The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and 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)-α-D-GlcpN3N1P), in addition to components lacking one of the terminal β-D-GlcpN and/or the β-D-Glcp residue (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 manBcore 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 terminal β-D-Glcp 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). 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 mannosyl transferase role predicted for WadC and the lack of GlcpN 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-GlcpN-(1→6)-β-D-GlcpN-(1→4)[β-D-GlcpN-(1→6)]-β-D-GlcpN-(1→3)-α-D-Manp-(1→5)] structure in virulence.
mutant, Gram-negative bacteria, Brucella melitensis, lipopolysaccharide (LPS), nuclear magnetic resonance (NMR), WadC
NCBI PubMed ID: 26867577Publication DOI: 10.1074/jbc.M115.701540Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: goran.widmalm@su.se
Institutions: Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden, the Instituto de Salud Tropical, Instituto de Investigacion Sanitaria de Navarra, and Departamento de Microbiologia y Parasitologia, Universidad de Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain, the Division of Structural Biochemistry, Leibniz-Center for Medicine and Biosciences, Priority Area Asthma and Allergy, Research Center Borstel, Airway Research Center North, Member of the German Center for Lung Research, D-23845 Borstel, Germany, the Centre d'Immunologie de Marseille-Luminy Aix-Marseille University, UM2 Marseille, France, INSERM, U1104 Marseille, France, and CNRS, UMR7280 Marseille, France
Methods: 13C NMR, 1H NMR, virulence assays, SDS-PAGE, ELISA, 31P NMR, mild acid hydrolysis, Western blotting, de-O-acylation with hydrazine, composition analysis, NMR-1D, HR-ESI-MS
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