大肠杆菌离体重组系统转运FhaB突变体

doi:10.16352/j.issn.1001-6325.2022.04.003

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (4): 565-569.doi: 10.16352/j.issn.1001-6325.2022.04.003

大肠杆菌离体重组系统转运FhaB突变体

黄丕英, 蔡锟, 韦良婉, 王星媛, 王哲, 储引娣^*, 范恩国

中国医学科学院基础医学研究所北京协和医学院基础学院病原生物学系医学分子生物学国家重点实验室,北京 100005

收稿日期:2021-11-26 修回日期:2022-01-14 出版日期:2022-04-05 发布日期:2022-04-01
通讯作者: * cylah@ibms.pumc.edu.cn
基金资助:
国家自然科学基金(31770901);国家重点实验室专项资金(2060204); 中国医学科学院医学与健康科技创新工程(2021-I2M-1-016);高等学校学科创新引智计划2.0(BP0820029)

Escherichia coli in vitro recombinant system transports FhaB mutants

HUANG Pi-ying, CAI Kun, WEI Liang-wan, WANG Xing-yuan, WANG Zhe, CHU Yin-di^*, FAN En-guo

State Key Laboratory of Medical Molecular Biology, Department of Microbiology and Parasitology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China

Received:2021-11-26 Revised:2022-01-14 Online:2022-04-05 Published:2022-04-01
Contact: * cylah@ibms.pumc.edu.cn

摘要/Abstract

摘要： 目的探讨离体重组系统用于双组分转运系统机制研究的可行性。方法通过构建不同分子质量大小的FhaB突变体,制备成原生质球,加入到FhaC蛋白脂质体中,使用蛋白酶K消化的方法检测所构建的离体重组体系转运不同大小底物的能力。结果成功构建突变体后,检测到原生质球分泌的37、60和80 ku大小的条带,并且含FhaC的蛋白脂质体能转运这3种分子质量大小的底物,此次所转运的底物分子质量高达80 ku。结论体外构建的重组转运系统可以转运大分子质量的底物,可尝试使用该体系进行双组分系统机制的研究。

关键词: 双组分转运系统, 突变体, FhaC, FhaB

Abstract: Objective To explore the mechanism of two-partner secretion with an in vitro reconstitution system. Methods Spheroplasts were used to construct FhaB mutants with different molecular weights, and then combined with FhaC containing proteoliposomes to perform the translocation experiments followed by digestion with proteinase K to test the transportation of substrates with different sizes in vitro by this recombinant system. Results After successful construction of the mutant, 37 ku, 60 ku and 80 ku bands secreted by spheroplasts were detected, and FhaC-containing protein liposomes could transport these three substrates with molecular weight as high as 80 ku. Conclusions The recombinant transport system constructed in vitro may transport substrates with large molecular weight and the mechanism of the two-partner secretion can be studied by this system.

Key words: two-partner secretion, mutants, FhaC, FhaB

中图分类号:

R378.99

黄丕英, 蔡锟, 韦良婉, 王星媛, 王哲, 储引娣, 范恩国. 大肠杆菌离体重组系统转运FhaB突变体[J]. 基础医学与临床, 2022, 42(4): 565-569.

HUANG Pi-ying, CAI Kun, WEI Liang-wan, WANG Xing-yuan, WANG Zhe, CHU Yin-di, FAN En-guo. Escherichia coli in vitro recombinant system transports FhaB mutants[J]. Basic & Clinical Medicine, 2022, 42(4): 565-569.

参考文献

[1] Bernstein HD. Type V secretion in gram-negative bacteria[J]. EcoSal Plus, 2019, 8. doi:10.1128/ecosalplus.ESP-0031-2018.
[2] Fan E, Chauhan N, Udatha DBRKG, et al. Type V secretion systems in bacteria[J]. Microbiol Spectr, 2016, 4. doi: 10.1128/microbiolspec.VMBF-0009-2015.
[3] Jacob-Dubuisson F, Guérin J, Baelen S, et al. Two-partner secretion: as simple as it sounds?[J]. Res Microbiol, 2013, 164:583-595.
[4] 张艳娟, 王哲, 蔡锟, 等. 革兰阴性菌中分子伴侣在双组分蛋白分泌至周质空间过程中的作用[J]. 基础医学与临床, 2020, 40:456-459.
[5] Nash ZM, Cotter PA. Bordetella filamentous hemagglu-tinin, a model for the two-partner secretion pathway[J]. Microbiol Spectr, 2019, 7. doi: 10.1128/microbiolspec.PSIB-0024-2018.
[6] Coutte L, Antoine R, Drobecq H, et al. Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway[J]. EMBO J, 2001, 20:5040-5048.
[7] Fan E, Fiedler S, Jacob-Dubuisson F, et al. Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane[J]. J Biol Chem, 2012, 287:2591-2599.
[8] Jacob-Dubuisson F, Buisine C, Mielcarek N, et al. Amino-terminal maturation of the Bordetella pertussis filamentous haemagglutinin[J]. Mol Microbiol, 1996, 19:65-78.
[9] Fan E, Norell D, Müller M. An in vitro assay for substrate translocation by FhaC in liposomes[J]. Methods Mol Biol, 2015, 1329:111-125.
[10] Jacob-Dubuisson F, Villeret V, Clantin B, et al. First structural insights into the TpsB/Omp85 superfamily[J]. Biol Chem, 2009, 390:675-684.
[11] Nash ZM, Cotter PA. Regulated, sequential processing by multiple proteases is required for proper maturation and release of Bordetella filamentous hemagglutinin[J]. Mol Microbiol, 2019,112:820-836.
[12] Malinverni JC, Silhavy TJ. Assembly of outer membrane β-barrel proteins: the Bam complex[J]. EcoSal Plus, 2011, 4. doi: 10.1128/ecosalplus.4.3.8.
[13] Ieva R, Tian P, Peterson JH, et al. Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain[J]. Proc Natl Acad Sci U S A, 2011, 108:E383-E391.

大肠杆菌离体重组系统转运FhaB突变体

Escherichia coli in vitro recombinant system transports FhaB mutants

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价