Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid A

Abstract

Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.

Keywords: glycolipids; lipid A; lipopolysaccharides; oligosaccharides; vaccines.

Figure 1

Structural characterization of A. faecalis core OS and O‐antigen. a) Zoom of the superimposition of ¹H, ¹H,¹³C HSQC and ¹H,¹³C HMBC NMR spectra of the fully deacylated LOS from A. faecalis. Key inter‐residue long‐range correlations are indicated. Numbering of sugar residues is as reported in Table S1 and Figure 2. b) Structure of the core OS elucidated by NMR spectroscopy, reported using letters as indicated in Table S1 and Figure 2. c) Zoom of the superimposition of proton, HSQC and HMBC NMR spectra after mild acid hydrolysis performed on the LPS fraction. Key inter‐residue long‐range correlations are indicated as in Table S2. d) Structure of the main pentasaccharide repeating unit comprising the O‐antigen as deduced by NMR spectroscopy and reported using the letters in Table S2 and Figure 2.

Figure 2

Chemical structure of a) the LOS and b) the O‐antigen from A. faecalis. The letter labels used for NMR analysis are as reported in Tables S1 and S2 and Figure 1. N‐Acetyl groups were deduced by NMR spectroscopic investigation of the core OS product after mild acid hydrolysis.

Scheme 1

Synthesis of the hexaacylated Alcaligenes faecalis lipid A (hexa‐AfLA).

Figure 3

Hexaacylated A. faecalis lipid A (hexa‐AfLA) mediated NF‐κB activation in a) HEK‐Blue™ hTLR4 cells and b) HEK‐Blue™ Null2 cells was evaluated by a SEAP reporter assay. Hexa‐AfLA‐mediated cytokine induction activity in PMA‐differentiated THP‐1 cells was evaluated by ELISA: c) release of IL‐6, d) release of IL‐6 in the presence of E. coli LPS O111 (1 ng mL⁻¹). Results in (a–d) represent the mean±standard deviation of three independent experiments. *p<0.05, **p<0.005, ****p<0.0001 by Student's t‐test.