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Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria
分枝杆菌中利福平耐药性突变频率的测定和突变频谱分析   

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Abstract

Understanding the genetic safeguarding mechanism of Mycobacterium tuberculosis (Mtb) may help us to explain i), how Mtb survive the genetic assaults elicited by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host macrophages and ii), why some strains of Mtb, e.g., Mtb strains from East Asian lineage and Beijing sublineage, exhibit high mutation rate and are more likely to acquire drug resistant mutations (e.g., rifampicin-resistance mutation) during infection. Mutation frequency analysis is a basic methods to study the genetic safeguarding mechanism. Moreover, to study the molecular mechanism of mutation, it is necessary to analyse the mutation spectrum (For example, oxidized cytosine may induce CG to TA mutation.). This protocol describes a method to determine the mutation frequency and understand the mutation spectrum in both Mycobacterium smegmatis (Msm) and Mtb.

Keywords: Mycobacterium(结核分枝杆菌), Mutation frequency(突变频率), DNA damage(DNA损伤)

Materials and Reagents

  1. Mycobacterium smegmatis mc2 155 or Mycobacterium tuberculosis H37Rv
  2. Middlebrook 7H9 broth (BD Biosciences, catalog number: 271310 )
  3. Middlebrook 7H11 agar (BD Biosciences, catalog number: 283810 )
  4. Middlebrook OADC (BD Biosciences, catalog number: 212351 )
  5. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  6. Tween 80 (Sigma-Aldrich, catalog number: P1754 )
  7. Rifampicin (Sigma-Aldrich, catalog number: R3501 )
  8. 7H9 medium (see Recipes)
  9. 7H9OADC medium (see Recipes)
  10. 7H11 agar (see Recipes)
  11. 7H11OADC agar (see Recipes)
  12. LBG agar (see Recipes)
  13. PBST (see Recipes)
  14. TE buffer (see Recipes)

Equipment

  1. Incubation shaker
  2. Centrifuge
  3. 37 °C incubator
  4. 100-ml flask
  5. Sterile glass beads
  6. 96-well flat bottom plate
  7. Adhesive film
  8. 96-well PCR plate
  9. PCR instrument
  10. Petri dish (70 mm diameter)

Software

  1. BLAST or ClustalW

Procedure

  1. Determination of rifampicin-resistance mutation frequency
    1. Streak Mycobacterium smegmatis culture on 7H11 agar plate (does not contain rifampicin) and culture at 37 °C for 3 days until colonies are visible.
      For Mtb, streak it on 7H11OADC agar plate and culture at 37 °C for 4 weeks.
    2. Inoculate a single colony in 5 ml 7H9 medium (for Mtb, use 7H9OADC medium) and grow at 37 °C with shaking (150 rpm) until the culture is saturated (OD600~2.0).
    3. The culture is sub-cultured (1% v/v inoculation) into 20 ml 7H9 medium (for Mtb, use 7H9OADC medium) in a 100-ml flask and grown at 37 °C with shaking to exponential phase (OD600=0.4~0.5).
      Note: Culture at this growth stage can be used for stress induction, e.g., oxidative stress (H2O2 treatment) or starvation.
    4. For CFU determination, the culture is serially diluted 1: 100,000 in PBST. 50 μl of the 10-5 dilution is plated on LBG agar plate (for Mtb, use 7H11OADC agar plate). Incubate the plates at 37 °C for 4 days. CFU/ml = (the number of colonies on each plate) x 2 x 106.
      Note: At least three independent dilutions are required for each sample.
    5. 3 ml of the cell culture from step 3 is centrifuged at 3,000 x g for 5 min and the cell pellet is resuspended in 100 μl PBST (The bacilli may form clumps in liquids without Tween 80.). To isolate rifampicin-resistant mutants, the resuspended pellet is spread with sterile glass beads on LBG agar plates containing 250 μg/ml rifampicin and incubated at 37 °C for 4 days.
      For Mtb, the resuspended cell pellet is spread on 7H11OADC agar plate containing 10 μg/ml rifampicin and incubate at 37 °C for 4 weeks.
      Note: Glass or metal spreader is not recommended in this step because the mycobacterial cell may adsorb and clump onto the spreader during plating, which may affect the accuracy of the result.
    6. The spontaneous rifampicin-resistant mutation frequency is calculated by dividing the number of rifampicin-resistant colonies on each plate by the total viable cell count per plate (Figure 1).


