Structure and Biosynthesis of Free Lipid A Molecules That Replace Lipopolysaccharide in Francisella tularensis subsp. novicida

Abstract

Francisella novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15 % of the total phospholipids, as judged by 32 P i labeling. Although lipopolysaccharide is detectable in F. novicida U112, less than 5 % of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2′, and a secondary palmitoyl residue at position 2′. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4′-and 3′-positions of A1 and A2 are not derivatized, and Kdo is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an α-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An α-linked glucose moiety is attached to the 6′-position of A2. The lipid A released by mild acid hydrolysis of F. novicida lipopolysaccharide consists solely of component A1. F. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.Lipopolysaccharide (LPS) makes up the outer leaflet of the outer membranes of most Gramnegative bacteria (1-3). It consists of a hydrophobic moiety known as lipid A, a non-repeating core oligosaccharide, and a distal repeating oligosaccharide, termed the O-antigen (1-3). Lipid A of wild-type E. coli is a hexa-acylated disaccharide of glucosamine that is phosphorylated at the 1-and 4′-positions (1-3) (Fig. 1A). It is recognized by the TLR4/MD2 receptor of the innate immune system (4-8), which triggers an inflammatory response and helps to clear localized infections. However, a more generalized response to lipid A in the context of a systemic infection, accompanied by massive over-production of cytokines, can lead to Gramnegative septic shock and death (9,10).Contact: C. R. H. Raetz,; E-mail: raetz@biochem.duke.edu. SUPPORTING INFORMATION AVAILABLE The NOESY analysis of HA2 and the HMBC spectra of HA1 and HA2 are available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 17. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWild-type E. coli cells contain ∼ 0.15 lipid A residues per glycerophospholipid molecule (11). All of the lipid A residues are covalently attached to LPS, whereas free lipid A and lipid A precursors are not usually detectable by 32 P i labeling (12) or mass spectrometry (MS) (13). Heptose-deficie...

LPS remodeling is an evolved survival strategy for bacteria

“…To observe the alteration of lipid A structures during transmission from an environmental source to a mammalian host, the lipid A synthesized by Fn after growth at the representative temperatures, 18°C (environment), 25°C (insect), and 37°C (mammalian) was analyzed using matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectrometry (MS) in the negative ion mode (23). Three lipid A patterns were identified by MALDI-TOF MS, associated with growth at 18°C, 25°C, and 37°C ( OH-C18 (17,24,25). For lipid A extracted after growth at 25°C, the major peaks were identified at m/z 1609, 1637 (dominant), 1665, 1771, 1799, and 1827 ( Fig.…”

Attenuated virulence of aFrancisellamutant lacking the lipid A 4′-phosphatase

“…3A) shows the presence of two phosphorus atoms. In contrast, the lipid A present in wild-type cells contains only a single phosphorus atom (12). Selective decoupling (17,18) of the individual phosphorus atoms present in compound A4 demonstrates that one phosphate group is attached to the 4Ј position of the glucosamine disaccharide and the other to the 1 position of the proximal unit ( Fig.…”

“…Both of these substituents are necessary for robust signaling via TLR4 (13). Another unusual feature of both F. novicida and the live vaccine strain of F. tularensis is the fact that most of their lipid A is present in a ''free'' form (12), i.e., lacking the core oligosaccharide and O-antigen.…”

“…Despite their unusual lipid A structures and relatively low levels of intact LPS (12), F. novicida and F. tularensis encode the same lipid A biosynthetic enzymes present in E. coli (14,15), suggesting that F. novicida cells initially synthesize lipid A precursors containing the 4Ј-phosphate group but thereafter remove it. We have recently discovered a selective lipid A 4Ј-phosphatase in F. novicida, designated LpxF (16).…”

Lipopolysaccharide Engineering in Neisseria meningitidis

“…2B, m/z 1237.865 and m/z 1196.863) in this preparation. Given that 3-O-acylated fatty acids in LPS are especially labile to alkaline hydrolysis (28,29) and to answer this question, we performed a very mild alkaline hydrolysis of the LPS in 5% triethylamine at 45°C. First, the hexaacylated LPS from the lgtB Ϫ mutant was subjected to alkaline hydrolysis under these conditions to determine how specific these were for the cleavage of 3-O-acylated C12:0(3-OH) fatty acids.…”