<i>Salmonella</i>-Type Heptaacylated Lipid A Is Inactive and Acts as an Antagonist of Lipopolysaccharide Action on Human Line Cells

Tanamoto, Ken‐ichi; Azumi, Satoko

doi:10.4049/jimmunol.164.6.3149

Cited by 74 publications

(77 citation statements)

References 37 publications

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“…Good examples are the presence or absence of a single fatty acid (31)(32)(33) or of substituents on the C-1 and/or C-4 Ј phosphates (34). Such structural differences could be easily demonstrated by the present method on small cell samples.…”

Section: Resultsmentioning

confidence: 72%

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

Hamidi

Tîrşoaga

Новиков

et al. 2005

Journal of Lipid Research

151

145

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Endotoxins (lipopolysaccharides) are the main components of Gram-negative bacterial outer membranes. A quick and simple way to isolate their lipid region (lipid A) directly from whole bacterial cells was devised. This method using hot ammonium-isobutyrate solvent was applied to small quantities of cells and proved to be indispensable when a rapid characterization of lipid A structure by mass spectrometry was required. Biological activities of endotoxins are directly related to the lipid A structures, which vary greatly with cell growth conditions. This method is suitable for rough-and smooth-type bacteria and very efficient for screening variations in lipid A structures. Data are acquired in a few hours and avoid the use of phenol in extraction. Lipopolysaccharides (LPSs) are the major components of the external membrane of almost all Gram-negative bacteria (1, 2); they are known as endotoxins and may cause several pathophysiological symptoms, such as fever, diarrhea, blood pressure decrease, septic shock, and death (3).The LPS molecular architecture consists of three different regions. The innermost, hydrophobic region, lipid A, is responsible for the major toxic and beneficial properties of bacterial endotoxins (4). Lipid A is the least variable part of the molecule among the different species of a genus, and its structure generally consists of a diglucosamine backbone substituted with varying numbers (usually four to seven) of ester-or amide-linked fatty acids. Phosphate and/or other substituents are linked to carbons at the C-1 and C-4 Ј positions of the glucosamine disaccharide (5). A 2-keto-3-deoxyoctonate (Kdo) unit links the lipid A to a core oligosaccharide (OS) composed of ‫ف‬ 10 sugar residues divided into two regions: a best conserved inner core part and a distal outer core. The core is linked to a third outermost region of a highly immunogenic and variable O-chain polysaccharide (PS) or O-antigen made up of repeating OS units. The latter region of the LPS molecule is responsible for bacterial serological strain specificity (6) and is present only in smooth-type bacteria. The so-called rough-type bacteria produce LPSs lacking O-antigens.Endotoxin can be isolated from Gram-negative bacteria by different methods, the most efficient and commonly used one being the hot phenol-water extraction procedure introduced by Westphal and Lüderitz (7). It was later modified by different authors (8, 9), and specific methods were developed for rough-type endotoxin extractions (10, 11). However, each method requires several days for the extraction and purification of endotoxins and further steps to isolate the lipid A moiety. New methods have been described to extract LPS from small quantities of cells, such as by mini phenol extraction (12) or using an RNAisolating reagent, but these methods still require 2 or 3 days and the use of phenol (13).In early experiments, mineral acid hydrolysis was used to liberate lipid A from endotoxins, splitting the acidolabile ketosidic bond of Kdo. It was followed in 1963 b...

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Section: Resultsmentioning

confidence: 72%

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

Hamidi

Tîrşoaga

Новиков

et al. 2005

Journal of Lipid Research

151

145

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“…We previously reported that lipid A preparations from various Salmonella strains and synthetic Salmonella-type lipid A (compound 516) possess very little stimulatory activity in human macrophages, despite being potently active in murine macrophages (27). On the other hand, lipid A preparations from E. coli and synthetic E. coli-type lipid A (compound 506) were equally active in human and murine macrophages (27).…”

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confidence: 99%

MD-2, a Novel Accessory Molecule, Is Involved in Species-Specific Actions ofSalmonellaLipid A

2002

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Salmonella lipid A is inactive in human macrophages despite being potently active in murine macrophages. We investigated the molecular basis for this species-specific action of Salmonella lipid A. When murine CD14 (mCD14), mTLR4, and mMD-2 were all expressed in human monocytic THP-1 cells, these cells were capable of responding to Salmonella lipid A. Expressing each of these proteins separately did not impart such responsiveness. Expression of mTLR4 plus mMD-2, but not mCD14 plus mTLR4 or mCD14 plus mMD-2, conferred this responsiveness. In THP-1 cells expressing mCD14, mTLR4, and mMD-2, replacing mCD14 with human CD14 had no effect on responsiveness to Salmonella lipid A or synthetic Salmonella-type lipid A (compound 516). When mTLR4 was replaced with human TLR4, the responses to these lipid A preparations were decreased to half, and the replacement of mMD-2 decreased responsiveness to one-third, although the responses to Escherichia coli lipid A or synthetic E. coli-type lipid A (compound 506) were not affected. These results indicate that both TLR4 and MD-2 participate in the species-specific action of Salmonella lipid A.

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“…4). It is known that the presence of different molecular species in a lipid A preparation leads to quantitative differences in biological activities, and some molecular species have been shown to act as antagonists (5,31) for the other structures in the preparation (4). The presence of this molecular species in higher amounts could affect biological activities.…”

Section: Resultsmentioning

confidence: 99%

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Tîrşoaga

Hamidi

Perry

et al. 2007

Journal of Lipid Research

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Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.-Tirsoaga, A., A.

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Salmonella-Type Heptaacylated Lipid A Is Inactive and Acts as an Antagonist of Lipopolysaccharide Action on Human Line Cells

Cited by 74 publications

References 37 publications

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

MD-2, a Novel Accessory Molecule, Is Involved in Species-Specific Actions ofSalmonellaLipid A

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

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