      Figure 1. The antimutator role of MazG in Msm (A) and Mtb (B). The frequencies conferring resistance to rifampicin in wild-type (wt), mazG-null (ΔmazG) and the complemented mutant (compl) strains were determined in exponential phase (OD600~0.5) with or without oxidative stress and in the stationary growth phase. Oxidative stress was induced by treating exponential phase cultures with 10 mM H2O2 for 5 h (Msm) or 24 h (Mtb). Stationary phase was at the 5th-day or 28th-day of culture for Msm or Mtb, respectively. The numbers shown are mean ± S.E. of 3 independent experiments totalling 15 cultures of Msm and 6 of Mtb.

  2. Mutation spectrum analysis
    1. A single rifampicin-resistant Msm colony is inoculated into 1 ml 7H9 media containing 100 μg/ml rifampicin in a 96-well flat bottom plate. The plate is then sealed with an adhesive film and incubated at 37 °C for 7 days. Note that at least 30 colonies are required per bacterial sample for statistically significant results.
      To inoculate rifampicin-resistant Mtb colony, use 7H9OADC with 4 μg/ml rifampicin, incubate at 37 °C for 3 weeks.
    2. Cells are then pelleted (4,000 x g, 5 min) and suspended in 50 μl TE buffer and the cell suspension is transferred to a 96-well PCR plate.
    3. The plate is heated in a PCR instrument at 95 °C for 20 min.
    4. The PCR plate is then centrifuged at 12,000 x g for 5 min and the supernatant is transferred to a new 96-well PCR plate.
    5. A region of the genome sequence containing the cluster I region of rpoB is amplified using primers Rpo-forward (5’-CGACCACTTCGGCAACCG-3’) and Rpo-reverse (5’-CGATCAGACCGATGTTGG-3’). For Mtb, using primer RpoTB-forward (5’-ATCACACCGCAGACGTTG-3’) and RpoTB-reverse (5’-TGCATCACAGTGATGTAGTCG-3’).
      PCR mix: 2 μl DNA template, 0.4 ul of each primer (10 μM), 1 U Pfu DNA polymerase, 0.6 μl DMSO, 0.4 μl dNTPs (10 mM each), H2O to 20 μl.
      PCR cycling: 95 °C 60 s, 30 cycles of 95 °C 20 s, 58 °C 10 s and 72 °C 40 s.
      Note: Most of the rifampicin-resistance mutations are occurred in the cluster I region of rpoB. If no mutation is detected in this region, it may need to amplify the whole rpoB gene (~3.5 Kbp) for sequencing.
    6. The PCR products are then bi-directionally sequenced and the mutation spectrum of the sequenced region is identified by BLAST or ClustalW.
    7. Calculate the mutation frequency of each specific mutation spectrum (Table 1).
      Mutation frequency of a given mutation spectrum = (the proportion of the mutation spectrum in strain A) x (the mutation frequency of strain A). For example, the mutation frequency of Msm is 10 x 10-8, the proportion of CG-TA mutation in Msm derived rifampicin-resistant mutants is 80%, therefore, the CG-TA mutation frequency will be 0.8 x 10 x 10-8=8 x 10-8.

      Table 1. mazG-null Msm exhibited elevated CG to TA mutation under oxidative stress conditions and in stationary phase. Spontaneous rifampicin-resistant colonies were collected from 3 independent experiments . Cluster I region of rpoB were PCR-amplified using Pfu DNA polymerase and sequenced bi-directionally. All of the sequenced colonies contain single non-synonymous mutations. wt, wild-type Msm; ΔmazG, mazG-null Msm.

