Bacteroides thetaiotaomicron is one of the most extensively studied symbionts of the human gut. Despite its widespread distribution among human populations, still very little is known about the role of its cell envelope in the crosstalk with the immune system. Due to the extraordinary characteristic of B. thetaiotaomicron to express multiple capsular polysaccharides on its surface, research activities focused on defining how these polymers affect immune responses. This resulted in the drawback of neglecting another immunostimulatory cell surface glycoconjugate, the lipopolysaccharide (LPS). By taking advantage of an acapsular mutant of B. thetaiotaomicron, here we describe the characterization of the structure of the rough-type LPS produced by this gut mutualist. This was made up of a mono-phosphorylated and hypoacylated lipid A and of a highly charged core oligosaccharide. In vitro studies revealed a weak ability to engage the MD-2/TLR4 pathway, while it was able to promote TLR2-mediated response.

lipid A, lipopolysaccharide (LPS), gut microbiota, Bacteroides thetaiotaomicron, commensal LPS, structure to function

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8
0,6,6,5,2,2,2,2,2,2	aDGlcpA	97.6	71.5	72.0	72.8	75.0	177.3
0,6,6,5,2,2,2,2,2	bDGlcp	101.6	77.1	75.3	69.8	74.8	60.7
0,6,6,5,2,2,2,2	bDGlcp	101.8	76.7	74.4	69.8	74.8	61.2
0,6,6,5,2,2,2,6,4	aDGlcp	97.3	71.7	72.6	69.4	71.74	60.6
0,6,6,5,2,2,2,6	aDGlcpN	95.1	54.0	69.8	76.8	72.2	61.6
0,6,6,5,2,2,2	aDManp	98.2	79.0	71.0	67.1	71.5	66.2
0,6,6,5,2,2,3	P
0,6,6,5,2,2	aDManp	97.5	79.0	73.6	65.2	72.1	61.5
0,6,6,5,2	bDGlcp	101.9	77.9	75.6	69.5	74.4	60.6
0,6,6,5,3	aD4dthrHexp4enA	97.3	69.3	65.4	106.7	?	?
0,6,6,5	aDManp	98.4	75.2	77.0	67.2	72.1	60.5
0,6,6,4,0	xXEtN	61.9	40.0
0,6,6,4	P
0,6,6	aXKdop	?	?	34.2	70.9	72.5	?	70.9	63.3
0,6	bDGlcpN	99.5	55.5	72.1	69.6	74.3	62.3
0	aDGlcpN	90.3	54.3	69.8	70.0	72.8	68.7
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
0,6,6,5,2,2,2,2,2,2	aDGlcpA	5.28	3.41	3.90	3.64	4.51	-
0,6,6,5,2,2,2,2,2	bDGlcp	4.88	3.33	3.51	3.32	3.59	3.86
0,6,6,5,2,2,2,2	bDGlcp	4.78	3.37	3.49	3.33	3.55	3.47-3.63
0,6,6,5,2,2,2,6,4	aDGlcp	5.36	3.36	3.69	3.40	3.95	3.61-3.82
0,6,6,5,2,2,2,6	aDGlcpN	5.08	3.19	3.82	3.39	3.66	3.82
0,6,6,5,2,2,2	aDManp	5.36	4.29	3.85	3.61	3.71	3.80-3.92
0,6,6,5,2,2,3	P
0,6,6,5,2,2	aDManp	5.29	4.24	4.16	3.70	3.63	3.82-3.93
0,6,6,5,2	bDGlcp	4.61	3.35	3.46	3.37	3.54	3.63
0,6,6,5,3	aD4dthrHexp4enA	5.33	3.88	4.02	5.80	-	-
0,6,6,5	aDManp	5.31	4.49	4.10	3.62	3.65	3.65
0,6,6,4,0	xXEtN	4.00	3.18
0,6,6,4	P
0,6,6	aXKdop	-	-	1.95-2.13	4.49	4.24	?	3.86	3.54-3.83
0,6	bDGlcpN	4.93	2.89	3.52	3.47	3.54	3.44-3.53
0	aDGlcpN	5.51	3.25	3.78	3.27	4.07	3.77-4.13
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8
0,6,6,5,2,2,2,2,2,2	aDGlcpA	97.6/5.28	71.5/3.41	72.0/3.90	72.8/3.64	75.0/4.51	
0,6,6,5,2,2,2,2,2	bDGlcp	101.6/4.88	77.1/3.33	75.3/3.51	69.8/3.32	74.8/3.59	60.7/3.86
0,6,6,5,2,2,2,2	bDGlcp	101.8/4.78	76.7/3.37	74.4/3.49	69.8/3.33	74.8/3.55	61.2/3.47-3.63
0,6,6,5,2,2,2,6,4	aDGlcp	97.3/5.36	71.7/3.36	72.6/3.69	69.4/3.40	71.74/3.95	60.6/3.61-3.82
0,6,6,5,2,2,2,6	aDGlcpN	95.1/5.08	54.0/3.19	69.8/3.82	76.8/3.39	72.2/3.66	61.6/3.82
0,6,6,5,2,2,2	aDManp	98.2/5.36	79.0/4.29	71.0/3.85	67.1/3.61	71.5/3.71	66.2/3.80-3.92
0,6,6,5,2,2,3	P
0,6,6,5,2,2	aDManp	97.5/5.29	79.0/4.24	73.6/4.16	65.2/3.70	72.1/3.63	61.5/3.82-3.93
0,6,6,5,2	bDGlcp	101.9/4.61	77.9/3.35	75.6/3.46	69.5/3.37	74.4/3.54	60.6/3.63
0,6,6,5,3	aD4dthrHexp4enA	97.3/5.33	69.3/3.88	65.4/4.02	106.7/5.80		
0,6,6,5	aDManp	98.4/5.31	75.2/4.49	77.0/4.10	67.2/3.62	72.1/3.65	60.5/3.65
0,6,6,4,0	xXEtN	61.9/4.00	40.0/3.18
0,6,6,4	P
0,6,6	aXKdop			34.2/1.95-2.13	70.9/4.49	72.5/4.24	?/?	70.9/3.86	63.3/3.54-3.83
0,6	bDGlcpN	99.5/4.93	55.5/2.89	72.1/3.52	69.6/3.47	74.3/3.54	62.3/3.44-3.53
0	aDGlcpN	90.3/5.51	54.3/3.25	69.8/3.78	70.0/3.27	72.8/4.07	68.7/3.77-4.13
	P

