While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer(Bf)), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer(Bf) has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.

human gut microbiota, galactosylceramide

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12	C13	C14	C15	C16	C17
1	aDGalp	100.0	69.1	69.3	70.2	71.6	61.0
2	lR3HOiMar	170.8	44.5	67.9	37.0	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	38.9	27.9	22.9
	Subst	67.4	53.4	69.6	34.2	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	25.6-31.7	38.9	27.9	22.9


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9	H10	H11	H12	H13	H14	H15	H16	H17
1	aDGalp	4.64	3.46-3.50	3.65-3.68	3.50-3.53	3.55-3.59	3.37-3.48
2	lR3HOiMar	-	2.17	3.73-3.79	1.25-1.33	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.06-1.14	1.42-1.52	0.82
	Subst	3.51-3.56	3.69-3.73	3.42-3.45	1.18-1.43	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.17-1.24	1.06-1.14	1.42-1.52	0.82


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9	C10/H10	C11/H11	C12/H12	C13/H13	C14/H14	C15/H15	C16/H16	C17/H17
1	aDGalp	100.0/4.64	69.1/3.46-3.50	69.3/3.65-3.68	70.2/3.50-3.53	71.6/3.55-3.59	61.0/3.37-3.48
2	lR3HOiMar		44.5/2.17	67.9/3.73-3.79	37.0/1.25-1.33	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	38.9/1.06-1.14	27.9/1.42-1.52	22.9/0.82
	Subst	67.4/3.51-3.56	53.4/3.69-3.73	69.6/3.42-3.45	34.2/1.18-1.43	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	25.6-31.7/1.17-1.24	38.9/1.06-1.14	27.9/1.42-1.52	22.9/0.82

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(C)CCCCCCCCCCC[C@@H](O)CC(=O)N[C@@H](CO[C@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O)[C@H](O)CCCCCCCCCCCC(C)C

718.07 g/mol (C₄₀H₇₉NO₉)