基于非靶向脂质组学分析脂蛋白Rv1411c对结核分枝杆菌脂质代谢的影响

doi:10.19982/j.issn.1000-6621.20230427

中国防痨杂志 ›› 2024, Vol. 46 ›› Issue (5): 538-548.doi: 10.19982/j.issn.1000-6621.20230427

基于非靶向脂质组学分析脂蛋白Rv1411c对结核分枝杆菌脂质代谢的影响

孙玉亭, 全舒婷, 孙柏旭, 田雪, 綦辉, 焦伟伟, 申阿东(), 孙琳()

国家儿童医学中心/首都医科大学附属北京儿童医院/北京市儿科研究所/儿科学国家重点学科/儿科重大疾病研究教育部重点实验室/国家呼吸系统疾病临床医学研究中心/儿童呼吸道感染性疾病研究北京市重点实验室,北京 100045

收稿日期:2023-11-28 出版日期:2024-05-10 发布日期:2024-04-29
通信作者: 孙琳,Email:chinatka@163.com; 申阿东,Email:shenad16@hotmail.com
基金资助:
国家自然科学基金(82170007);北京市自然科学基金(7222055);保定市科技计划项目(2272P010)

Analysis of effects of lipoprotein Rv1411c on Mycobacterium tuberculosis lipid metabolism based on non-targeted lipomics

Sun Yuting, Quan Shuting, Sun Baixu, Tian Xue, Qi Hui, Jiao Weiwei, Shen Adong(), Sun Lin()

Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Beijing 100045, China

Received:2023-11-28 Online:2024-05-10 Published:2024-04-29
Contact: Sun Lin, Email: chinatka@163.com; Shen Adong, Email: shenad16@hotmail.com
Supported by:
National Natural Science Foundation of China(82170007);Beijing Natural Science Foundation(7222055);Baoding City Science and Technology Planning Project(2272P010)

摘要/Abstract

摘要：

目的: 基于非靶向脂质组学探索结核分枝杆菌(Mycobacterium tuberculosis, MTB)脂蛋白Rv1411c在该菌脂质代谢中的作用。方法: 构建敲除编码脂蛋白Rv1411c基因的MTB(ΔRv1411c)菌株,测定ΔRv1411c与MTB标准株H37Rv的吸光度值(A₆₀₀值)并绘制两者生长曲线。提取ΔRv1411c与H37Rv菌液上清脂质成分,使用液相色谱-质谱(LC-MS)联用技术及非靶向脂质组学分析平台对菌株的差异脂质进行分析。采用偏最小二乘法判别分析(PLS-DA)、京都基因和基因组百科全书(KEGG)富集分析等方法分析两者的差异脂质代谢产物及差异脂质分子的富集通路。结果: 通过色谱分离及质谱分析,从ΔRv1411c与H37Rv菌株中成功鉴定出归属于28种脂质亚类的460个不同的脂质分子,再通过PLS-DA模型分析获得其中归属于17种脂质亚类的158个差异脂质分子[变量权重值(VIP)>1]。以变异倍数(FC)>1.5或<0.67且P<0.05为筛选标准,共筛选出36个具有显著差异的脂质分子,其中有12个脂质分子上调,24个脂质分子下调。经KEGG通路富集分析,差异脂质代谢产物主要富集于以下8种通路,即:甘油磷脂代谢、亚麻酸代谢、α-亚麻酸代谢、糖基磷脂酰肌醇(GPI)-锚的生物合成、甘油脂代谢、鞘脂代谢、花生四烯酸代谢及不饱和脂肪酸的生物合成通路;其中又以甘油磷脂的代谢通路最为重要,其次为甘油脂代谢及糖基磷脂酰肌醇(GPI)-锚的生物合成通路。结论: 脂蛋白Rv1411c可显著影响MTB的脂质代谢,可能参与MTB脂质代谢的调控。

