Liquid chromatography-mass spectrometry analysis of major lipid species in Bacteroides thetaiotaomicron VPI 5482

Abstract

Bacteroides thetaiotaomicron colonizes the human gastrointestinal tract and establishes a symbiotic relationship with the host, contributing to reducing intestinal inflammation and enhancing resistance against foreign pathogens. Recent reports have revealed that diverse lipid species such as glycerophospholipids, sphingolipids, and N-acyl amines exist in B. thetaiotaomicron and play essential roles in the immune process. In this research, total lipids obtained from B. thetaiotaomicron were purified via thin-layer chromatography, and the species and molecular structures of visible lipids in different hydrophobic regions were qualitatively characterized by high-performance liquid chromatography-mass spectrometry. The results indicated that seven lipid species were primarily displayed on the plate, including phosphatidylethanolamine, ethanolamine phosphoryl dihydroceramide, inositol phosphoryl dihydroceramide, glycyl-serine phosphoryl dihydroceramide, phosphatidylglycerol, cardiolipin, and glycyl-serine phosphoryl diacylglycerol. The phosphatidylethanolamine, ethanolamine phosphoryl dihydroceramide, and inositol phosphoryl dihydroceramide species corresponding to ion peaks at m/z 676.48, 691.53, and 796.53 exhibited significantly high abundance compared to other species, suggesting their prevalent presence in total lipids. The molecular structures of phosphatidylethanolamine and ethanolamine phosphoryl dihydroceramide were derived from the modification of diacylglycerol and dihydroceramide with phosphoethanolamine, while the structure of inositol phosphoryl dihydroceramide was derived from the modification of dihydroceramide with phosphoinositol. The phosphatidylglycerol and cardiolipin species corresponding to m/z 721.51 and 1323.94 have been detected in the membrane lipids of B. thetaiotaomicron, although they were not mentioned in previous studies. These findings are important for understanding the molecular mechanisms of B. thetaiotaomicron colonization in mammalian gut.

References

[1] KimK, ChoeD, SongY, KangM, LeeSG, LeeDH, ChoBK. Engineering Bacteroides thetaiotaomicron to produce non-native butyrate based on a genome-scale metabolic model-guided design. Metab Eng, 2021, 68: 174-86

[2] HooperLV, MidtvedtT, GordonJI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr, 2002, 22: 283-307

[3] BrownEM, KeX, HitchcockD, JeanfavreS, Avila-PachecoJ, NakataT, ArthurTD, FornelosN, HeimC, FranzosaEA, WatsonN, HuttenhowerC, HaiserHJ, DillowG, GrahamDB, FinlayBB, KosticAD, PorterJA, VlamakisH, ClishCB, XavierRJ. Bacteroides-derived sphingolipids are critical for maintaining intestinal homeostasis and symbiosis. Cell Host Microbe, 2019, 25: 668-e680667

[4] DeldayM, MulderI, LoganET, GrantG. Bacteroides thetaiotaomicron ameliorates colon inflammation in preclinical models of crohn’s disease. Inflamm Bowel Dis, 2019, 25: 85-96

[5] HansenR, SandersonIR, MuhammedR, AllenS, TzivinikosC, HendersonP, GervaisL, JefferyIB, MullinsDP, O’HerlihyEA, WeinbergJD, KitsonG, RussellRK, WilsonDC. A double-blind, placebo-controlled trial to assess safety and tolerability of (thetanix) Bacteroides thetaiotaomicron in adolescent Crohn’s disease. Clin Transl Gastroenterol, 2020, 12: e00287

[6] Jacobson AN, Choudhury BP, Fischbach MA. The biosynthesis of lipooligosaccharide from Bacteroides thetaiotaomicron. mBio. 2018;9.

[7] PitherMD, IllianoA, PagliucaC, JacobsonA, MantovaG, StornaiuoloA, ColicchioR, VitielloM, PintoG, SilipoA, FischbachMA, SalvatoreP, AmoresanoA, MolinaroA, Di LorenzoF. Bacteroides thetaiotaomicron rough-type lipopolysacchathee: The chemical structure and the immunological activity. Carbohydr Polym, 2022, 297: 120040

[8] Ryan E, Gonzalez Pastor B, Gethings LA, Clarke DJ, Joyce SA. Lipidomic analysis reveals differences in Bacteroides species driven largely by plasmalogens, glycerophosphoinositols and certain sphingolipids. Metabolites. 2023;13.

[9] HannunYA, ObeidLM. Sphingolipids and their metabolism in physiology and disease. Nat Rev Mol Cell Biol, 2018, 19: 673

[10] Lee-SarwarK, KellyRS, Lasky-SuJ, MoodyDB, MolaAR, ChengTY, ComstockLE, ZeigerRS, O’ConnorGT, SandelMT, BacharierLB, BeigelmanA, LaranjoN, GoldDR, BunyavanichS, SavageJH, WeissST, BrennanPJ, LitonjuaAA. Intestinal microbial-derived sphingolipids are inversely associated with childhood food allergy. J Allergy Clin Immunol, 2018, 142: 335-e338339

[11] LynchA, CrowleyE, CaseyE, CanoR, ShanahanR, McGlackenG, MarchesiJR, ClarkeDJ. The Bacteroidales produce an N-acylated derivative of glycine with both cholesterol-solubilising and hemolytic activity. Sci Rep, 2017, 7: 13270

[12] Bill MK, Brinkmann S, Oberpaul M, Patras MA, Leis B, Marner M, Maitre MP, Hammann PE, Vilcinskas A, Schuler SMM, Schäberle TF. Novel glycerophospholipid, lipo- and N-acyl amino acids from Bacteroidetes: isolation, structure elucidation and bioactivity. Molecules. 2021;26.

