1 in upcoming steps, yielding a final OD600 = 0.7.
  7. Add 100 μl washed cells to the wells of a sterile 96-well plate.
    Note: For consistency and statistical reliability, we recommend all samples to be run in at least triplicates.
  8. Add 100 μl buffer [20 mM Tris-HCl (pH 6.8)] containing divalent cations at varying concentrations.
    CaCl2: 0-100 mM
    MgCl2: 0-100 mM
    ZnCl2: 0-5 mM
    Note: Since many endolysins rely on the presence of divalent cations for their activity, it is critical to optimize this condition before moving on to other optimizations. Several other divalent cations might affect the activity of lysins, including but not limited to Mn2+, Fe2+, Co2+, Cu2+ and Ni2+.
  9. Add 1 μg of your peptidoglycan hydrolyzing protein (PlyPy).
    Note: The amount of lysin needed to be added is dependent on the activity of the lysin and needs to be determined empirically. A few μg lysin is a good starting point.
  10. Insert the 96-well plate into the SpectraMax Plus Reader, and incubate the plate at 37 °C for 60 min. Shake the plate and measure the OD600 every 30 sec.
  11. Plot the data in an Excel sheet and analyze the results.
  12. Repeat steps 9-12, including the necessary divalent cations at an empirically determined concentration in the buffer, but now optimize the conditions for other factors, including but not limited to:
    NaCl: 0-1,000 mM
    DTT: 0-20 mM
    Temperature: 20-50 °C
  13. To determine the optimal pH, exchange the 20 mM Tris-HCl (pH 6.8) used in steps 6, 7 and 9 to buffers of your choice, including but not limited to:
    20 mM sodium acetate (pH 5.0)
    20 mM sodium acetate (pH 5.5)
    20 mM sodium acetate (pH 6.0)
    20 mM Tris-HCl (pH 6.0)
    20 mM Tris-HCl (pH 6.5)
    20 mM Tris-HCl (pH 7.0)
    20 mM Tris-HCl (pH 7.5)
    20 mM Tris-HCl (pH 8.0)
    20 mM Tris-HCl (pH 8.8)
  14. To finally determine the activity of your lysin:
    1. Wash and resuspend your cells in activity buffer (optimized buffer based on your empirical values) to an OD600 ∼ 1.4.
    2. Add 100 μl cells and 100 μl activity buffer to each well in a 96-well plate.
      Note: Take into account that the maximum volume in the wells will be 200 μl. Therefore, less than 100 μl activity buffer will be added to wells that will also contain lysin (diluted in activity buffer). The exact volume depends on your initial concentration of the lysin.
    3. Add your lysin in serial dilutions, ranging from 0.1-51.2 μg/ml.
      Note: It is important to run all samples in triplicates to account for any outliers.
    4. Incubate for 15 min at 37 °C in a SpectraPlus Max Reader, shaking and measuring OD600 of the samples every 30 sec.
    5. Plot the data in an Excel sheet and analyze the results.
    6. The dilution closest corresponding to a 50% reduction of the starting OD corresponds to 1 Unit (Loeffler et al., 2001). Most characterized lysins have an activity of 0.5-1 U/μg.

Representative data



Figure 1. Activity determination of PlyPy. Washed S. pyogenes cells were incubated in activity buffer at 37 °C for 15 min with varying concentrations of PlyPy. The optical density (600) was measured every 30 sec in a SpectraPlus Max Reader. The mean value of triplicates is shown. For simplicity, a gray line has been added, representing the OD50% at 900 sec (15 min), thus corresponding to one Unit. In this representative figure, 2 μg/ml corresponds closely to one Unit; e.g. the activity of the lysin is 0.5 U/μg. Note the fast initial drop in optical density in samples with high concentration of lysin, stressing the necessity of reading your sample as soon as possible after adding the lysin.

Recipes

  1. PlyPy activity buffer
    20 mM Tris-HCl (pH 6.8)
    2 mM CaCl2
    100 mM NaCl
    Sterile filter the solution (0.22 μm) before usage

Acknowledgments

This protocol was published in a more abbreviated form in the full-length article by Lood et al. (2014). This study was supported in part by USPHS grant AI11822 and by a grant from Contrafect Inc. to V.A.F. and by a grant from the Tegger Foundation to R.L.

