Enzymatic Synthesis of an Undecorated Capsular Polysaccharide from Campylobacter jejuni

Abstract

The exterior surface of the human pathogen Campylobacter jejuni is coated with a capsular polysaccharide (CPS) that helps protect it from the host immune system. In C. jejuni NCTC 11168 the repeating linear polysaccharide is composed of d-ribose, N-acetyl-d-galactosamine and d-glucuronic acid that is further amidated with either ethanolamine or serinol. The CPS is also decorated with d-glycero-l-gluco-heptose and methyl phosphoramidate. We have now shown that the polymerization of the undecorated CPS requires the sequential activity of six unique enzymes that must act in concert with one another. The catalytic activity of these six enzymes enabled a robust synthetic strategy to be developed to facilitate the assembly and isolation of specific oligosaccharides of up to 10 units in length. Modifications to this strategy enabled the isolation of mixtures containing oligosaccharides containing at least 19 monomeric units. The oligosaccharides were isolated by anion exchange chromatography and chemically characterized using ESI mass spectrometry and ¹H NMR spectroscopy. These oligosaccharides will enable the reaction mechanisms for the decoration of the capsular polysaccharides to be determined and may facilitate the development of glycoconjugate vaccines.

1

Two repeating structural units in the capsular polysaccharide of C. jejuni NCTC 11168 (serotype HS:2). The repeating structural unit consists of d-ribose (highlighted in gray), N-acetyl-d-galactosamine (highlighted in orange), d-glucuronic acid (highlighted in yellow) and d-glycero-l-gluco-heptose (highlighted in blue). In structure 1 the glucuronic acid is amidated with serinol, whereas in structure 2 the amide is made from ethanolamine.

2

Gene cluster for the biosynthesis of the capsular polysaccharide in C. jejuni NCTC 11168 (serotype HS:2). The genes colored green are associated with the synthesis and transfer of the methyl phosphoramidate group to the polysaccharide chain. The genes colored blue are required for the synthesis and transfer of the d-glycero-l-gluco-heptose moiety to the capsular polysaccharide. The proteins encoded by the genes colored yellow are of unknown function. The salmon-colored gene are required for the biosynthesis of d-glucuronate, serinol-phosphate, and ethanolamine-phosphate. The gray colored gene is required for the conversion of UDP-NAc-d-galactopyranose to UDP-NAc-d-galactofuranose. The genes colored black are required for the polymerization of the CPS. Additional details are provided in the text.

3

Reaction scheme for the polymerization of d-ribose, d-GalfNAc, and d-GlcA during the biosynthesis of the CPS of C. jejuni, serotype HS:2. In this scheme we have shown the reaction catalyzed at the nonreducing end of the polymer by each enzyme in the cycle. Since three of these reactions (catalyzed by Cj1438_N, Cj1438, Cj1432_C, and Cj1432_N) are making the polymer longer, we have arbitrarily removed one sugar moiety from the reducing end of polymer for those steps to make the figure manageable.

4

Outline for the enzymatic synthesis of defined oligosaccharides containing d-ribose, d-GalfNAc, and the ethanolamine amide of d-GlcA. Additional details are found in the text.

5

ESI mass spectral data for the [M – H⁺]⁻ anions of the isolated oligomers 5, 8, and 10. (a) experimental data for oligomer 5; (b) calculated distribution for oligomer 5; (c) experimental data for oligomer 8; (d) calculated distribution for oligomer 8; (e) experimental data for oligomer 10; (f) calculated distribution for oligomer 10. Additional details are provided in the text.

6

¹H NMR spectra of isolated oligomers. (a) Oligomer 5; (b) oligomer 8; (c) oligomer 10. Shown in brackets are the relative integrations for the anomeric protons between 4.75 and 5.30 ppm, the –OMe group at ∼3.4 ppm and the –N-acetyl group at ∼1.9 ppm. Additional details are available in the text.

7

Reaction scheme for the generation of longer oligomeric products. Additional details are found in the text and in Figure .

8

ESI mass spectrometry of the unfractionated product mixture containing compounds 10 (decamer), 10–2 (tridecamer), 10–3 (hexadecamer), and 10–4 (nonadecamer). Additional details are provided in the text.

9

Expanded views of the isotopic clusters for the 13-mer (10–2), 16-mer (10–3), and 19-mer (10–4) oligomeric products identified in Figure . The measured and calculated mass spectra of oligomers 10–2 (a,b), 10–3 (c,d) and 10–4 (e,f).