Recipes

  1. 7H9 medium (100 ml)
    Dissolve 0.47 g of 7H9 broth in 80 ml dH2O
    Add 0.4 ml 50% glycerol and 0.5 ml 10% Tween 80
    dH2O to 100 ml
    Sterilized by autoclaving
    Stored at 4 °C
  2. 7H9OADC medium (100 ml)
    Dissolve 0.47 g of 7H9 broth in 80 ml dH2O
    Add 0.4 ml 50% glycerol and 0.5 ml 10% Tween 80
    dH2O to 90 ml
    Sterilized by autoclaving
    Cool to 50-55 °C and add 10 ml OADC
    Stored at 4 °C
  3. 7H11 agar (100 ml)
    Dissolve 2.1 g of 7H11 agar with 99 ml dH2O
    Add 1 ml 50% glycerol
    Sterilized by autoclaving
    Stored at 4 °C
  4. 7H11OADC agar (100 ml)
    Dissolve 2.1 g of 7H11 agar with 89 ml dH2O
    Add 1 ml 50% glycerol
    Sterilize by autoclaving
    Cool to 50-55 °C and add 10 ml OADC
    Stored at 4 °C
  5. LBG agar (100 ml)
    Dissolve 1 g tryptone, 0.5 g yeast extract, 1 g NaCl and 1.5 g agar with 99 ml dH2O
    Add 1 ml 50% glycerol
    Sterilized by autoclaving
    Stored at 4 °C
  6. PBST
    Dissolve the following in 800 ml of dH2O
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    5 ml of 10% Tween 80
    Adjust pH to 7.2
    Adjust volume to 1 L with dH2O
    Sterilized by autoclaving
  7. TE buffer
    10 mM Tris-HCl (pH 8.0)
    1 mM EDTA

Acknowledgments

This protocol was adapted from a previously published paper Lyu et al. (2013). This work was supported by grants from the National Natural Science Foundation of China (No. 30970077, 31121001, 31300126), the Research Unit Fund of Li Ka Shing Institute of Health Sciences (No. 7103506), the Hong Kong Health and Medical Research Fund (No. 12110622), the China Postdoctoral Science Foundation (No. 20110490754), the SIBS Postdoctoral Research Fund (No. 2011KIP509).

References

  1. Lyu, L. D., Tang, B. K., Fan, X. Y., Ma, H. and Zhao, G. P. (2013). Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP. PLoS Pathog 9(12): e1003814.

简介

了解结核分枝杆菌(Mtb)的遗传保护机制可以帮助我们解释i)如何Mtb存活由活性氧(ROS)和活性氮(RNS)产生的遗传攻击 宿主巨噬细胞和ii),为什么一些来自东亚谱系和北京亚系的Mtb菌株(例如eg)具有高突变率,并且更可能获得耐药突变(例如,利福平 - 抗性突变)。 突变频率分析是研究遗传保护机制的基本方法。 此外,为了研究突变的分子机制,有必要分析突变谱(例如,氧化的胞嘧啶可能诱导CG到TA突变)。 该协议描述了确定耻骨分枝杆菌(Msm)和Mtb中的突变频率和了解突变谱的方法。

关键字:结核分枝杆菌, 突变频率, DNA损伤

材料和试剂

  1. 耻垢分枝杆菌 mc 2 155或结核分枝杆菌 H37Rv
  2. Middlebrook 7H9肉汤(BD Biosciences,目录号:271310)
  3. Middlebrook 7H11琼脂(BD Biosciences,目录号:283810)
  4. Middlebrook OADC(BD Biosciences,目录号:212351)
  5. 甘油(Sigma-Aldrich,目录号:G5516)
  6. 吐温80(Sigma-Aldrich,目录号:P1754)
  7. 利福平(Sigma-Aldrich,目录号:R3501)
  8. 7H9培养基(见配方)
  9. 7H9OADC介质(参见配方)
  10. 7H11琼脂(见Recipes)
  11. 7H11OADC琼脂(见配方)
  12. LBG琼脂(见配方)
  13. PBST(参见配方)
  14. TE缓冲区(参见配方)