The spectrum also has 5 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

NCCOP(=O)(O)O[C@@H]1C[C@](OC[C@H]2O[C@@H](OC[C@H]3O[C@H](OP(=O)(O)O)[C@H](N)[C@@H](O)[C@@H]3O)[C@H](N)[C@@H](O)[C@@H]2O)(C(=O)O)O[C@H]([C@H](O)CO)[C@@H]1O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O[C@H]2OC(C(=O)O)=C[C@@H](O)[C@@H]2O)[C@@H]1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O[C@H]1O[C@H](CO)[C@@H](O)[C@H](OP(=O)(O)O)[C@@H]1O[C@H]1O[C@H](CO[C@H]2O[C@H](CO)[C@@H](O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2N)[C@@H](O)[C@H](O)[C@@H]1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O[C@H]1O[C@H](C(=O)O)[C@@H](O)[C@H](O)[C@H]1O

2473.889 g/mol (C₈₂H₁₃₉N₄O₇₅P₃)

Bacteroides thetaiotaomicron is one of the most extensively studied symbionts of the human gut. Despite its widespread distribution among human populations, still very little is known about the role of its cell envelope in the crosstalk with the immune system. Due to the extraordinary characteristic of B. thetaiotaomicron to express multiple capsular polysaccharides on its surface, research activities focused on defining how these polymers affect immune responses. This resulted in the drawback of neglecting another immunostimulatory cell surface glycoconjugate, the lipopolysaccharide (LPS). By taking advantage of an acapsular mutant of B. thetaiotaomicron, here we describe the characterization of the structure of the rough-type LPS produced by this gut mutualist. This was made up of a mono-phosphorylated and hypoacylated lipid A and of a highly charged core oligosaccharide. In vitro studies revealed a weak ability to engage the MD-2/TLR4 pathway, while it was able to promote TLR2-mediated response.

lipid A, lipopolysaccharide (LPS), gut microbiota, Bacteroides thetaiotaomicron, commensal LPS, structure to function

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,3	bDGalp	103.1	70.8	74.3	70.1	75.1	60.8
4,3,2	Ac
4,3,6	bDGlcp	104.0	73.2	76.3	69.3	75.5	60.0
4,3	bDGalpN	101.5	54.6	79.9	67.5	73.9	68.9
4,2	Ac
4	aDGlcpN	96.6	53.0	79.7	71.6	71.5	60.2
	aDGalpA	97.4	67.5	71.7	80.9	73.2	175.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,3	bDGalp	4.37	3.40	3.51	3.80	3.58	3.65
4,3,2	Ac
4,3,6	bDGlcp	4.39	3.32	3.69	3.41	3.37	3.56
4,3	bDGalpN	4.49	3.73	3.78	4.10	3.85	3.79-3.99
4,2	Ac
4	aDGlcpN	4.94	3.76	3.57	3.41	3.69	3.70
	aDGalpA	5.18	4.07	4.01	4.24	4.60	-


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,3	bDGalp	103.1/4.37	70.8/3.40	74.3/3.51	70.1/3.80	75.1/3.58	60.8/3.65
4,3,2	Ac
4,3,6	bDGlcp	104.0/4.39	73.2/3.32	76.3/3.69	69.3/3.41	75.5/3.37	60.0/3.56
4,3	bDGalpN	101.5/4.49	54.6/3.73	79.9/3.78	67.5/4.10	73.9/3.85	68.9/3.79-3.99
4,2	Ac
4	aDGlcpN	96.6/4.94	53.0/3.76	79.7/3.57	71.6/3.41	71.5/3.69	60.2/3.70
	aDGalpA	97.4/5.18	67.5/4.07	71.7/4.01	80.9/4.24	73.2/4.60	

