Brucella periplasmic cyclic β-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic β-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic β-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic β-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-β-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection.

Brucella abortus, virulence factor, mouse, colonization, protein A, adaptation

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
	bDGlcp	102.4-102.9	82.4-83.2	76.1-76.3	69.4-69.6	77.0	61.36-61.40


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
	bDGlcp	4.70-4.72	3.38-3.44	3.60	3.30	3.32	3.61-3.78


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
	bDGlcp	102.4-102.9/4.70-4.72	82.4-83.2/3.38-3.44	76.1-76.3/3.60	69.4-69.6/3.30	77.0/3.32	61.36-61.40/3.61-3.78

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](*)O[C@H](CO)[C@@H](O)[C@@H]1O

162.141 g/mol (C₆H₁₀R₂O₅, R = next and previous repeats, not counted in mol weight)