硫醇乙酰基转移酶mshD对结核分枝杆菌生长和应对压力刺激的研究

doi:10.19982/j.issn.1000-6621.20240184

摘要/Abstract

摘要：

目的: 探索硫醇乙酰基转移酶MshD对结核分枝杆菌在体外生长、应对压力刺激和活性氧(reactive oxygen species,ROS)水平的影响。方法: 利用CRISPR-NHEJ基因编辑技术,构建结核分枝杆菌mshD基因敲除株(ΔmshD株)和回补株(ΔmshD::mshD株)。分别检测H37Rv野生株(WT)、ΔmshD株和ΔmshD::mshD株在液体培养基和固体培养基中的生长情况,以及外源添加L-半胱氨酸和过氧化氢酶对菌株生长的影响;检测3种菌株对不同刺激剂如H₂O₂、二硫苏糖醇(DTT)、十二烷基硫酸钠(SDS)的敏感性,以及外源添加过氧化氢酶对压力条件处理后菌株的恢复情况;采用流式细胞术检测SDS处理3种菌株前后菌株的ROS水平。结果: 与WT和ΔmshD::mshD株相比,ΔmshD株在固体平板上的生长较为缓慢,外源添加过氧化氢酶可以恢复其生长趋势;ΔmshD株应对DTT [WT(6.96±2.02)%,ΔmshD(0.02±0.00)%,ΔmshD::mshD(6.64±0.77)%;F=29.700,P<0.001],H₂O₂[WT(0.23±0.06)%,ΔmshD(0.01±0.00)%,ΔmshD ::mshD(0.26±0.06)%;F=25.520,P=0.001]和SDS[WT(0.12±0.03)%,ΔmshD(0.01±0.00)%,ΔmshD::mshD(0.18±0.04)%;F=19.540,P=0.002]刺激的存活率更低,差异均有统计学意义;外源添加过氧化氢酶可以恢复其存活率。ΔmshD株自身ROS水平高于WT(WT:95.100±2.553,ΔmshD:106.000±4.000,ΔmshD::mshD:94.667±3.055;F=11.650,P=0.009),SDS处理ΔmshD株的自身ROS水平进一步升高(WT:436.000±8.000,ΔmshD:533.667±4.726,ΔmshD::mshD:441.333±2.517;F=292.900,P<0.001),差异均有统计学意义。结论: mshD基因缺失在固体培养基中生长减慢,mshD基因协助结核分枝杆菌抵抗各种应激压力,ΔmshD菌株内源性ROS水平增加。外源添加过氧化氢酶可一定程度恢复mshD基因敲除株生长和存活缺陷。

关键词: 分枝杆菌,结核, 乙酰基转移酶类, 过氧化氢酶, 活性氧

Abstract:

Objective: To investigate the influence of thiol acetyltransferase MshD on the growth dynamics, stress resilience, and reactive oxygen species (ROS) modulation in Mycobacterium tuberculosis (MTB) under in vitro conditions. Methods: Employing CRISPR-NHEJ gene editing, this study established an mshD gene knockout strain (ΔmshD) and a complemented strain (ΔmshD::mshD). We monitored the growth trajectories of the H37Rv wild-type strain (WT), the ΔmshD strain, and the ΔmshD::mshD strain in both liquid and solid media. Additionally, we assessed the impact of exogenously added L-cysteine and catalase on the growth of these strains. The sensitivity of the strains to various stressors, including H₂O₂, dithiothreitol (DTT), and sodium dodecyl sulfate(SDS), and their recovery post-stress intervention with exogenous catalase were evaluated. Furthermore, flow cytometry was utilized to measure the ROS levels in the strains both prior to and following SDS exposure. Results: The ΔmshD strain exhibited significantly slower growth on solid media compared to the WT and ΔmshD::mshD strains. However, the introduction of exogenous catalase reinstated their growth patterns to near-normal levels. In survival assays, the ΔmshD strain showed markedly reduced resilience against DTT (WT: (6.96±2.02) %, ΔmshD: (0.02±0.00) %, ΔmshD::mshD: (6.64±0.77) %; F=29.700, P<0.001), H₂O₂ (WT: (0.23±0.06) %, ΔmshD: (0.01±0.00) %, ΔmshD::mshD: (0.26±0.06) %; F=25.520, P=0.001), and SDS (WT: (0.12±0.03) %, ΔmshD: (0.01±0.00) %, ΔmshD::mshD: (0.18±0.04) %; F=19.540, P=0.002), with all differences reaching statistical significance. Catalase supplementation notably restored the survival rate of the ΔmshD strain. Additionally, ROS levels in the ΔmshD strain were elevated compared to WT (WT: 95.100±2.553, ΔmshD: 106.000±4.000, ΔmshD::mshD: 94.667±3.055; F=11.650, P=0.009) and further increased following SDS exposure (WT: 436.000±8.000, ΔmshD: 533.667±4.726, ΔmshD::mshD: 441.333±2.517; F=292.900, P<0.001), underlining significant oxidative stress responses. Conclusion: Deletion of the mshD gene impairs growth in solid culture mediums and diminishes MTB’s ability to withstand various stressors. The ΔmshD strain exhibited elevated endogenous ROS levels. Importantly, the exogenous application of catalase was able to partially rectify the growth and survival deficits observed in the ΔmshD strain.

