分枝杆菌利福平耐药基因<italic style="font-style: italic">rpoB</italic>高通量突变文库的构建

陈勇; 冯思源; 徐霖; 田国宝

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0413

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分枝杆菌利福平耐药基因rpoB高通量突变文库的构建

基础研究 | 更新时间：2024-02-19

- 分枝杆菌利福平耐药基因rpoB高通量突变文库的构建
- Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB
- 中山大学学报（医学科学版） 2023年44卷第4期页码：634-641
- 作者机构：
  
  1.成都医学院医学检验系，四川成都 610500
  2.中山大学中山医学院免疫学与微生物学系，广东广州 510080
- 作者简介：
  
  陈勇，研究方向：临床细菌耐药与感染，E-mail：cy-yongchen@outlook.com
- 基金信息：
  
  国家自然科学基金(81830103;82061128001)
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0413
  中图分类号： R378
- 纸质出版日期：2023-07-20，
  
  收稿日期：2023-03-17，
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陈勇,冯思源,徐霖等.分枝杆菌利福平耐药基因rpoB高通量突变文库的构建[J].中山大学学报（医学科学版）,2023,44(04):634-641.

CHEN Yong,FENG Si-yuan,XU lin,et al.Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(04):634-641.
陈勇,冯思源,徐霖等.分枝杆菌利福平耐药基因rpoB高通量突变文库的构建[J].中山大学学报（医学科学版）,2023,44(04):634-641. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0413.

CHEN Yong,FENG Si-yuan,XU lin,et al.Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(04):634-641. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0413.

摘要

目的

建立利福平耐药基因

rpoB

高通量突变文库。

方法

通过同源重组和L5

attB

噬菌体整合位点交换构建耻垢分枝杆菌Δ

rpoB

attB

：：

rpoB

菌株；基于L5

attB

质粒交换系统和抗性选择培养基，挑取48个克隆以验证质粒替换的效率；针对利福平耐药决定区（RRDR）区域内以每３个碱基为单位设计简并性引物，通过PCR扩增获得该区域81个碱基的全覆盖式突变文库；文库片段与载体进行无缝克隆转化到大肠杆菌中形成大肠杆菌突变文库；基于质粒交换原理，将突变质粒库转化至耻垢分枝杆菌

ΔrpoB attB

：：

rpoB

菌株中，替换原有L5

attB

位点质粒，形成耻垢分枝杆菌突变文库；通过基因组提取、建库和高通量测序明确文库的基因型种类。

结果

与野生型

rpoB

基因（5 600 bp）相比，敲除株的扩增片段为2 200 bp，证明耻垢分枝杆菌

ΔrpoB attB

：：

rpoB

条件性敲除株构建成功；质粒替换的成功率为100%；大肠杆菌文库和耻垢分枝杆菌文库中单氨基酸突变类型均为540种，大肠杆菌文库多点突变5 301种，耻垢分枝杆菌文库多点突变853种，大肠杆菌文库和耻垢分枝杆菌文库相关系数为0.84。

结论

开发了一种针对分枝杆菌必需基因

rpoB

突变体文库的构建策略，成功建立了利福平耐药基因

rpoB

的突变文库。

Abstract

Objective

To establish a mutation library of rifampicin resistance gene

rpoB

Methods

The Δ

rpoB attB：：rpoB

strain of

Mycobacterium smegmatis

（

M. smegmatis

） be constructed by homologous recombination and L5

attB

phage integration site exchange. Based on the L5

attB

plasmid exchange system and resistance selection medium， 48 clones are selected to verify plasmid replacement efficiency. Degenerate primers are designed every 3 bases in the rifampicin resistance determining region （RRDR）， and a full-coverage mutation library of 81 bases in RRDR region is obtained by PCR amplification. The library fragments are seamlessly cloned into the vector and transformed into

Escherichia coli

（

E. coli

）to form an

E. coli

mutation library. Based on the principle of plasmid exchange， the mutant plasmid library is transformed into the

M. smegmatis

strain

ΔrpoB attB：：rpoB

， and the original L5

attB

site plasmid is replaced to form the

M. smegmatis

mutant library. The genotype of the library are determined by genome extraction， library construction and high-throughput sequencing.

Results

Compared with the wild-type

rpoB

gene （5 600 bp）， the amplified fragment of the

rpoB

knockout strain is 2 200 bp， which proved that the Δ

rpoB

attB：

：

rpoB

conditional knockout strain of

M. smegmatis

is successfully constructed. The success rate of plasmid replacement is 100%. There were 540 kinds of single amino acid mutations in both

E. coli

library and

M. smegmatis

library， 5 301 kinds of multi-point mutations in

E. coli

library， and 853 kinds of multi-point mutations in

M. smegmatis

library. The correlation coefficient between

E. coli

library and

M. smegmatis

library is 0.84.

Conclusions

We have developed a strategy to construct a library of mutants targeting the essential mycobacterial gene

rpoB

， and successfully established a mutant library of rifampicin resistance gene

rpoB.

关键词

分枝杆菌利福平耐药突变文库rpoB

Keywords

mycobacteriumrifampicin resistancelibrary of mutationsrpoB

references

Jamil SM， Oren E， Garrison GW， et al. Diagnosis of tuberculosis in adults and children［J］. Annals ATS， 2017， 14（2）： 275-278.

