Structural studies of lipopolysaccharide-defective mutants from brucella melitensis identify a core oligosaccharide critical in virulence

Abstract

The structures of the lipooligosaccharides from Brucella melitensis mutants affected in theWbkDand ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β- D-Glcp-(1→4)-α-Kdop-(2→4)[β-D-GlcpN-(1→6)-β-D-GlcpN- (1→4)[β-D-GlcpN-(1→6)]-β-D-GlcpN-(1→3)-α-D-Manp- (1→5)]-α-Kdop-(2→6)-β-D-GlcpN3N4P-(1→6)-β-D-GlcpN3N1P), in addition to components lacking one of the terminal β-DGlcpN and/or the β-D-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of man- Bcore gives rise to a deep-rough pentasaccharide core (β-D-Glcp- (1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-D-GlcpN3N4P- (1→6)-α-D-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminalβ-DGlcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). Wealso report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted forWadCand the lack of GlcpNresidues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the β- D-GlcpN-(1→6)-β-D-GlcpN-(1→4)[β-D-GlcpN-(1→6)]-β-DGlcpN-( 1→3)-α-D-Manp-(1→5) structure in virulence.