设备

  1. 孵育振动器
  2. 离心机
  3. 37℃孵育器
  4. 100 ml烧瓶
  5. 无菌玻璃珠
  6. 96孔平底板
  7. 胶粘剂
  8. 96孔PCR板
  9. PCR仪
  10. 培养皿(直径70mm)

软件

  1. BLAST或ClustalW

程序

  1. 利福平耐药突变频率的测定
    1. 条纹在7H11琼脂平板上的耻垢分枝杆菌(Mycobacterium smegmatis)培养物(不是 含有利福平)并在37℃下培养3天直到菌落为止 可见。
      对于Mtb,将其在7H11OADC琼脂平板上划线,并在37℃下培养4周
    2. 接种单菌落在5ml 7H9培养基(对于Mtb,使用7H9OADC 培养基),并在37℃下振荡(150rpm)生长直至培养物 饱和(OD 600〜2.0)。
    3. 将培养物继代培养(1%v/v 接种)到20ml 7H9培养基(对于Mtb,使用7H9OADC培养基)中 并在37℃下振荡生长至指数期 (OD <600> = 0.4〜0.5)。
      注意:在该生长阶段的培养可用于应激诱导,例如氧化应激(H 2 2 治疗)或饥饿。
    4. 对于CFU测定,将培养物连续稀释1:100,000in PBST。将50μl的10 -5稀释液涂布在LBG琼脂板(用于Mtb, 使用7H11OADC琼脂板)。在37℃下孵育平板4天。 CFU/ml =(每个平板上的菌落数)×2×10 6 。 注意:每个样品至少需要三次稀释。
    5. 将3ml来自步骤3的细胞培养物在3,000xg离心5分钟 min,将细胞沉淀重悬于100μlPBST(杆菌可以 在没有吐温80的液体中形成团块。分离 利福平抗性突变体,重悬的颗粒被扩散 无菌玻璃珠在含有250μg/ml利福平的LBG琼脂平板上 并在37℃下孵育4天。
      对于Mtb,重悬细胞 将沉淀铺在含有10μg/ml利福平的7H11OADC琼脂平板上 并在37℃下孵育4周。
      注意:玻璃或金属吊具是 不推荐在此步骤中,因为分枝杆菌细胞可能吸附 并在电镀期间聚集在铺展器上,这可能影响 结果的准确性。
    6. 自发性利福平耐药 突变频率是通过除以数目来计算的 每个板上的利福平抗性集落由总活细胞 每板数(图1)。


      图1. MazG的antimutator角色  Msm(A)和Mtb(B)。赋予抗性的频率 利福平在野生型(wt),mazG -null(ΔmazG)和补充 在指数期(OD 600〜0.5)中测定突变(compl)菌株, 有或没有氧化应激和在静止生长期。 通过用处理指数期培养物诱导氧化应激  (Msm)或24小时(Mtb)的10mM H 2 O 2 O 2。固定相位于 分别为Msm或Mtb的培养的第5天或第28天。号码 显示为平均值±S.E。 的3个独立实验,共15个 Msm的培养物和Mtb的6。

  2. 突变谱分析
    1. 将单个利福平抗性Msm菌落接种到1ml 7H9中 在96孔平底板中含有100μg/ml利福平的培养基。 然后将板用粘性膜密封并在37℃下温育 7天。 注意,每个细菌至少需要30个菌落 具有统计显着性结果的样本 为了接种利福平抗性Mtb菌落,使用7H9OADC与4μg/ml利福平,在37℃孵育3周。
    2. 然后将细胞沉淀(4,000×g,5分钟),并悬浮于50μlTE中 缓冲液,将细胞悬浮液转移到96孔PCR板中
    3. 将板在PCR仪中在95℃加热20分钟
    4. 然后将PCR板在12,000×g离心5分钟,将上清液转移至新的96孔PCR板。
    5. 使用引物Rpo-forward(5'-CGACCACTTCGGCAACCG-3')和包含rpoB 的I区的基因组序列的区域扩增 Rpo-反向(5'-CGATCAGACCGATGTTGG-3')。对于Mtb,使用引物 RpoTB正向(5'-ATCACACCGCAGACGTTG-3')和RpoTB反向 (5'-TGCATCACAGTGATGTAGTCG-3') PCR混合物:2μlDNA模板,0.4μl 每种引物(10μM),1U pfu DNA聚合酶,0.6μlDMSO,0.4μldNTP (各10mM),H 2 O至20μl。
      PCR循环:95℃60秒,95℃20秒,58℃10秒和72℃40秒的30个循环。
      注意:  大多数利福平 - 抗性突变发生在簇中 rpoB的I区。 如果在这个区域没有检测到突变,它可能 需要扩增整个rpoB基因(〜3.5 Kbp)进行测序。
    6. 的   然后对PCR产物进行双向测序和突变 通过BLAST或ClustalW鉴定测序区域的光谱
    7. 计算每个特定突变光谱的突变频率(表1)。
      突变   给定突变频率的频率=(比例 菌株A的突变谱)×(菌株A的突变频率)。 例如,Msm的突变频率为10×10 -8 -8 /比例   的M-M衍生的利福平抗性突变体中的CG-TA突变是80% 因此,CG-TA突变频率将为0.8×10×10 -8 -8 = 8× 10 -8