[13] Lynch A, Tammireddy SR, Doherty MK, Whitfield PD, Clarke DJ. The glycine lipids of Bacteroides thetaiotaomicron are important for fitness during growth in vivo and in vitro. Appl Environ Microbiol. 2019;85.

[14] FrankfaterCF, SartorioMG, ValguarneraE, FeldmanMF, HsuFF. Lipidome of the Bacteroides genus containing new peptidolipid and sphingolipid families revealed by multiple-stage mass spectrometry. Biochem, 2023, 62: 1160-80

[15] SartorioMG, ValguarneraE, HsuFF, FeldmanMF. Lipidomics analysis of outer membrane vesicles and elucidation of the inositol phosphoceramide biosynthetic pathway in Bacteroides thetaiotaomicron. Microbiol Spectr, 2022, 10: e0063421

[16] Wieland BrownLC, PenarandaC, KashyapPC, WilliamsBB, ClardyJ, KronenbergM, SonnenburgJL, ComstockLE, BluestoneJA, FischbachMA. Production of α-galactosylceramide by a prominent member of the human gut microbiota. PLoS Biol, 2013, 11: e1001610

[17] NicholsFC, ClarkRB, MaciejewskiMW, ProvatasAA, BalsbaughJL, DewhirstFE, SmithMB, RahmlowA. A novel phosphoglycerol serine-glycine lipodipeptide of Porphyromonas gingivalis is a TLR2 ligand. J Lipid Res, 2020, 61: 1645-57

[18] Olsen I, Nichols FC. Are sphingolipids and serine dipeptide lipids underestimated virulence factors of Porphyromonas gingivalis? Infect Immun. 2018;86.

[19] Lamichhane S, Sen P, Alves MA, Ribeiro HC, Raunioniemi P, Hyötyläinen T, Orešič M. Linking gut microbiome and lipid metabolism: moving beyond associations. Metabolites. 2021;11.

[20] BlighEG, DyerWJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol, 1959, 37: 911-7

[21] WangX, RibeiroAA, GuanZ, McGrathSC, CotterRJ, RaetzCR. Structure and biosynthesis of free lipid a molecules that replace lipopolysaccharide in Francisella tularensis subsp. novicida. Biochem, 2006, 45: 14427-40

[22] RaetzCR, DowhanW. Biosynthesis and function of phospholipids in Escherichia coli. J Biol Chem, 1990, 265: 1235-8

[23] LiC, TanBK, ZhaoJ, GuanZ. In vivo and in vitro synthesis of phosphatidylglycerol by an Escherichia coli cardiolipin synthase. J Biol Chem, 2016, 291: 25144-53

[24] XuJ, BjursellMK, HimrodJ, DengS, CarmichaelLK, ChiangHC, HooperLV, GordonJI. A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science, 2003, 299: 2074-6

[25] LaiY, HayashiN, LuTK. Engineering the human gut commensal Bacteroides thetaiotaomicron with synthetic biology. Curr Opin Chem Biol, 2022, 70: 102178

[26] RizzaV, TuckerAN, WhiteDC. Lipids of Bacteroides melaninogenicus. J Bacteriol, 1970, 101: 84-91

[27] Fiebig A, Schnizlein MK, Pena-Rivera S, Trigodet F, Dubey AA, Hennessy M, Basu A, Pott S, Dalal S, Rubin D, Sogin ML, Murat Eren A, Chang EB, Crosson S. Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient. bioRxiv. 2023.

[28] LuYH, GuanZ, ZhaoJ, RaetzCR. Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli. J Biol Chem, 2011, 286: 5506-18

[29] SohlenkampC, GeigerO. Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiol Rev, 2016, 40: 133-59

[30] PizzutoM, LonezC, Baroja-MazoA, Martínez-BanaclochaH, TourlomousisP, GangloffM, PelegrinP, RuysschaertJM, GayNJ, BryantCE. Saturation of acyl chains converts cardiolipin from an antagonist to an activator of toll-like receptor-4. Cell Mol Life Sci, 2019, 76: 3667-78

[31] BaeM, CassillyCD, LiuX, ParkSM, TusiBK, ChenX, KwonJ, FilipčíkP, BolzeAS, LiuZ, VlamakisH, GrahamDB, BuhrlageSJ, XavierRJ, ClardyJ. Akkermansia muciniphila phospholipid induces homeostatic immune responses. Nature, 2022, 608: 168-73

[32] SalyersAA. Bacteroides of the human lower intestinal tract. Annu Rev Microbiol, 1984, 38: 293-313

[33] PanevskaA, SkočajM, KrižajI, MačekP, SepčićK. Ceramide phosphoethanolamine, an enigmatic cellular membrane sphingolipid. Biochim Biophys Acta Biomembr, 2019, 1861: 1284-92

[34] NicholsFC, HousleyWJ, O’ConorCA, ManningT, WuS, ClarkRB. Unique lipids from a common human bacterium represent a new class of toll-like receptor 2 ligands capable of enhancing autoimmunity. Am J Pathol, 2009, 175: 2430-8

Funding

Research Program of State Key Laboratory of Food Science and Resources, Jiangnan University(SKLF-ZZB-202417)