Novel Modification of Lipid A of Francisella tularensis

Abstract

We have investigated the lipid A of Francisella tularensis subsp. holarctica strain 1547-57, a type B strain, by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, nanoelectrospray quadrupole ion-trap mass spectrometry, and chemical methods. In accordance with the previously published structures of the lipid A from F. tularensis live vaccine strain (LVS) (ATCC 29684) (E. Vinogradov et al., Eur. J. Biochem. 269:6112-6118, 2002), all of the major lipid A forms from strain 1547-57 were tetraacylated. As in the LVS strain, the major fatty acids detected in the F. tularensis 1547-57 lipid A sample included 3-hydroxyoctadecanoic acid, 3-hydroxyhexadecanoic acid, hexadecanoic acid, and tetradecanoic acid. However, several of the lipid A components present in strain 1547-57 were of higher molecular weight than the previously published structures. A major component with an M r of 1,666 was found to contain three C 18:0 (3-OH) fatty acids, one C 16:0 fatty acid, one phosphate group, and one 161-Da moiety. This 161-Da moiety could be removed from the lipid A by treatment with aqueous hydrofluoric acid and was identified as galactosamine following peracetylation and analysis by gas chromatography-mass spectrometry. Detailed investigations of the M r -1,666 species by ion-trap mass spectrometry with multiple stages of fragmentation suggested that the galactosamine-1-phosphate was linked to the reducing terminus of the lipid A. Similar to the modification of lipid A with arabinosamine, lipopolysaccharide species from F. tularensis containing a phosphate-linked galactosamine could potentially influence its intracellular survival by conferring resistance to antimicrobial peptides.Francisella tularensis is an encapsulated gram-negative bacterium that causes tularemia, a severe disease of humans and other mammals (12,24). Severity of disease depends on the host immune response and the route of infection, including intradermal inoculation via zoonotic transmission, ingestion of contaminated meat or water, and inhalation (7). F. tularensis is a facultative intracellular bacterium, and several studies have shown that a cell-mediated immune response may be required for controlling the infection (22,24). Recently, interest in F. tularensis has increased because of its suitability for use as an agent of biological warfare (6).F. tularensis possesses a lipopolysaccharide (LPS) that, when compared to Escherichia coli LPS, is not biologically active (1,8,23). The only biological activity attributed to F. tularensis LPS in vitro is the ability to activate complement (8). The relatively nonendotoxic nature of the LPS is putatively attributed to the unusual structure of the lipid A molecule. A recent study showed that the lipid A molecule of F. tularensis live vaccine strain (LVS) is not only tetraacylated but also lacks phosphate substituents (27). These structural features may contribute to its low toxicity. It has been shown that mutations affecting the degree of acylation or the acylation pattern of lipid...

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.…”

“…We and others have previously demonstrated for Francisella that temperature plays an important role in altering the composition of lipid A (15,17,18). Upon growth at low temperatures (25°C or lower), a mannose residue was added to the nonreducing glucosamine on the lipid A backbone and extensive heterogeneity in the composition and position of the acyl chains (18) was observed.…”

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

“…Because it is not extensively glycosylated, F. novicida lipid A is extracted together with the glycerophospholipids by the method of Bligh and Dyer. Small amounts of LPS are indeed detectable in F. novicida, as well as other strains of Francisella (18,22), and their lipid A has been partially characterized by other investigators (Fig. 1).…”

“…1C) and, in addition, described an unprecedented galactosamine phosphate substituent at the 1 position of lipid A isolated from F. tulalrensis 1547-57 ( Fig. 1D) (22). Because NMR spectroscopy was not utilized by Phillips et al (22), the anomeric configurations of the galactosamine and the proximal glucosamine residues could not be determined (Figs.…”

“…The LPS of F. tularensis has been isolated (17), and the structures of its O-antigen, core and lipid A domains have been partially characterized (18)(19)(20)(21)(22). Some differences and ambiguities in the structures reported for F. tularensis lipid A are noteworthy (Fig.…”