Elsevier

Carbohydrate Research

Volume 346, Issue 13, 27 September 2011, Pages 1885-1897
Carbohydrate Research

Expression of a new disialyllacto structure in the lipopolysaccharide of non-typeable Haemophilus influenzae

https://doi.org/10.1016/j.carres.2011.05.020Get rights and content

Abstract

The structure of lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strains 1008 and 1247 has been investigated by mass spectrometry and NMR analyses on O-deacylated LPS and core oligosaccharide material. Both strains express the conserved triheptosyl inner core, [l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-Lipid A] with PCho→6)-β-d-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses the common structural motifs of H. influenzae; globotetraose [β-d-GalpNAc-(1→3)-α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and its truncated versions globoside [α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and lactose [β-d-Galp-(1→4)-β-d-Glcp-(1→] linked to the terminal heptose of the inner core and GlcI. A genetically distinct NTHi strain, 1008, expresses identical structures to strain 1247 with the exception that it lacks GalNAc. A lpsA mutant of strain 1247 expressed LPS of reduced complexity that facilitated unambiguous structural determination of the oligosaccharide from HepI. By CE-ESI-MS/MS we identified disialylated glycoforms indicating disialyllactose [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-β-d-Gal-(1→4)-β-d-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.

Highlights

► We determined the LPS structures of two non-typeable H. influenzae strains. ► The proximal and the distal inner core heptoses were found to be elongated. ► NTHi strain 1008 contains globoside elongations from two sites of the inner core. ► Globotetraose and globoside were found in the NTHi strain 1247. ► The NTHi 1247lpsA mutant indicated disialyllactose to extend from GlcI.

Introduction

The Gram-negative bacterium Haemophilus influenzae colonizes the nasopharynx and to a lesser extent the trachea and bronchi of human. H. influenzae exists in encapsulated and unencapsulated (non-typeable) forms. Non-typeable H. influenzae (NTHi) is a common cause of otitis media as well as acute and chronic lower respiratory tract infections.1, 2 A major virulence factor of H. influenzae is the outer surface expressed lipopolysaccharide (LPS):3 since it lacks the O-polysaccharide it is sometimes referred to as lipooligosaccharide. The LPS of H. influenzae consists of a conserved glucose substituted tri-heptosyl-moiety namely l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp linked to lipid A via a single α-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-4-phosphate. Each of the heptosyl residues can provide a point of further attachment of oligosaccharide and non-carbohydrate substituents, such as phosphocholine (PCho), glycine (Gly) and O-acetyl groups (Ac).4 The outer core region of NTHi LPS mimics host glycolipids and common structural motifs are globotetraose [β-d-GalNAcp-(1→3)-α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and its truncated versions thereof, globoside [α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and lactose [β-d-Galp-(1→4)-β-d-Glcp-(1→] as well as sialylactose [α-Neu5Ac-(2→3)−β-d-Galp-(1→4)-β-d-Glcp-(1→].4 Decoration of the LPS with N-acetyl-neuraminic acid (sialic acid, Neu5Ac) is an important factor for pathogenic behaviour of the bacterium in vivo.5, 6

Our previous studies have focused on the conservation and variability of patterns of LPS expressed in a representative set of NTHi clinical isolates obtained from otitis media patients, and relating this to the role of LPS in commensal and virulence behaviour. In this study we present LPS structures expressed in NTHi strains 1247 and 1008. We demonstrate that the two genetically distinct strains have almost identical LPS structures, the only difference being the absence of β-d-GalpNAc groups in strain 1008. Using a genetically defined isogenic mutant of strain NTHi strain 1247, 1247lpsA, which had oligosaccharide extensions from the proximal heptose (HepI) only, allowed us to identify disialyllactose linked to the first glucose (GlcI) attached to HepI. This is the first time that disialyllactose has been detected for H. influenzae in that molecular environment.

Section snippets

Results

NTHi strains 1247 and 1008 are clinical isolates of distinct ribotypes originating from otitis media. A lpsA mutant of NTHi strain 1247, predicted to lack all elongation from the distal heptose (HepIII), was constructed in order to facilitate detailed structural analysis of the simplified LPS molecule.7 The two NTHi wild type strains and the 1247lpsA mutant were grown in liquid media and the LPS was isolated by extraction using the phenol/chloroform/light petroleum method.

In earlier

Discussion

Studying the role of H. influenzae LPS in health and disease is part of our ongoing research and has focused upon the systematic structural elucidation of LPS from 25 representative NTHi isolates. These strains were obtained from infants with otitis media in Finland.17 Two of these clinical isolates are NTHi strains 1247 and 1008, strains that are not closely related by ribotyping or gene sequence analyses.17

LPS from both strains contains the triheptosyl inner-core moiety l-α-d-Hepp-(1→2)-[P

Bacterial cultivation and preparation of LPS

NTHi strains 1008 and 1247 are clinical isolates from infants with otitis media that originated from a pneumococcal vaccine study in Finland.17 Bacteria were cultured at 37 °C in brain–heart infusion broth supplemented with haemin (10 μg ml−1) and nicotinamide adenine dinucleotide (2 μg ml−1), then were lyophilized and the LPS was extracted by using phenol-chloroform-light petroleum, as described by Galanos et al.25 with the modification of LPS precipitation using diethyl ether-acetone (1:5, v/v; 6 

Acknowledgements

We thank the members of the Finnish Otitis Media Study group at the National Health Institute in Finland for the provision of NTHi nasopharyngeal isolates. Sofia J. Gulin is acknowledged for technical assistance. The Swedish NMR Centre is gratefully acknowledged for giving access to their facilities for the investigation of NTHi OS 1247. This project was supported by a grant from the Swedish Research Council (EKHS). DWH and ERM were supported by the Medical Research Council, UK.

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  • Present address: Molecular Infectious Diseases Group, University of Oxford Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford 0X3 9DU, UK.

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