关键词: 分枝杆菌,结核, 脂蛋白类, 脂类代谢, 脂质组

Abstract:

Objective: To explore the effects of lipoprotein Rv1411c on Mycobacterium tuberculosis (MTB) lipid metabolism based on non-targeted lipomics. Methods: The H37Rv-Rv1411c gene knocked-out strain (ΔRv1411c) was constructed and its optical density values (A₆₀₀) was measured and growth curve was drawn to be compared with that of the MTB standard strain (H37Rv). The lipid compositions of the two strains were extracted from the culture supernatant and were further analyzed using liquid chromatography-mass spectrometry technology (LC-MS) and non-targeted lipomics analysis platform. Partial leastsquares discriminant analysis (PLS-DA) and Kyoto Encylopaedia of Genes and Genomes (KEGG) enrichment analysis were used to explore metabolic product differences and pathways between the two groups. Results: Based on the LC-MS analysis, 460 lipid metabolites which belonged to 28 classes were identified. Then 158 lipid metabolites belonged to 17 classes with variable importance in projection (VIP) >1 were further selected based on PLS-DA model. Using the fold change (FC)>1.5 or<0.67 and P<0.05 as screening criteria, 36 lipids with significant difference were confirmed. Among them, 12 lipids were upregulated and 24 lipids were downregulated. KEGG pathway analysis suggested that the differentiating metabolites were mainly involved in eight metabolic pathways: glycerophospholipid metabolism pathway, linolenic acid-metabolism pathway, alpha-linolenic acid metabolism pathway, glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway, glycerolipid metabolism pathway, sphingolipid metabolism pathway, arachidonic acid metabolism pathway and biosynthesis of unsaturated fatty acids pathway. Glycerophospholipid metabolism pathway had the highest impact factor, followed by glycerolipid metabolism and glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway. Conclusion: Lipoprotein Rv1411c can significantly affect the lipid metabolism of MTB and may participate in the regulation of MTB lipid metabolism.

Key words: Mycobacterium tuberculosis, Lipoproteins, Lipid metabolism, Lipidome

中图分类号:

孙玉亭, 全舒婷, 孙柏旭, 田雪, 綦辉, 焦伟伟, 申阿东, 孙琳. 基于非靶向脂质组学分析脂蛋白Rv1411c对结核分枝杆菌脂质代谢的影响[J]. 中国防痨杂志, 2024, 46(5): 538-548. doi: 10.19982/j.issn.1000-6621.20230427

Sun Yuting, Quan Shuting, Sun Baixu, Tian Xue, Qi Hui, Jiao Weiwei, Shen Adong, Sun Lin. Analysis of effects of lipoprotein Rv1411c on Mycobacterium tuberculosis lipid metabolism based on non-targeted lipomics[J]. Chinese Journal of Antituberculosis, 2024, 46(5): 538-548. doi: 10.19982/j.issn.1000-6621.20230427

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引物名称	序列(5'-3')	备注
LFP	TTTTTTTTCCATAAATTGGACTCATTCGCCCGATGTT	左臂上游引物
LRP	TTTTTTTTCCATTTCTTGGTCCTCGACCAGCGGCTTC	左臂下游引物
RFP	TTTTTTTTCCATAGATTGGGATACCATCAACGGCCAGA	右臂上游引物
RRP	TTTTTTTTCCATCTTTTGGAACGCTGCCGAGCTCCTG	右臂下游引物
LYZFP	CTCAGGCAGCTTGTGCGTCTT	敲除验证引物
LYZRP	GTGGACCTCGACGACCCTAG	敲除验证引物
RYZFP	TGGATCTCTCCGGCTTCACC	敲除验证引物
RYZRP	CAACCGAAGAACGCCACC	敲除验证引物