Abdel-Aziz M， Wright A， Laszlo A， et al. Epidemiology of antituberculosis drug resistance （the global project on anti-tuberculosis drug resistance surveillance）： an updated analysis［J］. Lancet， 2006， 368（9553）： 2142-2154.

Muda MR， Harun SN， Syed-Sulaiman SA， et al. Population pharmacokinetics analyses of rifampicin in adult and children populations： a systematic review［J］. Br J Clin Pharmacol， 2022， 88（7）： 3132-3152.

Sharma P， Singh R. GeneXpert MTB/RIF based detection of rifampicin resistance and common mutations in rpoB gene of mycobacterium tuberculosis in tribal population of district anuppur， madhya pradesh， India［J］. J Clin Diagn Res， 2020， 14（9）： LM01-LM03.

Brandis G， Wrande M， Liljas L， et al. Fitness-compensatory mutations in rifampicin-resistant RNA polymerase［J］. Mol Microbiol， 2012， 85（1）： 142-151.

Evangelopoulos D， Prosser GA， Rodgers A， et al. Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes［J］. Nat Commun， 2019， 10（1）： 4177.

Bacon J， Hatch KA， Allnutt J. Application of continuous culture for measuring the effect of environmental stress on mutation frequency in mycobacterium tuberculosis［J］. Methods Mol Biol， 2010， 642： 123-140.

Castaneda-Garcia A， Martin-Blecua I， Cebrian-Sastre E， et al. Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies［J］. Sci Adv， 2020， 6（7）： eaay4453.

Yadon AN， Maharaj K， Adamson JH， et al. A comprehensive characterization of PncA polymorphisms that confer resistance to pyrazinamide［J］. Nat Commun， 2017， 8（1）： 1-10.

Feng SY， Liang LJ， Shen C， et al. A CRISPR-guided mutagenic DNA polymerase strategy for the detection of antibiotic-resistant mutations in M. tuberculosis［J］. Mol Ther Nucleic Acids， 2022， 29： 354-367.

Pashley CA， Parish T. Efficient switching of mycobacteriophage L5-based integrating plasmids in mycobacterium tuberculosis［J］. Fems Microbiol Lett， 2003， 229（2）： 211-215.

Borgers K， Vandewalle K， Festjens N， et al. A guide to mycobacterium mutagenesis［J］. Febs J， 2019， 286（19）： 3757-3774.

GoudeRenan， David M. Roberts and tanya parish， electroporation of mycobacteria ［M］. Methods in Molecular Biology， 2015：117-130.

Kim JH， Wei JR， Wallach JB， et al. Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase［J］. Nucleic Acids Res， 2011， 39（6）： 2210-2220.

Cascioferro A， Boldrin F， Serafini A， et al. Xer site-specific recombination， an efficient tool to introduce unmarked deletions into mycobacteria［J］. Appl Environ Microbiol， 2010， 76（15）： 5312-5316.

Wu KJ， Boutte CC， Ioerger TR， et al. Mycobacterium smegmatis HtrA blocks the toxic activity of a putative cell wall amidase［J］. Cell Rep， 2019， 27（8）： 2468-2479. e3.

World Health Organization. Global tuberculosis report 2022［EB/OL］. （2022-10-15）［2022-12-20］. https：//linkinghub.elsevier.com/retrieve/pii/S2666-5247（22）00359-7https://linkinghub.elsevier.com/retrieve/pii/S2666-5247(22)00359-7.

Mariam DH， Mengistu Y， Hoffner SE， et al. Effect of rpoB mutations conferring rifampin resistance on fitness of mycobacterium tuberculosis［J］. Antimicrob Agents Chemother， 2004， 48（4）： 1289-1294.

Gagneux S， Long CD， Small PM， et al. The competitive cost of antibiotic resistance in mycobacterium tuberculosis［J］. Science， 2006， 312（5782）： 1944-1946.

Kurepina N， Chudaev M， Kreiswirth BN， et al. Mutations compensating for the fitness cost of rifampicin resistance in escherichia coli exert pleiotropic effect on RNA polymerase catalysis［J］. Nucleic Acids Res， 2022， 50（10）： 5739-5756.

Comas I， Borrell S， Roetzer A， et al. Whole-genome sequencing of rifampicin-resistant mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes［J］. Nat Genet， 2012， 44（1）： 106-110.

Windels EM， Michiels JE， Fauvart M， et al. Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates［J］. ISME J， 2019， 13（5）： 1239-1251.

Ostrer L， Ji YD， Khodursky A. Identification and characterization of pleiotropic high-persistence mutations in the beta subunit of the bacterial RNA polymerase［J］. Antimicrob Agents Chemother， 2021， 65（11）： e0052221.

Feng SY， Wu ZX， Liang WF， et al. Prediction of antibiotic resistance evolution by growth measurement of all Proximal mutants of beta-lactamase［J］. Mol Biol Evol， 2022， 39（5）： msac086.

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分枝杆菌利福平耐药基因rpoB高通量突变文库的构建

Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB

DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0413

摘要

Abstract

关键词

Keywords

references