Reinvestigation of the structure of Brucella O-antigens

Abstract

O-Specific polysaccharides of Brucella contain two antigenic determinants, called A and M. Most of the strains express epitope A with a small amount of epitope M, whereas Brucella melitensis strain 16 M expresses longer polymer consisting mostly of M-type epitopes. Proposed explanation was that epitope A is defined by 1-2-linked homopolymer of N-formylperosamine (Rha4NFo), while epitope M is a pentasaccharide with four 2- and one 3-substituted Rha4NFo. We reinvestigated both types of structures by 2D NMR and showed that M-epitope is a tetrasaccharide, missing one of the 2-linked Rha4NFo as compared to the previously proposed structure. Polysaccharide from B. melitensis 16 M contains a fragment of 1-2-linked polymer, capped with M-type polymer. Other strains contain one or two M-type units at the non-reducing end of the 1-2-linked O-chain.

Keywords: 2-amino-2,6-dideoxy-d-glucose (quinovosamine); 4-amino-4,6-dideoxy-d-mannose (perosamine); 4-formamido-4,6-dideoxy-d-mannose (N-formylperosamine); Brucella; LPS; MS; NMR; QuiN; Rha4N; Rha4NFo; Structure; lipopolysaccharide.

Fig. 1

Anomeric region of the ¹H NMR spectra of B. melitensis strains 3 (upper trace), B. suis 4 (middle) and B. melitensis 16M (lower trace). Sugar labels are as on Fig. 1. The presence of unit D in the B. melitensis 16M polysaccharide was interpreted in earlier publications as an indication of pentasaccharide repeats. In fact, it belongs to a 1–2-linked homopolymer.

Fig. 2

Anomeric region of the overlapped COSY (green), TOCSY (red) and NOESY (black) spectra of the B. melitensis 16M polysaccharide. Single number labels are correlations between indicated proton and H-1 of the same residue. Note that unit D shows no NOE to other spin systems, because it belongs to a 1–2-linked homopolymer.

Fig. 3

MALDI mass spectrum of the native polysaccharide of B. suis 4. Numbers above peaks indicate the number of Rha4N residues in the molecular species. Masses are [M+22]⁺. Tall peaks contain one less formyl group than the number of Rha4N residues, next lower mass peaks to the left of each of them correspond to the lack of additional formyl group (28 amu). Mass of the core without Rha4N residues = 893.3 Da. For example, peak at m/z 1407.6 corresponds to a core with 3 Rha4N residues and 2 formyl groups.

Fig. 4

MALDI mass spectrum of the N-deformylated polysaccharide from B. melitensis 16M. Numbers above peaks indicate the number of Rha4N residues in the molecular species. Masses are [M+1]⁺. Mass of the core without Rha4N residues = 893.3 Da. Note the absense of any clustering due to the presence of the tetrasaccharide repeats.