Dipartimento di Chimica Organica e Biochimica, Universita di Napoli Federico II, Napoli, Italy
This is the first structural elucidation of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia vietnamiensis, a clinically important member of Burkholderia cepacia complex, a group of over 10 opportunistic species that are highly problematic in cystic fibrosis. We have characterized a novel LOS structure extracted from two clonal strains of B. vietnamiensis isolated from a cystic fibrosis patient who underwent lung transplantation. Strains were selected from the pretransplantation and post-transplantation periods and endotoxin was extracted. Subsequent analysis interestingly revealed identical oligosaccharidic sequences, but variation in lipid A moieties. Further, both LOS fractions were tested for their immunostimulatory activity on human myelomonocytic U937 cells and for signaling on an HEK293 cell line stably expressing both TLR 4 and MD-2. We observed an increase in lipid A acylation and a resultant increase in biological activity in bio-reporter assays of TNF-α secretion in the post-transplantation strain.
13C NMR, 1H NMR, GLC-MS, NMR-2D, SDS-PAGE, MALDI-TOF MS, composition analysis, NMR-1D
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7 C8
4 aXKop ? ? 70.8 71.8 71.7 71.9 71.8 63.8
5,3,2 aLRhap 97.4 68.8 70.2 72.3 69.7 17.4
5,3,3,3,3 bDGalp 105.3 70.5 72.3 68.5 75.0 60.8
5,3,3,3,2 Ac
5,3,3,3 aDGalpN 92.6 47.9 77.6 68.5 71.2 61.5
5,3,3,2 Ac
5,3,3 bDGalpN 100.6 51.4 74.9 70.0 74.3 60.8
5,3,7,7 %aXLDmanHepp 101.8 70.0 70.4 70.4 71.9 68.4 62.7
5,3,7 aXLDmanHepp 100.9 68.8 71.6 71.6 72.0 70.4 70.4
5,3 aXLDmanHepp 98.1 77.7 79.5 68.8 72.9 70.4 70.5
5,4 bDGlcp 102.1 73.6 75.6 70.2 77.2 61.4
5 aXLDmanHepp 98.5 71.7 71.8 72.7 72.2 68.7 63.7
aXKdo? ? ? 34.8 71.6 68.4 71.5 71.8 63.7
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8
4 aXKop - - 3.74 3.91 4.00 3.56 3.97 3.68-3.88
5,3,2 aLRhap 4.69 3.86 3.76 3.26 3.87 1.16
5,3,3,3,3 bDGalp 4.32 3.40 3.53 3.81 3.62 3.63-3.70
5,3,3,3,2 Ac
5,3,3,3 aDGalpN 4.97 4.29 3.67 4.16 3.70 3.72-3.84
5,3,3,2 Ac
5,3,3 bDGalpN 4.70 4.00 3.69 3.84 3.58 3.65-3.72
5,3,7,7 %aXLDmanHepp 4.81 3.85 3.76 3.76 3.56 4.02 3.57-3.87
5,3,7 aXLDmanHepp 4.80 3.87 3.72 3.72 3.53 3.95 3.61
5,3 aXLDmanHepp 5.29 4.13 3.97 3.88 3.64 3.96 3.60
5,4 bDGlcp 4.45 3.17 3.38 3.26 3.36 3.69-3.85
5 aXLDmanHepp 5.18 3.95 4.03 4.20 4.11 3.96 3.67-3.90
aXKdo? - - 1.97-2.01 4.00 4.13 3.71 3.92 3.67-3.89
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7 C8/H8
4 aXKop 70.8/3.74 71.8/3.91 71.7/4.00 71.9/3.56 71.8/3.97 63.8/3.68-3.88
5,3,2 aLRhap 97.4/4.69 68.8/3.86 70.2/3.76 72.3/3.26 69.7/3.87 17.4/1.16
5,3,3,3,3 bDGalp 105.3/4.32 70.5/3.40 72.3/3.53 68.5/3.81 75.0/3.62 60.8/3.63-3.70
5,3,3,3,2 Ac
5,3,3,3 aDGalpN 92.6/4.97 47.9/4.29 77.6/3.67 68.5/4.16 71.2/3.70 61.5/3.72-3.84
5,3,3,2 Ac
5,3,3 bDGalpN 100.6/4.70 51.4/4.00 74.9/3.69 70.0/3.84 74.3/3.58 60.8/3.65-3.72
5,3,7,7 %aXLDmanHepp 101.8/4.81 70.0/3.85 70.4/3.76 70.4/3.76 71.9/3.56 68.4/4.02 62.7/3.57-3.87
5,3,7 aXLDmanHepp 100.9/4.80 68.8/3.87 71.6/3.72 71.6/3.72 72.0/3.53 70.4/3.95 70.4/3.61
5,3 aXLDmanHepp 98.1/5.29 77.7/4.13 79.5/3.97 68.8/3.88 72.9/3.64 70.4/3.96 70.5/3.60
5,4 bDGlcp 102.1/4.45 73.6/3.17 75.6/3.38 70.2/3.26 77.2/3.36 61.4/3.69-3.85
5 aXLDmanHepp 98.5/5.18 71.7/3.95 71.8/4.03 72.7/4.20 72.2/4.11 68.7/3.96 63.7/3.67-3.90
aXKdo? 34.8/1.97-2.01 71.6/4.00 68.4/4.13 71.5/3.71 71.8/3.92 63.7/3.67-3.89
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