Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Host organism: Homo sapiens
Associated disease: brucellosis [ICD11:
1B95 
, ICD11:
XN22N ];
infection due to Brucella melitensis [ICD11:
XN7ZW ]
The structure was elucidated in this paperNCBI 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
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
Structure type: oligomer
Location inside paper: abstract, p.7734, fig.4B, BmEPR
Compound class: core oligosaccharide
Contained glycoepitopes: IEDB_130650,IEDB_130659,IEDB_130701,IEDB_141807,IEDB_144983,IEDB_151531,IEDB_152206,IEDB_983930,SB_44,SB_67,SB_72
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
Comments, role: deacylated LOS (17% nonasaccharide) obtained from Brucella melitensis BmEPR and mutant strain Bm_wbkD.
Related record ID(s): 11261, 11624, 11625, 11627
NCBI Taxonomy refs (TaxIDs): 29459
Show glycosyltransferases
NMR conditions: in D2O; pH 8 at 298 K
[as TSV]
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7 C8
0,6,6,4 aXKdop ? 101.74 35.29 66.77 67.37 72.96 71.03 64.85
0,6,6,5,3,4,6 bDGlcpN 104.18 57.26 76.30-76.33 70.55 76.87 61.62
0,6,6,5,3,4 bDGlcpN 104.03 57.45 76.01 70.48 75.70 69.66
0,6,6,5,3,6 bDGlcpN 103.77 57.16 76.30-76.33 70.55 76.87 61.57
0,6,6,5,3 bDGlcpN 102.12 ? ? ? ? ?
0,6,6,5 aDManp 101.40 68.90 80.47 65.66 73.41 61.66
0,6,6 aXKdop ? 100.45 35.25 74.96 74.36 ? ? ?
0,6,4 P
0,6 bDGlcpN3N 103.81 55.61 58.63 71.44 76.39 63.66
0 aDGlcpN3N 94.28 54.76 54.87 69.25 72.48 70.34
P
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8
0,6,6,4 aXKdop - - 1.802-2.161 4.042 4.022 3.702 3.994 3.782-3.983
0,6,6,5,3,4,6 bDGlcpN 4.426 2.682 3.384 3.464 3.460 3.752-3.934
0,6,6,5,3,4 bDGlcpN 4.442 2.748 3.405 3.467 3.668 3.851-4.267
0,6,6,5,3,6 bDGlcpN 4.465 2.723 3.373 3.409 3.460 3.748-3.913
0,6,6,5,3 bDGlcpN 4.626 ? ? ? ? ?
0,6,6,5 aDManp 5.275 4.310 4.089 3.874 4.089 3.887
0,6,6 aXKdop - - 1.996-2.099 4.177 4.254 ? ? ?
0,6,4 P
0,6 bDGlcpN3N 4.535 2.753 3.112 3.764 3.724 3.472-3.782
0 aDGlcpN3N 5.419 2.898 3.126 3.503 4.127 3.738-4.290
P
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7 C8/H8
0,6,6,4 aXKdop 35.29/1.802-2.161 66.77/4.042 67.37/4.022 72.96/3.702 71.03/3.994 64.85/3.782-3.983
0,6,6,5,3,4,6 bDGlcpN 104.18/4.426 57.26/2.682 76.30-76.33/3.384 70.55/3.464 76.87/3.460 61.62/3.752-3.934
0,6,6,5,3,4 bDGlcpN 104.03/4.442 57.45/2.748 76.01/3.405 70.48/3.467 75.70/3.668 69.66/3.851-4.267
0,6,6,5,3,6 bDGlcpN 103.77/4.465 57.16/2.723 76.30-76.33/3.373 70.55/3.409 76.87/3.460 61.57/3.748-3.913
0,6,6,5,3 bDGlcpN 102.12/4.626 ?/? ?/? ?/? ?/? ?/?
0,6,6,5 aDManp 101.40/5.275 68.90/4.310 80.47/4.089 65.66/3.874 73.41/4.089 61.66/3.887
0,6,6 aXKdop 35.25/1.996-2.099 74.96/4.177 74.36/4.254 ?/? ?/? ?/?
