Arsanis Biosciences GmbH, Vienna, Austria, Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
The Escherichia coli lineage ST131-O25b:H4 is a globally spread multi-drug resistant clone responsible for a significant proportion of extraintestinal infections. Driven by the high medical need associated with this successful pathogenic lineage, we generated murine monoclonal antibodies against its lipopolysaccharide (LPS) O25b antigen in order to develop quick diagnostic tests. Murine mAbs were generated by immunization of mice with whole killed non-encapsulated ST131-O25b E. coli cells and screening hybridoma supernatants for binding to purified LPS molecules obtained from an E. coli ST131-O25b clinical isolate. The mAbs selected for further study bound to the surface of live E. coli O25b strains irrespective of the capsular type expressed, while they could not bind to bacteria or purified LPS from other serotypes - including the related classical O25 antigen (O25a). Using these specific mAbs we have developed a latex bead-based agglutination assay that has greater specificity, more rapid and simpler than the currently available typing methods. The high specificity of these mAbs can be explained by the novel structure of the O25b repeating unit elucidated in this paper. Based on comparative analysis by NMR and mass spectrometry, the N-acetyl-fucose in the O25a O-antigen had been replaced by O-acetyl-rhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by alignment of corresponding genetic loci, and were confirmed by trans-complementation of a rough mutant by the sub-serotype specific fragments of the rfb operons.
13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, SDS-PAGE, DNA techniques, ELISA, 31P NMR, MALDI-TOF MS, composition analysis, NMR-1D, serological methods, genetic methods, immunoblotting
LOS structure of the fraction 3 isolated from LPS Escherichia coli ST131-O25b:H4 (strain 81009)
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7 C8
5,3,3,3,2,6,7,3,3,3 aLRhap 101.9 71.1 71.0 72.8 69.6 17.3
5,3,3,3,2,6,7,3,3,6 bDGlcp 103.5 74.0 76.5 70.5 76.8 61.5
5,3,3,3,2,6,7,3,3 aDGlcp 95.5 72.1 80.7 68.4 71.7 68.9
5,3,3,3,2,6,7,3,2 Ac 173.8 21.1
5,3,3,3,2,6,7,3 aLRhap 99.6 68.9 73.3 71.2 69.5 17.2
5,3,3,3,2,6,7,2 Ac 175.5 23.2
5,3,3,3,2,6,7 bDGlcpN 101.3 56.2 83.1 69.2 76.8 61.5
5,3,3,3,2,6 aXLDmanHepp 100.2 70.8 71.6 66.9 72.5 68.1 72.7
5,3,3,3,2 aDGlcp 96.8 72.1 74.1 70.2 70.9 65.5
5,3,3,3 aDGlcp 97.7 76.3 71.7 70.0 72.1 60.7
5,3,3,6 aDGalp 98.9 70.2 70.2 70.1 71.7 62.0
5,3,3 aDGlcp 102.3 71.0 81.3 69.5 71.5 65.6
5,3,4,0,0 xXEtN 63.2 40.9
5,3,4,0 %xXP?
5,3,4 P
5,3,7 aXLDmanHepp 100.2 70.8 71.6 66.9 ? ? ?
5,3 aXLDmanHepp 103.7 70.5 80.5 69.4 73.1 67.9 68.5
5,4,0,0 xXEtN 63.2 40.9
5,4,0 %xXP?
5,4 P
5 aXLDmanHepp 100.1 69.9 78.6 70.5 ? ? 63.8
aXKdop ? 96.2 34.1 66.3 73.3 72.8 70.0 64.9
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8
5,3,3,3,2,6,7,3,3,3 aLRhap 5.09 4.04 3.78 3.44 4.02 1.25
5,3,3,3,2,6,7,3,3,6 bDGlcp 4.50 3.31 3.50 3.39 3.45 3.72-3.92
5,3,3,3,2,6,7,3,3 aDGlcp 4.95 3.66 3.79 3.61 4.05 3.89-4.14
5,3,3,3,2,6,7,3,2 Ac - 2.13
5,3,3,3,2,6,7,3 aLRhap 4.92 5.20 3.99 3.62 4.10 1.28
5,3,3,3,2,6,7,2 Ac - 2.05
5,3,3,3,2,6,7 bDGlcpN 4.59 3.84 3.59 3.55 3.45 3.76-3.93
5,3,3,3,2,6 aXLDmanHepp 4.88 3.97 3.79 3.85 3.55 4.16 3.79-4.00
5,3,3,3,2 aDGlcp 5.17 3.60 3.74 3.50 4.15 3.65-3.91
5,3,3,3 aDGlcp 5.46 3.67 3.85 3.54 4.06 3.81-3.89
5,3,3,6 aDGalp 5.03 3.82 3.91 4.04 3.95 3.75-3.75
5,3,3 aDGlcp 5.21 3.67 3.94 3.82 4.12 3.77-4.10
5,3,4,0,0 xXEtN 4.21 3.30
5,3,4,0 %xXP?
