title: Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence.
abstract: The structures of the lipooligosaccharides fromBrucella melitensismutants affected in the WbkD and ManBcoreproteins have been fully characterized using NMR spectroscopy. The results revealed that disruption ofwbkDgives 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-Glcpresidues (48 and 17%, respectively). These structures were identical to those of the R-LPS fromB. melitensisEP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption ofmanBcoregives 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-Glcpresidue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcoreproteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion ofB. melitensis wadCremoves 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 GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential inB. melitensisvirulence, the core deficiency in thewadCmutant 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.
authors: From the Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.
authors: Carolina
authors: C
authors: Fontana
authors: the Instituto de Salud Tropical, Instituto de Investigación Sanitaria de Navarra, and Departamento de Microbiología y Parasitología, Universidad de Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain.
authors: Raquel
authors: R
authors: Conde-Álvarez
authors: From the Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.
authors: Jonas
authors: J
authors: Ståhle
authors: 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.
authors: Otto
authors: O
authors: Holst
authors: the Instituto de Salud Tropical, Instituto de Investigación Sanitaria de Navarra, and Departamento de Microbiología y Parasitología, Universidad de Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain.
authors: Maite
authors: M
authors: Iriarte
authors: the Centre d'Immunologie de Marseille-Luminy Aix-Marseille University, UM2 Marseille, France.
authors: Yun
authors: Y
authors: Zhao
authors: the Centre d'Immunologie de Marseille-Luminy Aix-Marseille University, UM2 Marseille, France, INSERM, U1104 Marseille, France, and CNRS, UMR7280 Marseille, France.
authors: Vilma
authors: V
authors: Arce-Gorvel
authors: the Centre d'Immunologie de Marseille-Luminy Aix-Marseille University, UM2 Marseille, France, INSERM, U1104 Marseille, France, and CNRS, UMR7280 Marseille, France.
authors: Seán
authors: S
authors: Hanniffy
authors: the Centre d'Immunologie de Marseille-Luminy Aix-Marseille University, UM2 Marseille, France, INSERM, U1104 Marseille, France, and CNRS, UMR7280 Marseille, France.
authors: Jean-Pierre
authors: JP
authors: Gorvel
authors: the Instituto de Salud Tropical, Instituto de Investigación Sanitaria de Navarra, and Departamento de Microbiología y Parasitología, Universidad de Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain.
authors: Ignacio
authors: I
authors: Moriyón
authors: From the Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden, goran.widmalm@su.se.
authors: Göran
authors: G
authors: Widmalm
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017347:Brucella melitensis / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D002006:Brucellosis / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D005260:Female; D008070:Lipopolysaccharides / Q000235:genetics / Q000378:metabolism*; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D009097:Multienzyme Complexes; D009844:Oligosaccharides; D037521:Virulence Factors; C093300:ManB protein, bacteria; D009713:Nucleotidyltransferases; D008359:Mannose-6-Phosphate Isomerase
keywords: Brucella melitensis; Gram-negative bacteria; WadC; glycosyltransferase; lipopolysaccharide (LPS); mutant; nuclear magnetic resonance (NMR)