A Typical Preparation of Francisella tularensis O-Antigen Yields a Mixture of Three Types of Saccharides

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, United States.
Biochemistry (Impact Factor: 3.02). 11/2011; 50(50):10941-50. DOI: 10.1021/bi201450v
Source: PubMed


Tularemia is a severe infectious disease in humans caused by the Gram-negative bacterium Francisella tularensis (Ft). Because of its low infectious dose, high mortality rate, and the threat of its large-scale dissemination in weaponized form, development of vaccines and immunotherapeutics against Ft is essential. Ft lipopolysaccharide (LPS), which contains the linear graded-length saccharide component O-antigen (OAg) attached to a core oligosaccharide, has been reported as a protective antigen. Purification of LPS saccharides of defined length and composition is necessary to reveal the epitopes targeted by protective antibodies. In this study, we purified saccharides from LPS preparations from both the Ft subspecies holarctica live vaccine strain (LVS) and the virulent Ft subspecies tularensis SchuS4 strain using liquid chromatography. We then characterized the fractions using high-resolution mass spectrometry and tandem mass spectrometry. Three types of saccharides were observed in both the LVS and SchuS4 preparations: two consisting of OAg tetrasaccharide repeats attached to one of two core oligosaccharide variants and one consisting of tetrasaccharide repeats only (coreless). The coreless OAg oligosaccharides were shown to contain Qui4NFm (4,6-dideoxy-4-formamido-D-glucose) at the nonreducing end and QuiNAc (2-acetamido-2,6-dideoxy-O-D-glucose) at the reducing end. Purified homogeneous preparations of saccharides of each type will allow mapping of protective epitopes in Ft LPS.

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    • "Group 4 capsules are composed of similar (often identical) O-antigen sugars as those found in the LPS, contain acetimido sugars in their repeat unit structures, and are greater than 100 kDa in size (Whitfield, 2006). The capsule of Francisella has been shown to have a molecular weight of 100–250 kDa and contain the core sugar tetrasaccharide repeat of <2-acetamido-2,6-dideoxy-o-glucose (o-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (o-Qui4NFm), and 2-acetamido-2-deoxy-o-galacturnoamide (o-GalNAcAN), with the o-GalNAcAN unit present at twice the concentration of the other two sugars (Apicella et al., 2010; Wang et al., 2011). This is the exact composition of the sugars present in the Francisella repeating O-antigen subunits of the LPS (Vinogradov et al., 2002; Thomas et al., 2007). "
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