乙酰化修饰在影响细菌对核糖体靶向抗生素敏感性中的作用

doi:10.3969/j.issn.1674-8115.2021.03.004

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (3): 308-313.doi: 10.3969/j.issn.1674-8115.2021.03.004

乙酰化修饰在影响细菌对核糖体靶向抗生素敏感性中的作用

来亚男¹(), 姚玉峰¹, 郭晓奎², 倪进婧¹()

^1.上海交通大学基础医学院免疫学与微生物学系，上海 200025
^2.上海交通大学医学院-国家热带病研究中心全球健康学院，上海 200025

收稿日期:2020-04-05 出版日期:2021-03-28 发布日期:2021-04-06
通讯作者: 倪进婧 E-mail:laiyanan1994@163.com;nijinjing45@163.com
作者简介:来亚男（1994—），女，硕士生；电子信箱：laiyanan1994@163.com。
基金资助:
国家自然科学基金(31700121);上海市高水平地方高校创新团队(SSMU-ZLCX20180202)

Acetylation involved in bacterial susceptibility to ribosome-targeting antibiotics

Ya-nan LAI¹(), Yu-feng YAO¹, Xiao-kui GUO², Jin-jing NI¹()

^1.Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2020-04-05 Online:2021-03-28 Published:2021-04-06
Contact: Jin-jing NI E-mail:laiyanan1994@163.com;nijinjing45@163.com
Supported by:
National Natural Science Foundation of China(31700121);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZLCX20180202)

摘要/Abstract

摘要：

目的·探究乙酰化修饰在细菌应对核糖体靶向抗生素胁迫下的作用。方法·通过肉汤稀释法检测嘌呤霉素、巴龙霉素、四环素和壮观霉素对于鼠伤寒沙门菌（Salmonella enterica serovar Typhimurium，S. Typhimurium）14028S的最低抑菌浓度（minimum inhibitory concentration，MIC）。配置浓度低于各抗生素MIC的固体培养基。点板实验检测S. Typhimurium 14028S和3种乙酰化修饰相关基因敲除株［蛋白质乙酰转移酶（protein acetyltransferase，Pat）编码基因敲除株Δpat、烟酰胺腺嘌呤二核苷酸依赖性去乙酰化酶（NAD⁺-dependent protein deacylase，CobB）编码基因敲除株ΔcobB、乙酸激酶（acetate kinase，AckA）编码基因敲除株ΔackA］在不同抗生素浓度的固体培养基上的生长差异。结果·乙酰化程度升高（ΔackA），增加S. Typhimurium对嘌呤霉素的敏感性，但会减弱S. Typhimurium对巴龙霉素的敏感性；而乙酰化程度升高（ΔackA、ΔcobB）或降低（Δpat）均不影响S. Typhimurium对四环素和壮观霉素的敏感性。结论·乙酰化修饰影响细菌对核糖体靶向抗生素的敏感性，这种改变可能是通过核糖体蛋白的乙酰化修饰来调控的。

关键词: 乙酰化修饰, 核糖体, 核糖体靶向抗生素

Abstract:

Objective·To investigate the effects of acetylation on the bacterial susceptibility to ribosome-targeting antibiotics.

Methods·The minimum inhibitory concentrations (MICs) of puromycin, paromomycin, tetracycline and spectinomycin for Salmonella enterica serovar Typhimurium (S. Typhimurium) 14028S were determined by the broth dilution method. The solid mediums with concentration lower than MIC was prepared. The spot dilution assay was employed to determine the susceptibility of S. Typhimuriumstrain 14028S, protein acetyltransferase (pat) knockout strain (Δpat), NAD⁺-dependent protein deacylase (cobB) knockout strain (ΔcobB) and acetate kinase (ackA) knockout strain (ΔackA) to four kinds of ribosome-targeting antibiotics.

Results·Increase of acetylation (ΔackA) promoted the susceptibility of S. Typhimurium to puromycin, but promoted the resistance of S. Typhimurium to paromomycin. However, the sensitivity of S. Typhimurium to tetracycline and spectinomycin was not affected by the increase of acetylation (ΔackA or ΔcobB) or decrease of acetylation (Δpat).

Conclusion·Acetylation is involved in the susceptibility of S. Typhimurium to ribosome-targeting antibiotics, suggesting that acetylation of ribosomal proteins may contribute to bacterial antibiotic sensitivity.

Key words: acetylation, ribosome, ribosome-targeting antibiotics

中图分类号:

来亚男, 姚玉峰, 郭晓奎, 倪进婧. 乙酰化修饰在影响细菌对核糖体靶向抗生素敏感性中的作用[J]. 上海交通大学学报(医学版), 2021, 41(3): 308-313.

Ya-nan LAI, Yu-feng YAO, Xiao-kui GUO, Jin-jing NI. Acetylation involved in bacterial susceptibility to ribosome-targeting antibiotics[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 308-313.

图/表 6

表1 抗生素稀释液的配制

Tab 1 Preparation of antibiotic dilution

Stage	Concentration/(mg·L^-1)	Source	Antibiotic volume/mL	LB medium volume/mL	Concentration obtained/(mg·L^-1)	Concentration in test/(mg·L^-1)
1	1 280	Stock	1	9	128	64
2	128	Stage 1	1	1	64	32
3	128	Stage 1	1	3	32	16
4	128	Stage 1	1	7	16	8
5	16	Stage 4	1	1	8	4
6	16	Stage 4	1	3	4	2
7	16	Stage 4	1	7	2	1
8	2	Stage 7	1	1	1	0.5
9	2	Stage 7	1	3	0.5	0.25
10	2	Stage 7	1	7	0.25	0.125
11	0.25	Stage 10	1	1	0.125	0.062 5
12	0.25	Stage 10	1	3	0.062 5	0.031 25

图1 S. Typhimurium 14028S和各乙酰化修饰相关基因敲除株的生长曲线

Fig 1 Growth curves of S. Typhimurium 14028S and acetylation related gene mutants

图2 嘌呤霉素的点板稀释实验结果Note: The 10-4-100 dilutions of S. Typhimurium 14028S and three mutant strains were spotted onto 256, 128 and 64 mg·L-1 puromycin-containing plates. The plate medium without puromycin was used as control.

Fig 2 Spot dilution assay of puromycin

图3 巴龙霉素的点板稀释实验结果Note: The 10-4-100 dilutions of S. Typhimurium 14028S and three mutant strains were spotted onto 32, 16 and 8 mg·L-1 paromomycin-containing plates. The plate medium without paromomycin was used as control.

Fig 3 Spot dilution assay of paromomycin

图4 四环素点板稀释实验Note: The 10-4-100 dilutions of S. Typhimurium 14028S and three mutant strains were spotted onto 2, 1 and 0.5 mg·L-1 tetracycline-containing plates. The plate medium without tetracycline was used as control.

Fig 4 Spot dilution assay of tetracycline

图5 壮观霉素点板稀释实验结果Note: The 10-4-100 dilutions of S. Typhimurium 14028S and three mutant strains were spotted onto 64, 32 and 16 mg·L-1 spectinomycin-containing plates. The plate medium without spectinomycin was used as control.

Fig 5 Spot dilution assay of spectinomycin

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乙酰化修饰在影响细菌对核糖体靶向抗生素敏感性中的作用

Acetylation involved in bacterial susceptibility to ribosome-targeting antibiotics

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