References

  1. Loeffler, J. M., Nelson, D. and Fischetti, V. A. (2001). Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294(5549): 2170-2172.
  2. Lood, R., Raz, A., Molina, H., Euler, C. W. and Fischetti, V. A. (2014). A highly active and negatively charged streptococcus pyogenes lysin with a rare d-alanyl-l-alanine endopeptidase activity protects mice against Streptococcal bacteremia. Antimicrob Agents Chemother 58(6): 3073-3084.

简介

细菌病毒(噬菌体)通过称为内溶素:肽聚糖降解酶的酶的机制降解细菌肽聚糖层而逃脱并杀死它们的宿主。 本文包括的方法可用于任何内溶素的初始表征,而不管具体的催化结构域(只要活性导致光密度降低),以确定其最佳酶(裂解)活性。 该方案对于化脓链球菌噬菌体内溶素PlyPy是特异性的,但可以适用于任何肽聚糖降解酶。

关键字:噬菌体裂解酶, 裂解, 溶解活性, 细胞壁水解酶, 肽聚糖裂解

材料和试剂

  1. 化脓性链球菌( pyogenes )菌株MGAS 315
  2. 纯化的噬菌体溶素PlyPy(或其他感兴趣的溶素)
  3. 脑心浸液(BHI)肉汤(BD,目录号:237500)
  4. 琼脂(BD,目录号:214010)
  5. Tris(Thermo Fisher Scientific,目录号:BP152-1)
  6. HCl(Thermo Fisher Scientific,目录号:A144S-500)
  7. (赛默飞世尔科技,目录号:C70-500)。
  8. (AMRESCO,目录号:0288-100G)。
  9. ZnCl 2(Fluka,目录号:96468-50G)
  10. DTT(Sigma-Aldrich,目录号:D0632-10G)
  11. 乙酸钠(Thermo Fisher Scientific,目录号:S210-500)
  12. NaCl(Thermo Fisher Scientific,目录号:S271-3)
  13. PlyPy活动缓冲区(参见配方)

设备

  1. 37℃孵育器
  2. 分光光度计(Bio-Rad Laboratories,SmartSpec ,型号:3000)
  3. 台式离心机(Eppendorf,型号:5810R)
  4. pH计(Thermo Fisher Scientific,Accumet TM,型号:15)
  5. 15ml管(Falcon ,目录号:352097)
  6. 50ml管(Falcon ,目录号:352098)
  7. 比色杯(Sarstedt AG,目录号:67.742)
  8. 96孔平底板(Falcon ,目录号:35-1172)
  9. 96孔板读数器(Molecular Devices,SpectraMax Plus Reader)

程序

注意:在BHI琼脂平板(或支持您的细菌生长的其他平板,包括血液琼脂和THY琼脂平板)上的条纹化脓性链球菌菌株MGAS 315,并允许在37℃下孵育过夜直到单个菌落可见。