      表1 mazG -null Msm表现出升高的CG至TA突变 在氧化应激条件下和在稳定期。 自发 从3个独立的收集利福平抗性集落 实验。 使用pfu DNA将PCR扩增rpoB 的I区   聚合酶并双向测序。 所有的排序 集落含有单个非同义突变。 wt,野生型Msm; Δ mazG , mazG -null Msm。

食谱

  1. 7H9培养基(100ml) 将0.47g 7H9肉汤溶解在80ml dH 2 O中 加入0.4ml 50%甘油和0.5ml 10%吐温80 dH 2 O至100毫升
    高压灭菌
    灭菌 储存在4°C
  2. 7H9OADC培养基(100ml) 将0.47g 7H9肉汤溶解在80ml dH 2 O中 加入0.4ml 50%甘油和0.5ml 10%吐温80 dH 2 O至90ml
    高压灭菌
    灭菌 冷却至50-55℃,加入10ml OADC
    储存在4°C
  3. 7H11琼脂(100ml) 将2.1g 7H11琼脂与99ml dH 2 O溶解 加入1ml 50%甘油
    高压灭菌
    灭菌 储存在4°C
  4. 7H11OADC琼脂(100ml) 将2.1g 7H11琼脂与89ml dH 2 O溶解 加入1ml 50%甘油
    高压灭菌
    灭菌 冷却至50-55℃,加入10ml OADC
    储存在4°C
  5. LBG琼脂(100ml) 将1g胰蛋白胨,0.5g酵母提取物,1g NaCl和1.5g琼脂与99ml dH 2 O溶解。
    加入1ml 50%甘油
    高压灭菌
    灭菌 储存在4°C
  6. PBST
    将以下物质溶解在800ml dH 2 O 2中 8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    5ml 10%吐温80 将pH调节至7.2
    使用dH <2> O调节音量至1 L
    高压灭菌
    灭菌
  7. TE缓冲区
    10mM Tris-HCl(pH8.0) 1mM EDTA

致谢

该协议改编自先前发表的论文Lyu等人。(2013)。 这项工作得到中国国家自然科学基金(30970077,31121001,31300126),李嘉诚健康科学研究所研究单位基金(7103506),香港健康和医学研究基金 (No. 12110622),中国博士后科学基金(No. 20110490754),SIBS博士后研究基金(No. 2011KIP509)。

参考文献

  1. Lyu,L.D.,Tang,B.K.,Fan,X.Y.,Ma,H。和Zhao,G.P。(2013)。 分枝杆菌MazG通过清除5-OH-dCTP来保护基因稳定性。 PLoS Pathog 9(12):e1003814。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lyu, L. and Zhao, G. (2014). Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria. Bio-protocol 4(13): e1168. DOI: 10.21769/BioProtoc.1168.
  2. Lyu, L. D., Tang, B. K., Fan, X. Y., Ma, H. and Zhao, G. P. (2013). Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP. PLoS Pathog 9(12): e1003814.
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