Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

Abstract

This article is available online at http://www.jlr.org of H. pylori outer membrane. The H. pylori LPS consists of a lipid A region, a core region, and an O-chain polysaccharide (also known as the O-antigen) ( 3 ). Lipid A, the hydrophobic moiety of LPS and a glucosamine-based saccharolipid, is the principal structural component responsible for the range of biological activities of LPS ( 4, 5 ).Generally, lipid A is a glucosamine disaccharide that carries phosphates at positions 1 and 4 ′ and usually has four primary (glucosamine-linked) hydroxyacyl chains and one or more secondary acyl chains ( 3, 6, 7 ). However, H. pylori lipid A is different from that of other bacterial species, including both phosphorylation and acylation patterns ( 8 ). The lipid A of H. pylori contains a phosphoethanolamine ( P Etn) group directly linked to the 1-position of the disaccharide backbone. This is in contrast to the P Etn groups found in other pathogenic Gram-negative bacteria, which are attached to the lipid A phosphate group to form a pyrophosphate linkage ( 9, 10 ). In addition, the predominant absence of ester-bound 4'-phosphate and the presence of tetraacyl lipid A with fatty acids of 16 to 18 carbons in length differentiate H. pylori lipid A from that of other Gram-negative bacteria. H. pylori synthesizes two types of lipid A molecules: hexaacyl-and tetraacyl-lipid A. Hexaacyllipid A has two phosphates or phosphoethanolamines on the lipid A disaccharide backbone, whereas tetraacyllipid A contains only one phosphate ( 8 ). It has also been reported that H. pylori does not survive long before it is deacylated at the 3 ′ position from hexaacyl structure to form the tetraacyl major lipid A species ( 11 ). The toxicity of H. pylori tetraacyl-lipid A on human monocytes is ‫ف‬ 4-fold lower than that of the hexaacyl form ( 12 ). It has been suggested that the phosphorylation and acylation patterns in lipid A of H. pylori LPS are responsible for its low biological activity ( 13,14 ).

Characterization of lipid A profiles from Shigella flexneri variant X lipopolysaccharide

“…(c). Whereas hepta‐acylated forms of lipid A have been rarely detected in other Gram‐negative bacteria, in this report we show evidence for the first time of its presence in Shigella flexneri .…”

Surface Acoustic Wave Nebulization Facilitating Lipid Mass Spectrometric Analysis

“…Lipid A from Francisella novicida grown at 37 °C was extracted using a microwave-based [36] or Bligh-Dyer extraction method [37], Each lipid was dissolved into a solution in CHCI 3 /CH 3 OH (2:1 v:v for lipid A and 1:10 for phospholipid) and introduced to the SAWN assembly by simple pipetting with a micropipet. Each droplet contained 2–3 μL of sample and generated a plume that lasted a few seconds.…”

Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

“…Cells of Escherichia coli O116 and Salmonella enterica serovar Riogrande were from the NRCC collection (19). H. pylori 26695/hp0826:kan isogenic mutant strain expressing a deep-rough inner core LPS was described previously (36). Francisella novicida cells were grown as described previously (31).…”

“…Lipid A from E. coli, F. novicida, and Salmonella serovar Riogrande were prepared by hydrolyzing LPS in 1% acetic acid at 100°C for 1 h. The resultant lipid A pellet was washed twice with water and extracted with a mixture of chloroform-methanol-water (12:6:1, vol/vol/vol). Lipid A from H. pylori was isolated from whole cells as described previously (36).…”

“…Mass spectrometry (MS) has been widely used to gain information about the heterogeneity, i.e., the number of different species of lipid A families and a distribution of fatty acids on each glucosamine residue (2,3,9,16,20,23,28,29,30,32,35,36). Detailed structural information, including the phosphorylation pattern of lipid A, can be obtained by tandem mass spectrometry.…”