Key words: Mycobacterium tuberculosis, Acetyltransferases, Catalase, Reactive oxygen species

中图分类号:

R52
R378.91

张蓝月, 王颖超, 刘唯夷, 尚雪恬, 贾红彦, 朱传智, 张宗德, 潘丽萍. 硫醇乙酰基转移酶mshD对结核分枝杆菌生长和应对压力刺激的研究[J]. 中国防痨杂志, 2024, 46(8): 935-941. doi: 10.19982/j.issn.1000-6621.20240184

Zhang Lanyue, Wang Yingchao, Liu Weiyi, Shang Xuetian, Jia Hongyan, Zhu Chuanzhi, Zhang Zongde, Pan Liping. Study on the effect of thiol acetyltransferase mshD on the growth and stress response of Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2024, 46(8): 935-941. doi: 10.19982/j.issn.1000-6621.20240184

图/表 7

表1

表2

表3

WT、ΔmshD和ΔmshD::mshD菌株的体外生长速率比较

测定时间 (d)	A₆₀₀值( $x ˉ$ ±s)			F值	P值
测定时间 (d)	WT	ΔmshD	ΔmshD::mshD	F值	P值
0	0.040±0.001	0.040±0.001	0.040±0.001	1.000	0.422
3	0.223±0.007	0.212±0.006	0.237±0.013	5.120	0.050
8	0.593±0.013	0.636±0.029	0.663±0.032	5.484	0.044
10	0.839±0.038	0.818±0.034	0.885±0.008	4.160	0.074
14	1.493±0.083	1.437±0.035	1.543±0.048	2.417	0.170

表3

图1

表4

WT、ΔmshD和ΔmshD::mshD菌株经多种应激压力处理前后存活情况比较

菌株类型	菌落形成单位(CFU, $x ˉ$ ±s)				处理后存活率(%, $x ˉ$ ±s)
菌株类型	处理前 (×10⁵)	DTT处理 (×10⁴)	H₂O₂处理 (×10³)	SDS处理 (×10³)	DTT	H₂O₂	SDS
WT	900.00±72.11	626.67±181.48	206.67±50.33	106.67±30.55	6.96±2.02	0.23±0.06	0.12±0.03
ΔmshD	680.00±91.65	1.20±0.20	8.67±3.06	9.33±3.06	0.02±0.00	0.01±0.00	0.01±0.00
ΔmshD::mshD	913.33±90.18	606.67±70.24	240.00±52.92	160.00±40.00	6.64±0.77	0.26±0.06	0.18±0.04
F值	7.110	30.030	26.340	20.660	29.700	25.520	19.540
P值	0.026	<0.001	0.001	0.002	<0.001	0.001	0.002

表4

表5

WT、ΔmshD和ΔmshD::mshD株经多种体外应激压力处理前后在补充过氧化氢酶平板上的存活情况比较

菌株类型	菌落形成单位(CFU, $x ˉ$ ±s)				处理后存活率(%, $x ˉ$ ±s)
菌株类型	处理前 (×10⁵)	DTT处理 (×10⁴)	H₂O₂处理 (×10³)	SDS处理 (×10³)	DTT	H₂O₂	SDS
WT	1013.33±80.83	720.00±60.00	293.33±70.24	126.67±23.09	7.11±0.59	0.29±0.07	0.13±0.02
ΔmshD	906.67±50.33	573.33±94.52	273.30±64.29	126.67±30.55	6.32±1.04	0.30±0.07	0.14±0.03
ΔmshD::mshD	980.00±40.00	673.33±70.24	293.33±70.24	180.00±52.92	6.87±0.72	0.30±0.07	0.18±0.05
F值	2.512	2.893	0.0857	2.000	0.742	0.025	1.835
P值	0.161	0.132	0.919	0.216	0.515	0.976	0.239

表5

表6

WT、ΔmshD和ΔmshD::mshD株的活性氧水平

处理条件	平均荧光强度( $x ˉ$ ±s)			F值	P值
处理条件	WT	ΔmshD	ΔmshD::mshD	F值	P值
未处理	95.100±2.553	106.000±4.000	94.667±3.055	11.650	0.009
SDS	436.000±8.000	533.667±4.726	441.333±2.517	292.900	<0.001

表6

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材料类型	相关特点	来源
菌株
E.coli DH5α	克隆菌株	北京全式金生物技术有限公司
结核分枝杆菌	H37Rv野生株	本实验室保存
ΔmshD	敲除了mshD的结核分枝杆菌菌株	本研究
ΔmshD::mshD	在ΔmshD菌株中回补mshD的结核分枝杆菌菌株	本研究
质粒
pYC1759	表达NHEJ辅助元件的质粒,含卡那霉素抗性元件	文献[11]
pYC1876	表达sgRNA的质粒,含潮霉素抗性元件	文献[11]
874-L5	含L5整合酶,含潮霉素抗性元件	文献[11]
874-L5-mshD	把mshD基因放在874-L5的hsp60启动子下,含潮霉素抗性元件	本研究

引物名称	引物序列(5'~3')
mshD cx F	ggccgggatggtatgcaaga
mshD cx R	agctcctgcttacgacttct
874 cx F	ctgattctgtggataaccgt
874 cx R	gacctgggcaccgcattgcc
XbaI-Hsp60 F	tccgtcaggatggccttctagacggtgaccacaacgcgc
mshD-Hsp60 R	cgccagtcaagcgccgtcacgcaattgtcttggccattgc
Hsp60-mshD F	gcaatggccaagacaattgcgtgacggcgcttgactggcg
NotI-mshD R	cgaccgagcgcaacgcgtgctcagttatccgtgccagcca