Novel modification of lipid A of Francisella tularensis - PubMed (original) (raw)

Novel modification of lipid A of Francisella tularensis

Nancy J Phillips et al. Infect Immun. 2004 Sep.

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.

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Figures

FIG. 1.

FIG. 1.

GC-MS total ion chromatogram of the FAMEs derived from the lipid A of F. tularensis strain 1547-57.

FIG. 2.

FIG. 2.

Sections of three MALDI-TOF mass spectra of the F. tularensis strain 1547-57 lipid A sample, acquired in reflectron mode. (A) The negative-ion MALDI-TOF MS. (B) The positive-ion MALDI-TOF MS. (C) The positive-ion MALDI-TOF MS of the lipid A sample after HF treatment for 40 h.

FIG. 3.

FIG. 3.

Negative-ion nanoelectrospray MS_n_ spectra of the lipid A from F. tularensis strain 1547-57. (A) MS/MS of m/z 1,665.2. (B) MS3 of m/z 1,408.9. (C) MS3 of m/z 1,203.9. (D) MS3 of m/z 947.8.

FIG. 4.

FIG. 4.

Section of the positive-ion MALDI spectrum of the F. tularensis 1547-57 lipid A sample after reduction by treatment with NaBD4 for 4 h, acquired in reflectron mode. Insets show expansions of the indicated peaks. Asterisks mark the monoisotopic molecular ions of reduced species that have incorporated three deuterium atoms.

FIG. 5.

FIG. 5.

Proposed structure of the 1,666-_M_r component of the F. tularensis strain 1547-57 lipid A sample. The diglucosamine backbone bears four fatty acids: three C18:0(3-OH) fatty acids, and one C16:0 fatty acid. The acylation pattern shown is consistent with the arrangement determined for the lipid A of F. tularensis LVS (ATCC 29684) (27). The further modification of the _M_r-1,666 component of F. tularensis strain 1547-57 lipid A with a galactosamine-1-phosphate on the reducing terminus represents a novel structural feature not previously observed. The anomeric configuration of the galactosamine-1-phosphate linkage has not been determined.

FIG. 6.

FIG. 6.

Sections of the MALDI-TOF mass spectra of the lipid A from F. tularensis LVS recorded in negative-ion (A) and positive-ion (B) reflectron modes. For comparison to the F. tularensis strain 1547-57 lipid A sample, refer to Fig. 2. For proposed compositions, see the text and Table 1.

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