Analyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

Abstract

Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of Escherichia coli and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to E. coli strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.

Lipopolysaccharide and the gut microbiota: considering structural variation

“…Recently, Okahashi et al. [165] created a method for analyzing lipid A using LC‐quadrupole time‐of‐flight MS, which was developed and used in the analyses of E. coli and Bacteroidetes strains. Briefly, an ammonia‐containing alkaline solvent was used to produce sharp lipid A peaks with high sensitivity.…”

“…The difficulty in the analysis of intact lipid A by LC-MS is due to the diverse physical properties arising from different numbers of acyl chains and phosphate groups [164]. Recently, Okahashi et al [165] created a method for analyzing lipid A using LC-quadrupole time-of-flight MS, which was developed and used in the analyses of E. coli and Bacteroidetes strains. Briefly, an ammoniacontaining alkaline solvent was used to produce sharp lipid A peaks with high sensitivity.…”

LipidA-IDER to Explore the Global Lipid A Repertoire of Drug-Resistant Gram-Negative Bacteria

“…Variations from the classical hydrophobic components were observed in minor lipid A forms, including (i) myristoylation at the C2 secondary position (Ec-40) and (ii) incorporation of an unsaturated C16:1 secondary FA chain at the C2′ position (Ec-26, Ec-35) . LipidA-IDER also differentiated isomers with different acylation positions (Figure E,F) based on their distinct fragmentation patterns (Figure G).…”

“…Numerous studies demonstrated the feasibility to determine the complex structure of lipid A using negative ESI-MS 2 with collision-induced dissociation (CID) − (for additional references, refer to Supporting information Tables S1 and S2). Figure A shows the general annotation of lipid A structure for simple and intuitive assignment of MS 2 fragment ions generated in this work.…”

“…In total, 55 potential lipid A species (assigned ID numbers Ec-1 to Ec-55 in Supporting Information Table S9) were resolved at level 3 identification, in contrast to 44 lipid A with a similar or higher level of structural resolution reported previously. 28,37,40,46 Besides mono-, di-, and triphosphorylated lipid A, PEtN (Ec-52, Ec-55) and Ara4N (Ec-53) modifications were detected in E. coli strain EC958, a globally disseminated virulent and multidrug-resistant E. coli O 25b:H4-ST131 clone 51 (Figure 3D). Variations from the classical hydrophobic components were observed in minor lipid A forms, including (i) myristoylation at the C2 secondary position (Ec-40) 37 and (ii) incorporation of an unsaturated C16:1 secondary FA chain at the C2′ position (Ec-26, Ec-35).…”