Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence *

摘要

The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManB_(core)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 -Glc p -(1→4)-α-Kdo p -(2→4)[β- d -Glc p N-(1→6)-β- d -Glc p N-(1→4)[β- d -Glc p N-(1→6)]-β- d -Glc p N-(1→3)-α- d -Man p -(1→5)]-α-Kdo p -(2→6)-β- d -Glc p N3N4 P -(1→6)-α- d -Glc p N3N1 P ), in addition to components lacking one of the terminal β- d -Glc p N and/or the β- d -Glc p 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 manB_(core) gives rise to a deep-rough pentasaccharide core (β- d -Glc p -(1→4)-α-Kdo p -(2→4)-α-Kdo p -(2→6)-β- d -Glc p N3N4 P -(1→6)-α- d -Glc p N3N1 P ) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β- d -Glc p residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManB_(core)proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also 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 for WadC and the lack of Glc p N residues 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 -Glc p N-(1→6)-β- d -Glc p N-(1→4)[β- d -Glc p N-(1→6)]-β- d -Glc p N-(1→3)-α- d -Man p -(1→5) structure in virulence.