In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz

Abstract

Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria/host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach which enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared N-Acetyl-D-galactosamine-diphospho-undecaprenyl, the E. coli O86 oligosaccharide repeating unit was assembled via sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy via a chaperone co-expression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of additional substrates revealed a defined mode of recognition for Wzy towards the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well-defined system for further underpinning molecular details of this biosynthetic pathway.

Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils

“…The roles played by K. pneumoniae CPS are varied and strain specific (23,25,26). A gene known as wzy is required for capsule biosynthesis in many species of bacteria, including K. pneumoniae (27)(28)(29). Previous studies performed with serotype K1 K. pneumoniae strains demonstrated that a wzy-like gene known as magA confers resistance to serum complement and phagocytosis (28,30,31).…”

Structure-Guided Investigation of Lipopolysaccharide O-Antigen Chain Length Regulators Reveals Regions Critical for Modal Length Control

“…This segment is disordered in all the available crystal structures of the chain length regulators (35). A recent reconstitution of the O-antigen polymerization in vitro demonstrates no need for participation of any proteins other than WzzB and Wzy to obtain O-antigen polymers with medium modal lengths (39). Wzy has a predicted large periplasmic loop (15,36).…”

Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein–Polysaccharide Interactions

“…Carbohydrate ligands become more flexible with increasing length, resulting in weak affinities challenging the understanding of how polysaccharides bind to proteins transiently. For example, in processes such as polysaccharide synthesis and hydrolysis 27 , chain length control 28 or transport 29 , short-lived polysaccharide binding may take place with rapid ligand off-rates. The computational approach fills the gap between the experimentally accessible descriptions of small oligosaccharides in protein binding sites and the need for models of large proteinpolysaccharide complexes.…”