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@@H](O[C@H]2[C@@H](C(=O)O)O[C@H](O)[C@H](O)[C@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@@H]1O[C@H](CO[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H]1NC(C)=O

924.809 g/mol (C₃₄H₅₆N₂O₂₇)

Bacteroides thetaiotaomicron is one of the most extensively studied symbionts of the human gut. Despite its widespread distribution among human populations, still very little is known about the role of its cell envelope in the crosstalk with the immune system. Due to the extraordinary characteristic of B. thetaiotaomicron to express multiple capsular polysaccharides on its surface, research activities focused on defining how these polymers affect immune responses. This resulted in the drawback of neglecting another immunostimulatory cell surface glycoconjugate, the lipopolysaccharide (LPS). By taking advantage of an acapsular mutant of B. thetaiotaomicron, here we describe the characterization of the structure of the rough-type LPS produced by this gut mutualist. This was made up of a mono-phosphorylated and hypoacylated lipid A and of a highly charged core oligosaccharide. In vitro studies revealed a weak ability to engage the MD-2/TLR4 pathway, while it was able to promote TLR2-mediated response.

lipid A, lipopolysaccharide (LPS), gut microbiota, Bacteroides thetaiotaomicron, commensal LPS, structure to function

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@@H](O[C@H]2[C@@H](C(=O)O)O[C@H](O[C@@H]3[C@H](O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](OP(=O)(O)O)[C@@H]4O[C@H]4O[C@H](CO[C@H]5O[C@H](CO)[C@@H](O[C@H]6O[C@H](CO)[C@@H](O)[C@H](O)[C@H]6O)[C@H](O)[C@H]5N)[C@@H](O)[C@H](O)[C@@H]4O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O[C@H]4O[C@H](C(=O)O)[C@@H](O)[C@H](O)[C@H]4O)[C@@H](O[C@@H]4[C@H](OP(=O)(O)OCCN)C[C@](OC[C@H]5O[C@@H](OC[C@H]6O[C@H](OP(=O)(O)O)[C@H](N)[C@@H](O)[C@@H]6O)[C@H](N)[C@@H](O)[C@@H]5O)(C(=O)O)O[C@@H]4[C@H](O)CO)O[C@H](CO)[C@H]3O)[C@H](O)[C@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@@H]1O[C@H](CO[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H]1NC(C)=O

3222.574 g/mol (C₁₁₀H₁₈₇N₆O₉₆P₃)

Bacteroides thetaiotaomicron is one of the most extensively studied symbionts of the human gut. Despite its widespread distribution among human populations, still very little is known about the role of its cell envelope in the crosstalk with the immune system. Due to the extraordinary characteristic of B. thetaiotaomicron to express multiple capsular polysaccharides on its surface, research activities focused on defining how these polymers affect immune responses. This resulted in the drawback of neglecting another immunostimulatory cell surface glycoconjugate, the lipopolysaccharide (LPS). By taking advantage of an acapsular mutant of B. thetaiotaomicron, here we describe the characterization of the structure of the rough-type LPS produced by this gut mutualist. This was made up of a mono-phosphorylated and hypoacylated lipid A and of a highly charged core oligosaccharide. In vitro studies revealed a weak ability to engage the MD-2/TLR4 pathway, while it was able to promote TLR2-mediated response.

lipid A, lipopolysaccharide (LPS), gut microbiota, Bacteroides thetaiotaomicron, commensal LPS, structure to function

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCCCCCCCCCCCC[C@@H](O)CC(=O)O[C@H]1[C@H](O)[C@@H](CO[C@]2(C(=O)O)C[C@@H](OP(=O)(O)OCCN)[C@@H](O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O[C@H]4O[C@H](C(=O)O)[C@H](O[C@H]5O[C@H](CO)[C@@H](O)[C@H](O[C@@H]6O[C@H](CO[C@@H]7O[C@H](CO)[C@@H](O)[C@H](O)[C@H]7O)[C@H](O)[C@H](O[C@@H]7O[C@H](CO)[C@H](O)[C@H](O)[C@H]7O)[C@H]6NC(C)=O)[C@H]5NC(C)=O)[C@H](O)[C@H]4O)[C@@H]3O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](OP(=O)(O)O)[C@@H]3O[C@H]3O[C@H](CO[C@H]4O[C@H](CO)[C@@H](O[C@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4N)[C@@H](O)[C@H](O)[C@@H]3O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O[C@H]3O[C@H](C(=O)O)[C@@H](O)[C@H](O)[C@H]3O)[C@@H]([C@H](O)CO)O2)O[C@@H](OC[C@H]2O[C@H](OP(=O)(O)O)[C@H](NC(=O)C[C@H](O)CCCCCCCCCCCC(C)C)[C@@H](OC(=O)C[C@H](O)CCCCCCCCCCCCC)[C@@H]2O)[C@@H]1NC(=O)C[C@@H](CCCCCCCCCCCC(C)C)OC(=O)CCCCCCCCCCCC(C)C

4492.672 g/mol (C₁₉₁H₃₃₉N₆O₁₀₅P₃)