5,3,4 P
5,3,7 aXLDmanHepp 5.00 3.93 3.89 3.86 ? ? ?
5,3 aXLDmanHepp 5.11 4.37 4.12 4.41 3.81 4.27 3.76
5,4,0,0 xXEtN 4.21 3.30
5,4,0 %xXP?
5,4 P
5 aXLDmanHepp 5.21 4.01 4.08 4.43 ? ? 3.64-?
aXKdop - - 1.91-2.26 4.12 4.17 3.82 3.70 3.47-3.94
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7 C8/H8
5,3,3,3,2,6,7,3,3,3 aLRhap 101.9/5.09 71.1/4.04 71.0/3.78 72.8/3.44 69.6/4.02 17.3/1.25
5,3,3,3,2,6,7,3,3,6 bDGlcp 103.5/4.50 74.0/3.31 76.5/3.50 70.5/3.39 76.8/3.45 61.5/3.72-3.92
5,3,3,3,2,6,7,3,3 aDGlcp 95.5/4.95 72.1/3.66 80.7/3.79 68.4/3.61 71.7/4.05 68.9/3.89-4.14
5,3,3,3,2,6,7,3,2 Ac 21.1/2.13
5,3,3,3,2,6,7,3 aLRhap 99.6/4.92 68.9/5.20 73.3/3.99 71.2/3.62 69.5/4.10 17.2/1.28
5,3,3,3,2,6,7,2 Ac 23.2/2.05
5,3,3,3,2,6,7 bDGlcpN 101.3/4.59 56.2/3.84 83.1/3.59 69.2/3.55 76.8/3.45 61.5/3.76-3.93
5,3,3,3,2,6 aXLDmanHepp 100.2/4.88 70.8/3.97 71.6/3.79 66.9/3.85 72.5/3.55 68.1/4.16 72.7/3.79-4.00
5,3,3,3,2 aDGlcp 96.8/5.17 72.1/3.60 74.1/3.74 70.2/3.50 70.9/4.15 65.5/3.65-3.91
5,3,3,3 aDGlcp 97.7/5.46 76.3/3.67 71.7/3.85 70.0/3.54 72.1/4.06 60.7/3.81-3.89
5,3,3,6 aDGalp 98.9/5.03 70.2/3.82 70.2/3.91 70.1/4.04 71.7/3.95 62.0/3.75-3.75
5,3,3 aDGlcp 102.3/5.21 71.0/3.67 81.3/3.94 69.5/3.82 71.5/4.12 65.6/3.77-4.10
5,3,4,0,0 xXEtN 63.2/4.21 40.9/3.30
5,3,4,0 %xXP?
5,3,4 P
5,3,7 aXLDmanHepp 100.2/5.00 70.8/3.93 71.6/3.89 66.9/3.86 ?/? ?/? ?/?
5,3 aXLDmanHepp 103.7/5.11 70.5/4.37 80.5/4.12 69.4/4.41 73.1/3.81 67.9/4.27 68.5/3.76
5,4,0,0 xXEtN 63.2/4.21 40.9/3.30
5,4,0 %xXP?
5,4 P
5 aXLDmanHepp 100.1/5.21 69.9/4.01 78.6/4.08 70.5/4.43 ?/? ?/? 63.8/3.64-?
aXKdop 34.1/1.91-2.26 66.3/4.12 73.3/4.17 72.8/3.82 70.0/3.70 64.9/3.47-3.94
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