培养时间 (d)	吸光度值($\bar{x}±s$)		t值	P值
培养时间 (d)	H37Rv菌株	ΔRv1411c菌株	t值	P值
3	0.269±0.087	0.240±0.054	0.492	0.648
6	0.527±0.098	0.585±0.025	0.983	0.381
9	0.718±0.072	0.707±0.015	0.252	0.814
12	0.859±0.086	0.898±0.073	0.607	0.577
15	1.014±0.074	1.037±0.056	0.437	0.685
18	1.175±0.073	1.219±0.028	0.969	0.387
21	1.243±0.055	1.270±0.037	0.711	0.516
24	1.314±0.060	1.350±0.054	0.772	0.483
27	1.341±0.056	1.370±0.050	0.666	0.542

主成分	准确度	R²值	Q²值
1	0.92	0.88	0.36
2	0.83	0.99	0.34
3	0.83	1.00	0.34
4	0.83	1.00	0.34
5	0.83	1.00	0.34

趋势/脂质分子	FC值	log₂(FC)值	t值	P值	VIP值	趋势/脂质分子	FC值	log₂(FC)值	t值	P值	VIP值
上调						PE 40:6e	0.44	-1.19	-3.43	0.006	2.01
SM d40:0	3.19	1.67	5.87	<0.001	2.41	PC 30:0e	0.55	-0.86	-3.38	0.007	2.00
FA 18:2	2.45	1.29	5.19	<0.001	2.33	TAG 52:6	0.67	-0.59	-3.20	0.010	1.94
PE 40:5e	1.93	0.95	3.33	0.008	1.98	DAG 39:6e	0.62	-0.68	-3.06	0.012	1.90
PE 38:7e	2.07	1.05	2.98	0.014	1.88	PE 36:6e	0.16	-2.68	-3.02	0.013	1.89
SM d34:0	1.66	0.73	2.96	0.014	1.87	PC 38:6	0.66	-0.59	-3.00	0.013	1.88
FA 18:1	2.62	1.39	2.90	0.016	1.85	PC 38:2	0.49	-1.03	-3.00	0.013	1.88
PC 36:5e	3.00	1.59	2.70	0.022	1.77	PC 32:2e	0.52	-0.95	-2.83	0.018	1.82
PC 39:2e	1.80	0.85	2.65	0.024	1.76	DAG 36:1	0.50	-0.99	-2.74	0.021	1.79
PC 42:6	2.55	1.35	2.37	0.039	1.64	PI 36:4	0.32	-1.66	-2.63	0.025	1.75
SM d36:1	1.80	0.85	2.34	0.041	1.63	PC 36:3e	0.50	-0.99	-2.48	0.033	1.69
TAG 56:6	1.60	0.67	2.36	0.040	1.63	LPC 18:1e	0.56	-0.83	-2.45	0.034	1.67
PC 40:3	2.06	1.04	2.31	0.044	1.61	PE 34:2	0.42	-1.24	-2.38	0.039	1.64
下调						PS 36:1	0.26	-1.97	-2.36	0.040	1.63
PE 34:1	0.23	-2.13	-13.96	<0.001	2.67	PE 34:2e	0.20	-2.33	-2.35	0.041	1.63
PC 32:1e	0.48	-1.07	-7.18	<0.001	2.50	SM d40:1	0.42	-1.25	-2.34	0.042	1.62
PE 36:2	0.18	-2.46	-5.38	<0.001	2.36	LPE 20:5	0.43	-1.21	-2.31	0.043	1.61
PE 35:1	0.13	-2.93	-4.41	0.001	2.22	PE 36:3e	0.40	-1.33	-2.31	0.044	1.61
PE 35:2	0.45	-1.16	-4.20	0.002	2.18	GM3 d42:1	0.15	-2.74	-2.28	0.046	1.60

基于非靶向脂质组学分析脂蛋白Rv1411c对结核分枝杆菌脂质代谢的影响

Analysis of effects of lipoprotein Rv1411c on Mycobacterium tuberculosis lipid metabolism based on non-targeted lipomics

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