耻垢分枝杆菌ClpC和ClpX敲低表达菌株的构建及表型分析<sup>*</sup>

doi:10.13523/j.cb.2103030

中国生物工程杂志

2021, Vol. 41

Issue (6): 13-22 DOI: 10.13523/j.cb.2103030

研究报告

耻垢分枝杆菌ClpC和ClpX敲低表达菌株的构建及表型分析^*

白嘉诚¹,迟明哲¹,胡亚文¹,郝梦²,张雪莲^1,^**(

)

1 复旦大学生命科学学院遗传工程国家重点实验室上海 200433
2 宁夏回族自治区第四人民医院银川 750021

Construction and Biological Characteristics of ClpC and ClpX Knock-down Strains in Mycobacterium smegmatis

BAI Jia-cheng¹,CHI Ming-zhe¹,HU Ya-wen¹,HAO Meng²,ZHANG Xue-lian^1,^**(

)

1 State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China
2 The Fourth Hospital of Ningxia Hui Autonomous Region, Yinchuan 750021, China

全文: PDF(1345 KB) HTML

摘要：

蛋白质平衡稳定对细菌的生长繁殖以及应对宿主免疫压力十分重要。Clp蛋白酶复合体在结核分枝杆菌的蛋白质降解和平衡稳定中发挥重要作用。Clp蛋白酶中负责识别底物蛋白并将其解折叠的蛋白质有两种:ClpC和ClpX。为初步探究分枝杆菌中ClpC和ClpX各自的功能特点,运用CRISPRi的方法成功构建了耻垢分枝杆菌的ClpC和ClpX诱导型敲低表达菌株,并对其生长相关表型进行分析。结果显示:与野生菌株相比,ClpC和ClpX的低表达均能严重影响耻垢分枝杆菌的生长。ClpC低表达可导致菌株丧失生物膜的形成能力,而ClpX低表达则导致菌株无法维持正常细胞形态,电镜显示细胞壁不完整且细胞呈丝状化,提示ClpC和ClpX可能在分枝杆菌中具有不同的生理功能。可为后期深入开展ClpC和ClpX对分枝杆菌生理调控功能研究及新型抗结核药物筛选提供基础。

关键词： 耻垢分枝杆菌; ClpC; ClpX

Abstract:

Protein homeostasis through protein quality control is critical for bacterial growth and adaptation to host immune stress. The Clp protease plays an important role in protein degradation and homeostasis in Mycobacterium tuberculosis. There are two proteins in the Clp protease that are responsible for recognizing unfolding substrate proteins:ClpC and ClpX. Then unfoled substract is transferred into the chamber of ClpP, where proteolysis is carried out by protease. In order to explore the respective functional characteristics of ClpC and ClpX in mycobacterium,Mycobacterium smegmatis was selected as the experimental strain, and knockdown strains of ClpC and ClpX were constructed successfully by the CRISPRi method. The results showed that the ClpC and ClpX knockdown strains showed significant growth phenotypes differences compared with wild Mycobacterium smegmatis. Low expression of both ClpC and ClpX severely affected the growth of Mycobacterium smegmatis. ClpC low expression resulted in the loss of biofilm formation ability of the strain, while that of ClpX caused incomplete cell wall and bacterial filamentation indicating that ClpC and ClpX may have different physiological functions in mycobacterium.

Key words: Mycobacterium smegmatis ClpC ClpX

收稿日期: 2021-03-15 出版日期: 2021-07-06

ZTFLH:

Q93

基金资助: * 国家自然科学基金(81971898);国家重点研发计划(2016YFA0500601);新疆建设兵团重点科技攻关项目(2020AB015)

通讯作者: 张雪莲 E-mail: xuelianzhang@fudan.edu.cn

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引用本文:

白嘉诚,迟明哲,胡亚文,郝梦,张雪莲. 耻垢分枝杆菌ClpC和ClpX敲低表达菌株的构建及表型分析^*[J]. 中国生物工程杂志, 2021, 41(6): 13-22.

BAI Jia-cheng,CHI Ming-zhe,HU Ya-wen,HAO Meng,ZHANG Xue-lian. Construction and Biological Characteristics of ClpC and ClpX Knock-down Strains in Mycobacterium smegmatis. China Biotechnology, 2021, 41(6): 13-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2103030 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I6/13

表1 本研究使用的引物

表2 不同细菌间主要蛋白酶降解系统分析

图1 重组pLJR962质粒构建及DNA测序图谱

图2 耻垢分枝杆菌ClpC和ClpX敲低表达菌株转录水平验证

图3 耻垢分枝杆菌ClpC和ClpX敲低表达菌株固体平板生长点板

图4 耻垢分枝杆菌ClpC和ClpX敲低表达菌株生长曲线

图5 耻垢分枝杆菌ClpC和ClpX敲低表达菌株菌落形态

图6 耻垢分枝杆菌ClpC和ClpX敲低表达菌株生物膜

图7 耻垢分枝杆菌ClpC和ClpX敲低表达菌株细胞形态

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