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

doi:10.19982/j.issn.1000-6621.20230427

Abstract

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

CLC Number:

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

Figures/Tables 15

References 31

[1]	Chatterjee D. The mycobacterial cell wall: structure, biosynthesis and sites of drug action. Curr Opin Chem Biol, 1997, 1(4): 579-588. doi:10.1016/s1367-5931(97)80055-5. pmid: 9667898
[2]	孙丕梅. 结核分枝杆菌中的分枝菌酸. 中国防痨杂志, 2010, 32(7): 44-49.
[3]	Becker K, Sander P. Mycobacterium tuberculosis lipoproteins in virulence and immunity-fighting with a double-edged sword. FEBS Letters, 2016, 590(21): 3800-3819. doi:10.1002/1873-3468.12273. pmid: 27350117
[4]	Sander P, Rezwan M, Walker B, et al. Lipoprotein processing is required for virulence of Mycobacterium tuberculosis. Mol Microbiol, 2004, 52(6): 1543-1552. doi:10.1111/j.1365-2958.2004.04041.x. pmid: 15186407
[5]	Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998, 393(6685): 537-544. doi:10.1038/31159.
[6]	Post FA, Manca C, Neyrolles O, et al. Mycobacterium tuberculosis 19-kilodalton lipoprotein inhibits Mycobacterium smegmatis-induced cytokine production by human macrophages in vitro. Infect Immun, 2001, 69(3): 1433-1439. doi:10.1128/iai.69.3.1433-1439.2001. pmid: 11179309
[7]	Fulton SA, Reba SM, Pai RK, et al. Inhibition of major histocompatibility complex Ⅱ expression and antigen processing in murine alveolar macrophages by Mycobacterium bovis BCG and the 19-kilodalton mycobacterial lipoprotein. Infect Immun, 2004, 72(4): 2101-2110. doi:10.1128/iai.72.4.2101-2110.2004. pmid: 15039332
[8]	Pandey AK, Sassetti CM. Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci U S A, 2008, 105(11): 4376-4380. doi:10.1073/pnas.0711159105.
[9]	Miner MD, Chang JC, Pandey AK, et al. Role of cholesterol in Mycobacterium tuberculosis infection. Indian J Exp Biol, 2009, 47(6): 407-411.
[10]	Sulzenbacher G, Canaan S, Bordat Y, et al. LppX is a lipoprotein required for the translocation of phthiocerol dimycocero-sates to the surface of Mycobacterium tuberculosis. EMBO J, 2006, 25(7): 1436-1444. doi:10.1038/sj.emboj.7601048. pmid: 16541102
[11]	Bigi F, Gioffré A, Klepp L, et al. The knockout of the lprG-Rv1410 operon produces strong attenuation of Mycobacterium tuberculosis. Microbes Infect, 2004, 6(2): 182-187. doi:10.1016/j.micinf.2003.10.010.
[12]	Bigi F, Espitia C, Alito A, et al. A novel 27 kDa lipoprotein antigen from Mycobacterium bovis. Microbiology (Reading), 1997, 143 (Pt 11): 3599-3605. doi:10.1099/00221287-143-11-3599.
[13]	Gaur RL, Ren K, Blumenthal A, et al. LprG-mediated surface expression of lipoarabinomannan is essential for virulence of Mycobacterium tuberculosis. PLoS Pathog, 2014, 10(9): e1004376. doi:10.1371/journal.ppat.1004376.
[14]	Shukla S, Richardson ET, Athman JJ, et al. Mycobacterium tuberculosis lipoprotein LprG binds lipoarabinomannan and determines its cell envelope localization to control phagolysosomal fusion. PLoS Pathog, 2014, 10(10): e1004471. doi:10.1371/journal.ppat.1004471.
[15]	Drage MG, Tsai HC, Pecora ND, et al. Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) binds triacylated glycolipid agonists of Toll-like receptor 2. Nat Struct Mol Biol, 2010, 17(9): 1088-1095. doi:10.1038/nsmb.1869.
