Structural characterisation of the capsular polysaccharide expressed by Burkholderia thailandensis strain E555:: wbiI (pKnock-KmR) and assessment of the significance of the 2-O-acetyl group in immune protection

Marc Bayliss,Matthew I Donaldson,Sergey A Nepogodiev,Giulia Pergolizzi,Andrew E Scott,Nicholas J Harmer,Robert A Field,Joann L Prior

doi:10.1016/j.carres.2017.09.011

Abstract

Burkholderia pseudomallei and its close relative B. mallei are human pathogens that are classified as Tier 1 bio-threat agents. Both organisms have previously been shown to constitutively produce a capsular polysaccharide (CPS) that is both a virulence determinant and protective antigen. Extraction and purification of CPS for use as a potential vaccine candidate requires containment level 3 laboratories which is expensive and time-consuming. B. thailandensis strain E555 is closely related to B. pseudomallei and B. mallei, but is non-pathogenic to humans and based on immunological cross-reactivity has previously been shown to express a B. pseudomallei-like CPS. In this study, capsular polysaccharide isolated from an O-antigen deficient strain of B. thailandensis E555 was identified by 1H and 13C NMR spectroscopy as -3-)-2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(-1, and identical to that produced by B. pseudomallei. This was further substantiated by anti-CPS monoclonal antibody binding. In connection with the production of CPS fragments for use in glycoconjugate vaccines, we set out to assess the importance or otherwise of the CPS 2-OAc groups in immune protection. To this end conjugates of the native and de-O-acetylated CPS with the Hc fragment of tetanus toxin (TetHc) were used as vaccines in a mouse model of melioidosis. The level of protection provided by deacetylated CPS was significantly lower than that from native, acetylated CPS. In addition, sera from mice vaccinated with the deacetylated CPS conjugate did not recognise native CPS. This suggests that CPS extracted from B. thailandensis can be used as antigen and that the acetyl group is essential for protection.

Highlights

An additional spin system present in capsular polysaccharide (CPS) 1H NMR spectra can be identified as a set of signals belonging to a-1,3-mannan, a polymer which is known to be produced by B. pseudomallei and B. mallei strains and is present in purified B. thailandensis CPS [9]
The manno-heptose-based CPS expressed by B. pseudomallei and B. mallei has shown to be partially protective in animal models of melioidosis, as have anti-CPS monoclonal antibodies in passive transfer studies [13,14]
As B. pseudomallei and B. mallei are classified by the Advisory Committee on Dangerous Pathogens (ACDP) as hazard group 3 pathogens, extraction of CPS in containment level 3 laboratories is required: this is expensive and time consuming, several studies have described the extraction of CPS from non-pathogenic, select-agent excluded strains of B. pseudomallei [16,17]

Summary

Introduction

B. mallei, a close relative of B. pseudomallei, is the etiologic agent of glanders, a disease primarily of horses and related solipeds, where it is associated with a high mortality rate without antibiotic therapy [6]. B. pseudomallei and B. mallei are classified as Tier 1 select agents by the Department of Health and Human Services [8]; no vaccines are available for either melioidosis or glanders. Both B. pseudomallei and B. mallei organisms produce multiple cell surface glycans, one of which is common to both species; capsular polysaccharide (CPS) with the repeating homopolymer structure based on -3)-2-O-acetyl-6-deoxy-b-D-manno-heptopyranose-(1- [9]. Several studies have utilised the 6-deoxyheptan CPS extracted from non-pathogenic, select-agent excluded strains of B. pseudomallei [16,17]

Methods

Results

Conclusion