Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers for use as haptens in glycoconjugate vaccines.

N Hegerle,M.M Levine,G Ramachandran,J.E Galen,S.M Tennant,R Simon,J Bose

doi:10.1111/jam.13772

N Hegerle, M.M Levine + Show 5 more

Open Access

https://doi.org/10.1111/jam.13772

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Abstract

AimsO‐polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram‐negative bacteria to produce homogenous O‐polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O‐polysaccharide chain length regulators of the Wzx‐/Wzy‐dependent pathway.Method and ResultsThe O‐polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram‐negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O‐polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa.ConclusionsOverexpression of Wzz proteins in Gram‐negative bacteria using the Wzx/Wzy‐dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O‐polysaccharide population of a defined size.Significance and Impact of the StudyThe methods presented herein represent a cost‐effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O‐polysaccharides in glycoconjugate vaccine development.

Highlights

Lipopolysaccharides (LPS) are the main constituents of the Gram-negative bacterial (GNB) outer membrane
Salmonella Typhimurium wzzB and fepE were cloned into pSEC10 downstream of the ompC promoter and confirmed by sequencing (Fig. 1a,b)
P. aeruginosa wzz2 was cloned into the pUCP19 plasmid downstream of the lac promoter and confirmed by sequencing (Fig. 1c). wzz2 expression was induced by the addition of 0Á1 mol lÀ1 Isopropyl b-D-1-thiogalactopyranoside (IPTG) to the growth medium

Summary

Introduction

Lipopolysaccharides (LPS) are the main constituents of the Gram-negative bacterial (GNB) outer membrane. The basic structure of LPS is comprised of three distinct units. The lipid A serves as a membrane anchor and possesses endotoxic properties. The core polysaccharide is covalently attached to lipid A and is mostly conserved within. The final component is the Opolysaccharide (OPS) that extends from the core and is a variable polymer of repeating saccharide units for which the chemical structure defines the associated bacterial serogroup (Erridge et al 2002). While most GNB (e.g. Salmonella enterica, Shigella flexneri, Pseudomonas aeruginosa, Klebsiella pneumoniae) possess OPS, some GNB (e.g. Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae) produce lipo-oligosaccharide or LOS, that is short and characterized by only a few saccharide repeats

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