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Results: 4

1.
Figure 4

Figure 4. Proposed pathway for Bacteroides sphingolipid biosynthesis.. From: Production of ?-Galactosylceramide by a Prominent Member of the Human Gut Microbiota.

BF2461, a putative serine palmitoyltransferase, would catalyze the pyridoxal-phosphate-dependent conjugation of serine and a long-chain acyl-CoA to form 3-ketodihydrosphingosine, which would undergo a ketoreductase-catalyzed conversion to dihydrosphingosine. At this branchpoint, dihydrosphingosine could either be phosphorylated by the putative sphingosine kinase BF2462 to form S1P, or it could undergo N-acylation to yield the observed dihydroceramide intermediate (compound 2). This common C34 scaffold would then be the substrate for two alternative head group modifications: glycosylation to form α-GalCerBf, or phosphorylethanolamine group transfer to form CPE.

Laura C. Wieland Brown, et al. PLoS Biol. 2013 July;11(7):e1001610.
2.
Figure 1

Figure 1. Chemical structures of the B. fragilis sphingolipids and related molecules.. From: Production of ?-Galactosylceramide by a Prominent Member of the Human Gut Microbiota.

(A) B. fragilis produces the phosphosphingolipid ceramide phosphoryl-ethanolamine (CPE, top) and the corresponding free ceramide (ceramideBf, middle), which are similar in structure to the most abundant (4,5-dehydro) and third-most abundant (4,5-dihydro) forms of sphingomyelin in human plasma (bottom). (B) B. fragilis produces the glycosphingolipid α-galactosylceramide (α-GalCerBf, top), which is similar in structure to the sponge-derived α-galactosylceramide agelasphin-9b (middle) and a widely used derivative of agelasphin-9b, KRN7000 (bottom). Chemical groups that vary among the molecules in each column are colored red and blue for B. fragilis and non–B. fragilis sphingolipids, respectively. CPE, ceramideBf, and α-GalCerBf were each purified as inseparable mixtures of varying lipid chain length. The proposed structures of the most abundant species are shown here.

Laura C. Wieland Brown, et al. PLoS Biol. 2013 July;11(7):e1001610.
3.
Figure 2

Figure 2. B. fragilis Δ2461 is deficient in the production of sphingolipids.. From: Production of ?-Galactosylceramide by a Prominent Member of the Human Gut Microbiota.

HPLC-MS traces of crude lipid extracts of (A) wild-type B. fragilis and (B) the sphingolipid-deficient mutant ΔBF2461 are shown. The traces shown are the total ion count (black) and the extracted ion traces of sphingolipid masses for ceramide (m/z [M-H]: 540.5, 554.5, 568.5, 582.6; green), CPE (m/z [M-H]: 663.5, 677.5, 691.5, 705.5; brown), α-GalCerBf (m/z [M-H]: 702.6, 716.6, 730.6, 744.6; blue), and phosphatidylethanolamine (m/z [M-H]: 648.5, 662.5, 676.5, 690.5). Peaks corresponding to the three sphingolipids, but not the phospholipid phosphatidylethanolamine, are absent in B. fragilis Δ2461. (C) High-resolution mass spectra of CPE, ceramideBf, and α-GalCerBf collected in the negative ion mode. The insets show a zoomed-in view of the dominant field of peaks for each compound. (D) A table showing the calculated and observed masses for the dominant mass ions for each compound. See S1.1 in Supporting Information S1 for details.

Laura C. Wieland Brown, et al. PLoS Biol. 2013 July;11(7):e1001610.
4.
Figure 3

Figure 3. α-GalCerBf binds CD1d and activates NKT cells.. From: Production of ?-Galactosylceramide by a Prominent Member of the Human Gut Microbiota.

(A) Hybridomas were stained with anti-CD3 antibodies and empty mCD1d tetramers or CD1d tetramers loaded with α-GalCerBf or KRN7000. Flow cytometry plots are pregated on DAPI events in lymphocyte gate stained with CD3 antibodies and the specified tetramer. Plots representative of three independent experiments are shown. (B) Hybridomas were cultured with BMDCs pre-pulsed with LPS or LPS + α-GalCerBf in the presence of control Ig or anti-CD1d blocking antibodies. IL-2 secretion was measured in supernatants 16 h later. Data are representative of three independent experiments. (C) Plates were coated with CD1d monomers and loaded with the specified amounts of α-GalCerBf. Hybridomas were then incubated for 16–18 h and IL-2 was measured in the supernatants by ELISA. Data are representative of three independent experiments. (D) Liver mononuclear cells were cultured with splenocytes plus increasing amounts of α-GalCerBf in the presence or absence of anti-CD1d blocking antibodies. IFN-γ secretion was measured in supernatants on day 5. Data are representative of three independent experiments. (E and F) Representative flow cytometry plots and pooled data of PBMCs cultured for 13–14 d with 0.1 µg/ml KRN7000, 1 µg/ml α-GalCerBf, or 1 µg/ml ceramideBf. Dot plots show all events in the lymphocyte gate stained with 6B11 (specific for Vα24) and CD3 antibodies. Gate shows percentage of Vα24+CD3+ NKT cells pre- and postexpansion. Pooled data showing six individual donors tested in three independent experiments. *p = 0.0078, **p = 0.0020 compared to control day 13 culture. (G–I) Bone-marrow-derived dendritic cells were pulsed in vitro with LPS only or LPS + α-GalCerBf for 24 h. The 0.4×106 cells were transferred to WT mice, which were treated with control Ig or anti-CD1d blocking antibody prior to cell transfer. Liver mononuclear cells were analyzed 16–18 h later. Data shown were pooled from three independent experiments. (G) Expression of CD25 and CD69 on gated CD3+tetramer+ cells. Representative flow cytometry plots and pooled data showing fold change of CD25 and CD69 surface expression compared to NKT cells isolated from mice transferred with LPS-pulsed BMDCs. (H) Representative flow cytometry plots and pooled data of intracellular IFN-γ expression on gated CD3+tetramer+ cells. (I) Serum IFN-γ levels.

Laura C. Wieland Brown, et al. PLoS Biol. 2013 July;11(7):e1001610.

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