Structural analysis of Francisella tularensis lipopolysaccharide

Abstract

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a β‐GlcN‐(1–6)‐GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O‐1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O‐3 and O‐4 of β‐GlcN, and no substituent at O‐4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure: where Δ‐GalNA‐(1–3)‐β‐QuiNAc represents a modified fragment of the O‐chain repeating unit. Two shorter oligosaccharides lacking the O‐chain fragment were also identified. A minor amount of the disaccharide β‐GlcN‐(1–6)‐α‐GlcN‐1‐P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end. The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2‐N‐(3‐O‐acyl4‐acyl2)‐β‐GlcN‐(1–6)‐2‐N‐acyl1−3‐O‐acyl3‐GlcN where acyl1, acyl2 and acyl3 are 3‐hydroxyhexadecanoic or 3‐hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH‐1 of the reducing end glucosamine, and OH‐3 and OH‐4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.

NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus

“…Compositional analysis by GLC-MS of both core oligosaccharide fractions resulted in identical sugar residues being present in comparable proportions. As expected, the core oligosaccharide fraction of the mutant did not contain contaminating amylopectin (21) and sugars from the O-chain (9). Thus, the Y1C12 mutant was found to be representative for further structural analysis of the complete rough-type C. canimorsus LPS (3).…”

Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS

“…However, the O-antigenspecific glycose N-acetyl quinovosamine (QuiNAc) was not detected in WbtI G191V LPS, confirming that the O antigen was not present in the mutant. The presence of a complete core structure (Vinogradov et al, 2002) in WbtI G191V , but no O antigen, was confirmed by MALDI-MS ( Fig. 2; details in the legend).…”

“…Most of the work on F. tularensis surface components has focused on the LPS. The LPS of F. tularensis types A and B is unusual in that the O antigen consists entirely of dideoxyglycoses, the core oligosaccharide contains mannose in place of heptose (Vinogradov et al, 2002(Vinogradov et al, , 1991, and lipid A of the live vaccine strain (LVS) is tetraacylated and lacks phosphate (Vinogradov et al, 2002), while lipid A from a virulent type B isolate also contains a phosphatelinked galactosamine (Phillips et al, 2004). Furthermore, the LPS does not signal through TLR4, is not an agonist for TLR4, and does not induce an inflammatory response Cole et al, 2006;Hajjar et al, 2006), which is probably due to the atypical structure of lipid A.…”

A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence

“…The lipopolysaccharide (LPS) of F. tularensis possesses an atypical lipid A moiety and core structure [15]. Mice deficient in Toll-like receptor (TLR) 4, which binds LPS, do not gain increased resistance to F. tularensis, succumbing to disease as rapidly as do wild-type mice [16].…”