  1. 接种一个殖民地的MGAS 315在5ml BHI在15毫升管。在37℃下孵育过夜
  2. 通过将45ml BHI加入50ml试管中预热BHI,并在37℃下孵育过夜
  3. 将5毫升过夜培养的MGAS 315转移到预热的BHI(1:10稀释)中,并温育直到OD 600〜0.3。培养物达到OD OD 600的时间根据使用的菌株而变化,但对于S通常需要3-4小时。化脓。
    注意:细胞可以在其他OD 值,但细菌通常更容易受内源性添加赖氨酸的影响,在中间对数期与固定相细胞相比。因此,建议在进行任何进一步的实验之前,对细菌进行生长曲线以确定中期对数期。
  4. 通过离心收集细胞(4,000×g; 15分钟; 4℃)
  5. 弃去上清液,并在20mM Tris-HCl(pH6.8)(4,000×g; 15分钟; 4℃)中洗涤细胞三次。
    注意:其他缓冲液可能证明对于其他肽聚糖降解酶更有效,但pH范围6-8中的缓冲液通常是良好的起点,因为细菌通常容忍该pH,并且大多数溶素在该pH内具有高活性范围。
  6. 将洗涤的细胞在20mM Tris-HCl(pH 6.8)中重悬至OD 600〜1.4。
    注意:为了获得最佳实验分辨率,建议最终OD 600 为0.5-1.0。洗涤的细胞将在后续步骤中以1:1稀释,得到最终OD 600 = 0.7。
  7. 将100μl洗涤的细胞加入无菌96孔板的孔中 注意:为了一致性和统计信息的可靠性,我们建议所有样品至少运行三次。
  8. 加入含有不同浓度二价阳离子的100μl缓冲液[20mM Tris-HCl(pH6.8)] CaCl 2:0-100mM
    MgCl 2:0-100mM
    ZnCl 2:0-5mM
    注意:由于许多内溶素依赖于二价阳离子的存在用于其活性,因此在进行其它优化之前优化该条件是至关重要的。几种其它二价阳离子可能影响溶素的活性,包括但不限于Mn ,Fe em em> 2 +
  9. 加入1μg的肽聚糖水解蛋白(PlyPy) 注意:需要添加的溶素的量取决于溶素的活性,并且需要根据经验确定。几μg溶素是一个好的起点。
  10. 将96孔板插入SpectraMax Plus读数器,并在37℃孵育板60分钟。摇动平板并每30秒测量OD <600>。
  11. 在Excel表格中绘制数据并分析结果。
  12. 重复步骤9-12,包括在缓冲液中根据经验确定的浓度的必要二价阳离子,但现在优化其他因素的条件,包括但不限于:
    NaCl:0-1,000mM
    DTT:0-20mM
    温度:20-50℃
  13. 要确定最佳pH,请将步骤6,7和9中使用的20 mM Tris-HCl(pH 6.8)交换为您选择的缓冲液,包括但不限于:
    20mM乙酸钠(pH5.0) 20mM乙酸钠(pH 5.5) 20mM乙酸钠(pH6.0) 20mM Tris-HCl(pH6.0) 20mM Tris-HCl(pH6.5) 20mM Tris-HCl(pH7.0) 20mM Tris-HCl(pH7.5) 20mM Tris-HCl(pH8.0) 20mM Tris-HCl(pH8.8)
  14. 最后确定你的lysin的活性:
    1. 在活性缓冲液(根据经验值优化缓冲液)中洗涤并重悬细胞至OD <600> <1.4。
    2. 向96孔板的每个孔中加入100μl细胞和100μl活性缓冲液 注意:考虑到孔中的最大体积将是200μl。因此,将小于100μl活性缓冲液加入也包含溶素(在活性缓冲液中稀释)的孔中。准确的体积取决于溶素的初始浓度。
    3. 加入溶素连续稀释,范围从0.1 - 51.2μg/ml 注意:重复运行所有样品一式三份,以解释任何异常值。
    4. 在SpectraPlus Max读数器中在37℃孵育15分钟,摇动并每30秒测量样品OD 600。
    5. 在Excel表格中绘制数据并分析结果。
    6. 对应于起始OD的50%减少的最接近的稀释度对应于1单位(Loeffler等人,2001)。最特征的溶素具有0.5-1U /μg的活性。

代表数据



图1. PlyPy的活性测定。洗涤的。 化脓性细胞在活性缓冲液中在37℃下用不同浓度的PlyPy孵育15分钟。 在SpectraPlus Max读数器中每30秒测量光密度(600)。 显示一式三份的平均值。 为了简单起见,添加了灰色线,表示在900秒(15分钟)的OD 50%,因此对应于一个单位。 在该代表性图中,2μg/ml接近一个单位; 例如,lysin的活性为0.5U /μg。 注意,具有高浓度溶素的样品中光密度的快速初始下降,强调在加入溶素后尽快阅读样品的必要性。

食谱

  1. PlyPy活动缓冲区
    20mM Tris-HCl(pH 6.8)
    2mM CaCl 2 2 / 100 mM NaCl
    使用前对溶液(0.22μm)进行无菌过滤

致谢

该协议在Lood等人的全篇文章(2014年)中以更为简化的形式出版。 本研究部分由USPHS授权AI11822和Contrafect公司授予V.A.F. 并通过由Tegger基金会授予R.L.

参考文献

  1. Loeffler,J.M.,Nelson,D。和Fischetti,V.A。(2001)。 用噬菌体细胞壁水解酶快速杀死肺炎链球菌。< a> Science 294(5549):2170-2172。
  2. Lood,R.,Raz,A.,Molina,H.,Euler,C.W.and Fischetti,V.A。(2014)。 具有罕见的d-丙氨酰-1-丙氨酸内肽酶活性的高活性和带负电荷的化脓性链球菌溶素 保护小鼠免受链球菌性菌血症。抗微生物剂化疗 58(6):3073-3084。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lood, R. and Fischetti, V. A. (2014). Activity Assays for Bacteriophage Endolysin PlyPy. Bio-protocol 4(18): e1233. DOI: 10.21769/BioProtoc.1233.
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