0,6,4 P
0,6 bDGlcpN3N 103.81/4.535 55.61/2.753 58.63/3.112 71.44/3.764 76.39/3.724 63.66/3.472-3.782
0 aDGlcpN3N 94.28/5.419 54.76/2.898 54.87/3.126 69.25/3.503 72.48/4.127 70.34/3.738-4.290
P
1H NMR data:
| Linkage | Residue | H1 | H2 | H3 | H4 | H5 | H6 | H7 | H8 |
|---|
| 0,6,6,4 | aXKdop |
|
| 1.802
2.161 | 4.042 | 4.022 | 3.702 | 3.994 | 3.782
3.983 |
| 0,6,6,5,3,4,6 | bDGlcpN | 4.426 | 2.682 | 3.384 | 3.464 | 3.460 | 3.752
3.934 | |
| 0,6,6,5,3,4 | bDGlcpN | 4.442 | 2.748 | 3.405 | 3.467 | 3.668 | 3.851
4.267 | |
| 0,6,6,5,3,6 | bDGlcpN | 4.465 | 2.723 | 3.373 | 3.409 | 3.460 | 3.748
3.913 | |
| 0,6,6,5,3 | bDGlcpN | 4.626 | ? | ? | ? | ? | ? | |
| 0,6,6,5 | aDManp | 5.275 | 4.310 | 4.089 | 3.874 | 4.089 | 3.887 | |
| 0,6,6 | aXKdop |
|
| 1.996
2.099 | 4.177 | 4.254 | ? | ? | ? |
| 0,6,4 | P | |
| 0,6 | bDGlcpN3N | 4.535 | 2.753 | 3.112 | 3.764 | 3.724 | 3.472
3.782 | |
| 0 | aDGlcpN3N | 5.419 | 2.898 | 3.126 | 3.503 | 4.127 | 3.738
4.290 | |
| | P | |
|
13C NMR data:
| Linkage | Residue | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 |
|---|
| 0,6,6,4 | aXKdop | ? | 101.74 | 35.29 | 66.77 | 67.37 | 72.96 | 71.03 | 64.85 |
| 0,6,6,5,3,4,6 | bDGlcpN | 104.18 | 57.26 | 76.30
76.33 | 70.55 | 76.87 | 61.62 | |
| 0,6,6,5,3,4 | bDGlcpN | 104.03 | 57.45 | 76.01 | 70.48 | 75.70 | 69.66 | |
| 0,6,6,5,3,6 | bDGlcpN | 103.77 | 57.16 | 76.30
76.33 | 70.55 | 76.87 | 61.57 | |
| 0,6,6,5,3 | bDGlcpN | 102.12 | ? | ? | ? | ? | ? | |
| 0,6,6,5 | aDManp | 101.40 | 68.90 | 80.47 | 65.66 | 73.41 | 61.66 | |
| 0,6,6 | aXKdop | ? | 100.45 | 35.25 | 74.96 | 74.36 | ? | ? | ? |
| 0,6,4 | P | |
| 0,6 | bDGlcpN3N | 103.81 | 55.61 | 58.63 | 71.44 | 76.39 | 63.66 | |
| 0 | aDGlcpN3N | 94.28 | 54.76 | 54.87 | 69.25 | 72.48 | 70.34 | |
| | P | |
|
![](nmr_drawer.py?width=750&height=130&nucleus=C&rotate_labels=false&spectrum=[{"s":101.74,"l":"2","c":[0,0,225]},{"s":35.29,"l":"3","c":[0,0,225]},{"s":66.77,"l":"4","c":[0,0,225]},{"s":67.37,"l":"5","c":[0,0,225]},{"s":72.96,"l":"6","c":[0,0,225]},{"s":71.03,"l":"7","c":[0,0,225]},{"s":64.85,"l":"8","c":[0,0,225]},{"s":104.18,"l":"1","c":[230,0,30]},{"s":57.26,"l":"2","c":[230,0,30]},{"s":76.32,"l":"3","c":[230,0,30]},{"s":70.55,"l":"4","c":[230,0,30]},{"s":76.87,"l":"5","c":[230,0,30]},{"s":61.62,"l":"6","c":[230,0,30]},{"s":104.03,"l":"1","c":[0,165,0]},{"s":57.45,"l":"2","c":[0,165,0]},{"s":76.01,"l":"3","c":[0,165,0]},{"s":70.48,"l":"4","c":[0,165,0]},{"s":75.7,"l":"5","c":[0,165,0]},{"s":69.66,"l":"6","c":[0,165,0]},{"s":103.77,"l":"1","c":[120,0,225]},{"s":57.16,"l":"2","c":[120,0,225]},{"s":76.32,"l":"3","c":[120,0,225]},{"s":70.55,"l":"4","c":[120,0,225]},{"s":76.87,"l":"5","c":[120,0,225]},{"s":61.57,"l":"6","c":[120,0,225]},{"s":102.12,"l":"1","c":[0,192,255]},{"s":101.4,"l":"1","c":[220,0,180]},{"s":68.9,"l":"2","c":[220,0,180]},{"s":80.47,"l":"3","c":[220,0,180]},{"s":65.66,"l":"4","c":[220,0,180]},{"s":73.41,"l":"5","c":[220,0,180]},{"s":61.66,"l":"6","c":[220,0,180]},{"s":100.45,"l":"2","c":[100,160,0]},{"s":35.25,"l":"3","c":[100,160,0]},{"s":74.96,"l":"4","c":[100,160,0]},{"s":74.36,"l":"5","c":[100,160,0]},{"s":103.81,"l":"1","c":[210,210,0]},{"s":55.61,"l":"2","c":[210,210,0]},{"s":58.63,"l":"3","c":[210,210,0]},{"s":71.44,"l":"4","c":[210,210,0]},{"s":76.39,"l":"5","c":[210,210,0]},{"s":63.66,"l":"6","c":[210,210,0]},{"s":94.28,"l":"1","c":[210,110,0]},{"s":54.76,"l":"2","c":[210,110,0]},{"s":54.87,"l":"3","c":[210,110,0]},{"s":69.25,"l":"4","c":[210,110,0]},{"s":72.48,"l":"5","c":[210,110,0]},{"s":70.34,"l":"6","c":[210,110,0]}])
The spectrum also has 10 signals at unknown positions (not plotted). |
There is only one chemically distinct structure:
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