Department of Immunochemistry, L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
Hafnia alvei, a gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. Various 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-containing fragments different from known structures of core oligosaccharides were previously found among fractions obtained by mild acid hydrolysis of some H. alvei lipopolysaccharides (LPSs). However, the positions of these segments in the LPS structure were not known. Analysis of de-N,O-acylated LPS by nuclear magnetic resonance spectroscopy and mass spectrometry allowed the determination of the location of a Kdo-containing trisaccharide in the structure of H. alvei PCM 32 LPS. It was established that the trisaccharide {L-α-D-Hepp-(1→4)-[α-D-Galp6OAc-(1→7)]-α-Kdop-(2→} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between →4,7)-α-Kdop and →2)-Glcp in the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes of H. alvei LPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides of H. alvei 2, 32, 600, 1192, 1206, and 1211 LPSs. The weaker cross-reactions with LPSs of strains 974, 1188, 1198, 1204, and 1214 suggest the presence of similar structures in these LPSs, as well. Thus, we have identified new examples of endotoxins among those elucidated so far. This type of core oligosaccharide deviates from the classical scheme by the presence of the structural Kdo-containing motif in the outer-core region.
13C NMR, 1H NMR, methylation, NMR-2D, de-O-acylation, SDS-PAGE, sugar analysis, ELISA, 31P NMR, ESI-MS, mild acid hydrolysis, MALDI-TOF MS, NMR-1D, serological methods, immunoblotting, de-N-acylation
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7 C8
0,6,6,4 aXKdop ? 103.3 36.6 67.1 68.1 73.3 ? 64.5
0,6,6,5,3,3,3,2,4 aXLDmanHepp 98.7 71.6 71.1 67.6 73.2 ? 64.5
0,6,6,5,3,3,3,2,7 aDGalp 101.8 69.6 70.7 70.7 70.3 64.5
0,6,6,5,3,3,3,2 aXKdop ? 100.0 34.5 72.3 64.0 71.2 78.9 62.8
0,6,6,5,3,3,3 aDGlcp 96.8 74.0 73.1 72.6 72.2 62.7
0,6,6,5,3,3 aDGlcp 102.4 71.9 80.3 72.3 73.5 62.1
0,6,6,5,3,4 P
0,6,6,5,3,7 aXLDmanHepp 101.7 71.5 72.2 67.6 73.0 ? 64.5
0,6,6,5,3 aXLDmanHepp 103.8 71.3 79.4 69.2 74.5 68.9 70.1
0,6,6,5,4 P
0,6,6,5 aXLDmanHepp 100.0 72.5 78.2 70.4 74.6 70.4 64.3
0,6,6 aXKdop ? 101.0 35.6 72.3 69.6 73.1 ? 65.2
0,6 bDGlcpN 103.1 57.5 75.6 71.2 75.8 62.7
0 aDGlcpN 94.0 56.4 73.2 71.3 73.3 70.3
P
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8
0,6,6,4 aXKdop - - 1.76-2.14 4.16 4.05 3.65 ? 3.94
0,6,6,5,3,3,3,2,4 aXLDmanHepp 5.08 3.94 3.85 3.82 3.65 ? ?
0,6,6,5,3,3,3,2,7 aDGalp 5.20 3.82 3.88 3.99 4.15 3.73
0,6,6,5,3,3,3,2 aXKdop - - 1.88-2.30 4.20 4.33 4.39 3.94 3.73-4.00
0,6,6,5,3,3,3 aDGlcp 5.46 3.70 3.80 3.27 4.18 3.57-3.93
0,6,6,5,3,3 aDGlcp 5.23 3.54 4.01 3.68 3.81 3.73-3.89
0,6,6,5,3,4 P
0,6,6,5,3,7 aXLDmanHepp 4.89 3.95 3.85 3.84 3.64 ? ?
0,6,6,5,3 aXLDmanHepp 5.16 4.43 4.05 4.34 3.74 4.27 3.65-3.68
0,6,6,5,4 P
0,6,6,5 aXLDmanHepp 5.22 4.01 4.03 4.32 4.26 4.03 ?
0,6,6 aXKdop - - 1.90-2.21 4.14 4.21 3.62 ? 3.56-3.91
0,6 bDGlcpN 4.68 2.86 3.46 3.47 3.57 3.56
0 aDGlcpN 5.48 3.00 3.74 3.45 4.08 3.81-4.19
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 36.6/1.76-2.14 67.1/4.16 68.1/4.05 73.3/3.65 ?/? 64.5/3.94
0,6,6,5,3,3,3,2,4 aXLDmanHepp 98.7/5.08 71.6/3.94 71.1/3.85 67.6/3.82 73.2/3.65 ?/? 64.5/?
0,6,6,5,3,3,3,2,7 aDGalp 101.8/5.20 69.6/3.82 70.7/3.88 70.7/3.99 70.3/4.15 64.5/3.73
0,6,6,5,3,3,3,2 aXKdop 34.5/1.88-2.30 72.3/4.20 64.0/4.33 71.2/4.39 78.9/3.94 62.8/3.73-4.00
0,6,6,5,3,3,3 aDGlcp 96.8/5.46 74.0/3.70 73.1/3.80 72.6/3.27 72.2/4.18 62.7/3.57-3.93
0,6,6,5,3,3 aDGlcp 102.4/5.23 71.9/3.54 80.3/4.01 72.3/3.68 73.5/3.81 62.1/3.73-3.89
0,6,6,5,3,4 P
0,6,6,5,3,7 aXLDmanHepp 101.7/4.89 71.5/3.95 72.2/3.85 67.6/3.84 73.0/3.64 ?/? 64.5/?
0,6,6,5,3 aXLDmanHepp 103.8/5.16 71.3/4.43 79.4/4.05 69.2/4.34 74.5/3.74 68.9/4.27 70.1/3.65-3.68
0,6,6,5,4 P
0,6,6,5 aXLDmanHepp 100.0/5.22 72.5/4.01 78.2/4.03 70.4/4.32 74.6/4.26 70.4/4.03 64.3/?
0,6,6 aXKdop 35.6/1.90-2.21 72.3/4.14 69.6/4.21 73.1/3.62 ?/? 65.2/3.56-3.91
0,6 bDGlcpN 103.1/4.68 57.5/2.86 75.6/3.46 71.2/3.47 75.8/3.57 62.7/3.56
0 aDGlcpN 94.0/5.48 56.4/3.00 73.2/3.74 71.3/3.45 73.3/4.08 70.3/3.81-4.19
P
report error
Found 1 record.
Displayed record 1
]; septicemia [ICD11: MA15.Y 
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