[16]	Martinot AJ, Farrow M, Bai L, et al. Mycobacterial Metabolic Syndrome: LprG and Rv1410 Regulate Triacylglyceride Levels, Growth Rate and Virulence in Mycobacterium tuberculosis. PLoS Pathog, 2016, 12(1): e1005351. doi:10.1371/journal.ppat.1005351.
[17]	Li J, Luu LDW, Wang X, et al. Metabolomic analysis reveals potential biomarkers and the underlying pathogenesis involved in Mycoplasma pneumoniae pneumonia. Emerg Microbes Infect, 2022, 11(1): 593-605. doi:10.1080/22221751.2022.2036582.
[18]	Zhai W, Wu F, Zhang Y, et al. The Immune Escape Mechanisms of Mycobacterium Tuberculosis. Int J Mol Sci, 2019, 20(2): 340. doi:10.3390/ijms20020340.
[19]	Layre E. Trafficking of Mycobacterium tuberculosis Envelope Components and Release Within Extracellular Vesicles: Host-Pathogen Interactions Beyond the Wall. Front Immunol, 2020, 11: 1230. doi:10.3389/fimmu.2020.01230.
[20]	Kang PB, Azad AK, Torrelles JB, et al. The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exp Med, 2005, 202(7): 987-999. doi:10.1084/jem.20051239.
[21]	Quigley J, Hughitt VK, Velikovsky CA, et al. The Cell Wall Lipid PDIM Contributes to Phagosomal Escape and Host Cell Exit of Mycobacterium tuberculosis. mBio, 2017, 8(2): e00148-17. doi:10.1128/mBio.00148-17.
[22]	Gago G, Diacovich L, Gramajo H. Lipid metabolism and its implication in mycobacteria-host interaction. Curr Opin Microbiol, 2018, 41: 36-42. doi:10.1016/j.mib.2017.11.020. pmid: 29190491
[23]	Neyrolles O, Guilhot C. Recent advances in deciphering the contribution of Mycobacterium tuberculosis lipids to pathogenesis. Tuberculosis (Edinb), 2011, 91(3): 187-195. doi:10.1016/j.tube.2011.01.002.
[24]	Cook GM, Greening C, Hards K, et al. Energetics of pathogenic bacteria and opportunities for drug development. Adv Microb Physiol, 2014, 65: 1-62. doi:10.1016/bs.ampbs.2014.08.001. pmid: 25476763
[25]	Daniel J, Maamar H, Deb C, et al. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog, 2011, 7(6): e1002093. doi:10.1371/journal.ppat.1002093.
[26]	Lee W, VanderVen BC, Fahey RJ, et al. Intracellular Mycobacterium tuberculosis exploits host-derived fatty acids to limit metabolic stress. J Biol Chem, 2013, 288(10): 6788-6800. doi:10.1074/jbc.M112.445056.
[27]	Agarwal P, Gordon S, Martinez FO. Foam Cell Macrophages in Tuberculosis. Front Immunol, 2021, 12: 775326. doi:10.3389/fimmu.2021.775326.
[28]	Trivedi OA, Arora P, Sridharan V, et al. Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. Nature, 2004, 428(6981): 441-445. doi:10.1038/nature02384.
[29]	Baran M, Grimes KD, Sibbald PA, et al. Development of small-molecule inhibitors of fatty acyl-AMP and fatty acyl-CoA ligases in Mycobacterium tuberculosis. J Med Chem, 2020, 201: 112408. doi:10.1016/j.ejmech.2020.112408.
[30]	Pitarque S, Larrouy-Maumus G, Payré B, et al. The immunomodulatory lipoglycans, lipoar-abinomannan and lipomannan, are exposed at the mycobacterial cell surface. Tuberculosis (Edinb), 2008, 88(6): 560-565. doi:10.1016/j.tube.2008.04.002.
[31]	Fischer K, Chatterjee D, Torrelles J, et al. Mycobacterial lysocardiolipin is exported from phagosomes upon cleavage of cardiolipin by a macrophage-derived lysosomal phospholipase A2. J Immunol, 2001, 167(4): 2187-2192. doi:10.4049/jimmunol.167.4.2187. pmid: 11490004

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